Archive for the ‘Crispr’ Category

There are different types of biotechnology protocols for genome/gene editing (GE), but the preferred one is the Clustered Regularly Interspaced Short Palynodromic Repeat (CRISPR) Cas9 system. Advantages include precision, the ability to design variants tailored to needs, and optimal operational cost and time.

CRISPR was first identified in 1987 as a bacterial defence system against viral infections. The Nobel Prize-winning research (Chemistry, 2020) of Jennifer Doudna and Emmanuelle Charpentier (2012) provided a novel translational biotech protocol to precisely target and cleave a specific DNA sequence in any living organism, facilitating its transfer to any other living organism.

Since then, CRISPR-edited biotech applications have revolutionised human welfare initiatives in healthcare (new medicines, treatment of viral infections, clinical diagnostics, cure of inherited genetic disorders such as retinal degeneration, boosting immunity, powerful DNA vaccines, etc.), agriculture (crop and livestock transformation for high yield, pests/parasite eradication), energy (bio-fuels), biodiversity conservation, and climate change. However, CRISPR applications in human reproductive biology could pose serious threats to human identity and social order if not handled with prudence.

The scope of CRISPR-edited biotech applications demonstrates that the Biological Cellular Universe/Space is as enchanting and intellectually challenging as the external universe/space, contributing to our knowledge base and driving global industrial markets (corporate science) for socio-economic development. Yet, it has remained in the shadows of the physical sciences dominated technology ecosystem.

Interestingly, a publication database survey by P D Ramos and others in 2023 revealed low awareness of the impact of CRISPR among the general public. Communicating the importance of CRISPR and other global techno-science developments to stakeholders should be prioritised.

Increasing CRISPR capabilities in human reproductive biology have ignited intense ethical debates across the globe. The 2018 reports of the birth of a CRISPR-edited human child jolted and compelled intellectuals to take serious note of the adverse impact on biological human identity and ethical practices. A new era of bio-politics is upon us.

Ethical challenges: Techno-science developments such as AI, CRISPR, and their regulatory frameworks are in a perpetual arms race, with the latter more often lagging. The chances of maverick scientists attempting to transform human embryos with malicious intentions should be anticipated and prevented. The apprehensions of Oppenheimer, Neil Bohr, and Fermi (their discoveries in nuclear fission led to the making of an atomic bomb) are equally applicable to CRISPR-edited applications, including human babies.

At the heart of the ethical landscape is the precautionary principle invoked since 1970 for environmental threats due to genetically modified crop plants. Agricultural biotechnologists and environmentalists are engaged in heated debates about whether the principle is being applied excessively, stifling innovation and delaying human welfare, or whether it should be strictly enforced until all the safety concerns are addressed.Notwithstanding, 72 countries have accepted biotech crops, including the United States (71.5 million hectares), Brazil (52.8 million hectares), Argentina (24.0 million hectares), Canada (12.5 million hectares), and India (11.9 million hectares). The global agricultural biotechnology market is estimated to reach $110 billion by 2030.

The theoretical trajectories of ongoing research at the MIT Centre for Brains, Minds, and Machines on human brain-AI interfaces and the CRISPR-edited birth of a human baby seem to converge on the development of a human-like robot or robot-like human, with potential performance competencies surpassing human cognitive abilities. If realised, it would be one of the greatest ethical threats that humanity has ever faced.

Science philosopher and visionary C P Snow, in his book, The Two Cultures and the Science Revolution, called upon humanist studies to counter the technologico-Benthamite culture, which misuses science in ways that cheapen, impoverish, and dehumanise life. According to Yoshua Bengio, extreme governance measures would be needed to prevent misadventures in AI. This is also true for CRISPR or any other technology with a potential threat to ethical practices.

Regulatory Landscape: WHO is set to create a Global Regulatory Framework to verify the science behind CRISPR applications and to regulate human gene editing. In the US, the Food and Drug Administration regulates gene therapy products through a risk-based approach, evaluating each therapy on a case-by-case basis to ensure that it meets rigorous safety standards. The European Medicines Agency and the National Medical Products Administration in China have established similar frameworks. Argentina, Canada, Brazil, Australia, South Africa, and Japan have also taken similar initiatives; a global consensus remains elusive.

In India, genetically modified organisms and their products are regulated by the Environment (Protection) Act (1986) and the Rules for the manufacture, use, import, export, and storage of hazardous microorganisms, genetically engineered organisms, or cells (1989).

Harnessing the benefits of the techno-science revolution would be a meaningful mission only when its inherent fundamentalism is dealt with strongly by a robust policy framework with accountability and self-regulation legally assigned to all stakeholders. (The writer is a former professor and registrar, Bangalore University)

(The writer is a formerprofessor and registrar, Bangalore University)

Published 17 July 2024, 20:39 IST

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Ethics and benefits of gene editing - Deccan Herald

Type VI CRISPR-Cas systems, of which Cas13 is the defining member, provide adaptive immunity in prokaryotes by targeting RNA transcripts of invading mobile genetic elements. Cas13-mediated RNA targeting occurs through the action of a Cas13 protein and its CRISPR RNA (crRNA), which function together as an RNA-guided ribonuclease.

All Cas13s possess two higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains; these have been shown to dimerise intramolecularly to form the active site in response to target-transcript recognition.

HEPN domains, which are not exclusive to Cas13, are known for their high diversity and poor sequence conservation, which impedes standard homology searches and makes evolutionary analyses challenging. As a result, compared to other widely study Cas endonucleases, e.g., Cas9 and Cas12, few distinct Cas13 subtypes have been identified to date, and their evolutionary origins remain poorly understood.

To address those challenges, a team led by Nobel Laureate Jennifer Doudna built an automated structural-search pipeline that combines the speed of machine learning-based search methods with the sensitivity of traditional structure alignment programmes. Using this pipeline, they identified an ancestral clade of Cas13 (Cas13an) and traced Cas13 origins to defence-associated ribonucleases (1).

The team shows that in spite of its small size compared to other Cas13s, Cas13an can selectively target green fluorescent protein (GFP) transcripts when co-expressed with GFP-targeting crRNAs in E. coli, and that the HEPN domain is essential for this activity.

Cas13an also provided defence against diverse bacteriophages when co-expressed with selected phage-targeting crRNAs in E. coli. The study revealed that Cas13an employs a single active site for both CRISPR RNA processing and RNA-guided cleavage in contrast to the larger Cas13s, revealing two modes of activity for the ancestral nuclease domain.

The findings, which were published in Science today, add to the current understanding of CRISPR-Cas evolution and should create new opportunities for precise RNA editing.

References

1. P. H. Yoon et al., Science 10.1126/science.adq0553 (2024).

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Structure-guided discovery reveals ancient clade of Cas13 ribonucleases - CRISPR Medicine News

Prosperity Consulting Group LLC purchased a new position in CRISPR Therapeutics AG (NASDAQ:CRSP Free Report) during the 1st quarter, according to its most recent Form 13F filing with the Securities and Exchange Commission (SEC). The firm purchased 2,966 shares of the companys stock, valued at approximately $202,000.

Several other large investors also recently bought and sold shares of the business. ARK Investment Management LLC boosted its holdings in shares of CRISPR Therapeutics by 19.2% during the 4th quarter. ARK Investment Management LLC now owns 8,536,104 shares of the companys stock worth $534,360,000 after purchasing an additional 1,372,986 shares during the last quarter. Norges Bank acquired a new position in CRISPR Therapeutics during the fourth quarter worth $38,661,000. Vestmark Advisory Solutions Inc. acquired a new position in CRISPR Therapeutics during the fourth quarter worth $10,848,000. PBCay One RSC Ltd purchased a new stake in CRISPR Therapeutics in the 4th quarter valued at $10,329,000. Finally, Trexquant Investment LP acquired a new stake in shares of CRISPR Therapeutics in the 4th quarter worth $8,287,000. Hedge funds and other institutional investors own 69.20% of the companys stock.

A number of brokerages recently issued reports on CRSP. Cantor Fitzgerald reissued a neutral rating on shares of CRISPR Therapeutics in a report on Thursday, May 9th. Piper Sandler restated an overweight rating and set a $105.00 price target on shares of CRISPR Therapeutics in a report on Monday, June 17th. Wells Fargo & Company decreased their price objective on CRISPR Therapeutics from $70.00 to $65.00 and set an equal weight rating on the stock in a report on Thursday, May 9th. JMP Securities reissued a market outperform rating and set a $86.00 target price on shares of CRISPR Therapeutics in a research note on Thursday, May 9th. Finally, Robert W. Baird lifted their target price on CRISPR Therapeutics from $46.00 to $52.00 and gave the company a neutral rating in a research report on Thursday, May 9th. Three research analysts have rated the stock with a sell rating, eight have issued a hold rating and eight have assigned a buy rating to the company. According to data from MarketBeat, the company currently has a consensus rating of Hold and an average price target of $75.71.

In other news, COO Julianne Bruno sold 3,366 shares of the businesss stock in a transaction dated Friday, June 21st. The stock was sold at an average price of $56.09, for a total value of $188,798.94. Following the completion of the sale, the chief operating officer now directly owns 6,745 shares of the companys stock, valued at approximately $378,327.05. The transaction was disclosed in a document filed with the Securities & Exchange Commission, which can be accessed through the SEC website. In other news, COO Julianne Bruno sold 3,366 shares of the firms stock in a transaction that occurred on Friday, June 21st. The shares were sold at an average price of $56.09, for a total transaction of $188,798.94. Following the transaction, the chief operating officer now directly owns 6,745 shares in the company, valued at $378,327.05. The sale was disclosed in a legal filing with the SEC, which is accessible through this hyperlink. Also, CEO Samarth Kulkarni sold 19,582 shares of the companys stock in a transaction on Monday, April 15th. The shares were sold at an average price of $59.91, for a total transaction of $1,173,157.62. Following the completion of the sale, the chief executive officer now owns 208,122 shares of the companys stock, valued at approximately $12,468,589.02. The disclosure for this sale can be found here. 4.10% of the stock is currently owned by insiders.

CRSP stock opened at $53.10 on Friday. The business has a 50 day moving average price of $56.57 and a 200 day moving average price of $64.18. The stock has a market cap of $4.51 billion, a price-to-earnings ratio of -19.52 and a beta of 1.70. CRISPR Therapeutics AG has a fifty-two week low of $37.55 and a fifty-two week high of $91.10.

CRISPR Therapeutics (NASDAQ:CRSP Get Free Report) last issued its quarterly earnings data on Wednesday, May 8th. The company reported ($1.43) earnings per share for the quarter, missing the consensus estimate of ($1.35) by ($0.08). The firm had revenue of $0.50 million for the quarter, compared to analyst estimates of $25.53 million. The companys revenue was down 99.5% on a year-over-year basis. During the same period in the previous year, the company earned ($0.67) EPS. As a group, sell-side analysts anticipate that CRISPR Therapeutics AG will post -5.51 EPS for the current year.

(Free Report)

CRISPR Therapeutics is a gene-editing company focused on developing transformative gene-based medicines for serious diseases using its proprietary CRISPR/Cas9 platform. CRISPR/Cas9 is a revolutionary gene-editing technology that allows for precise, directed changes to genomic DNA. CRISPR Therapeutics has established a portfolio of therapeutic programs across a broad range of disease areas including hemoglobinopathies, oncology, regenerative medicine and rare diseases.

Want to see what other hedge funds are holding CRSP? Visit HoldingsChannel.com to get the latest 13F filings and insider trades for CRISPR Therapeutics AG (NASDAQ:CRSP Free Report).

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Prosperity Consulting Group LLC Invests $202000 in CRISPR Therapeutics AG (NASDAQ:CRSP) - Defense World

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Tracking-seq reveals the heterogeneity of off-target effects in CRISPRCas9-mediated genome editing - Nature.com

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Mice

All mice were housed at the China Agricultural University Laboratory Animals Resource Center, where they were subjected to a standard lightdark cycle of 12h each, and maintained at a temperature of 2022C. All animal experiments were conducted in Sen Wus laboratory and were approved by the Institutional Animal Care and Use Committee of China Agricultural University (Approval Number: SKLAB-2012-11). We have complied with all relevant ethical regulations for animal use. The 5 weeks old female ICR mice were procured from Beijing Vital River Laboratory Animal Technology.

The OG cell lines were maintained on feeder cells using a basic serum/LIF medium. This medium comprised DMEM (Gibco, 10829018), 15% FBS (Gibco, 10099), 1% penicillin/streptomycin (Gibco, 15104122), 1% non-essential amino acids (Gibco, 11140050), 1% GlutaMAX (Gibco, 35050079), 106 units/L of mouse LIF (Millipore, ESG1106) and 100mM -mercaptoethanol (Gibco, 15104122). The feeder cells were treated with mitomycin C (Amresco, MJ594) to inhibit their growth and were cultured in DMEM (Gibco, 11960) supplemented with 10% FBS (Gibco, 10099), 1% penicillin/streptomycin (Gibco, 15104122), 1% non-essential amino acids (Gibco, 11140050) and 1% sodium pyruvate (Gibco, 15104122). Other small molecules mentioned in our article were procured from Selleck and added individually to the basal serum/LIF medium at varying concentrations. Subculturing of all cell lines was performed every 23 days at a ratio ranging from 1:6 to 1:10 using Tryple (Gibco, 12605028). To establish the MERVL-tdTomato reporter cell lines, the OG cell lines underwent transfection via electroporation and were subsequently selected using 1g/ml puromycin for 7 days. Clones were then isolated and confirmed through polymerase chain reaction (PCR) analysis. All cell lines were maintained in a humidified incubator at 37C with 5% CO2.

The CRISPR-Cas9 DNA library contained 130,209 sgRNAs about 20,611 genes constructed by our laboratory. To obtain the mutant cell library, we transfected 108 OG cells with the MERVL reporter by electroporation (2B Nucleofector System, Lonza). After 24h transfection, the cells were selected with 350ng/l G418 (InvivoGen, ant-gn-5) for 7 days. We expanded these cells to 3108 for primary screen. We collected 2C-positive cells using FACS and amplified them for consecutive screen. Each round of screening was repeated three times. Medium needed to be changed every day.

Genomic DNA were extracted from 108 cells of the mutant cell pool and 510106 cells of 2C-postive from the consecutive screen. The integrated sgRNA sequences were amplified by PCR using Gotaq DNA Polymerase (Mei5bio, MF002) with the left primer (5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNTGAAAGTATTTCGATTTCTTGG-3) and the right primer (5- CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTTGATAACGGACTAGCCTTATT-3). Then PCR products were purified and sequenced by Illumina HiSeq TM4000. We used MAGeCK package to analyze our screening results and took Z-score into consideration. Ggplot2 was used to summarize these results.

To generate CRISPR KO cells, we designed two sgRNAs for each target gene and cloned them into the pSg6 Plasmid. Subsequently, we transfected 106 mESCs using the Lonza 2B Nucleofector System. Twenty-four hours after transfection, cells were subjected to selection with 350ng/l G418 for a duration of 7 days. Following selection, individual clones were isolated into a 48-well plate, and their targeted regions were verified. Detailed sequences of the sgRNAs can be found in TableS1.

Total RNA was extracted to amplify the CDS of Mdm2 by PCR. Subsequently, the amplified fragment was cloned into the PB vector under the control of the EF1 promoter. A total of 4g of the vector was transfected into 106 mESCs using the Lonza 2B Nucleofector System. Following transfection, mESCs were cultured for 24h and then subjected to selection with 350ng/l G418 for a period of 7 days. Clones were subsequently isolated and expanded in a 48-well plate to allow for the extraction of RNA and protein.

Total RNA was extracted from cells using the RaPure Total RNA Kits (Magene, R4011) following the manufacturers protocol. Subsequently, 1g of RNA was reverse transcribed into cDNA using the ABScript III RT Master Mix (Abclonal, RK20429). qPCR was conducted using the 2x RealStar Green Power Mix (Genestar, A311-10) on a Roche PCR machine. The relative quantification of each gene was achieved by normalizing to Gapdh. A complete list of primers utilized for qPCR can be found in TableS2.

Total RNA was purified using magnetic beads with Oligo (dT) to selectively isolate mRNA. RNA-seq libraries were subsequently constructed and assessed using a combination of TIANGEN Biotech and the Agilent 2100 BioAnalyzer. The sequencing process generated 150bp paired-end reads using PE150 on an Illumina platform. To analyze the data, clean reads were mapped to the Mus musculus genome using HISAT2. Read counts were quantified using HTSeq-count (v0.6.0) and then normalized to obtain the Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) values. Differentially expressed genes (DEGs) were identified using edgeR with criteria including an absolute log2 (fold change)>2 and a p value<0.05. Subsequently, the datasets were further analyzed using the R programming language.

Cell sorting and analysis were carried out using the FACS CaliburTM flow cytometer (BD, San Jose, CA, USA). Data visualization was conducted using FlowJo software version 10. For our gating strategy, we employed wild-type (WT) mESCs with the MERVL reporter.

Collect 293T cells cultured in 10cm culture dishes using 1mL of IP lysis buffer (Beyotime, P0037), and transfer them to 1.5mL centrifuge tubes. Let them lyse on ice for 30min. After centrifugation at maximum speed (4C, 21,100g, 10min), collect the supernatant for subsequent Co-IP experiments. Supernatants were incubated with Pierce beads (Thermo Scientific, 88802) for 68h at 4C with rotation. Antibodies used include FLAG (Sigma, F1804, 1:200 mouse) and HA (Beyotime, AH158, 1:200 mouse), IgG (Beyotime, A7028, 1:200 mouse). The protein solution collected was denatured at 95C for 10min using 10% SDSPAGE for western blot analysis.

The cells were lysed using IP lysis buffer supplemented with 100 PMSF and incubated on ice for 30min. The supernatant was obtained by centrifuging at 4C and 20,000g for 15min. To determine protein concentration, we utilized the BCA protein assay kit (Beyotime, P0012) according to the manufacturers instructions. Equal amounts of protein were denatured using 10% SDSPAGE. For western blotting, the following primary antibodies were used: MDM2 (Abcam, ab259265, 1:1000 rabbit), SUZ12 (Cell Signal Technology, 3737, 1:1000 rabbit), ACTIN (Beyotime, AA128, 1:1000 mouse), TUBULIN (Beyotime, AF2835, 1:1000 mouse), HA (Beyotime, AH158, 1:1000 mouse), FLAG (Sigma, F1804, 1:1000 mouse), H3 (Elabscience, E-AB-22003, 1:1000 mouse) and H3K27me3 (Sigma, 07-449, 1:10,000 rabbit).

Embryos were subjected to fixation with 4% paraformaldehyde for a duration of 30min and subsequent permeabilization using 0.5% Triton X-100 for 30min at room temperature. Blocking of embryos was treated with 1% BSA in PBS supplemented with 0.1% Tween 20, lasting for 60min at room temperature. Incubation with CDX2 primary antibodies (Biogenex, MU392A, 1:200 mouse) occurred overnight at 4C, followed by incubation with secondary antibodies (Invitrogen, A32723, 1:500) for 1h at room temperature. Lastly, the nuclei of the embryos were stained with DAPI (Beyotime, P0131), and images were acquired using the fluorescence microscope.

Cells were fixed by incubating with 70% ethanol at 20C overnight. The next day, the fixed cells were centrifuged at 4C, 300g, and washed once with PBS. Afterward, RNase A treatment was performed at 37C for 30min. Finally, the cells were stained with propidium iodide (PI) at 4C for 30min. Cell cycle analysis was performed using a BD flow cytometer, and the data were analyzed with Modfit LT software to determine the distribution of cells across different phases of the cell cycle.

To inhibit the expression of Mdm2 in pre-ZGA embryos, zygotes were cultured in KSOM or HM medium supplemented with Nutlin-3 (Selleck, S1061) at a final concentration of 5M. DMSO was included as a negative control. The embryos were cultured at 37C and monitored daily until they reached the blastocyst stage.

CUT&Tag assay was conducted using the NovoNGS CUT&Tag 3.0 High-Sensitivity Kit (Novoprotein, N259). Approximately 1105 mESCs were incubated with 10L of Binding ConA beads. Primary antibodies targeting FLAG (Sigma, F1804, 1:50 mouse) and H3K27me3 (Sigma, 07-449, 1:100, rabbit) were incubated overnight at 4C. Subsequently, secondary antibodies were added, and the mixture was incubated at room temperature for 1h. Following this, the samples were treated with 1L of Transposome and incubated at room temperature for 1h. DNA was then collected for subsequent PCR analysis. The libraries were amplified and subjected to sequencing using the Illumina NovaSeq PE150 platform following the manufacturers instructions.

The raw data underwent quality filtering using Trimmomatic to obtain clean data. These clean data were then aligned to the mm10 genome using Bowtie2. For the identification of peaks, we utilized MACS2. Heatmaps were generated using Deeptools, and ChIPseeker was employed to annotate the promoters.

We conducted a re-analysis of publicly available datasets. H3K27me3 ChIP-seq data for mESCs and SUZ12 KO mESCs were obtained from GSE103685. Additionally, H3K27me3 modification data for 2C embryos were acquired from GSE73952, and RNA-seq data for mouse and pig pre-implantation embryos were retrieved from GSE71434 and GSE163709.

The statistical differences were analyzed by the Students t-test when two independent groups were compared. Data are displayed in a bar graph with error bars representing the meanSD and individual sample points shown. GraphPad Prism was used for the statistical analysis of data. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. Three independent biological replicates were included and the figure legends specify the sample sizes.

Detailed information on the research design can be found in the Nature Portfolio Reporting Summary associated with this article.

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A CRISPR/Cas9 screen in embryonic stem cells reveals that Mdm2 regulates totipotency exit | Communications Biology - Nature.com

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A CRISPR activation screen identifies FBXO22 supporting targeted protein degradation - Nature.com

Dublin, July 05, 2024 (GLOBE NEWSWIRE) -- The "Plant Breeding and CRISPR Plant Market - Global Industry Size, Share, Trends, Opportunity, and Forecast, 2019-2029F" report has been added to ResearchAndMarkets.com's offering.

Global Plant Breeding and CRISPR Plant Market was valued at USD 11.16 billion in 2023 and is anticipated to project robust growth in the forecast period with a CAGR of 6.91% through 2029

Global agriculture landscape is undergoing a transformative phase with advancements in plant breeding and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technologies. These innovative approaches hold the promise of revolutionizing crop development, addressing the challenges of a growing population, climate change, and sustainable agriculture. In this article, we delve into the intricacies of the Global Plant Breeding and CRISPR Plant Market, examining key drivers, challenges, and the potential impact on global food security.

Global Plant Breeding and CRISPR Plant Market represent a frontier in agricultural innovation, offering solutions to pressing global challenges. As technology continues to evolve and stakeholders navigate regulatory landscapes, the potential for these advancements to drive sustainable agriculture and enhance global food security is substantial. The coming years are likely to witness further breakthroughs and collaborations that will shape the future of plant breeding and CRISPR technologies in the agricultural sector.

Precision Breeding

Precision breeding has emerged as a pivotal trend propelling the Plant Breeding and CRISPR Plant Market into a new era of innovation, particularly in the realm of disease resistance. The ability to edit genes using CRISPR technology precisely and specifically has revolutionized the development of crops with enhanced resistance to diseases, marking a paradigm shift in the agricultural landscape.

In the face of evolving and increasingly complex plant pathogens, precision breeding offers a targeted approach to fortify crops against specific diseases. Unlike conventional breeding methods, which may introduce unintended changes, CRISPR technology allows scientists to pinpoint and modify the genes responsible for conferring resistance. This precision not only accelerates the development timeline but also ensures the preservation of desirable traits in the modified crops.

The market's focus on precision breeding for disease resistance is driven by the imperative to address global food security challenges. Crop losses due to diseases can have severe economic and humanitarian impacts, making the development of resilient varieties a top priority. Biotechnology companies, researchers, and agricultural stakeholders are investing heavily in precision breeding techniques to create crops capable of withstanding the onslaught of pathogens, thereby ensuring stable yields and securing the global food supply.

Gene Editing Application

Gene editing applications, particularly the revolutionary CRISPR-Cas9 technology, are steering the Plant Breeding and CRISPR Plant Market towards unprecedented heights, with a laser focus on enhancing disease resistance in crops. This transformative approach to genetic modification has become a driving force, offering a level of precision and efficiency that traditional breeding methods struggle to match.

The ability to precisely edit specific genes responsible for disease resistance is a game-changer for agricultural sustainability. CRISPR-Cas9 enables researchers and biotech companies to tailor crops with enhanced immunity to specific pathogens, safeguarding against the economic and food security risks posed by plant diseases.

One of the key catalysts for the market's enthusiasm towards gene editing for disease resistance is the rapid development timeline. Traditional breeding methods often entail years of crossbreeding and selection processes, whereas CRISPR allows for targeted modifications in a fraction of the time. This accelerated pace is critical in responding to emerging and evolving plant pathogens.

The demand for disease-resistant crops is driven by the imperative to ensure stable and secure food production amidst a backdrop of changing climates and global uncertainties. As gene editing applications become increasingly refined and accessible, the market witnesses a surge in investments, collaborative research efforts, and commercialization strategies aimed at bringing disease-resistant varieties to farms worldwide.

Integration of Bioinformatics

The integration of bioinformatics into plant breeding and CRISPR technologies is ushering in a new era of precision and efficiency in the pursuit of disease-resistant crops. This strategic amalgamation of biological data analysis and genetic information has become a pivotal driver, shaping the landscape of the Plant Breeding and CRISPR Plant Market.

Bioinformatics enables researchers to analyze vast datasets with unprecedented speed and accuracy, expediting the identification of genes associated with disease resistance. This data-driven approach enhances the selection of target genes for modification, ensuring a more focused and effective genetic editing process.

The intricate relationship between bioinformatics and disease resistance is particularly crucial in addressing the constant threat of evolving pathogens. By deciphering the genetic basis of plant-pathogen interactions, scientists can design crops with tailored resistance mechanisms, bolstering the overall resilience of agricultural systems.

Moreover, the integration of bioinformatics streamlines the identification of potential off-target effects during CRISPR-mediated gene editing. This not only ensures the precision of genetic modifications but also addresses regulatory concerns and enhances the overall safety profile of genetically modified crops.

Regional Insights

The Asia-Pacific region stands at the forefront of driving the Plant Breeding and CRISPR Plant Market, fueled by its diverse agricultural landscape and the urgent need to address food security challenges. Countries like China and India are making substantial investments in research and development, leveraging CRISPR technology to enhance the traits of staple crops. Rice, a dietary staple for a significant portion of the global population, has been a focal point, with initiatives aimed at improving yield, nutritional content, and resilience to pests and diseases.

Furthermore, Asia-Pacific nations are actively collaborating with international biotech companies and research institutions. These collaborations not only facilitate the exchange of knowledge but also contribute to the development of region-specific crop varieties tailored to local agricultural needs.

In Europe, stringent regulatory frameworks have not deterred the pursuit of innovation in the Plant Breeding and CRISPR Plant Market. The region's commitment to sustainable agriculture aligns seamlessly with the goals of these technologies. Countries like the United Kingdom and Germany are investing heavily in research initiatives, focusing on developing crops with increased resistance to environmental stressors, reduced reliance on chemical inputs, and improved nutritional profiles.

The European Union's recent decision to regulate gene-edited crops based on the characteristics of the final product rather than the technology used provides a more nuanced regulatory approach. This shift has invigorated the industry, encouraging companies and researchers to explore the full potential of CRISPR technology in plant breeding.

North America, particularly the United States and Canada, is a key driver in the Plant Breeding and CRISPR Plant Market. The region's strong emphasis on technological innovation, coupled with a robust agricultural sector, has created an environment conducive to the adoption of these advanced breeding technologies.

Major biotechnology companies headquartered in North America are leading the charge in developing and commercializing genetically modified crops. The focus extends beyond staple crops to include cash crops like soybeans and corn, where CRISPR technology is employed to enhance traits such as drought resistance, pest tolerance, and improved yields.

Additionally, collaborations between North American research institutions and international partners contribute to the global pool of knowledge, further advancing the capabilities of Plant Breeding and CRISPR technologies.

Key Attributes:

Competitive Landscape

Company Profiles: Detailed analysis of the major companies present in the Global Plant Breeding and CRISPR Plant Market.

Report Scope:

Plant Breeding and CRISPR Plant Market, By Type:

Plant Breeding and CRISPR Plant Market, By Trait:

Plant Breeding and CRISPR Plant Market, By Application:

Plant Breeding and CRISPR Plant Market, By Region:

For more information about this report visit https://www.researchandmarkets.com/r/zbh2fy

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$16.7 Billion Plant Breeding and CRISPR Plant Market - Global Industry Size, Share, Trends, Opportunity, and Forecast ... - GlobeNewswire

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Tunable translation-level CRISPR interference by dCas13 and engineered gRNA in bacteria - Nature.com

According to latest report, the U.S. CRISPR and cas genes market size was estimated at USD 1.85 billion in 2023 and is projected to hit around USD 8.59 billion by 2033, growing at a CAGR of 16.6% during the forecast period from 2024 to 2033.

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Therapeutic applications of CRISPR and Cas genes, rising significance for gene editing, and the introduction of anti-CRISPR proteins are boosting market growth. Moreover, CRISPR and Cas genes provide growth prospects for developing novel cancer therapies that boost market expansion.

U.S. CRISPR and Cas genes market accounted for a 63.0% share in the global CRISPR and Cas genes market in 2023. CRISPR technology is preferred for treating various diseases, including cancer and inflammatory and infectious diseases, making it favorable for future biomedical therapeutics. It revolutionizes cancer treatment by enhancing Chimeric Antigen Receptor T-cell (CAR-T) immunotherapy. Unlike traditional methods, the next generation of CAR-T therapies utilizes CRISPR to improve the precision and efficiency of therapeutic and manufacturing processes. This advancement allows for accurate delivery of CAR genes into T-cell DNA, a significant improvement over viral vectors that randomly insert genes. These features of CRISPR-Cas increase its demand in the market.

Furthermore, the increasing investment from government bodies, funding agencies, and biotechnology companies in genomic research is poised to impact the CRISPR market significantly. International funding organizations such as the NIH and Wellcome Trust play a crucial role by providing financial support for genetic studies. This substantial funding is expected to drive the utilization of genetic editing tools in the country, shaping the landscape of the CRISPR market. Furthermore, a report published in June 2022 stated that the U.S. government granted USD 639.5 million to Human Genome Research Institute for R&D in 2022. The institute further requested USD 629.6 million as funding for 2023.

U.S. CRISPR And Cas Genes Market Key Takeaways

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Global CRISPR & Cas Genes Market Size and share 2024 to 2033

The global CRISPR And Cas Genes market size was USD 3.11 billion in 2023, calculated at USD 3.64 billion in 2024 and is expected to reach around USD 15.15 billion by 2033, expanding at a CAGR of 17.16% from 2024 to 2033. North America dominated the market and accounted for the highest revenue share of 39.17% in 2023.

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What is CRISPR, the powerful genome-editing tool?

CRISPR-Cas9 is a simple yet powerful tool for editing genomes. It enables researchers to easily alter DNA sequences and modify gene function.

CRISPR is a powerful tool for editing genomes, meaning it allows researchers to easily alter DNA sequences and modify gene function. It has many potential applications, including correcting genetic defects, treating and preventing the spread of diseases, and improving the growth and resilience of crops. However, despite its promise, the technology also raises ethical concerns.

In popular usage, "CRISPR" (pronounced "crisper") is shorthand for "CRISPR-Cas9." CRISPRs are specialized stretches of DNA, and the protein Cas9 where Cas stands for "CRISPR-associated" is an enzyme that acts like a pair of molecular scissors, capable of cutting strands of DNA.

CRISPR technology was adapted from the natural defense mechanisms of bacteria and archaea, a domain of relatively simple single-celled microorganisms. These organisms use CRISPR-derived RNA, a molecular cousin to DNA, and various Cas proteins to foil attacks by viruses. To foil attacks, the organisms chop up the DNA of viruses and then stow bits of that DNA in their own genome, to be used as a weapon against the foreign invaders should those viruses attack again. When the components of CRISPR are transferred into other, more complex, organisms, those components can then manipulate genes, a process called "gene editing."

U.S. CRISPR And Cas Genes Market Concentration & Characteristics

The presence of several companies in the U.S. CRISPR And CAS Genes market contributed to its fragmented landscape. Moreover, companies are leveraging CRISPR editing in the human genome, offering growth opportunities in academic and pharmaceutical research.

By introducing innovative CRISPR-based therapies, companies have the potential to attract customers, drive revenue growth, and enhance patient outcomes significantly. Utilizing CRISPR technology in the development of advanced therapies can assist companies in differentiating themselves in the market, meet patients' evolving requirements, and contribute to personalized medicine. This offers a promising path for using cutting-edge therapies and advancement in personalized medicine.

Companies like CRISPR Therapeutics, Vertex Pharmaceuticals, Intellia Therapeutics, and Regeneron Pharmaceuticals have engaged in strategic partnerships to drive innovation in precision medicine. These collaborations involve sharing resources, expertise, and technologies to develop novel treatments that target genetic disorders at their root cause. Numerous biotechnological and pharmaceutical companies focus on M&A activities that showcase crucial aspects of strategic management that enable companies to facilitate growth, restructuring, and enhancing competitive positions within the industry

Various biotechnology and CRISPR-based therapeutics companies focus on providing services such as cell line engineering and design tools that help in research activities undertaken by institutions and other companies. This approach increases their customer base and enables them to sustain their position in the industry.

Increasing focus on regional expansion by key manufacturers serves a wide range of customers and capitalizes on geographical industry growth opportunities. This approach allows companies to strengthen their presence in different regions, adapt to local market needs, and enhance their market share by targeting diverse customer segments.

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By Product & Service Insights

In 2023, the product segment held the largest market share of 79.11%. This growth was fueled by the efforts of institutions and key companies to increase research and development activities. This comprises kits& enzymes such as CAS9, CRISPR libraries, antibodies, design tools, and more. Moreover, Vector-based CAS9 products facilitate research experiments, signifying the use of CRISPR/Cas for gene editing. Such applications shift the focus of manufacturers to develop these products, further expanding market growth. Epic Bio unveiled the GEMS (Gene Expression Modulation System) platform in 2022, which offers precise gene expression modification. The GEMS platform contains a vast library of newly discovered modulators combined with advanced functional and computational genomics capabilities. It enables the rapid design of guide RNAs highly targeted to specific genes.

From 2024 to 2033, the services segment is expected to experience the most rapid compound annual growth rate. This growth is fueled by the rising number of licensing agreements with biotech firms, which offer services ranging from cell line engineering and beyond. Moreover, CRISPR-based gene editing companies focus on offering advanced services to fulfill the unmet demand for this market. For instance, in August 2022, Creative Biogene introduced one-stop microbial genome editing services for knock-in, knockout, and foreign gene insertion using a variety of bacteria. These microbial genome editing services maximize customers microbial gene sequence efficiency and minimize off-target effects using the advanced CRISPR/Cas 9 platform.

By Application Insights

The biomedical application segment held the largest share of 92.25% in 2023, owing to the increasing popularity of CRISPR/Cas9 genome editing technology in several areas of biomedical sciences. The market is experiencing profitable growth opportunities thanks to the emergence of different CRISPR-based therapies to treat chronic conditions such as cancer, diabetes, blood disorders, and infectious diseases. Moreover, regulatory approval of these therapies is driving the segments growth. For instance, in December 2023, the US FDA approved Casgevy therapy for sickle cell anemia. Casgevy is the first FDA-approved therapy utilizing CRISPR/Cas9, a genome editing technology.

The agriculture segment will exhibit the quickest compound annual growth rate (CAGR) between 2024 and 2033. The rise in awareness of CRISPR-based gene editing in the agriculture sector has led to increasing adoption improvement in crop production. There is an increase in research studies on agriculture products using CRISPR technology. This trend will expand the growth trajectory of this segment.

By End-use Insights

The biotechnology and pharmaceutical companies segment dominated the market with a share of 50.19% in 2023, owing to the increasing development of CRISPR-based products by major biotechnology and pharmaceutical companies for drug and therapy development. The growing adoption of CRISPR technology in these sectors significantly influences the market landscape and drives revenue growth. Additionally, the rise in the number of companies offering gene and gene-editing products and services in recent years is further fueling the revenue growth within this segment.

The academic and government research institutes are anticipated to grow at a CAGR of 16.1% over the forecast period. The increased research and numerous genomics and gene editing studies in academic and government institutes have increased the demand for CRISPR-Cas9. This surge in research activities focusing on gene engineering and its applications is anticipated to drive rapid growth in the forecast period.

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U.S. Genome Editing Market: The U.S. genome editing market size was estimated at USD 3.55 billion in 2023 and is projected to hit around USD 16.49 billion by 2033, growing at a CAGR of 16.6% during the forecast period from 2024 to 2033.

Gene Expression Market : The global gene expression market size was estimated at USD 13.85 billion in 2023 and is projected to hit around USD 37.35 billion by 2033, growing at a CAGR of 37.35% during the forecast period from 2024 to 2033.

Genome Editing Market: The global genome editing market size was valued at USD 8.45 billion in 2023 and is anticipated to reach around USD 40.48 billion by 2033, growing at a CAGR of 16.96% from 2024 to 2033.

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U.S. Gene Synthesis Market: The U.S. Gene Synthesis market size was valued at USD 700.90 million in 2023 and is projected to surpass around USD 3,118.73 million by 2033, registering a CAGR of 16.1% over the forecast period of 2024 to 2033.

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U.S. CRISPR And Cas Genes Market Recent Developments

U.S. CRISPR And Cas Genes Market Top Key Companies:

U.S. CRISPR And Cas Genes Market Report Segmentation

This report forecasts revenue growth at country levels and provides an analysis of the latest industry trends in each of the sub-segments from 2021 to 2033. For this study, Nova one advisor, Inc. has segmented the U.S. CRISPR And Cas Genes market.

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U.S. CRISPR And Cas Genes Market Size to Hit USD 8.59 Billion by 2033 - BioSpace

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a gene-editing technology that allows scientists to modify DNA with unprecedented precision. Discovered in the early 2010s, CRISPR technology leverages a natural defense mechanism used by bacteria to protect against viral infections. The system uses a guide RNA to direct the Cas9 enzyme to a specific location in the genome, where it creates a double-strand break. This break can then be repaired by the cells natural mechanisms, allowing for the addition, deletion, or modification of genetic material. CRISPR companies are seeing more and more success in the clinic and the market is growing.

CRISPR has rapidly become one of the most powerful tools in genetic engineering, enabling precise changes to the DNA. Its applications are not limited to medicine, which will be our focus in this article, as it also allows the creation of crops with desirable traits in agriculture for instance.

In recent years, the field of CRISPR technology has improved and different forms of the technology are now being leveraged by biotech companies. Prime editing and base editing are innovative CRISPR-related technologies aiming to improve the versatility and precision of therapies.

After CRISPR Therapeutics and Vertex Pharmaceuticals collaborative success leading to CASGEVYs approval by the U.S. Food and Drug Administration (FDA) and Editas Medicines promising efforts to treat blindness, here are eight companies keeping the CRISPR field dynamic.

Beam Therapeutics was founded in 2017, and is headquartered in Cambridge, Massachusetts. The CRISPR technology company develops precision genetic medicines using its proprietary base editing technology.

The company went public on NASDAQ in February 2020 and has raised a total of $689 million since its creation according to Crunchbase.

Beams base editing technology distinguishes itself by focusing on single-base alterations, which can correct mutations at the nucleotide level. This precision reduces the risk of off-target effects and enhances the potential for treating a wide range of genetic disorders. The companys base editing platform includes the REPAIR (adenosine to inosine) and RESCUE (cytosine to uracil) systems for RNA editing, enabling targeted genetic modifications.

Beam Therapeutics has several key candidates in various stages of development:

Eligo Bioscience is a French company founded in 2014. The company focuses on precision gene editing of the microbiome to treat diseases driven by bacterial genes. Eligo Bioscience leverages its proprietary Gene Editing of the Microbiome (GEM) platform to develop therapies that target and modify specific bacterial populations. Eligo Bioscience recently raised $30 million in a series B funding led by Sanofi Ventures.

The companys GEM platform uses engineered bacteriophages to deliver CRISPR-Cas systems directly to specific bacteria within the microbiome. This approach allows for the precise elimination of pathogenic bacteria or the correction of harmful bacterial genes without disrupting the overall balance of the microbiome. By targeting bacterial genes in vivo, Eligos technology aims to address various diseases associated with microbiome dysbiosis, including antibiotic-resistant infections and chronic diseases.

Unlike broad-spectrum antibiotics, which indiscriminately kill bacteria and disrupt the microbiome, Eligos technology selectively targets pathogenic bacteria or genes within the microbiome. This precision reduces collateral damage to beneficial bacteria which helps maintain a healthy microbiome.

In January, Xavier Duportet, chief executive officer (CEO) of the company, was our guest on the Beyond Biotech podcast to talk about its flagship product EB005 targeting acne vulgaris. This candidate is on track to reach the clinic and expand its application to oncology.

Founded in 2018 and headquartered in South San Francisco, California, Epic Bio is focused on developing therapies to modulate gene expression in vivo using its proprietary Gene Expression Modulation System (GEMS) platform. The company launched in 2022 with a $55 million series A round.

Epic Bios approach combines a miniature DNA-binding protein called CasMINI with customized guide RNAs and a wide array of modulator proteins. CasMINI, licensed from Stanford University, is the smallest Cas protein to date, less than half the size of Cas9 and Cas12a, allowing for efficient delivery using adeno-associated virus (AAV) vectors. This platform enables precise gene modulation, expanding the potential for treating a variety of genetic diseases.

The CasMINI protein is engineered to function effectively in cells and is small enough to be delivered in vivo using AAV vectors. This compact size and robust functionality make it possible to target a wide range of tissues and organs with high precision.

Epic Bios pipeline is still preclinical and targets a wide variety of diseases. The companys lead candidate targets facioscapulohumeral muscular dystrophy (FSHD), a genetic muscle disorder characterized by progressive muscle weakness and wasting. The company also develops candidates for heterozygous familial hypercholesterolemia (HeFH), a genetic disorder characterized by high cholesterol levels, and retinitis pigmentosa, a group of inherited disorders that cause progressive retinal degeneration, leading to vision loss.

Locus Biosciences is a biotechnology company founded in 2015 and headquartered in Morrisville, North Carolina. The company specializes in developing precision antibacterial therapies using CRISPR-Cas3-enhanced bacteriophage technology, known as crPhage. Locus Biosciences most recent funding is a $35 million series B round in 2022.

The company employs a CRISPR-Cas3 system for its antibacterial therapies. Unlike the more commonly used Cas9, Cas3 destroys the DNA of target bacteria irreversibly, making it highly effective against antibiotic-resistant strains. This technology is delivered using engineered bacteriophages, viruses that specifically target bacteria, allowing the preservation of the microbiome.

The CRISPR-Cas3 system sets Locus apart by offering a genetic chainsaw approach, which differs from the genetic scissors approach of CRISPR-Cas9. Cas3s ability to degrade large segments of DNA makes it particularly effective for combating multi-drug resistant bacteria.

Locus lead candidate LBP-EC01 is currently in phase 2/3 and targets Escherichia coli (E. coli) infections. E. coli is a type of bacteria commonly found in the intestines of humans and animals. While most strains are harmless and part of the normal gut flora, some can cause serious infections. E. coli infections can occur through the consumption of contaminated food or water or by contact with animals or person-to-person spread.

LBP-SA01, another candidate in the companys pipeline, targets staphylococcus aureus infections. While it often exists harmlessly, it can cause a wide range of infections if it enters the body through a cut or a wound.

Founded in 2017 and headquartered in Brisbane, California, the company leverages its proprietary CRISPR platform for therapeutics and diagnostics. Like Caribou Biosciences we mentioned last week, this CRISPR company was co-founded by Nobel laureate Jennifer Doudna.

Mammoth Biosciences has raised substantial funding, including a $150 million series D financing round in 2021, which has elevated its status to a unicorn with a valuation of over $1 billion.

Mammoth Biosciences focuses on the discovery and engineering of novel CRISPR systems, specifically the ultra-small Cas14 and Cas (phi) enzymes. These systems are smaller and have an increased temperature stability, and faster reaction, which enhance their effectiveness in in vivo genome editing and diagnostics.

The use of Cas14 and Cas enzymes allows Mammoth Biosciences to develop CRISPR-based solutions that are more efficient and versatile. The smaller size of these enzymes enables easier delivery into cells, especially for diseases that affect the central nervous system.

Mammoth Biosciences is developing both therapeutic and diagnostic products. The companys therapeutic pipeline is still in the preclinical and research stages, and the indications of its candidates are mostly undisclosed.

Additionally, Mammoth has its diagnostic platform, the DETECTR platform, which is a CRISPR-based detection system.

Prime Medicine was founded in 2019 and is headquartered in Cambridge, Massachusetts. The company focuses on developing gene editing therapies using its proprietary prime editing technology. Prime editing aims to address the root causes of genetic diseases by precisely correcting mutations at their source.

The company launched with $315 million in financing, comprising a $115 million series A round followed by a $200 million series B round.

Prime Medicine utilizes prime editing, a novel gene editing technology that acts like a DNA word processor to search and replace disease-causing genetic sequences. Unlike traditional CRISPR methods, prime editing does not create double-strand breaks in DNA, which reduces the risk of unintended modifications. This technology can correct a wide range of genetic mutations, making it an interesting and promising tool for developing therapies for genetic disorders.

The technology employs a fusion protein combining a Cas protein with a reverse transcriptase enzyme and a guide RNA (pegRNA) to direct the correction process. This approach allows for highly specific and predictable edits at the targeted genomic location, minimizing off-target effects.

Prime Medicine is advancing several preclinical programs targeting various genetic diseases: Wilsons disease, preventing the body from properly eliminating excess copper and leading to severe brain and liver issues, glycogen storage disease, and retinitis pigmentosa, among others.

Primes most advanced program, however, is an ex vivo therapy in phase 1/2 targeting chronic granulomatous disease, an inherited immunodeficiency disorder that affects the bodys ability to fight certain infections.

Scribe Therapeutics is a molecular engineering company founded in 2018 and headquartered in Alameda, California. The company focuses on developing advanced CRISPR-based genetic medicines and collaborates with industry leaders such as Biogen or Sanofi.

The company recently completed a $100 million Series B financing round led by Avoro Ventures and Avoro Capital Advisors.

Scribe Therapeutics leverages its CRISPR by design platform, which includes custom-engineered CRISPR enzymes. By optimizing the CRISPR enzymes for greater efficiency, Scribes XE technology can achieve more precise and robust gene edits.Scribes XE platform features advancements in delivery technologies, such as viral vectors and lipid nanoparticles, that are optimized for delivering CRISPR components into target cells and tissues in vivo.

The CRISPR company works on several therapeutic areas hand in hand with key players in the industry. Scribe is collaborating with Biogen to develop CRISPR-based therapies for amyotrophic lateral sclerosis (ALS). In partnership with Sanofi, Scribe is also working on genetically modifying natural killer (NK) cell therapies for cancer treatment. The XE platforms high specificity and efficacy make it ideal for engineering these cells to target and eliminate cancer cells effectively.

SNIPR Biome was founded in 2017 and is headquartered in Copenhagen, Denmark. The company specializes in developing CRISPR-based microbial gene therapies aimed at precisely targeting and eradicating pathogenic bacteria, including antibiotic-resistant strains.

SNIPR Biome has raised notable funding including one of Europes largest series A rounds, securing $50 million.

SNIPR Biomes primary technology involves CRISPR-Guided Vectors (CGV), which deliver CRISPR components into bacterial cells via engineered bacteriophages. These vectors create double-stranded breaks in the DNA of target bacteria, leading to rapid and specific bacterial killing. This approach is designed to preserve beneficial microbiota while targeting harmful pathogens, particularly those resistant to conventional antibiotics.

SNIPR001 is the companys lead candidate, a CRISPR therapy targeting E. coli, including antibiotic-resistant strains. SNIPR001 is designed to prevent bloodstream infections in patients undergoing hematopoietic stem cell transplants, who are particularly vulnerable to such infections. Positive interim results from phase 1 clinical trials showed that SNIPR001 was well-tolerated and effectively reduced gut E. coli levels in treated individuals.

The CRISPR technology market is experiencing robust growth and substantial investments. In 2024, the global market was valued at approximately $3.78 billion and is projected to reach around $9.34 billion by 2029, growing at a compound annual growth rate (CAGR) of 19.9%. This promising outlook for the CRISPR market is driven not only by the recent success of companies like Vertex and CRISPR Therapeutics but also by the emergence of more refined versions of the CRISPR technology.

While there is no doubt CRISPR has a bright future ahead, the market faces several challenges. The high costs associated with CRISPR technology are one of the main obstacles to its democratization in the future. Ethical concerns regarding genetic modifications and regulatory hurdles are also significant obstacles as ethics and law always move slower than technology.

More broadly, the gene editing and engineering scenes are moving fast, and technologies such as epigenetic editing and gene writing with companies such as Chroma Medicine and Tessera Therapeutics show significant potential.

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Seven CRISPR companies to watch in 2024 - Labiotech.eu

Direct in vivo CRISPR gene editing in the mouse pancreas

To functionally test putative PDAC cancer genes in vivo, we employed a multiplexed CRISPR/Cas9 genome editing approach to generate knock-out clones directly in the pancreatic epithelium of tumor-prone mice. We used conditional Lox-Stop-Lox-(LSL)-KrasG12D and LSL-Cas9-GFP mice crossed to the pancreas-specific PDX1-Cre driver line (termed KC mice) and injected an adeno-associated virus that expresses a sgRNA and the H2B-RFP fluorescent marker (AAV-sgRNA-RFP) (Fig.1a). Cre-mediated excision of Lox-Stop-Lox cassettes resulted in expression of oncogenic KrasG12D, Cas9 and GFP and formation of hundreds of cytokeratin19 positive (CK19) pancreatic intraepithelial neoplasia (PanIN) precursor lesions, which can be lineage-traced by virtue of red fluorescence (Supplementary Fig.1a, b). To validate the efficiency of CRISPR/Cas9-mediated mutagenesis, we injected sgRNAs targeting GFP, which revealed a knock-out efficacy of 786% (Supplementary Fig.1c).

A Experimental design of the in vivo PDAC CRISPR screen, showing gene selection from long-tail mutations, pancreatic injection of AAV libraries and tumor sequencing. B Tumor-free survival of Pdx1-Cre;LSL-KrasG12D;LSL-Cas9-GFP mice transduced with a sgRNA library targeting putative pancreatic cancer genes (n=23) or a control sgRNA library (n=13) C Representative whole-mount, H&E and immunofluorescent images of an H2B-RFP+ pancreatic PDAC-library tumor: Scale bar 2mm. H&E image: scale bar 250m. Representative immunofluorescence image shows H2B-RFP and CK19 expression. Scale bar 50m. Similar results were observed in all collected tumor. D Representative pie charts showing tumor suppressor genes with enriched sgRNAs in tumor DNA obtained from three different pancreatic tumors and a control-transduced pancreas with multifocal PanINs. E Bar graph showing putative tumor suppressor genes with enriched sgRNAs in tumor DNA obtained from the PDAC mouse model (sgRNA enriched per tumors are indicated by color).

KC mice exhibited rapid growth of pre-invasive PanINs precursor lesions but showed very slow progression to invasive PDAC with a median latency of 14 months (Fig.1b). Additional genetic alterations such as loss of transformation-related protein 53 (Trp53), p16Ink4a, Lkb1 or inactivation of TGF- signaling was previously shown to cooperate with KrasG12D and induces rapid PDAC development within 3-5 month16,17,18,19,20. To test whether our direct in vivo CRISPR approach can reveal genetic interactions, we recapitulated cooperation between oncogenic KrasG12D and loss of p53 (Trp53). Indeed, Cas9-mediated ablation of Trp53 in KC mice triggered rapid PDAC formation with a median latency of 14 weeks, while littermates transduced with scrambled control sgRNAs remained cancer-free for over 1 year (Supplementary Fig.1d). This is in line with previous efforts using CRISPR/Cas9 gene editing in KRasG12D mice21,22 and demonstrates that this approach can be used to test for genetic cooperation between PDAC genes.

In pancreatic cancer, 125 genes show recurrent somatic mutations6,7. To assess these genes in vivo, we established a sgRNA library targeting the corresponding mouse orthologs (4 sgRNAs/gene; 500 sgRNAs) and a library of 420 non-targeting control sgRNAs (Supplementary Data1). Of note, we did not include sgRNAs targeting well-established PDAC driver genes such as Trp53 or Smad416,17,18,19,20.

Next, we optimized the parameters for an in vivo CRISPR screen. Using a mixture of AAV-GFP and AAV-RFP, we determined the viral titer that transduces the pancreatic epithelium at clonal density (MOI<1). Higher viral titers were associated with double infections, whereas a 15% overall transduction level minimized double infections while generating necessary clones sufficient to screen (Supplementary Fig.1e). Using multicolor Rosa26-Lox-Stop-Lox(R26-LSL)-Confetti Cre-reporter mice, we next determined the viral titer required to generate thousands of discrete clones within the pancreatic epithelium (Supplementary Fig.1f). Thus, at a transduction level of 15% and a pool of 500 sgRNAs, each sgRNA would be introduced into at least 50 CK19+ epithelial cells within a single pancreas.

To uncover long-tail genes that cooperate with oncogenic KRasG12D and accelerate PDAC development, we injected the experimental and the control AAV-sgRNA libraries into the pancreas of 23 and 13 KC mice, respectively. Next-generation sequencing confirmed efficient AAV transduction of all sgRNAs (Supplementary Fig.2a). Importantly, KC mice transduced with the long-tail PDAC sgRNA library developed pancreatic cancer significantly faster than littermates transduced with the control sgRNA library (31 versus 59 weeks; p<0.0001) (Fig.1b and c). In addition, 13/23 (56%) KC mice transduced with the long-tail PDAC sgRNA library developed liver and/or lung metastasis, while only 1/13 (~8%) littermate mice transduced with the control sgRNA library developed metastasis (Supplementary Fig.2b-e), indicating the existence of strong tumor suppressors within the long-tail of PDAC associated genes.

To identify these PDAC driver genes, we examined the sgRNA representation in 151 tumors. 78% of tumors showed strong enrichment for single sgRNAs, indicating a clonal origin. In contrast, the pancreas of control-transduced mice with multifocal PanINs showed enrichment of several non-template control sgRNAs (Fig.1d). We prioritized genes that were targeted by 2 sgRNAs and knocked out in multiple tumors and/or metastatic foci, resulting in 8 candidate tumor suppressor genes (Fig.1e, Supplementary Data2). These candidates included well-known PDAC tumor suppressor genes, such as Cdkn2a23, Rnf4324, Fbxw725 or NF226, as well as genes with poorly understood function, such as Usp15 and Scaf1.

Pancreatitis is a risk factor for the development of PDAC in humans and cooperates with oncogenic KRas mutations to induce PDAC formation in mice23,27. Therefore, we repeated our screen and treated mice with chronic, low doses of cerulein to induce mild pancreatitis. As expected, cerulein treatment significantly accelerated PDAC development in KC mice transduced with the PDAC sgRNA library (17 versus 32 weeks median survival, p<0.0001), and a trend towards faster PDAC development in KC mice transduced with the control library (Supplementary Fig.2f). In line with the previous screen, Cdkn2a was the top-scoring gene followed by Rnf43 and the newly identified genes, Usp15 and Scaf1 (Supplementary Fig.2g), further supporting their function as strong suppressors of pancreatic cancer in KC mice.

The multi-domain deubiquitinase USP15 regulates diverse processes, such as the p53 tumor suppressor pathway28, MAPK signaling29, Wnt/beta-catenin signaling30, TGF- signaling31,32,33, NfKb signaling32,34,35 and chromosome integrity36,37, either through regulated de-ubiquitination of direct substrates such as MDM2, APC, SMADs or TGF- receptors, or de-ubiquitination-independent functions such as through protein-protein interactions38.

To validate the tumor suppressive function of Usp15, we first injected KC mice individually with one library or one newly designed sgRNA. All transduced mice developed highly proliferative pancreatic tumors with much shorter latencies compared to mice transduced with the non-targeting control sgRNAs (sgCrtl) (Fig.2a). In fact, age-matched control KC mice only exhibited PanINs at the time when USP15 knockout mice exhibit aggressive PDACs (Fig.2b). All tested tumors exhibited efficient CRISRP/Cas9-mediated mutagenesis of Usp15 (Supplementary Fig.3a and b).

A Tumor-free survival of Pdx1-Cre;LSL-KrasG12D;LSL-Cas9-GFP mice injected with CRISPR AAV-sgRNAs targeting the indicated gene or non-targeting control sgRNA (sgCtrl, n=6). Two independent sgRNAs were used (sgUsp15_1 n=9, sgUsp15_2 n=9). Log-Rank test (Mantel-Cox). B Representative H&E images showing multifocal PanINs in sgCtrl transduced pancreas and PADC tumors in sgUsp15 transduced pancreas. Scale bar 100m. C Tumor-free survival of Pdx1-Cre;LSL-KrasG12D mice with the indicated Usp15 genotype where + indicates the wildtype allele and indicates a conditionally deleted allele. KrasG12D +/- Usp15 +/+ (n=12); KrasG12D +/- Usp15 +/- (n=11); KrasG12D +/- Usp15 -/- (n=13). Log-Rank test (Mantel-Cox). D Representative H&E images of mice with the indicated genotype showing multifocal PanINs and PADC tumors. Scale bar 100m. E Cell proliferation curves of KC cells transduced with the indicated sgRNA obtained using the IncuCyte live-cell imaging. Cells were grown for five days and data are expressed as cell confluence percentage (%; meanSD, n=3 independent experiments, Two-way ANOVA (sgUsp15_1 p=3.43e-8: sgUsp15_2 p=4.89e-9), Dunnetts multiple comparison. F Cell proliferation curves of KC cells expressing ubiquitin variants inhibiting Usp15 (Ubv15.1a and Ubv15.1/d) or wildtype ubiquitin (Ubwt) as control. (%; meanSD, n=3 independent experiments, Two-way ANOVA (Ubv15.1a p=2.74e-6: Ubv15.1/d p=2.06e-7), Dunnetts multiple comparison. G Tumor-free survival of NSG (NOD Scid Gamma) mice after orthotopic injection sgCtrl (n=5) or sgUsp15_1/2 (n=5; n=5) KC cells. Two independent sgRNAs were used. Log-Rank test (Mantel-Cox). H Dose-response curves for KPC sgCtrl or sgUsp15 cells treated with the indicated concentration of Olaparib (meanSD, n=3 independent experiments). Two-way ANOVA (sgUsp15_1 p=0.0349: sgUsp15_2 p=0.0431), Dunnetts multiple comparison.

To further confirm the tumor suppressive role and rule out any confounding effect of Cas9 endonuclease expression, we generated conditional Usp15fl/fl; KRasG12D; Pdx1-Cre. This conventional knock-out approach recapitulated our CRISPR/Cas9 findings (Fig.2c and d), validating our in vivo CRISPR approach. Interestingly, Usp15fl/+ heterozygous mice also manifested significantly shorter disease-free survival (Fig.2c). To assess whether tumor development was due to Usp15 loss of heterozygosity, we used fluorescence-activated cell sorting (FACS) to isolate tumor cells from Usp15 homozygous, heterozygous and wild-type KRasG12D tumors. Western Blot analysis revealed Usp15 expression in Usp15 heterozygous tumor cells, albeit at a reduced level compared to control tumors (Supplementary Fig.3c), indicating Usp15 functions as a haploinsufficient tumor suppressor.

Next, we established primary PDAC cell lines from KC mice as well as KC mice with concomitant expression of the hotspot p53R270H mutant (KPC) and used CRISPR/Cas9 to knock-out Usp15 (Supplementary Fig.3d). Loss of Usp15 significantly increased proliferation of these KC cells (Fig.2e), while it did not affect KPC cells (Supplementary Fig.3e), presumably, because those cells are at the maximal proliferation rate. Similar results were obtained using ubiquitin variants (UbVs) that bind and block the catalytic domain of Usp1538, indicating that this tumor suppressive function is de-ubiquitination dependent (Fig.2f). Upon orthotopic injection, Usp15 knock-out KC cells also formed allograft tumor faster than non-targeting control cells (Fig.2g). Together, these data show that Usp15 regulates tumor cell proliferation in a cell-autonomous manner and loss of Usp15 increases a cells ability to form allograft tumors.

Consistent with a previous report36, we also found that loss of Usp15 sensitizes pancreatic cancer cells to Poly-(ADP-ribose) polymerase inhibition (PARPi) by Olaparib. This increased drug sensitivity was stronger in KPC cells than KC cells and was also seen in response to Gemcitabine, one of the most commonly used chemotherapies to treat pancreatic cancer (Fig.2h and Supplementary Fig3f, Fig.4a and b). KC cells were overall more sensitive to Gemcitabine likely due to the intact p53 response (Supplementary Fig3g). Importantly, loss of USP15 also sensitized allograft tumors in vivo towards Olaparib treatment (Supplementary Fig.4c). In addition, we found that Olaparib and Gemcitabine treatment significantly increases expression of Usp15 in KC and KPC cells (Supplementary Fig.4d). In line with its haploinsufficient tumorigenic effect, heterozygous loss of Usp15 also significantly increased proliferation and sensitized to Olaparib treatment, but not as pronounced as complete Usp15 loss (Supplementary Fig.4e and f). As such, Usp15 appears to function as a double-edged sword in pancreatic cancer, where the loss of Usp15 enhances tumor progression in the initial stages of tumorigenesis but sensitizes to certain treatment regimens in the later stages.

Given the wide range of USP15 substrates and USP15-regulated pathways with well-known functions in cancer, we set out to elucidate USP15s exact role in PDAC suppression. First, we transcriptionally profiled primary KC cells transduced with sgRNAs targeting Usp15 or non-template controls sgRNAs. Inactivation of Usp15 resulted in dramatic changes in gene expression compared to scrambled control KrasG12D tumor cells (794 differentially expressed genes (DEG), false discovery rate (FDR, Benjamini-Hochberg)<0.05 and absolute log2 fold-change > 1, Fig.3a and Supplementary Data3). Gene set enrichment analyses (GSEA) revealed significantly upregulated gene sets associated with xenobiotic detoxification, glutathione metabolism, anabolic processes, and oxidative phosphorylation (Fig.3b and Supplementary Data3). These findings are in line with USP15s known role in negatively regulating NRF239 (encoded by the NFE2L2 gene), the master regulator of glutathione metabolism and the redox balance of a cell. In addition, NRF2 expression is induced by oncogenic KRAS and known to stimulate proliferation and suppress senescence of PDAC cells40. Indeed, Usp15 knock-out cells exhibited significantly increased levels of Nrf2 (Supplementary Fig.5a).

A Volcano Blot showing differential expressed genes between Usp15-knockout compared to sgCtrl control KC cells. Wald test and Benjamini-Hochberg (BH)-adjusted P-value. Two independent sgRNAs, two biological duplicates. B Bar graph showing gene set enrichment analysis (GSEA) of Usp15-knockout compared to sgCtrl control KC cells. GSEA nominal p-values. Two independent sgRNAs, two biological duplicates. C GSEA plots and Heatmaps of log2 counts per million for selected differentially expressed pathways and genes in sgUsp15 versus sgCtrl control KC cells. GSEA nominal p-values. Two independent sgRNAs, two biological duplicates. D Expression levels of genes related to TNF signaling evaluated by RT-qPCR. Results were normalized with Gapdh and are expressed in fold change compared to Ctrl (meanSEM, n=3 independent experiments). Cells were incubated with 10ng/mL TNF-for 30min. Two-sided T-test, Rel-B p=0.043; TRAF-1 p=0.037/p=0.034; NFKB1 p=0.036; Rel-B p=0.042; TRAF-1 p=0.039; CXCL2 p=0.028; CXCL3 p=0.047/p=0.043; NFKB1 p=0.038/p=0.040; NFKB2 p=0.039/p=0.043.

GSEA also revealed depleted genes sets associated with inflammatory responses, TNF, TGF and p53 signaling (Fig.3b-d and Supplementary Fig.5b), all pathways with well-known tumor suppressive function in PDAC development17,41. Quantitative RT-PCR confirmed reduced expression of TNF and TGF responsive genes at baseline as well as TNF/TGF-stimulated conditions (Fig.3d and Supplementary Fig.5c). In addition, loss of USP15 reduced TNFinduced cell death and TGF-induced migration (Supplementary Fig.5d and e). Together, these data indicate that Usp15 functions as a strong haploinsufficient PDAC tumor suppressor potentially by regulating tumor suppressive cytokine signaling pathway.

Our second new hit, SCAF1 (SR-Related CTD Associated Factor 1), is a member of the human SR (Ser/Arg-rich) superfamily of pre-mRNA splicing factors. It interacts with the CTD domain of the RNA polymerase II (RNAPII) and is thought to be involved in pre-mRNA splicing42. Its close homologs SCAF4 and SCAF8 were recently shown to be essential for correct polyA site selection and RNAPII transcriptional termination in human cells43. SCAF1 was also one of the top-scoring hits in a screen for genes that can restore homologous recombination in BRCA1-deficient cells and thus conferred resistance to PARP inhibition44. However, the molecular function of SCAF1 remains completely elusive.

First, we validated the tumor suppressive function of Scaf1 by injecting KC mice individually with one library or one newly designed sgRNA. All transduced mice developed highly proliferative pancreatic cancer with much shorter latencies compared to mice transduced with the non-targeting control sgRNAs (Fig.4a, b). Of note, both Scaf1 sgRNAs induced high CRISPR/Cas9-mediated mutagenesis and resulted in significantly reduced Scaf1 mRNA expression (Supplementary Fig.6a, b). Similar to Usp15 knockout cells, we also found that primary Scaf1 knockout KC cells exhibited increased proliferation in culture and formed tumors faster when injected orthotopically into mice compared to scrambled control KC cells (Fig.4c and d). Scaf1 knockout cells also exhibited significantly increased sensitivity to Olaparib in vitro and in vivo (Fig.4e and Supplementary Fig.6c and d), again phenocopying Usp15 knockout cells.

A Tumor-free survival of Pdx1-Cre;LSL-KrasG12D;LSL-Cas9-GFP mice injected with CRISPR AAV targeting the indicated gene or non-targeting control sgRNA (sgCtrl n=6). Two independent sgRNAs were used (sgScaf1_1 n=8, sgScaf1_2 n=7). Log-Rank test (Mantel-Cox). B Representative H&E images showing multifocal PanINs in sgCtrl-transduced pancreas and PADC tumors in sgScaf1-transduced pancreas. Scale bar 100m. C Cell proliferation curves of KC sgCtrl and sgScaf1 cells were obtained using the IncuCyte live-cell imaging and data are expressed as cell confluence percentage (%; meanSD, n=3 independent experiments). Two-way ANOVA (sgScaf1_1 p=0.0039: sgScaf1_2 p=0.00042), Dunnetts multiple comparison. D Tumor-free survival of NSG mice orthotopically injected with sgCtrl (n=5) or sgScaf1_1/2 (n=5; n=5) KC cells. Two independent sgRNAs were used. Log-Rank test (Mantel-Cox). E Dose-response curves for KPC sgCtrl or sgScaf1 cells treated with the indicated concentration of Olaparib (meanSD, n=3 independent experiments). Two-way ANOVA (sgScaf1_1 p=0.0084: sgScaf1_2 p=0.0028), Dunnetts multiple comparisons. F Representative Western Blot of Usp15 in KC cells transduced with the indicated sgRNAs and treated as indicated. This experiment was repeated independently two times with similar results. G Cell growth curves of KC sgCtrl and sgScaf1cells expressing the listed isoform of USP15 or an empty vector (EV). Data are expressed as cell confluence percentage (%; meanSD, n=3 independent experiments); two-way ANOVA (sgScaf1+EV p=0.0342), Dunnetts multiple comparison. Dose-response curves for KC sgCtrl and sgScaf1 cells expressing the listed isoforms of USP15 or EV and treated with Olaparib. (%; meanSD, n=3 independent experiments; two-way ANOVA (sgScaf1+EV p=0.00129), Sidak multiple comparison H Tumor-free survival of NSG mice orthotopically injected with sgCtrl (n=5) or sgScaf1 KC cells expressing the listed isoforms of USP15 (n=5: n=5) or EV (n=5; n=5). Log-Rank test (Mantel-Cox).

Interestingly, we found a connection between Scaf1 and Usp15. Scaf1 knockout cells exhibited reduced expression of full-length Usp15 (molecular weight of ~125kDa) and showed expression of a 25kDa short Usp15 isoform under homeostatic as well as Olaparib and gemcitabine treatment (Fig.4f and Supplementary Fig.6e and f). SCAF1 KO tumors also exhibited lower levels of full-length USP15 (Supplementary Data Fig.6g). To examine a potential function of this truncated isoform, we cloned and transduced the long and the short isoforms into primary Usp15 knock-out KC cells (Supplementary Fig.6h). While full-length Usp15 was able to supress the hyperproliferative phenotype of Usp15 knock-out cells, the short isoform failed to suppress the cell proliferation (Supplementary Fig.7a). Similarly, re-expressing the full-length but not the short Usp15 isoform reversed the sensitivity of Usp15 knock-out KC cells to Olaparib and gemcitabine (Supplementary Fig.7b). In addition, overexpression of the full-length or the short Usp15 isoform did not alter proliferation of wildtype KC cells (Supplementary Fig.7c), indicating that the short isoform does not exhibit dominant negative functions. However, expression of the long but not the short Usp15 isoform or a catalytically-dead USP15 isoform suppressed the hyperproliferation and Olaparib sensitivity as well as the increased in vivo tumorigenesis of Scaf1 knock-out cells (Fig.4g and h and Supplementary Fig.7c-f). Together these data indicate that the short isoform has no tumor suppressive functions or alters the response to PARP inhibition and that Scaf1s tumor suppressive function is at least in part routed by regulating the expression of full-length Usp15.

To further elucidate the effects of Scaf1, we transcriptionally profiled Scaf1 knockout KC cells. Inactivation of Scaf1 resulted in 625 differentially expressed genes (DEG) (false discovery rate (FDR)<0.05 and absolute log2 fold-change > 1, Fig.5a and Supplementary Data3) compared to scrambled control KrasG12D tumor cells. GSEA revealed significantly upregulated gene sets associated with nucleotide metabolism, glutathione metabolism, microtubule polymerization, and oxidative phosphorylation as well as downregulation gene sets associated with TNF signaling, one-carbon metabolism, xenobiotic catabolic processes, mTorc1/mTOR signaling, hypoxia and p53 signaling (Fig.5b and Supplementary Fig.7g). In addition, we found a trend towards downregulated TGF signaling (Fig.5b and Supplementary Data3), reminiscent of the pathways altered in Usp15 knock-out cells.

A Volcano Blot showing differential expressed genes between Scaf1-knockout compared to control KC cells. Wald test and Benjamini-Hochberg (BH)-adjusted P-value. Two independent sgRNAs, two biological duplicates. B Bar graph showing gene set enrichment analysis of Scaf1-knockout compared to control KC cells. GSEA nominal p-values. Two independent sgRNAs, two biological duplicates. C Bar graph showing gene set enrichment analysis of Usp15-knockout and Scaf1-knockout compared to sgCtrl control KC cells treated with Olaparib (1M). GSEA nominal p-values. Two independent sgRNAs, two biological duplicates. D Expression levels of genes related to HH signaling evaluated by RT-qPCR in the indicated KC cell lines. Results were normalized with Gapdh and are expressed in fold change to CTRL (meanSEM, n=3 independent experiments). Cells were treated with 100nM Smoothened Agonist (SAG) and 1M Olaparib. Two-sided T-test, for sgUSP15: NRP2 p=0.021/p=0.018; PTCH1 p=0.037/p=0.033; GLI1 p=0.033; NRP2 p=0.042/p=0.037; for sgScaf1: PTCH1 p=0.046; PTCH2 p=0.040/p=0.042/p=0.033; GLI1 p=0.037/p=0.031; NRP2 p=0.042.

Lastly, we set out to elucidate how Usp15 and Scaf1 regulate the response of pancreatic cancer cells to PARP inhibition. Interestingly, transcriptional profiling and GSEA following Olaparib treatment revealed that both Usp15 and Scaf1 knock-out cells, exhibited downregulation of hedgehog signaling, TGF signaling and axon guidance by netrin as well as upregulation of glycolysis as the top dysregulated pathways compared to Olaparib-treated control KC cells (Fig.5c and Supplementary Data4). Together, this indicates a common mechanism leading to increased sensitivity to PARP inhibition shared between Usp15 and Scaf1 knock-out cells. Indeed, quantitative RT-PCR confirmed reduced expression of hedgehog target genes at baseline as well as upon sonic hedgehog stimulation (Fig.5d). Thus, Scaf1 and Usp15 knockout cells share several alterations such as upregulated TNF signaling and downregulated TGF, hedgehog and p53 signaling but also several distinct pathways.

To extend our findings from mouse to human cancers, we analyzed 295 PDAC samples from The Cancer Genome Atlas45,46,47. Mutations and homozygous deletion of USP15 and Scaf1 are rare as expected for long-tail mutation and were found in only 2.4% and 1.4% of PDAC samples, respectively. However, an additional 25% and 13% of PDAC cases showed shallow deletions of USP15 and SCAF1, respectively, indicative of heterozygous loss of these genes (Fig.6a). Focal USP15 and SCAF1 copy-number losses have been identified in independent large-scale genome studies48,49. In addition, allelic copy number loss also coincided with reduced expression of USP15 and SCAF1 and patients with deep or shallow USP15 or SCAF1 deletions showed a significant trend towards a shorter overall survival (Fig.6b and Supplementary Fig.8a). Given our genetic and biochemical data linking SCAF1 and USP15, we next considered patients with deep or shallow USP15 or SCAF1 deletions as a group (=37% of patients) and found a significantly shorter overall survival (Supplementary Fig.8b). This raises the possibility that USP15 and potentially also SCAF1 function in a haploinsufficient manner, which is in line with the increased tumorigenesis found in the Usp15fl/+; KRasG12D; Pdx1-Cre mice.

A Oncoprint of the indicated genes in PDAC samples (n=293, TCGA). B Kaplan-Meier survival analyses of PDAC patients with deep or shallow USP15 or SCAF1 deletion. (n=293, TCGA) Log-Rank test (Mantel-Cox). C Tumor-free survival of NSG mice orthotopically injected with sgCtrl (n=5), sgUsp15_1/2 (n=5; n=5) or sgSCAF1_1/2 (n=5; n=5) PANC-1 cells. Two independent sgRNAs were used. Log-Rank test (Mantel-Cox) D Dose-response curves for sgCtrl, sgUsp15 or sgSCAF1 PANC-1 cells treated with Olaparib. (%; meanSD, n=3 independent experiments) two-way ANOVA (sgUsp15_1 p=0.0242; sgUsp15_2 p=0.0387; sgScaf1_1 p=0.0281; sgScaf1_2 p=0.0371), Dunnetts multiple comparison. E sgUSP15 and sgOR2W5 PDO Competition Assay. sgUSP15 and control sgOR2W5 patient-derived organoids were disassociated into single cells and mixed in a 20:80% ratio. Organoid cultures were passaged, and a sample was collected every ~7 days. Percentage of DNA indels is tracked overtime by sanger-sequencing and TIDE analysis.

Next, we assessed the expression of USP15 in 4 human pancreatic cancer cell lines. While, PANC1 and HPAFII exhibited expression of the small as well as the long USP15 isoform, MiaPACA2 and BXPC3 cells only exhibited low-level expression of the long USP15 isoform, indicating that USP15 is also downregulated in some human pancreatic cancer cell lines (Supplementary Fig.8c).

To functionally test USP15 and SCAF1, we genetically ablated these genes in human PANC1 cells (Supplementary Fig.8d and e). Importantly, genetic ablation of SCAF1 resulted in increased expression of the short USP15 isoform, indicating that this mechanism is conserved from mouse to human cells (Supplementary Fig.8f). Similarly, to our autochthonous mouse experiments, we also found that loss of USP15 or SCAF1 in PANC1 cells resulted in accelerated tumorigenesis and increased sensitivity to Olaparib and Gemcitabine (Fig.6c, d and Supplementary Fig.8g). We also observed increased NRF2 protein levels in USP15 knockout PANC1 cells, which showed further elevated upon inhibition of TXNRD1/2 and antioxidant imbalance by auranofin treatment50 (Supplementary Fig.8h), akin to our findings in mouse KC cells. USP15 knockout PANC1 cells also exhibited increased sensitivity to auranofin treatment (Supplementary Fig.8i).

Lastly, we genetically ablated USP15 in patient-derived organoids (PDOs) from 3 different pancreatic cancer patients using Cas9 ribonucleotide particles. We set up competitive growth assays to assess the relative fitness of USP15 knockout PDOs compared to OR2W5 knockout PDOs. Of note, the OR2W5 olfactory receptor is not expressed in pancreatic PDOs and thus serves as control. We mixed the USP15 knockout and the OR2W5 knockout PDOs at a 1:4 ratio and followed their relative growth by quantifying the percent of USP15 and OR2W5 mutations over time using Sanger sequencing. Within ~10 passages, we observed that the PDO cultures were almost completely taken over by USP15 knockout cells (Fig.6e). Together, these data demonstrate the tumor suppressive function of USP15 and SCAF1 in pancreatic cancer by modulating several important signaling pathways and that loss of USP15 and SCAF1 sensitizes to Gemcitabine and Olaparib.

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In vivo CRISPR screens reveal SCAF1 and USP15 as drivers of pancreatic cancer - Nature.com

Researchers from the Broad Institute of MIT, Harvard, and Princeton University have developed a low-cost paper strip test that uses CRISPR to distinguish between the two main types of seasonal influenza, A and B, as well as subtypes H1N1 and H3N2.

Furthermore, the test can also identify strains that resist antiviral treatment and could potentially be adapted to detect swine and avian flu strains, including H5N1, currently causing an outbreak in cattle.

Results from the test, described in an article in The Journal of Molecular Diagnostics, show that the test, which was designed to be rapid, affordable, and easy to deploy at point-of-need is also accurate at distinguishing the types and subtypes of influenza, achieving results in 100% concordance with traditional polymerase chain reaction (PCR) assays.

The test is based on a technology called SHINE, which was developed in 2020 by the lab of co-author Pardis Sabeti, an institute member at the Broad and a professor at Harvard University and the Harvard T.H. Chan School of Public Health, as well as a Howard Hughes Medical Institute investigator. SHINE uses CRISPR enzymes to identify specific sequences of viral RNA in samples.

The teams aim was to create low-cost, rapid tests that could be deployed in clinics or in the field rather than in hospitals or diagnostic labs, and that didnt require expensive equipment to run. Accessible, efficient testing technology not only improves clinical care, but also may potentially improve outbreak management, making it easier for scientists to collect samples strategically to better monitor viral spread.

In contrast to typical diagnostic assays such as PCR which may require lengthy processing times, extensive training for personnel, and specialized equipment, SHINE testing can be performed in about 90 minutes at room temperature, and only requires an inexpensive heat block to warm the reaction.

The researchers first used SHINE to test for SARS-CoV-2, later adapting it to distinguish between the Delta and Omicron variants. They began adapting the assay to test for influenza viruses in 2022.

In the future, the team said in a story from the Broad Institute, the assay could be adapted to distinguish between different viruses with similar symptoms, such as influenza and SARS-CoV-2.

See more here:
CRISPR-based paper test could improve influenza testing access, outbreak surveillance - LabPulse

A team of scientists created a new gene-editing tool that they claim is more accurate than the industry standard, CRISPR.

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Researchers from the University of Sydney, Australia, developed what is called SeekRNA, a new gene-editing tool that uses a programmable ribonucleic acid (RNA) strand capable of identifying and inserting itself into specific sites in genetic sequences. The team behind the project is being led by Dr. Sandro Ataide in the School of Life and Environmental Sciences, and their findings have already been published in Nature Communications.

The team explained that while CRISPR is the industry standard when it comes to genetic engineering, having revolutionized multiple industries such as medicine, agriculture, and biotechnology, it doesn't come without any problems. According to Dr. Ataide, SeekRNA differentiates itself from CRISPR in various ways, such as by not requiring any extra components to be cut and pasted into genetic sequences. SeekRNA is a stand-alone cut-and-paste tool that has higher accuracy.

Furthermore, CRISPR relies on creating a break in both strands of target DNA, which is the double-helix strand that commonly depicts a DNA sequence. While CRISPR is certainly impressive in its own right it requires the use of proteins or the DNA repair machinery to insert the new DNA sequence into its designated location. This process can produce errors in the code.

"SeekRNA can precisely cleave the target site and insert the new DNA sequence without the use of any other proteins. This allows for a much cleaner editing tool with higher accuracy and fewer errors," said Dr. Ataide

"We are tremendously excited by the potential for this technology. SeekRNA's ability to target selection with precision and flexibility sets the stage for a new era of genetic engineering, surpassing the limitations of current technologies," Dr Ataide said.

"With CRISPR you need extra components to have a 'cut-and-paste tool', whereas the promise of seekRNA is that it is a stand-alone 'cut-and-paste tool' with higher accuracy that can deliver a wide range of DNA sequences."

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Scientists create gene-editing tool that may revolutionize DNA engineering - TweakTown

When researcher Arkasubhra Ghosh finally met Uditi Saraf, he hoped that there was still a chance to save her.

Ghosh and his collaborators were racing to design a one-off treatment that would edit the DNA in the 20-year-old womans brain cells and get them to stop producing toxic proteins. It was an approach that had never been tried before, with a long list of reasons for why it might not work.

CRISPR cures and cancer vaccines: researchers can help to shepherd them to market

But the team was making swift progress. The researchers were maybe six months away from being ready to give Uditi the therapy, Ghosh told her parents over breakfast at their home outside New Delhi last June. Even so, Uditis mother was not satisfied. Work faster, she urged him.

Then, Uditi was carried to the breakfast table, and Ghosh understood her urgency. Once a gregarious and energetic child and teenager, with a quick laugh and a mischievous streak, Uditi was now unable to walk or feed herself. She had become nearly blind and deaf. Her family tried to talk to her: These are the people who are making a therapy for you, they said loudly.

Shaken, Ghosh returned to his gene-therapy laboratory at Narayana Nethralaya Eye Hospital in Bengaluru, India, and got to work. If you need to put up tents in the lab, then we can do so, he told his students. Im not going to sleep.

Four months later, Uditi was gone.

The first therapy using CRISPR genome editing was approved in late 2023 to treat blood disorders that affect thousands of people worldwide. But the approach is also a source of hope to many people who have extremely rare genetic conditions, like the one Uditi had. Genome editing could one day become a radical way to address the diseases that are overlooked by pharmaceutical companies. Patients are waiting, families are waiting, says Jennifer Doudna, a molecular biologist at the University of California, Berkeley. So we need to get on with it.

How personalized cancer vaccines could keep tumours from coming back

Researchers are still laying the groundwork for this future. They are working out how best to design and manufacture the treatments, and how to deliver them to precise locations in the body. The cost is also a problem: the price of genome-editing therapy threatens to put it out of reach for many. Ghosh wants to bring those barriers down, and hes convinced that India will eventually be the country to do it.

But Uditis family could not wait the pace of scientific research was too slow. They needed a sprint, and a team of researchers willing to take on not only the scientific challenge, but also the emotional heft and high risk of failure involved in attempting something that had never been done. What we were trying to do was really almost in the realms of science fiction, Ghosh says.

And he remains convinced that, despite Uditis tragic death, the lessons learnt will help others on a similar path. It truly is a story of hope.

As a young girl, Uditi was always in a hurry. Seizing any excuse to celebrate whether it was a birthday or a festival she would buzz around the house getting ready hours ahead of everyone else, peppering her mother with urgent requests. She greeted family and friends with cuddles and kisses and brightened parties with her laughter and dancing.

For the first nine years, there was no hint of trouble. And when it began, it was just a flicker a few seconds here and there, when Uditi would zone out.

Shed switch back on again as if nothing had happened, and her mother, Sonam, wasnt sure if she should worry. But then Sonam saw nine-year-old Uditi drop a camera on the floor and become confused as to why it was no longer in her hand. A mothers hunch hardened: something was wrong.

Uditi Saraf with her mother, Sonam Saraf.Credit: Rajeev and Sonam Saraf

The physicians diagnosed her with epilepsy. When Uditis seizures became more pronounced and she began to struggle at school, Sonam and Rajeev, Uditis father, decided it must be something more. In 2017, they had part of Uditis genome sequenced.

It was a deviation from the standard treatment path, but the Sarafs were technologically savvy and financially well off. In India, as in many places in the world, genome sequencing was still uncommon, its roll-out slowed both by the costs and by the dearth of genetic information from people of Indian descent in genetic databases. Without such data, it can be difficult to interpret sequencing results.

Uditis results, however, were unambiguous: a single-base change in the gene that codes for a protein called neuroserpin caused tangled polymers to form in her brain cells, interfering with their function. Uditis neurons were dying.

This condition is called FENIB (familial encephalopathy with neuroserpin inclusion bodies), and the symptoms which can be similar to dementia usually manifest late in life. Elena Miranda, a cell biologist at the Sapienza University of Rome, runs the worlds only lab that focuses on the disease. She says that its possible that many cases of FENIB go unreported because physicians do not often sequence the genomes of older adults with dementia.

How CRISPR gene editing could help treat Alzheimers

But the most severe forms of FENIB strike early and are exceedingly rare. Miranda has known of only three other people with the same mutation that Uditi had. This form of the disease is very aggressive, she says.

Uditi and her parents embarked on a lonely journey familiar to many people with rare diseases. They had never heard of FENIB, and neither had Uditis physicians. Sonam did some research but couldnt bring herself to fully absorb what she found. We thought its not possible, she says. It cannot happen with our daughter.

The Sarafs studied what they could find online and tried the interventions available to them: Indian ayurvedic treatments, a ketogenic diet, special schools, seeing a slew of physicians and trying out various medicines. We shopped for doctors. We shopped for gods, says Sonam, but Uditis condition slowly worsened.

The three moved to upstate New York in 2018 to send Uditi to a school for people with disabilities. Her seizures intensified, and frequent muscle spasms made it hard for her to walk or drink from a glass. Her bright personality was dimming. The Sarafs discussed experimental treatments with Uditis new physician, epilepsy specialist Orrin Devinsky at NYU Langone Health in New York City. Devinsky mentioned a couple of options, one of which was CRISPR genome editing. Rajeev seized on the idea.

Uditis disease was caused by a mutation that converts a single DNA base from a G to an A. A variation on CRISPR genome editing, called base editing, could theoretically correct exactly this kind of mutation (see Precision gene repair).

Devinsky also emphasized the difficulties. At that time, base editing which was first reported in 2016 had never been tested in a clinical trial. The technique requires shuttling a relatively large protein and a snippet of RNA into affected cells. Researchers were struggling with how to perfect this delivery for many organs the brain being one of the most daunting.

Even if each of these hurdles were surmounted, at best, base editing might stop the production of neuroserpin clumps in some of Uditis neurons. The treatment was unlikely to reach all affected cells, and it was unlikely to clear the clumps that were already present or to regenerate neurons that had been lost.

But Rajeev and Sonam saw an opportunity for hope: perhaps such a therapy could slow down the progression of Uditis disease, buying time for scientists to develop another treatment that could repair the damage that had been done. The Sarafs were on board.

Devinsky assembled a team at NYU Langone Health with expertise in genome editing and neuroscience to conduct preliminary studies of the approach. The researchers pulled together what funding they could from other grants, and the Sarafs funded the rest. We will sell our house if we have to, Sonam said.

The pressure in the lab was intense, says team member Jayeeta Basu, a neuroscientist at NYU Langone Health. The team genetically engineered Uditis FENIB mutation into cells grown in the lab. When the cells initially didnt seem to behave as expected, Basu asked her graduate student to repeat the experiment five times. I was always pushing, she says. We had to be fast, but we also had to be diligent. There was no short cut.

Rajeev Saraf with his daughter Uditi.Credit: Rajeev and Sonam Saraf

In December 2019, the Sarafs moved back to India. Maintaining a home in the United States was expensive, and Uditi missed her extended family. Then the COVID-19 pandemic struck, and in January 2021, Uditi was hospitalized with severe COVID-19. She spent 20 days in the hospital and her health was never the same, says Sonam. Communication became increasingly difficult for Uditi and she began to pace the house incessantly, rarely even going to sleep.

The Sarafs decided to speed up the base-editing project by funding a second team in India.

Meanwhile, Devinsky had petitioned a US foundation to devise a different experimental treatment called antisense therapy for Uditi. The family flew from India to the United States twice for injections into her spine. The trips became traumatic as her ability to understand the world around her declined.

CRISPR 2.0: a new wave of gene editors heads for clinical trials

The treatments didnt work. And the experience taught Rajeev and Sonam how long it could take to get approval to try an experimental therapy in the United States. They decided Uditis base-editing therapy should also be manufactured and administered in India.

About an hour and a half away from their home, Debojyoti Chakraborty, a geneticist at the Council of Scientific and Industrial Researchs Institute of Genomics and Integrative Biology in New Delhi, had been making headlines for his efforts to devise a CRISPR-based treatment for a genetic blood disorder called sickle-cell disease.

Researchers in the United States were also developing genome-editing therapies for sickle-cell disease, but those therapies were expected to be expensive and potentially out of reach for much of the world. (The UK Medicines and Healthcare Products Regulatory Agency approved the first one, Casgevy, made by Vertex Pharmaceuticals in Boston, Massachusetts, and CRISPR Therapeutics in Zug, Switzerland, which costs US$2.2 million per patient.)

Most of the people with sickle-cell disease in India a country with one of the highest rates of the condition live in impoverished communities. Chakraborty and his colleagues hoped to develop a therapy that could be produced and administered at a fraction of the price that is charged in the United States, if not less.

Debojyoti Chakraborty is trying to develop affordable CRISPR-based treatments in India.Credit: RNA Biology Lab

Rajeev and Sonam went to the institute to talk to Chakraborty and the institutes director, chemist Souvik Maiti, who had been collaborating with Chakraborty on the CRISPR technology behind the sickle-cell project.

Move over, CRISPR: RNA-editing therapies pick up steam

Although the institute gets many requests for help from people with rare diseases and their caregivers, the Sarafs were unusual in that they would be able to help fund the work, says Maiti. Uditi was the only person in India known to have her neuroserpin mutation, and no government agency, company or philanthropy was likely to pay for the development of a treatment. Its very difficult, Maiti says. Even if our heart is telling us we should work on it, until there is funding, we cannot do it.

Even with funding, Maiti and Chakraborty took some time to discuss the project with Ghosh, who was building a facility in Bengaluru to produce viruses called adeno-associated viruses (AAVs), which are often used in gene therapies. Ghosh aimed for his facility to be one of the first in India to produce AAVs to the standards required for use in people.

There were a lot of unknowns in the base-editing project. And in addition to the work on stem cells in the lab, the team would need to do further experiments to determine which base-editing systems would work best, where and how to deliver its components into the body, and whether the process generated any unwanted changes to the DNA sequence. They would need to do experiments in mice to test the safety and efficacy of the treatment. They also needed to get Ghoshs facility approved by Indias regulators for producing the base-editing components.

Uditis illness had probably already progressed beyond the point at which the therapy could offer a notable benefit, but the family wanted them to try, reasoning that the work that they did on this project could help future endeavours to develop genome-editing therapies for genetic conditions.

It was not the first time Ghosh was swayed by a personal appeal: a few years before he met Uditi, Ghosh came to work and found two women waiting outside of his office. They would not leave, the women said, until he committed to finding a treatment for their young sons illness, a genetic condition called Duchenne muscular dystrophy, which can be fatal. The women pledged to help raise funds, and Ghosh found himself unable to say no. He has worked on the project and grown close to the families since then.

UK first to approve CRISPR treatment for diseases: what you need to know

Lab protocols for making medicines are notoriously strict, with each step carefully controlled to minimize the chance of contamination. When setting up his facility for manufacturing gene therapies, Ghosh scrutinized each step, looking for ways to cut costs without sacrificing safety, arguing his case to Indias regulators. He estimates that gene therapies for eye diseases that are developed in his lab will one day be available for one-hundredth of what they cost in the United States. We will certainly short circuit this entire field, Ghosh says.

India has earned a reputation for making complex drugs on a budget. During the COVID-19 pandemic, Indian manufacturers cranked out millions of doses of vaccines. Now, the country is manufacturing a malaria vaccine at a fraction of the cost of that in Europe, and it is developing sophisticated cell and gene therapies used in cancer treatments for much less than the price of those in the United States.

Chakraborty took the lead on Uditis project. He is a go-getter kind of person, says Riya Rauthan, who was then a PhD student in Chakrabortys lab. He is not bothered by who he needs to ask to get something done, he just does it.

To minimize interruptions, the team mapped out all of the experiments and the components they would need from start to finish. In India, many lab reagents have to be imported, and supply interruptions can delay projects by weeks or months. Everything had to be planned and ordered ahead of time, and Maiti worked to keep the supplies coming, seeking out vendors and negotiating prices. Time was more valuable than anything else, he says.

One of the most important reagents had to come from abroad: antibodies that could recognize the neuroserpin protein and its tangles. Few researchers use such antibodies, and the supply was uncertain. The team decided that the quickest way to get reliable antibodies was to ask Miranda in Rome to share the ones she had developed. She gladly did. This was a desperate approach, she says. But for me the priority was to try to help as much as I could.

Rauthan generated stem cells from samples of Uditis blood. Then, she and her colleagues coaxed those cells to become neurons, and used base editing on them in the lab.

Arkasubhra Ghosh is building an Indian facility to make viral vectors for gene therapy.Credit: Arkasubhra Ghosh

Ghosh worked on preparing the AAV that would be used to transport the CRISPR components into Uditis neurons. The team needed to determine which strain of AAV would work best some strains could trigger inflammation in the brain. Ghoshs lab tested several types of AAV in mice, to find out which one caused the least amount of inflammation and how best to administer it. The team eventually settled on one type called AAV9 and determined that it should be injected directly into Uditis brain.

Still, that was not the end of their challenges. AAV genomes can carry only an extra 4,700 DNA bases, but the gene that codes for the enzyme needed in base editing is longer than that. Ghosh and his students worked to divide up their genomic cargo so that it could fit in two separate viruses, and added sequences that would allow the two pieces to be spliced together again when they are expressed inside a cell. The team would inject both viruses at the same time.

The approach has been shown to work in mice but had not yet been tested in humans (J. M. Levy et al. Nature Biomed. Eng. 4, 97110; 2020).

By June 2023, the team seemed to be barrelling towards the finish line. Many of the researchers were working 10-to-12-hour days, and it was nearly time to test their therapy in mice. Ghosh was also scheduling a regulatory inspection to ensure that he would have the approvals he needed by the time the animal results were in. A surgeon had agreed to perform Uditis injection.

If all went well, they might be ready to treat Uditi in as little as six months, Chakraborty predicted.

In early October, a few months after Chakraborty and Ghosh had breakfast with Uditi and her parents, the team received a series of messages from Rajeev on their WhatsApp group. Uditi had become ill with pneumonia and had been taken to the hospital. Then she was in a coma and had been sent home there was nothing else the physicians could do for her.

Soon afterwards came the message they had all feared: Uditi was gone.

Ghosh thought immediately of the two little boys with Duchenne muscular dystrophy: What if Im too late for them, too?

Others in the lab also took the news hard. For clinicians, perhaps they become hardened, says Chakraborty. We dont have that experience. We were feeling agony.

Ten days after learning that Uditi died, Chakraborty presented the labs efforts at a local conference and finished his talk with a picture of Uditi, smiling. In the audience, Riya Patra, a graduate student in Ghoshs lab, began to cry. It was the first time shed let herself see a picture of the young woman shed tried so hard to save. Before, I had thought that if I saw her, maybe I would cry, she says. And I wouldnt be able to work anymore.

Is CRISPR safe? Genome editing gets its first FDA scrutiny

An estimated 100 million people in India have a rare disease. For decades, people affected by such conditions have cycled through hope and disappointment as researchers have inched closer to developing therapies that can help them at a genetic level. After a series of sporadic starts and failures, gene therapy has finally begun to find its footing. This has set the stage for CRISPR-based genome editing to rocket to the clinic.

When nine-year-old Uditi first dropped her camera, CRISPR was just an oddity a strange assembly of sequences found in microbial genomes, only studied by a few die-hard microbiologists. Four years before she was diagnosed with FENIB, researchers showed for the first time that a CRISPR-based system could cut DNA in human cells grown in the lab. And the first CRISPR-based genome-editing therapy was approved in the United Kingdom to treat sickle-cell disease the month after Uditi died.

In theory, many people with a genetic condition, no matter how rare, could benefit from these technologies. But the reality is harsher. It will take years to establish the techniques needed to create rapid, on-demand, bespoke CRISPR therapies. Most people with these conditions dont have that kind of time.

But researchers are working to streamline the process. Doudnas institute, for example, is working to standardize some aspects of genome-editing therapies, in part to make it cheaper and easier to develop such treatments for people with rare conditions. And the US National Institutes of Health has been trying to develop similar pipelines for gene therapies an effort that could help to inform genome-editing efforts. Its been really hard, says Doudna. But what were doing is going to have long-term impact.

In India, the work has continued. Rajeev has urged Chakraborty to finish the teams studies in mice, so that the next person with FENIB will not have to wait as long for a potential treatment. Some of the work will be completed, and the effort could benefit others with genetic conditions that affect the brain particularly in India. We are not really trying as aggressively as we did earlier, he says. But that technology has a lot of potential.

At Uditis memorial service, Rajeev tried to make sense of the timing. Uditi was always in a hurry, he told attendees. She always had to be first. She was only a few months away from receiving an experimental treatment, but she would not wait, not even for that. She could not let science win, he said. She was always ahead.

Link:
Hope, despair and CRISPR the race to save one woman's life - Nature.com

More than 30,000 people in the United States alone have already received personalized CART-T-cell therapy for cancer.Credit: Qilai Shen/Bloomberg/Getty

When researchers first began to test engineered immune cells designed to fight cancer about 20 years ago, there was a scepticism. The scientific potential might be clear, but what about the economics of such a complex and specialized therapy? Each dose would have to be made afresh, with cells from an individual being shipped to a centralized laboratory, genetically engineered using sophisticated techniques and shipped back for reinfusion. The process would take too long and be too expensive. Regulators would also surely struggle to ensure the safety of such an involved, individualized process.

Today, the chatter is very different. Engineered CAR T immune cells have so far been used to treat more than 30,000 people with cancer in the United States alone. CAR-T therapy is being tested for other conditions, including some severe autoimmune disorders. As for commercial success, in 2023, CAR T cells earned biotechnology companies US$8.4 billion worldwide.

Two News Features in this issue describe other complex, bespoke therapies that, a decade ago, would have been considered infeasible, if not impossible. One is an mRNA cancer vaccine tailored to an individuals tumour genome. The other is a CRISPR-based genome-editing therapy designed but sadly never used for one young woman with a rare neurological disorder.

How personalized cancer vaccines could keep tumours from coming back

Both approaches are fraught with challenges. As in the early days of CAR-T therapy, many of them are not scientific. But by guiding regulators and developing flexible platforms for producing bespoke treatments, researchers can help to shepherd therapies to the people who need them.

Researchers have long chased after vaccines that could rally the immune system against tumours, similarly to how vaccines rouse defences against pathogens. Companies can now sequence portions of a persons tumour and select those most likely to be visible to the immune system. The mRNA molecules corresponding to those regions are synthesized, then encapsulated in fatty particles and injected much like mRNA COVID-19 vaccines. From start to finish, the process takes as little as a month.

The technology behind these cancer vaccines is clinically more advanced than the genome editing used for some more specialized applications, for which researchers do not have the luxury of running large clinical trials. In one instance, scientists knew of only one person with the mutation they aimed to treat, using a technique called base editing that can make changes to specific DNA bases. It was, in effect, a treatment designed for a market of one person.

This kind of approach is called an n-of-1 therapy, a term that highlights the statistical challenges of interpreting results from a sample of one not to mention the commercial challenge of designing and selling a therapy with a one-person market. But the name is potentially misleading and stigmatizing. A cancer vaccine based on an individuals tumour could also be considered an n-of-1 therapy, yet this approach has attracted heavy investment from the pharmaceutical industry because the same process can be extended to many other people with cancer.

Hope, despair and CRISPR the race to save one womans life

The same thinking is needed for genome-editing therapies for rare disorders. Some genetic conditions that weaken or disable the immune system could be grouped together, and therapies for these diseases designed and administered in the same manner, even if the specific DNA changes made are different. Identical or similar measures such as levels of immune-cell function could be used to determine how well the treatment works.

But for-profit companies cannot be relied on to develop such platforms for CRISPR-based therapies as long as the perceived market remains small. Some academic researchers are focusing on developing such platforms for CRISPR-based therapies. More should join them or the chance to use genome editing to correct genetic disorders, the most severe of which are often rare, will be squandered.

Researchers can help regulatory agencies grappling with the new technologies. Regulators in the United States, the European Union, India and the United Kingdom have signalled a wish to aid the development of treatments for ultra-rare disorders. But they need help. Many regulations governing the manufacturing of therapies are grounded in regulatory paths forged years ago. Scientists can advise regulators on which technological advances have rendered certain cumbersome regulations unnecessary. This could speed up the development of treatments, as well as lower their costs.

Researchers around the world can engage in the same discussions with their regulators, and not just in typical hotspots for drug development, such as the United States and Europe. Such conversations will help to prepare for a future in which bespoke genetic therapies can be produced worldwide. They could also help to harmonize regulations between countries: an important goal for promoting the development of drugs for conditions that affect only a few individuals scattered around the globe.

As data accumulate from the treatment of people with rare genetic disorders, lessons learnt about the safety, effectiveness and manufacturing of bespoke therapies can be translated to treatments for more-common conditions. So the treatment of ultra-rare genetic disorders should not be devalued. Although a single disorder might affect only a few people, in aggregate, ultra-rare diseases affect millions. When it comes to personalized medicine, serving the interests of the few is in the interests of the many.

Continued here:
CRISPR cures and cancer vaccines: researchers can help to shepherd them to market - Nature.com

Over the past decade, CRISPR has taken the biomedical world and life sciences by storm for its ability to easily and precisely edit DNA. Here, Stanford University bioengineer Stanley Qi explains how CRISPR works, why its such an important tool, and how it could be used in the future including current developments in using CRISPR to edit the epigenome, which involves altering the chemistry of DNA instead of the DNA sequence itself.

CRISPR is not merely a tool for research. Its becoming a discipline, a driving force, and a promise that solves long-standing challenges from basic science, engineering, medicine, and the environment, said Qi, an associate professor in the Department of Bioengineering and institute scholar at Sarafan ChEM-H. Together, we can think innovatively about how to match needs with technologies to solve the most challenging problems.

(click the question to jump to the answer):

What is CRISPR

How does it work?

What are gene therapy and cell therapy, and how is CRISPR involved?

How does it differ from other gene-editing tools?

Why is it such a big deal?

How far has CRISPR technology come since it was created?

In 2019, Victoria Gray was the first person in the U.S. to receive CRISPR treatment for a genetic disease (sickle cell anemia). Now, CRISPR-based therapies are approved in the U.S. and the U.K. What is next?

Were you surprised when the 2020 Nobel Prize in chemistry went to CRISPRs developers?

Besides treatment for diseases, what are other real-world applications for CRISPR technology?

What are your views on some of the ethical concerns surrounding CRISPR?

Your group demonstrated that its possible to shrink CRISPR. Why is this significant?

What is your lab working on in terms of epigenome editing?

Are there limitations to what CRISPR can do?

What do you think CRISPR is capable of doing in the future?

How far are we from actually achieving those idealistic future goals?

The short answer: CRISPR is an immune system used by microbes to find and eliminate unwanted invaders.

Qi: CRISPR stands for clustered interspaced short palindromic repeats. Biologists use the term to describe the genetic appearance of a system that was discovered in microbes including bacteria and archaea as early as 1987. For a long time, no one really understood what it did, but around 2005, researchers discovered CRISPR is an immune system. Its used by microbes to help protect themselves from invading viruses. To stop the invaders, the microbes use CRISPR to recognize and eliminate specific trespassers.

Back to the list of questions

The short answer: When a virus or other invader enters a bacterial cell, the bacterium incorporates some of the trespassers DNA into its own genome so it can find and eliminate the virus during future infections.

Qi: Its similar to the human immune system. When a virus infects us, we generate an immune memory in the form of antibodies lots of them. Then, when the same virus infects us again, these antibodies quickly recognize the invaders and eliminate them.

When a virus infects a bacterial cell, CRISPR helps establish a memory a genetic one. The bacterium takes a piece of the viruss genome and inserts the DNA into its own genome. From that newly acquired DNA sequence, CRISPR creates a new guide RNA, a sequence that helps CRISPR find the invader via sequence complementarity (i.e., A binds to T and C binds to G). So, the next time when the virus infects that bacteria cell, the guide RNA rapidly recognizes the virus DNA sequence, binds to it, and destroys it.

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The short answer: Gene therapy can mean using CRISPR as a macromolecule drug to either fix a mutated gene or regulate a defective gene to treat a disease. Cell therapy means using CRISPR to make your bodys cells attack toxic cells or regenerate beneficial cells.

Qi: Gene therapy can mean two things: One is to fix a mutated gene, and the other is to regulate a genes expression into protein products. Our current understanding of gene therapy is still rapidly advancing, and the challenge is managing therapy safely and cheaply. Furthermore, were only looking at the simplest genetic diseases. For example, sickle cell anemia is a disease we know a lot about, and its often caused by a single mutation. So, we can configure CRISPR to fix it. But many more diseases are caused by widespread mutations, multiple mutations, and even multiple genes. In the future, gene therapy could go beyond a single mutation, and I am optimistic that in the next decades, gene therapy will become a pillar of medicine.

Cell therapy is a little different. For example, when people try to treat leukemia, a type of white blood cell tumor, sometimes chemotherapy drugs cant completely get rid of the tumor cells. In the past two decades, scientists have found that if they retrieve some of the patients T cells, which fight infections, these cells can be engineered as better fighters to recognize and eliminate tumorous cells. When the modified T cells are injected back into the patient, they can attack the tumors. However, cells are quite complicated. Sometimes, they go out of control when injected back into the patient, killing healthy cells along with the tumor cells. At other times, they may fail to work because they are suppressed by the tumor cells. CRISPR offers a powerful tool to enhance the efficacy and safety of these immune cells so that they are completely under our control for best clinical benefits.

Back to the list of questions

The short answer: CRISPR is much easier to program than other tools.

Qi: Before CRISPR, most gene-editing tools were a single protein. By changing the peptide sequence of these proteins, scientists could alter their targets. To change the target, you need to completely redesign the proteins sequence and then test if it even works, which is tedious, unpredictable, and time-consuming. These gene-editing tools were theoretically interesting, but they were difficult to use for large-scale studies and therapeutics.

Compared to that, CRISPR is elegant because the target recognition sequence is mostly encoded within an RNA rather than a protein, and redesigning this sequence is one of the simplest things you can do in molecular biology. It makes genome editing similar to operating a GPS: If you want to go to destination A, you just type the address, and to change to destination B, you just enter the new location. So, this tool dramatically reduces the burdens, cost, timing, while increasing the precision and accuracy of a gene-editing system.

Back to the list of questions

The short answer: CRISPR can precisely modify a piece of DNA or its chemistry (so-called epigenetics) in the human body, making it a potential tool for clinical uses in the biomedical sciences.

Qi: CRISPR is a molecule and tool desired by everyone who works in the life sciences, biomedical research, and clinical settings. Its high precision is unparalleled and enables many uses including gene therapy.

My dream has been to develop new biotechnologies and apply them to diseases without a cure. Genetic diseases make up a big part of this category. Traditional medicines small molecule drugs, surgery, and other methods dont work for these types of diseases. But CRISPR molecules have become highly promising as treatments because they allow us to precisely modify a piece of DNA in the human body. This could lead not only to relief but also to a cure.

Indeed, recent FDA approval of the first CRISPR drug, Casgevy, in treating sickle cell anemia and beta thalassemia speaks to its safety and potential for other diseases. Sickle cell anemia is a disease in which people have a mutation in their red blood cells. Normally, theres no treatment other than frequent blood transfusions or bone marrow transplants from a matched donor, which are expensive and damaging to a patients overall health. Using CRISPR, its possible to perform a one-time treatment to permanently correct the mutation. There are more than 8,000 genetic diseases like that, which can be potentially considered.

Back to the list of questions

The short answer: In about a decade, scientists went from wondering if this technology would even work in human cells to getting the first CRISPR drug approved uses in the clinic.

Qi: In 2010, I was working on CRISPR as a bioengineering graduate student at the University of California, Berkeley, under Adam Arkin, a synthetic biologist and bioengineer, and collaborated with Jennifer Doudna, a biochemist and structural biologist. In the early days, CRISPRs practical usefulness was not very publicly recognized. At that time, many counterarguments said CRISPR was just a bacterial system and most of these simply dont work in human cells which, to be fair, is true.

But after Jennifer Doudna and Emmanuelle Charpentier published their seminal 2012 paper on Cas9 one type of CRISPR that cuts DNA using a single protein and an engineered single guide RNA the research and published papers grew exponentially. Firstly, because its a system that everyone in the life sciences wants. Secondly, using CRISPR is super easy, flexible, and robust. Its not like other technologies that take multiple years and millions of dollars to set up CRISPR only takes a couple of weeks and a bit more than a few hundred dollars to set up now.

A lot of researchers significantly contributed to the rapid development. For example, within three years following its initial demonstration, structural biologists solved the high-resolution, three-dimensional structure of what Cas9 and other CRISPR proteins look like. Bioinformaticians have revealed many new species of Cas molecules beyond Cas9, many of which have novel functions. Biochemists engineered CRISPR to understand how fast and tightly it binds to DNA. Bioengineers, including me, engineered the proteins to make them work more efficiently and more specifically so they can work better in the human body for gene therapies. Also, clinical researchers started to use the tool to address particular diseases.

Furthermore, the applications of CRISPR went beyond gene editing. Epigenetic editing is an exciting development, although we still await clinical benefits. It was used for targeting the human 3-dimensional genome, visualizing the DNA dynamics, or even targeting another set of molecules, RNA, for gene regulation.

I dont think Im exaggerating to say that, essentially, CRISPR has been tested as a potential treatment option for every disease that we have clear knowledge about. CRISPR cant solve all of them, but because this tool is so powerful, easy to use, and so far-reaching, it has allowed everyone to combine their expertise with CRISPR.

Back to the list of questions

The short answer: This is very exciting. Future CRISPR drugs will address more incurable diseases, which provide a test case for CRISPRs efficacy and safety in different organs and patients.

Qi: Im super excited to see CRISPR becoming a drug to treat a disease as a one-time cure. When CRISPR first came out, there were concerns about whether these bacterial molecules could be used safely in humans and whether it was safe to cut and edit human DNA. While there are still questions regarding long-term effects (beyond the period of clinical trials in tested patients) it is very encouraging that CRISPR is safe and effective.

The next step is to expand the scope of CRISPR drugs. Medicine isn't made in one day. Different diseases are caused by different mechanisms. There are already more than dozens of CRISPR clinical trials for different diseases in the liver, immune cells, eyes, and muscles. Furthermore CRISPR epigenetic editing is expanding the scope of disease to treat more types of muscular dystrophy, retina disorders, and brain diseases.

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The short answer: Not at all. But I hope the award doesnt lead people to think CRISPR research is finished its still growing, and theres much more to explore in basic research, medicine, and beyond.

Qi: Im not surprised at all. Even before 2020, researchers had been discussing when the Nobel Committee would recognize CRISPR. So, when it happened, I was super excited.

Jennifer Doudna (University of California, Berkeley) and Emmanuelle Charpentier (Max Planck Unit for the Science of Pathogens) received the Nobel Prize in Chemistry only seven years after CRISPR was first reported as a molecular system for modifying the human genome.

I hope that giving the Nobel Prize to CRISPR wont give people the impression that the genome editing field is done. This is a field thats still growing in every corner of life sciences. Besides being explored as medicine in humans, it is expanding its influence in plants, microbes, and difficult-to-engineer organisms such as fungi. There are so many questions about how we can use CRISPR for safely controlling the genome, how to use it for novel and innovative research, and how to make it a clinical product that still need to be explored.

These are exciting frontiers of further increasing the safety of CRISPR-based therapies and expanding the scope of diseases treatable by this technology.

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The short answer: Some other uses are diagnostics, manufacturing, sustainability, and ecological engineering.

Qi: CRISPR can be used for diagnostics. It has been developed as a way to sensitively detect pathogens in the environment that are affecting our bodies.

There are also opportunities in manufacturing, such as making products that we care about using organisms like yeast and bacteria. Imagine that we could use CRISPR to engineer new microbes that could boost production like 10x more beer, for instance. And also, beer that tastes much better and can be catered to different peoples wants and needs.

Sustainability is also a big application for CRISPR via bioengineering. Creating sustainable, carbon-neutral methods of energy or food production is a challenge. Genome engineering may offer better manufacturing protocols through microbes that reduce greenhouse gases, plastic, and food waste.

Finally, we get to ecological engineering. For example, people are trying to eliminate certain invading or pathogenic mosquito species using CRISPR, but in my opinion, its long-term safety and impact still need careful evaluation. Other people are trying to revive extinct species. Recently, scientists announced they were trying to revive a woolly mammoth that can live in the Arctic cold.

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The short answer: My research group often thinks about the ethics of CRISPR. Some ethically questionable areas are disease prevention and eliminating pesky species, and some definite unethical areas are enhancement and creating designer babies.

Qi: The ethical side of CRISPR is something my research group thinks about every day. One of the fundamental principles of ethics is to do no harm. Sure, we want to do something great and helpful to people, but at the same time, we have to consider if were harming other people. Using that principle, we can consider a few cases.

One example is a designer baby, which is a scary topic. That is regarded as unethical because this may create a new human species. When the germ cells sperm and egg cells are edited, this not only affects that single person, but also the children that person could have in the future.

Another concern is in the division of treatment, which has three categories: cure, prevention, and enhancement. Curing someones disease is great. Prevention, which means someone is at risk of developing a problem, is a gray area. If someone has a high chance of getting an infectious disease, should we use gene therapy to permanently modify their DNA to reduce their risk? That question really depends on if we have other options. The last category enhancement is likely unethical. People talk about the possibility of targeting a gene to grow more muscle or make people smarter or better looking. But if research goes into this category, only some people may be able to afford it. This could amplify the imbalance of socioeconomic status. Another facet to consider is medical necessity. Is the therapy really necessary, or are there other ways to solve the problem through currently available drugs, diet, exercise, etc.?

Beyond medicine, some scientists may want to use CRISPR for ecological reasons, for example, eliminating mosquitoes. From my viewpoint, thats controversial because I think every species exists for a reason. If we try to eliminate mosquitoes, we might have a chain reaction that affects other life forms in the environment and can be irreversible. I hope in the future we can make this technology reversible like installing a switch so that if we make something that turns out to be less than ideal, we still have some way to reset it.

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The short answer: Its tricky to deliver CRISPR molecules into cells. Shrinking the size of the molecule helps it easily traverse inside of cells and get to its DNA target.

Qi: CRISPR is such a magic molecule, but that magic only works if CRISPR gets inside cells and touches the DNA. The question is obvious: How can we even make CRISPR get inside the cell?

Human cells are designed to resist any invading DNA. So the human body has many strategies to prevent foreign DNA from getting in.

Many delivery methods scientists used have limited power. We can use retooled viruses to deliver clinical products into cells, but they have a small capacity the Cas9 version of CRISPR usually doesnt fit inside the virus. Therefore, the currently approved CRISPR drug requires isolating patient cells, modifying them, and putting them back in. This process is costly and slow. If we want CRISPR to become a broadly useful medicine, then we need to make the molecule as small as possible.

Thats why we made this miniature CRISPR, which we call CasMINI, which is only half the size of Cas9. We also saw that it is easier to enter cells and works better than other CRISPR molecules because it can get inside more efficiently. This miniature CRISPR can revolutionize the way that we can perform editing in the body. Our hope is to address these technical barriers then test how miniature CRISPR can be delivered to different parts of the human body to treat various genetic diseases.

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The short answer: Were trying to use CRISPR to control gene function rather than editing genes to treat diseases.

Qi: Im excited about exploring how to treat diseases without modifying human DNA through epigenome editing. Its a different way of thinking about gene therapy. Unlike gene editing, epigenome editing is reversible, safer, and promising for complex diseases that can not be easily targeted by gene editing.

To enable epigenome editing, we developed the first nuclease-deactivated dCas9 in living cells, to programmably target and control gene expression, without altering the DNA sequence. For example, if a person doesnt have enough properly working proteins, we can use epigenome editing to increase the gene expression over a long term to make more proteins to compensate for this deficiency problem, thus restoring the function to normal in patients.

In other cases, someone may have a gene mutation that produces a toxic product, such as in many muscular dystrophies or neurological degenerative diseases. Rather than using CRISPR to modify DNA, we can use our epigenome editing technology to permanently silence the gene without modifying the DNA. I am excited to test this solution in the clinic as I believe this offers a safer strategy for treatment without altering DNA.

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The short answer: There are limitations to gene editing, but new technologies are trying to expand the power of CRISPR.

Qi: One major limitation is weve been using it for only 10 years. Often, time is the best test of all technologies. Only by collecting data over enough time in all scenarios will we be able to understand everything about these technologies, like how safe they are over the long term.

In testing in human subjects with patients, even though we didnt see off-target effects or immune responses, there are still question marks. We still need to constantly improve our understanding, as well as CRISPRs accuracy and precision in different human tissues and different patients, when treating a problem.

Also, right now, CRISPR is mostly used as molecular scissors to cut DNA. But sometimes, the problem genes affected function isnt caused by a DNA mutation. Sometimes, its a gene turning on or off abnormally that causes the problem. So in that case, CRISPR shouldnt be used as molecular scissors to cut DNA, but rather as a switch to restore the gene to work properly. Epigenetic editing tools can well address such challenges.

CRISPR is like a powerful hammer. But the question is: Where is the nail? What is the most suitable nail to work on? For example, as of today, we still dont know for sure which gene causes Alzheimers disease in many patients. To use CRISPR, we need to know which gene to target and which cell is the destination. We also need to know when to perform the treatment sometimes treatment can only be done in an early stage of a persons life.

Another big issue is the high costs associated with the current CRISPR medicine. How to reduce cost is a major question. Im glad that there are active conversations between academia and industrial partners to have multiple experts in the same room to come up with the best solution.

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The short answer: It could help improve the quality of life as we age, engineer useful organisms, and even serve as a universal vaccine against viruses.

Qi: Im excited by CRISPR possibly helping anti-aging, but less in the sense of making people live longer. No one can escape aging, and its a huge burden to our healthcare system and decreases the quality of life. My hope is that in the future, CRISPR isnt just being used to save lives, but also to improve the quality of life when people age.

I also hope CRISPR can become a way to engineer a lot of useful life forms. For example, there are microbes that can capture solar energy and convert it to electricity, and maybe those could be used to produce sustainable energy. Additionally, we could engineer food thats more nutritious, prevents obesity, and so on.

Another application could be vaccines. Even now, infectious diseases, like COVID-19, have dramatically changed everyones lives, which is unbelievable. So another dream is to develop cheap and safe genetic vaccines to fight all viruses, since thats their original role in bacteria. And maybe, in the future, we could receive a small dose of CRISPR that could completely kill any new virus. Its not easy, but given that this genetic system was designed as an antiviral system, theres a chance this could work.

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The short answer: Were close to some goals but may be far from some other idealistic goals.

Qi: When it comes to CRISPR and achieving those big dreams we have for it, we're at different stages. For some goals, it might feel like we're just starting out, but for others, we're getting pretty close. For example, I'm really excited about how we're starting to use CRISPR in real-life treatments for diseases, such as sickle cell anemia. This is a big step forward! I am also very excited about CRISPR epigenetic editing, a way to turn genes on or off without changing DNA sequence, which is getting ready for its big moment in clinical trials.

The reason weve come this far is thanks to a lot of people who believe in the power of safely editing our genes to make us healthier and are working hard every day to make that a reality. Its their passion and the demand for these solutions that keep pushing us forward. Im optimistic that many of the things were dreaming about with CRISPR could become real, sooner rather than later.

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What is CRISPR? A bioengineer explains | Stanford Report - Stanford University News

The successful development of the revolutionary gene-editing technology called CRISPR won the 2020 Nobel Prize in Chemistry, awarded to Emmanuelle Charpentier of the Max Planck Institute, and Jennifer Doudna of the University of California, Berkeley.

CRISPR offers the exciting possibility of developing new gene therapies treatments involving genome editing to prevent/treat human diseases that have a genomics basis, such as cystic fibrosis, diabetes, muscular dystrophy, sickle-cell anaemia and more. Unfortunately, CRISPR could also be used to achieve bad ends, such as being used in misguided eugenics programmes.

CRISPR stands for clustered regularly interspaced short palindromic repeats, and is a natural molecular technology used by many bacteria to fight off invading viruses. This molecular technology can also be used in mammalian cells to edit genetic DNA by adding, removing or changing DNA letters, and by turning genes on/off without altering their sequence.

Although there are other means of carrying out gene therapy, CRISPR has opened up new possibilities for treating human genetic diseases, is faster and much cheaper than other gene-editing technologies, and has revolutionised biotechnology. Last year the worlds first CRISPR therapy was approved to treat human patients with sickle-cell disease.

Any ideas of modifying the evolution of society through genetic manipulation resonate with the views of Francis Galton (18221911) of guiding human evolution through controlled breeding, also known as eugenics. Not only were Galtons ideas based on false evidence, but they led on to disastrous programmes of mass sterilisation and eventually to the genocidal nightmare of the Nazi gas ovens.

Engineering inheritable genetic changes to produce what might be seen as socially desirable traits that are not disease-related in future generations, such as light skin colour, is problematic

CRISPR should be used to pioneer cures for diseases while upholding human dignity and sanctity of life for all. We must be extremely cautious and conservative about using CRISPR to manipulate the genetic make-up of future generations. Human diversity of cultures, perspectives, abilities and identities are vitally important for global equilibrium and functioning, and we interfere with this diversity at our peril.

A reasonable person might think it would it be a good idea to use CRISPR to eliminate disease-related genes from the human genome, eg genes that predispose people to heart disease, obesity and type 2 diabetes. However, there is mounting evidence that these genes conferred advantages related to survival upon our ancestors prior to the Industrial Revolution, when nutrition was generally poor and patchy (see the views of Maria Esther Rubio Ruiz and others in International Journal of Evolutionary Biology).

[Worlds first gene-editing therapy for humans approved in UK]

Today, many people eat highly processed foods rich in fat/sugar while at the same time leading sedentary lives, a combination that interacts with these old survival-enhancing genes to cause deadly modern diseases. So, in a future world possibly bedevilled by famines and other privations, these bad genes could actually function as good genes. It would be unwise to eliminate them from the population now.

Also, engineering inheritable genetic changes to produce what might be seen as socially desirable traits that are not disease-related in future generations, such as light skin colour, is problematic. Many non-westerners use skin-whitening cream to induce fair complexions, believing fair skin looks better than dark skin. But genetically engineering fair skin in offspring could put them at a future disadvantage in terms of survival. If atmospheric ozone levels deplete significantly, genetically engineered fair-skinned people, who would otherwise be protected by skin melanin, would suffer increased incidence of skin cancer.

A global moratorium on heritable gene-editing was introduced in 2019. This moratorium will end soon

In 2018, maverick Chinese scientist He Jiankui carried out unregulated gene editing of human embryos, two of whom were later born as the twin girls Lulu and Nana. This genetic editing was designed to confer resistance to the Aids-inducing human immunodeficiency virus (HIV) by inactivating the gene CCR5, mimicking a natural mutation that protects a small number of people from HIV.

[ Dodos have been extinct for hundreds of years. Can scientists really bring them back to life?]

This work was greeted with worldwide concern. Scientists pointed out that too little is known about genes to make such changes safely, that other genes can be damaged in the process, and that these changes are passed on to future generations. A global moratorium on heritable gene-editing was introduced in 2019. This moratorium will end soon, and anxieties regarding what CRISPR technology might lead to are mounting.

While CRISPR technology promises wonderful advances in medicine, it also raises profound ethical questions. Going forward, we must remain vigilant in guarding human rights and ethical practices that support equality and dignity of all, regardless of their genetic make-up. No less than the aforementioned Doudna, one of the inventors of CRISPR technology, has called for a ban on heritable gene-editing until scientific, technical and ethical questions are answered and public consensus has developed.

William Reville is an emeritus professor of biochemistry at UCC

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Advantages - and dangers - of new gene-editing technology CRISPR - The Irish Times

Intellia Therapeutics said Sunday that the first 10 patients to receive a CRISPR-based treatment for hereditary angioedema, a genetic disease, saw their swelling attacks the conditions hallmark symptom virtually eliminated for an average of 20 months and counting.

The attacks were reduced by an average of 98%. One patient has remained attack-free for 26 months. Two patients who suffered particularly frequent attacks, experiencing 14 and 16.8 per month, respectively, have now gone more than 20 months since their last attack.

No patient has had an attack in the last 11 months of follow-up, according to the data presented at the European Academy of Allergy and Clinical Immunology Congress. All side effects were either grade 1 or 2 on a 5-grade scale.

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Intellia reports positive results for its CRISPR-based treatment - STAT

Last year, startup Pairwise started selling the first food in the US made with Crispr technology: a new type of mustard greens with an adjusted flavor. But chances are, most consumers never got to sample them. The company introduced the greens to the food service industryselect restaurants, cafeterias, hotels, retirement centers, and caterersin just a few cities. A single grocery store in New York City also stocked them.

Now, biotech giant Bayer has licensed the greens from Pairwise and plans to distribute them to grocery stores across the country. We hope to have product reaching kitchen and dinner tables in the fall of this year, says Anne Williams, head of protected crops in Bayers vegetable seeds division. She says Bayer is currently talking to farms and salad companies on how best to grow and package the greens.

Pairwise was looking to make salads more appetizing and nutritious, and the company targeted mustard greens because of their high nutritional value, which is similar to kale. But their peppery, bitter taste means theyre not often eaten raw. Instead, theyre usually cooked to make them more palatable. Pairwise aimed to tone down the flavor while keeping all the fiber, antioxidants, and other nutrients that mustard greens offer. The company used Crispr to remove several copies of a gene responsible for their pungency. We think people will really like the taste, Williams says.

Pairwise previously took the greens to farmers markets for taste-test trials and explained to shoppers that they were made with gene editing. Tasters were generally positive about the greens, according to Pairwise CEO Tom Adams. The company is now turning its attention to developing pitless cherries and seedless blackberries. We see our role in the food chain as inventing new products, he says.

The first Crispr-edited food available to consumers debuted in Japan in 2021 when Tokyo-based startup Sanatech Seed began selling a tomato with high levels of -aminobutyric acid, or GABA, a chemical made in the brain and also found naturally in some foods. The company claims that GABA can help lower blood pressure and promote relaxation.

At a May 28 event in the Netherlands, Sanatech president Shimpei Takeshita said the company has expanded distribution in Japan and has completed all the regulatory paperwork to introduce its tomato in the Philippines. Its also looking to bring its edited tomato to the US.

The mustard greens and high GABA tomato arent exactly genetically modified organisms, or GMOsnot in the traditional sense, at least. Typically, GMOs are crops that contain added genetic material from a different species entirely. By contrast, gene editing involves modifying an organisms own DNA.

Williams describes Crispr as a tool that speeds up breeding new plants, allowing scientists to make changes that could conceivably happen in nature, just much faster. In the US, the Department of Agriculture has decided that crops made with gene editing dont have to go through a lengthy regulatory review, reasoning that they do not contain foreign DNA and could have otherwise been developed through conventional breedingthat is, choosing parent plants with certain characteristics to produce offspring with those traits.

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Gene-Edited Salad Greens Are Coming to US Stores This Fall - WIRED

Treating neurons and brain organoids with a modified form of CRISPR rescues the effects of pathogenic variants in two high-confidence autism-linked genes, according to a new preprint. The approach boosts the expression of the genes, CHD8 and SCN2A, by targeting structures that regulate them.

SCN2A codes for an ion channel that helps propagate electrical signals across the brain. Variants in that gene are associated with seizures, autism and intellectual disability. CHD8 is involved in the remodeling of chromatin, the complex of DNA and proteins that makes up chromosomes. People with faulty copies of CHD8 typically have autism and a larger-than-average head.

The work suggests that the impact of autism-related variants could be reversed by altering gene expression, as opposed to altering genes directly, although more research is needed before the technique can find applications in the clinic, experts say.

This is another way in which we can regulate genes that are extremely important in [autism], so thats a major finding, says Kevin Bender, associate professor of neurology at the University of California, San Francisco, who was not involved in the study. But its really the first step in understanding whether this approach is broadly applicable.

In the past, Bender and his colleagues used the same version of CRISPR, which activates genes rather than editing them, to boost the expression of SCN2A in mice with a harmful variant in one copy of the gene. The treatment corrected problems in the animals neurons.

By increasing the expression of SCN2A in mutant neurons and brain organoids, the new study confirmed many of the previous findings. But the work also revealed, for the first time, that activating gene regulatory elements with CRISPR could counteract the effects associated with harmful variants in CHD8.

Because that gene controls the expression of thousands of other genes, to see that the upregulation of CHD8 is restorative in some way is really exciting, Bender says.

T

So Geschwind and his team used CRISPR to boost activity in noncoding regions of the genome called enhancers, which can regulate a genes transcription, in this case linked to SCN2A and CHD8.

Boosting enhancers of CHD8 in neurons and brain organoids that lacked a functional copy of that gene led to a reduction in organoid size and in the number of differentially expressed genes. This finding suggests the intervention can rescue cells: without it, CHD8 mutant organoids are larger and show an over-proliferation of neural progenitor cells in comparison with controls that have two functional gene copies.

To see that the upregulation of CHD8 is restorative in some way is really exciting.

Similarly, enhancing the expression of SCN2A reversed the problems observed in neurons and brain organoids that lack the gene, including impaired development, reduced excitability and sluggish responses to electrical currents.

The team posted their findings on the preprint server bioRxiv in March.

Identifying enhancers for SCN2A and CHD8 was a feat in itself, Bender says. Enhancers arent typically located close to the genes they regulate. To find them, scientists must undertake a treasure hunt, he says. Their ability to do that was really remarkableand theyve done it for two major [autism-linked] genes.

Unlike traditional CRISPR approaches, which cut DNA to delete or insert variants and can have off-target effects, CRISPR activation may be less likely to cause harm. But before the approach finds its way to the clinic, researchers will need to determine its safety profile and look for any unintended consequences, saysNadav Ahituv, professor of bioengineering and therapeutic sciences at the University of California, San Francisco, who was not involved in the study.

Ahituv, who has worked with Bender on activating SCN2A using CRISPR, is co-founder of a company that is developing CRISPR therapies that target SCN2A and SCN1A, which has also been linked to autism.

Researchers must also identify a suitable way to deliver the intervention to the brain, Geschwind says. People are working out in-utero gene delivery, so I dont think its that far off in the next decade, he says. Im fairly optimistic about this type of approach.

The new study, he adds, also avoids the need to invest significant resources and time into deciphering the functions and mechanisms of individual genes, as well as developing methods to counteract them. Instead, it uses a genes regulatory elements to activate its expression. To me, thats an exciting therapeutic shortcut.

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CRISPR gives autism-linked genes a boost, rescues functioning - The Transmitter: Neuroscience News and Perspectives

Plasmid design and construction

A summary of plasmid constructs are in Supplementary Table 1 and plasmid sequences are in Supplementary Data 2. Unless otherwise specified, cloning was performed by Gibson Assembly of PCR-amplified or commercially synthesized gene fragments (from Integrated DNA Technologies or Twist Bioscience) using NEBuilder Hifi Master Mix (NEB, E262), and final plasmids sequence-verified by Sanger sequencing of the open reading frame and/or commercial whole-plasmid sequencing service provided by Primordium.

To summarize, denAsCas12a-KRAB, multiAsCas12a-KRAB, multiAsCas12a and enAsCas12a-KRAB open reading frames were embedded in the same fusion protein architecture consisting of an N-terminal 6xMyc-NLS29 and C-terminal XTEN80-KRAB-P2A-BFP103. The denAsCas12a open reading frame was PCR amplified from pCAG-denAsCas12a(E174R/S542R/K548R/D908A)-NLS(nuc)-3xHA-VPR (RTW776) (Addgene, plasmid 107943 (ref. 30)). AsCas12a variants described were generated by using the denAsCas12a open reading frame as starting template and introducing the specific mutations encoded in overhangs on PCR primers that serve as junctions of Gibson assembly reactions. opAsCas12a (ref. 29) is available as Addgene plasmid 149723, pRG232. 6xMyc-NLS was PCR amplified from pRG232. KRAB domain sequence from KOX1 was previously reported42. The lentiviral backbone for expressing Cas12a fusion protein constructs expresses the transgene from an SFFV promoter adjacent to UCOE and is a gift from Marco Jost and Jonathan Weissman, derived from a plasmid available as Addgene 188765. XTEN80 linker sequence was taken from a previous study51 and was originally from Schellenberger et al.111. For constructs used in piggyBac transposition, the open reading frame was cloned into a piggyBac vector backbone (Addgene, 133568) and expressed from a CAG promoter. Super PiggyBac Transposase (PB210PA-1) was purchased from System Biosciences.

dAsCas12a-3xKRAB open reading frame sequence is from a construct originally referred to as SiT-ddCas12a-[Repr]27. We generated SiT-ddCas12a-[Repr] by introducing the DNase-inactivating E993A by PCR-based mutagenesis using SiT-Cas12a-[Repr] (Addgene, 133568) as template. Using Gibson Assembly of PCR products, we inserted the resulting ddCas12a-[Repr] open reading frame in-frame with P2A-BFP in a piggyBac vector (Addgene, 133568) to enable direct comparison with other fusion protein constructs cloned in the same vector backbone (crRNAs are encoded on separate plasmids as described below).

Fusion protein constructs described in Supplementary Fig. 8bf were assembled by subcloning the protein-coding sequences of AsCas12a and KRAB into a lentiviral expression vector using the In-Fusion HD Cloning system (TBUSA). AsCas12a mutants were cloned by mutagenesis PCR on the complete wild-type AsCas12a vector to generate the final lentiviral expression vector.

All individually cloned crRNA constructs and their expression vector backbone are listed in Supplementary Table 1. Unless otherwise specified, individual single and 3-plex crRNA constructs were cloned into the human U6 promoter-driven expression vector pRG212 (Addgene, 149722 (ref. 29)), which contains wildtype (WT) direct repeats (DR). Library 1, Library 2, and some 3-plex and all 4-plex, 5-plex and 6-plex As. crRNA constructs were cloned into pCH67, which is derived from pRG212 by replacing the 3 DR with the variant DR8 (ref. 28). For constructs cloned into pCH67, the specific As. DR variants were assigned to each position of the array as follows, in 5 to 3 order:

3-plex: WT DR, DR1, DR3, DR8

4-plex: WT DR, DR1, DR10, DR3, DR8

5-plex: WT DR, DR1, DR16, DR10, DR3, DR8

6-plex: WT DR, DR1, DR16, DR18, DR10, DR3, DR8

8-plex: WT DR, DR1, DR16, DR_NS1, DR17, DR18, DR10, DR3, DR8

10-plex: WT DR, DR1, DR16, DR_NS1, DR4, DR_NS2, DR17, DR18, DR10, DR3, DR8

DR sequences are as follows: WT DR=AATTTCTACTCTTGTAGAT, DR1=AATTTCTACTGTCGTAGAT, DR16=AATTCCTACTATTGTAGGT, DR_NS1=AATTCCTCCTCTTGGAGGT, DR4=AATTTCTACTATTGTAGAT, DR_NS2=AATTCCTCCTATAGGAGGT, DR17=AATTTCTCCTATAGGAGAT, DR18=AATTCCTACTCTAGTAGGT, DR10=AATTCCTACTCTCGTAGGT, DR3=AATTTCTACTCTAGTAGAT, DR8=AATTTCTCCTCTAGGAGAT. Sequences for DR variants were previously reported28, except for DR_NS1 and DR_NS2, which were newly designed based on combining previously reported variants28. The rationale for selecting specific DR variants was to minimize homology across variants and maintain high crRNA activity based on prior analysis28.

1-plex,3-plex, 8-plex, and 10-plex crRNA constructs were cloned by annealing sets of complementary oligos with compatible overhangs in spacer regions, phosphorylation by T4 polynucleotide kinase (NEB M0201S), and ligated with T4 DNA ligase (NEB M0202) into BsmbI site of vector backbones. 4-plex, 5-plex and 6-plex crRNA arrays were ordered as double-stranded gene fragments and cloned into the BsmbI site of vector backbones by Gibson Assembly using the NEBuilder HiFi DNA Assembly Master Mix (NEB, E2621). Functions for designing oligos or gene blocks for cloning crRNA arrays are available as an R package at https://github.com/chris-hsiung/bears01.

All spacer and PAM sequences are provided in Supplementary Table 1. For cloning individual crRNA constructs targeting TSSs, CRISPick (https://portals.broadinstitute.org/gppx/crispick/public) was used in the enAsCas12a CRISPRi mode (by providing gene name) or CRISPRko mode (by providing sequence for TSS-proximal regions) to design spacers targeting canonical (TTTV) or non-canonical PAMs generally located within 50-bp to +300-bp region around the targeted TSS whenever possible, but some sites farther from the annotated TSS can show successful CRISPRi activity and were used. We manually selected spacers from the CRISPick output by prioritizing the highest on-target efficacy scores while avoiding spacers with high off-target predictions. The same non-targeting spacer was used throughout the individual well-based experiments and was randomly generated and checked for absence of alignment to the human genome by BLAT112.

The hg19 genomic coordinates for MYC enhancers are e1 chr8:128910869-128911521, e2 chr8:128972341-128973219 and e3 chr8:129057272-129057795. DNA sequences from those regions were downloaded from the UCSC Genome Browser and submitted to CRISPick. The top three spacers targeting each enhancer were picked based on CRISPick on-target efficacy score, having no Tier I or Tier II Bin I predicted off-target sites, and considering proximity to peaks of ENCODE110 DNase hypersensitivity signal (UCSC Genome Browser113 accession # wgEncodeEH000484, wgEncodeUwDnaseK562RawRep1.bigWig) and H3K27Ac ChIP-seq signal (UCSC Genome Browser accession # wgEncodeEH000043, wgEncodeBroadHistoneK562H3k27acStdSig.bigWig). These DNase hypersensitivity and H3K27Ac ChIP-seq tracks were similarly used to nominate candidate enhancer regions at the CD55 locus, whose genomic sequences are provided in Supplementary Table 1.

C4-2B cells114 were gifted by F. Feng, originally gifted by L. Chung. All cell lines were cultured at 37C with 5% CO2 in tissue culture incubators. K562 and C4-2B cells were maintained in RPMI-1640 (Gibco, 22400121) containing 25mM HEPES, 2 mM L-glutamine and supplemented with 10% FBS (VWR), 100 U ml1 streptomycin, and 100mg ml1 penicillin. For pooled screens using K562 cells cultured in flasks in a shaking incubator, the culture medium was supplemented with 0.1% Pluronic F-127 (Thermo Fisher, P6866). HEK 293T cells were cultured in media consisting of DMEM, high glucose (Gibco 11965084, containing 4.5g ml1 glucose and 4mM L-glutamine) supplemented with 10% FBS (VWR) and 100units/mL streptomycin, 100mg ml1 penicillin. Adherent cells were routinely passaged and harvested by incubation with 0.25% trypsin-EDTA (Thermo Fisher, 25200056) at 37C for 510min, followed by neutralization with media containing 10% FBS.

Unless otherwise specified below, lentiviral particles were produced by transfecting standard packaging vectors (pMD2.G and pCMV-dR8.91) into HEK293T using TransIT-LT1 Transfection Reagent (Mirus, MIR2306). At <24h after transfection, culture medium was exchanged with fresh medium supplemented with ViralBoost (Alstem Bio, VB100) at 1:500 dilution. Viral supernatants were harvested ~4872h after transfection and filtered through a 0.45mm PVDF syringe filter and either stored in 4C for use within <2 weeks or stored in 80C until use. Lentiviral infections included polybrene (8g/ml). MOI was estimated from the fraction of transduced cells (based on fluorescence marker positivity) by the following equation115,116: MOI=ln(1 fraction of cells transduced).

For experiments described in Supplemental Fig. 8af, lentivirus was produced by transfecting HEK293T cells with lentiviral vector, VSVG and psPAX2 helper plasmids using polyethylenimine. Medium was changed ~68h post transfection. Viral supernatant was collected every 12h five times and passed through 0.45-m PVDF filters. Lentivirus was added to target cell lines with 8g ml1 polybrene and centrifuged at 650g for 25min at room temperature. Medium was replaced 15h after infection. An antibiotic (1g ml1 puromycin) was added 48h after infection.

For piggyBac transposition of fusion protein constructs, cells were electroporated with ~210ng of AsCas12a fusion protein plasmid and ~84ng of Super PiggyBac Transposase Expression Vector (PB210PA-1, Systems Biosciences) using the SF Cell Line 4D-Nucleofector X Kit (V4XC-2032, Lonza Bioscience) and the 4D-Nucleofector X Unit as per manufacturers instructions (FF-120 program for K562 cells; EN-120 program for C4-2B cells).

The following antibodies were used for flow cytometry at 1:100 dilution: CD55-APC (BioLegend, 311312), CD55-PE (BioLegend, 311308), CD81-PE (BioLegend, 349506), CD81-AlexaFluor700 (BioLegend, 349518), B2M-APC (BioLegend, 316311), KIT-PE (BioLegend, 313204), KIT-BrilliantViolet785 (BioLegend, 313238) and FOLH1-APC (BioLegend, 342508). Cells were stained with antibodies were diluted in FACS Buffer (PBS with 1% BSA) and washed with FACS Buffer, followed by data acquisition on the Attune NxT instrument in 96-well plate format unless otherwise specified. For CRISPRi experiments, all data points shown in figures are events first gated for single cells based on FSC/SSC, then gated on GFP-positivity as a marker for cells successfully transduced with crRNA construct, as exemplified in Supplementary Fig. 1. For CRISPRi experiments in C4-2B cells, propensity score matching on BFP signal was performed using the MatchIt v4.5.3R package.

For cell fitness competition assays, the percentage of cells expressing the GFP marker encoded on the crRNA expression vector is quantified by flow cytometry. log2 fold-change of percentage of GFP-positive cells was calculated relative to day 2 (for experiments targeting the Rpa3 locus in Supplementary Fig. 8) or day 6 (for experiments targeting the MYC locus in Fig. 6b). For experiments targeting the Rpa3 locus, flow cytometry was performed on the Guava Easycyte 10 HT instrument.

For all crRNAs in Library 1 and Library 2, we excluded in the analysis spacers with the following off-target prediction criteria using CRISPick run in the CRISPRi setting: 1) off-target match = MAX for any tier or bin, or 2) # Off-Target Tier I Match Bin I Matches > 1). The only crRNAs for which this filter was not applied are the non-targeting negative control spacers, which do not have an associated CRISPick output. All crRNA sequences were also filtered to exclude BsmbI sites used for cloning and three or more consecutive Ts, which mimic RNA Pol III termination signal.

To design crRNA spacers targeting gene TSSs for Library 1, we used the 50-bp to +300-bp regions of TSS annotations derived from capped analysis of gene expression data and can include multiple TSSs per gene67. We targeted the TSSs of 559 common essential genes from DepMap with the strongest cell fitness defects in K562 cells based on prior dCas9-KRAB CRISPRi screen67. We used CRISPick with enAsCas12a settings to target all possible PAMs (TTTV and 44 non-canonical PAMs) in these TSS-proximal regions. Except for the criteria mentioned in the previous paragraph, no other exclusion criteria were applied. For the TSS-level analyses shown in Fig. 4d,e, each gene was assigned to a single TSS targeted by the crRNA with the strongest fitness score for that gene.

Negative controls in Library 1 fall into two categories: 1) 524 intergenic negative controls, and 2) 445 non-targeting negative controls that do not map to the human genome. Target sites for intergenic negative controls were picked by removing all regions in the hg19 genome that are within 10kb of annotated ensembl genes (retrieved from biomaRt from https://grch37.ensembl.org) or within 3kb of any ENCODE DNase hypersensitive site (wgEncodeRegDnaseClusteredV3.bed from http://hgdownload.cse.ucsc.edu/goldenpath/hg19/encodeDCC/wgEncodeRegDnaseClustered/). The remaining regions were divided into 1-kb fragments. 90 such 1-kb fragments were sampled from each chromosome. Fragments containing 20 consecutive Ns were removed. The remaining sequences were submitted to CRISPick run under CRISPRi settings. The CRISPick output was further filtered for spacers that meet these criteria: 1) off-target prediction criteria described in the beginning of this section, and 2) on-target Efficacy Score 0.5 (the rationale is to maximize representation by likely active crRNAs to bias for revealing any potential cell fitness effects from nonspecific genotoxicity due to residual DNA cutting by multiCas12a-KRAB), 3) mapping uniquely to the hg19 genome by Bowtie117 using -m 1 and otherwise default parameters, 3) filtered once more against those whose uniquely mapped site falls within 10kb of annotated ensembl genes or any ENCODE DNase hypersensitive site.

Non-targeting negative control spacers were generated by 1) combining non-targeting negative controls in the Humagne C and D libraries (Addgene accession numbers 172650 and 172651), 2) taking 20-nt non-targeting spacers from the dCas9-KRAB CRISPRi_v2 genome-wide library67, removing the G in the 1st position and appending random 4-mers to the 3 end. This set of spacers were then filtered for those that do not map to the hg19 genome using Bowtie with default settings.

Sublibrary A (42,600 constructs designed): Test position spacers were encoded at each position of the 6-plex array, with remaining positions referred to as context positions and filled with negative control spacers. Test positions encodes one of 506 intergenic negative control spacers and 914 essential TSS-targeting spacers. The essential TSS-targeting spacers were selected from among all spacers targeting PAMs within 50-bp to +300-bp TSS-proximal regions of 50 common essential genes with the strongest K562 cell fitness defect in prior dCas9-KRAB CRISPRi screen67 and must have 0.7 CRISPick on-target efficacy score. Negative control context spacers consist of five 6-plex combinations; three of these combinations consist entirely of non-targeting negative controls, and two of the combinations consist entirely of intergenic negative controls.

Sublibrary B (6,370 constructs designed): crRNA combinations targeting cis-regulatory elements at the MYC locus were assembled from a subset of combinations possible from 15 starting spacers (3 targeting MYC TSS, 3 targeting each of 3 enhancers, and 3 intergenic negative control spacers). The three enhancer elements are described in the subsection Design of individual crRNAs. These 15 starting spacers were grouped into 5 3-plex combinations, each 3-plex combination exclusively targeting one of the four cis-regulatory elements, or consisting entirely of intergenic negative controls. Each 3-plex was then encoded in positions 13 of 6-plex arrays, and positions 46 were filled with all possible 3-plex combinations chosen from the starting 15 spacers. All 6-plex combinations were also encoded in the reverse order in the array.

All-negative control constructs (2,000 constructs designed): 1,500 6-plex combinations were randomly sampled from the intergenic negative control spacers described for Library 1. 500 6-plex combinations were randomly sampled from non-targeting negative control spacers described for Library 1.

Intergenic negative controls and non-targeting negative controls are defined the same as in Library 1.

As Library 2 was designed and cloned prior to the completion of the Library 1 screen, the majority of Library 2 contains constructs encoding for spacers in the test position that in hindsight do not produce strong phenotypes as single crRNAs in the Library 1 screen.

Both Library 1 and Library 2 were constructed from pooled oligonucleotide libraries designed to contain crRNA constructs designed for exploratory analysis for a separate unpublished study. Sequencing reads from those non-contributory constructs are present in the raw fastq files, do not affect interpretation of Library 1 and Library 2 screen cell fitness scores, and are excluded from analysis in the present study.

All PCRs were performed with NEBNext Ultra II Q5 Master Mix (NEB M0544). For Library 1, ~140 fmol pooled oligo libraries from Twist were subjected to 10 cycles of PCR amplification using primers specific to adaptor sequences flanking the oligos and containing BsmbI sites. The PCR amplicons were cloned into a crRNA expression backbone (pCH67) by Golden Gate Assembly with ~1:1 insert:backbone ratio using ~500 fmol, followed by bacterial transformation to arrive at an estimated 778 coverage in the final plasmid Library 1. For Library 2, 915 fmol of pooled oligo libraries from Twist was subjected to 18 cycles of PCR amplification and agarose gel purification of the correctly sized band before proceeding to Golden Gate Assembly. The estimated coverage of plasmid Library 2 from bacterial colony forming units is ~60. Additional details are described in Supplementary Information.

Primer sequences are provided in Supplementary Table 2. Sequences of the expected PCR amplicons for Illumina sequencing are in Supplementary Data 2. crRNA inserts were amplified from genomic DNA isolated from screens using 16 cycles of first round PCR using pooled 0-8nt staggered forward and reverse primers, treated with ExoSAP-IT (Thermo Fisher, 78201.1.ML), followed by 7 cycles of round 2 PCR to introduce Illumina unique dual indices and adaptors. Sequencing primer binding sites, unique dual indices, P5 and P7 adaptor sequences are from Illumina Adaptor Sequences Document #1000000002694 v16. PCR amplicons were subject to size selection by magnetic beads (SPRIselect, Beckman, B23318) prior to sequencing on an Illumina NovaSeq6000 using SP100 kit (PE100) for Library 1 or SP500 kit (PE250) for Library 2. Sequencing of plasmid libraries were performed similarly, except 7 cycles of amplification were each used for Round 1 and Round 2 PCR. The size distribution of the final library was measured on an Agilent TapeStation system. We noted that even after magnetic bead selection of Round 2 PCR-amplified Library 2 plasmid library (colonies from which were Sanger sequencing verified) and genomic DNA from screens, smaller sized fragments from PCR amplification during Illumina sequencing library preparation persisted. Thus, the majority of unmapped reads likely reflect undesired PCR by-products, though lentiviral recombination could contribute at an uncertain but relatively low frequency as well.

Library 1 screen: K562 cells engineered by piggyBac transposition to constitutively express denAsCas12a-KRAB or multiAsCas12a-KRAB were transduced with lentivirally packaged Library 1 constructs at MOI ~0.15. Transduced cells were then selected using 1g/ml puromycin for 2 days, followed by washout of puromycin. On Day 6 after transduction, initial (T0) time point was harvested, and the culture was split into 2 replicates that are separately cultured henceforth. 10 days later (T10), the final time point was harvested (8.6 total doublings for multiAsCas12a-KRAB cells, 9.15 total doublings for denasCas12a-KRAB cells). A cell coverage of >500 was maintained throughout the screen. Library 2 screen: K562 cells engineered by piggyBac transposition to constitutively express multiAsCas12a-KRAB were transduced with lentivirally packaged Library 2 constructs at MOI ~0.15. The screen was carried out similarly as described for Library 1 screen, except the screen was carried out for 14 days (T14) or 13.5 total doublings and maintained at a cell coverage of >2,000 throughout. Genomic DNA was isolated using the NucleoSpin Blood XL Maxi kit (Machery-Nagel, 740950.50).

Summary of library contents are in Supplementary Fig. 18.

For Library 1, reads were mapped to crRNA constructs using sgcount (https://noamteyssier.github.io/sgcount/), requiring perfect match to the reference sequence. For Library 2, reads were mapped using an algorithm (detailed in Supplementary Information) requiring perfect match to the reference sequence, implemented as casmap constructs command in a package written in Rust, available at https://github.com/noamteyssier/casmap.

Starting from read counts, the remainder of analyses were performed using custom scripts in R. Constructs that contained less than 1 reads per million (RPM) aligned to the reference library in either replicates at T0 were removed from analysis. From the constructs that meet this read coverage threshold, a pseudocount of 1 was added for each construct and the RPM recalculated and used to obtain a fitness score118 that can be interpreted as the fractional defect in cell fitness per cell population doubling:

$${gamma }=log_2left(frac{left({mathrm{RPMfinal}}/{mathrm{negctrlmedianRPMfinal}}right)}{left({mathrm{RPMinitial}}/{mathrm{negctrlmedianRPMinitial}}right)}right)Big/{mathrm{totaldoublings}},$$

where RPM is the read count per million reads mapped to reference (initial = at T0, final = at end of screen), negctrlmedian is the median of RPM of intergenic negative control constructs, totaldoublings is the total cell population doublings in the screen. For Library 1, data from a single T0 sample was used to calculate the fitness score for both replicates due to an unexpected global loss of sequencing read counts for one of two originally intended T0 replicate samples. For each screen replicate in Library 2, data from two separate sequencing library preps from the same Round 1 PCR material subjected to separate Round 2 PCRs and sequenced on separate runs were pooled together for analysis.

K562 cell lines engineered with the corresponding Cas12a protein constructs were transduced with crRNAs and sorted for transduced cells based on GFP-positivity. 200,000 cells were collected 14 or 15 days after crRNA transduction and genomic DNA was isolated using NucleoSpin Blood (Macherey-Nagel, 740951.50). For analysis of CD55 and CD81 loci, PCRs for loci of interest were run using Amplicon-EZ (Genewiz) partial Illumina adapters and amplicons were processed using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, 740609.250). Paired-end (2 250bp) sequencing was completed at GENEWIZ (Azenta Life Sciences). Raw fastq files were obtained from GENEWIZ and aligned to reference sequences using CRISPResso2 (ref. 119). Quantification diagrams were generated in R. For analysis at the KIT locus, cells were lysed using QuickExtract DNA Solution (Lucigen) and amplicons were generated using 15 cycles of PCR to introduce Illumina sequencing primer binding sites and 0-8 staggered bases to ensure library diversity. After reaction clean-up using ExoSAP-IT kit (Thermo Fisher, 78201), an additional 15 cycles of PCR was used to introduce unique dual indices and Illumina P5 and P7 adaptors. Libraries were pooled and purified by SPRIselect magnetic beads before paired-end sequencing using an Illumina MiSeq at the Arc Institute Multi-Omics Technology Center. Sequencing primer binding sites, unique dual indices (from Illumina TruSeq kits), P5 and P7 adaptor sequences are from Illumina Adaptor Sequences Document #1000000002694 v16. Bioinformatic analysis of indel frequencies and simulation of indel impacts on gene expression, accounting for DNA copy number of the target region in the K562 genome65, are detailed in Supplementary Information. Primer sequences are in Supplementary Table 2.

Genomic DNA was harvested from 20 million cells using the Qiagen Genomic Tips Kit (10243). As detailed in Supplementary Information, we used a custom protocol adapted from the Nanopore Cas9 Sequencing Kit users manual (SQK-CS9109, though this kit was not actually used) to enrich for genomic DNA surrounding crRNA target sites for Nanopore sequencing using Kit 14 chemistry. Cas9 guide spacer sequences are in Supplementary Table 1.

fastq files generated by MinKNOW version 23.07.15 (Oxford Nanopore Technologies) were aligned to the ~20-kb regions (defined by the outermost Cas9 sgRNA protospacer sites flanking each targeted locus) surrounding each crRNA target site in MinKNOW to generate bam files. Bam files for each sample were merged using samtool merge (samtools v1.6 (ref. 120)). Merged bam files were filtered for alignments that overlap the start and end coordinates of the protospacer region of the Cas12a crRNA using bamtools filter -region (bamtools v2.5.1 (ref. 121)). Filtered bam files were loaded into the Integrative Genomics Viewer 2.17.0 (ref. 122) for visualization of individual read alignments. pysamstats fasta type variation (pysamstats v1.1.2) was used to extract per base total read coverage and deletion counts. The fraction of aligned reads harboring a deletion at each base was plotted using custom scripts in R.

Approximately 200,000 to 1 million cells were harvested, resuspended in 300l RNA Lysis Buffer (Zymo, R1060), and stored at 70C until further processing for RNA isolation using the Quick-RNA Miniprep Kit (Zymo, R1055). 3 RNA-seq was batch processed together with samples unrelated to this study using a QuantSeq-Pool Sample-Barcoded 3 mRNA-Seq Library Prep Kit for Illumina (Lexogen cat#139) in accordance with the manufacturers instructions. 10ng of each purified input RNA was used for first-strand cDNA synthesis with an oligo(dT) primer containing a sample barcode and a unique molecular identifier. Subsequently, barcoded samples were pooled and used for second strand synthesis and library amplification. Amplified libraries were sequenced on an Illumina HiSeq4000 with 100-bp paired-end reads. The QuantSeq-Pool data was demultiplexed and preprocessed using an implementation of pipeline originally provided by Lexogen (https://github.com/Lexogen-Tools/quantseqpool_analysis). The final outputs of this step are gene level counts for all samples (including samples from multiple projects multiplexed together). Downstream analyses were performed using DESeq2 (ref. 123) for differential expression analysis, crisprVerse124 for off-target analysis, and custom R scripts for plotting as detailed in Supplementary Information.

For the CRISPRi experiments targeting the HBG1/HBG2 TSSs or HS2 enhancer, K562 cells engineered (by lentiviral transduction at MOI~5) for constitutive expression of multiAsCas12a-KRAB were transduced with crRNAs and sorted, followed by resuspension of ~200,000 to 1 million cells in 300l RNA Lysis Buffer from the Quick-RNA Miniprep Kit (Zymo, R1055) and stored in 70C. RNA isolation was performed following the kits protocols, including on-column DNase I digestion. 500ng RNA was used as input for cDNA synthesis primed by random hexamers using the RevertAid RT Reverse Transcription Kit (Thermo Fisher, K1691), as per manufacturers instructions. cDNA was diluted 1:4 with water and 2l used as template for qPCR using 250nM primers using the SsoFast EvaGreen Supermix (BioRad, 1725200) on an Applied Biosystems ViiA 7 Real Time PCR System. Data was analyzed using the ddCT method, normalized to GAPDH and no crRNA sample as reference. qPCR primer sequences are in Supplementary Table 2.

For co-transfection experiments, the day before transfection, 100,000 HEK293T cells were seeded into wells of a 24-well plate. The following day, we transiently transfected 0.6g of each protein construct and 0.3g gRNA construct per well (in duplicate) in Mirus TransIT-LT1 (MIR 2304) transfection reagent according to manufacturers instructions. Mixtures were incubated at room temperature for 30min and then added in dropwise fashion into each well. 24h after transfection, cells were replenished with fresh media. 48h after transfection, BFP and GFP-positive cells (indicative of successful delivery of protein and crRNA constructs) were sorted on a BD FACSAria Fusion and carried out for subsequent flow cytometry experiments.

Approximately 400,000 cells per sample were washed with 1ml cold PBS and resuspended in 400l Pierce RIPA Buffer supplemented with Halt Protease and Phosphatase inhibitor cocktail (Thermo Fisher, 1861281) on ice. Samples were rotated for 15min at 4C, followed by centrifugation at 20,000g for 15min to pellet cell debris. The supernatant was collected and mixed with 4x Bolt LSD Sample Buffer (Thermo Fisher, B0007) supplemented with 50mM DTT, followed by heating for 10min at 70C. Samples were electrophoresed on Bolt 4%12% Bis-Tris Plus Gels (Thermo Fisher), and transferred using the BioRad TurboTransfer system onto Trans-Blot Turbo Mini 0.2m Nitrocellulose Transfer Packs (1704158). Membranes were blocked with 6% BSA in TBST (Tris-buffered saline, 0.1% Tween 20) at room temperature for ~1h, followed by incubation at 4C overnight with antibodies against anti-HA-tag rabbit antibody (Cell Signaling Technology, 3724S) at 1:1,000 dilution and anti-GAPDH rabbit antibody (Cell Signaling Technology, 2118) at 1:3,000 dilution in 6% BSA in TBST. Membranes were washed with TBST at room temperature three times for 5min. each, followed by incubation with IRDye secondary antibody for 1h at room temperature, washed three times with TBST 5min for each and two times with PBS. Blots were imaged using Odyssey CLx (LI-COR).

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations - Nature.com

-Naimish Patel, M.D., appointed to Chief Medical Officer-

-Julianne Bruno, M.B.A., promoted to Chief Operating Officer-

ZUG, Switzerland and BOSTON, May 23, 2024 (GLOBE NEWSWIRE) -- CRISPR Therapeutics(Nasdaq: CRSP), a biopharmaceutical company focused on creating transformative gene-based medicines for serious diseases, today announced the appointment of Naimish Patel, M.D., as Chief Medical Officer, effective May 28, 2024. Dr. Patel is an experienced drug developer who has worked across a wide range of disease areas, including his most recent leadership role as the Global Development Therapeutic Area Head of Immunology and Inflammation at Sanofi. In addition, the Company also announced the promotion of Julianne Bruno, M.B.A., to Chief Operating Officer, effective as of May 23, 2024. She currently serves as the Companys Senior Vice President and Head of Programs & Portfolio Management.

Im thrilled to welcome a transformational leader of Naimishs caliber to the executive team at CRISPR Therapeutics, said Samarth Kulkarni, Ph.D., Chief Executive Officer and Chairman of CRISPR Therapeutics. His extensive drug development experience and proven leadership will be critical as we expand our portfolio and advance multiple assets in our pipeline.

Dr. Kulkarni added: Additionally, I am very pleased to announce Julie's promotion and I look forward to her continued contributions as we scale the Company. Since joining CRISPR Therapeutics in 2019, Julie has been a valuable member of the leadership team and has led several important and impactful cross-functional initiatives including our collaboration with Vertex. With this strengthened executive team, combined with our significant progress to date, CRISPR Therapeutics remains well positioned to rapidly advance our programs and deliver on our mission to develop transformative medicines for patients suffering from serious diseases.

CRISPR Therapeutics' compelling and innovative platform, exciting clinical assets and impressive manufacturing capabilities position the Company to potentially bring several transformative therapies to patients with significant unmet medical need, said Naimish Patel, M.D., I am incredibly excited to join the CRISPR leadership team and help bring these therapies to patients in need.

Dr. Patel joins CRISPR Therapeutics from Sanofi, where he most recently served as the Global Development Therapeutic Area Head of Immunology and Inflammation. Previously, he was the Global Program Head for Dupilumab at Sanofi, leading multiple waves of indication expansion including chronic obstructive pulmonary disease and eosinophilic esophagitis. During his time at Sanofi, Dr. Patel led the development of an industry-leading pipeline across key therapeutic areas including respiratory, dermatology, gastroenterology, and rheumatology. He also oversaw key business development and M&A activities during a rapid phase of pipeline expansion. Dr. Patel is a pulmonary and critical care physician with an extensive background in translational medicine and clinical trials.

Dr. Patel received a B.S. in Mechanical Engineering from MIT and an M.D. from McGill University. He completed his internal medicine training at Columbia-Presbyterian Hospital and his fellowship training in Pulmonary and Critical Medicine at Harvard Medical School. After completing his fellowship, Dr. Patel was a member of the faculty at Harvard and Beth Israel Deaconess Medical Center where he led an NIH-funded lab in translational immunology focused on innate defense functions of the lungs. He previously held positions in clinical development and discovery project leadership at AstraZeneca and Vertex Pharmaceuticals.

Julianne Bruno, M.B.A., has served as Senior Vice President and Head of Programs & Portfolio Management at CRISPR Therapeutics since March 2023. During her time at CRISPR Therapeutics since joining the Company in April 2019, she has taken on positions of increasing responsibility, including leading the hemoglobinopathies partnership with Vertex through the early clinical stage through approval. In addition, she has been responsible for program leadership of our immuno-oncology assets and the program management function across our franchises. Prior to joining CRISPR Therapeutics, Ms. Bruno worked at McKinsey & Company from August 2015 to March 2019 where she was a leader in the biotech practice and served a number of biotechnology companies on a wide range of commercial topics. She received her M.B.A. from The Wharton School and also holds an A.B. from Princeton University.

AboutCRISPR Therapeutics Since its inception over a decade ago, CRISPR Therapeutics has transformed from a research-stage company advancing programs in the field of gene editing, to a company that recently celebrated the historic approval of the first-ever CRISPR-based therapy and has a diverse portfolio of product candidates across a broad range of disease areas including hemoglobinopathies, oncology, regenerative medicine, cardiovascular, autoimmune and rare diseases. CRISPR Therapeutics advanced the first-ever CRISPR/Cas9 gene-edited therapy into the clinic in 2018 to investigate the treatment of sickle cell disease or transfusion-dependent beta thalassemia, and beginning in late 2023, CASGEVY (exagamglogene autotemcel) was approved in some countries to treat eligible patients with either of those conditions. The Nobel Prize-winning CRISPR science has revolutionized biomedical research and represents a powerful, clinically validated approach with the potential to create a new class of potentially transformative medicines. To accelerate and expand its efforts, CRISPR Therapeutics has established strategic partnerships with leading companies including Bayer and Vertex Pharmaceuticals. CRISPR Therapeutics AG is headquartered in Zug, Switzerland, with its wholly-owned U.S. subsidiary, CRISPR Therapeutics, Inc., and R&D operations based in Boston, Massachusetts and San Francisco, California, and business offices in London, United Kingdom. To learn more, visit http://www.crisprtx.com.

CRISPR THERAPEUTICS standard character mark and design logo are trademarks and registered trademarks ofCRISPR Therapeutics AG.The CASGEVY word mark and design are trademarks of Vertex Pharmaceuticals Incorporated. All other trademarks and registered trademarks are the property of their respective owners.

CRISPR Therapeutics Forward-Looking StatementThis press release may contain a number of forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995, as amended, including statements made by Drs. Kulkarni and Patel in this press release, as well as statements regarding CRISPR Therapeutics expectations about any or all of the following: (i) its plans for and its preclinical studies, clinical trials and pipeline products and programs, including, without limitation, manufacturing capabilities, status of such studies and trials, potential expansion into new indications and expectations regarding data generally; (ii) the data that will be generated by ongoing and planned clinical trials, and the ability to use that data for the design and initiation of further clinical trials; (iii) the sufficiency of its cash resources; (iv) the expected benefits of its collaborations; and (v) the therapeutic value, development, and commercial potential of CRISPR/Cas9 gene editing technologies and therapies. Without limiting the foregoing, the words believes, anticipates, plans, expects and similar expressions are intended to identify forward-looking statements. You are cautioned that forward-looking statements are inherently uncertain. Although CRISPR Therapeutics believes that such statements are based on reasonable assumptions within the bounds of its knowledge of its business and operations, forward-looking statements are neither promises nor guarantees and they are necessarily subject to a high degree of uncertainty and risk. Actual performance and results may differ materially from those projected or suggested in the forward-looking statements due to various risks and uncertainties. These risks and uncertainties include, among others: the efficacy and safety results from ongoing clinical trials will not continue or be repeated in ongoing or planned clinical trials or may not support regulatory submissions; clinical trial results may not be favorable; one or more of its product candidate programs will not proceed as planned for technical, scientific or commercial reasons; future competitive or other market factors may adversely affect the commercial potential for its product candidates; initiation and completion of preclinical studies for its product candidates is uncertain and results from such studies may not be predictive of future results of future studies or clinical trials; regulatory approvals to conduct trials or to market products are uncertain; uncertainties inherent in the operation of a manufacturing facility; it may not realize the potential benefits of its collaborations;uncertainties regarding the intellectual property protection for its technology and intellectual property belonging to third parties, and the outcome of proceedings (such as an interference, an opposition or a similar proceeding) involving all or any portion of such intellectual property; and those risks and uncertainties described under the heading "Risk Factors" in CRISPR Therapeutics most recent annual report on Form 10-K, quarterly report on Form 10-Q and in any other subsequent filings made by CRISPR Therapeutics with the U.S. Securities and Exchange Commission, which are available on the SEC's website at http://www.sec.gov. Existing and prospective investors are cautioned not to place undue reliance on these forward-looking statements, which speak only as of the date they are made. CRISPR Therapeutics disclaims any obligation or undertaking to update or revise any forward-looking statements contained in this press release, other than to the extent required by law.

Investor Contact: Susan Kim +1-617-307-7503 susan.kim@crisprtx.com

Media Contact: Rachel Eides +1-617-315-4493 rachel.eides@crisprtx.com

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CRISPR Therapeutics Strengthens Executive Leadership Team with Key Appointments - GlobeNewswire

CRISPR Therapeutics AG (NASDAQ:CRSP Get Free Report) shares traded down 3.1% during trading on Thursday after Citigroup lowered their price target on the stock from $89.00 to $84.00. Citigroup currently has a buy rating on the stock. CRISPR Therapeutics traded as low as $54.32 and last traded at $55.06. 521,944 shares traded hands during mid-day trading, a decline of 70% from the average session volume of 1,717,410 shares. The stock had previously closed at $56.80.

Several other analysts have also recently weighed in on the company. Needham & Company LLC dropped their price objective on CRISPR Therapeutics from $90.00 to $88.00 and set a buy rating for the company in a report on Thursday, May 9th. Chardan Capital increased their price objective on shares of CRISPR Therapeutics from $110.00 to $112.00 and gave the company a buy rating in a report on Thursday, February 22nd. Wells Fargo & Company lowered their target price on shares of CRISPR Therapeutics from $70.00 to $65.00 and set an equal weight rating for the company in a report on Thursday, May 9th. TheStreet raised CRISPR Therapeutics from a d+ rating to a c rating in a research note on Friday, February 23rd. Finally, Oppenheimer decreased their price objective on CRISPR Therapeutics from $102.00 to $95.00 and set an outperform rating for the company in a research note on Friday, May 10th. Three analysts have rated the stock with a sell rating, seven have assigned a hold rating and eight have issued a buy rating to the company. According to data from MarketBeat, the stock presently has a consensus rating of Hold and a consensus target price of $73.57.

Check Out Our Latest Report on CRSP

Hedge funds have recently modified their holdings of the company. Teacher Retirement System of Texas boosted its position in CRISPR Therapeutics by 5.0% during the 3rd quarter. Teacher Retirement System of Texas now owns 21,284 shares of the companys stock worth $966,000 after acquiring an additional 1,015 shares during the last quarter. California Public Employees Retirement System grew its position in CRISPR Therapeutics by 5.9% in the third quarter. California Public Employees Retirement System now owns 112,169 shares of the companys stock valued at $5,091,000 after purchasing an additional 6,207 shares in the last quarter. Private Advisor Group LLC increased its stake in CRISPR Therapeutics by 12.3% in the 3rd quarter. Private Advisor Group LLC now owns 5,654 shares of the companys stock valued at $254,000 after buying an additional 618 shares during the last quarter. SteelPeak Wealth LLC lifted its position in CRISPR Therapeutics by 14.6% during the 3rd quarter. SteelPeak Wealth LLC now owns 5,780 shares of the companys stock worth $262,000 after buying an additional 735 shares in the last quarter. Finally, Loring Wolcott & Coolidge Fiduciary Advisors LLP MA boosted its stake in shares of CRISPR Therapeutics by 137.9% during the 3rd quarter. Loring Wolcott & Coolidge Fiduciary Advisors LLP MA now owns 1,035 shares of the companys stock worth $48,000 after buying an additional 600 shares during the last quarter. 69.20% of the stock is currently owned by institutional investors.

The company has a market cap of $4.73 billion, a P/E ratio of -20.49 and a beta of 1.80. The company has a fifty day moving average price of $60.59 and a two-hundred day moving average price of $65.51.

CRISPR Therapeutics (NASDAQ:CRSP Get Free Report) last posted its quarterly earnings results on Wednesday, May 8th. The company reported ($1.43) earnings per share for the quarter, missing analysts consensus estimates of ($1.35) by ($0.08). The business had revenue of $0.50 million during the quarter, compared to analysts expectations of $25.53 million. During the same period last year, the company posted ($0.67) EPS. The firms quarterly revenue was down 99.5% on a year-over-year basis. Research analysts anticipate that CRISPR Therapeutics AG will post -5.64 EPS for the current year.

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CRISPR Therapeutics is a gene-editing company focused on developing transformative gene-based medicines for serious diseases using its proprietary CRISPR/Cas9 platform. CRISPR/Cas9 is a revolutionary gene-editing technology that allows for precise, directed changes to genomic DNA. CRISPR Therapeutics has established a portfolio of therapeutic programs across a broad range of disease areas including hemoglobinopathies, oncology, regenerative medicine and rare diseases.

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CRISPR Therapeutics (NASDAQ:CRSP) Trading Down 3.1% on Analyst Downgrade - Defense World

Pictured: 3D illustration of the CRISPR-Cas9 system editing a stretch of DNA/iStock,Meletios Verras

Excision BioTherapeutics attempt to use a CRISPR-based gene editing therapy to cure HIV has failed an early-stage study, according to several media reports on Friday.

Results from the Phase I trial of five patients showed that Excisions CRISPR therapeutic did not strongly suppress the HIV virus. Three patients who were taken off of antiretroviral therapy soon developed viral rebound and needed to resume conventional treatments, according to reporting by STAT News.

Still, Excisions approach did show signs of promise. One patient was able to keep the virus at bay for 16 weeks after stopping antiretroviral treatment before rebounding. Typically, HIV levels resurge after around four weeks. The experimental CRISPR treatment also appears to have lowered the number of infected cells in this patient, according to STAT.

In response to the disappointing outcome, CEO Daniel Dornbusch told STAT that Excision is encouraged by the safety data in the Phase I study, which suggests that in-vivo CRISPR can be done in a safe way in humans. The company will use these findings to work on a new gene editing candidate that it can apply to other chronic viral infections, such as herpes and hepatitis B.

According to its website, Excisions lead candidate is EBT-101, a CRISPR-based gene editor that works by cutting out the HIV pro-viral DNA from all infected cells. The candidate is delivered via an AAV9 vector and uses two guide RNAs that can home in on three target sites in the HIV genome, minimizing the likelihood of viral escape.

The company is positioning EBT-101 as a potentially curative treatment for chronic HIV and is rapidly advancing toward clinical trials.

In October 2023, Excision released safety data from its first-in-human Phase I/II study of EBT-101, showing that there were no serious adverse events or dose-limiting toxicities in all three patients that had been dosed with the gene editor at the time. The study found four mild toxicities that could be potentially related to EBT-101, though all were resolved without intervention.

The early data also showed that EBT-101 was present at detectable levels in the blood four weeks after treatment.

In addition to targeting HIV, Excision is also leveraging its proprietary CRISPR-based gene-editing platform against herpes 1 and 2, for which it is advancing EBT-104, and hepatitis B, for which it is developing EBT-107.

Several biotech companies have responded to the HIV crisis, including industry powerhouses Gilead and GSK. Gilead owns the oral antiretroviral pill Biktarvy (bictegravir/emtricitabine/tenofovir alafenamide), while GSK and partner ViiV Healthcare market the oral pill Tivicay (dolutegravir) and long-acting injectable Cabenuva (cabotegravir/rilpivirine).

While these virus-suppressive treatments are effective, a true cure remains elusive and the field continues to face several substantial challenges. In March 2024, the World Health Organization warned of a growing virus resistance against Tivicay.

Meanwhile, gene therapies which have strong curative potential still pose potential safety concerns, particularly unintended edits and off-target effects.

Tristan Manalac is an independent science writer based in Metro Manila, Philippines. Reach out to him on LinkedIn or email him at tristan@tristanmanalac.com or tristan.manalac@biospace.com.

Excerpt from:
Excisions CRISPR-Based HIV Treatment Fails to Show Curative Potential in Early Study - BioSpace