Archive for the ‘Crispr’ Category

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5 ways CRISPR will save your life Red Bull Past tense, because of Clustered Regularly Interspaced Short Palindromic Repeats, or CRISPR for short. CRISPR works much like a DNA-level pair of scissors and glue stick. It dramatically lowers the bar for biotech innovation, making it 99 percent ... and more »

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5 ways CRISPR will save your life - Red Bull

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Crispr Patent-Holders Move Toward Easing Access to Gene-Editing Technology Wall Street Journal (subscription) A holder of key patents to the Crispr gene-editing technology is willing to join a world-wide joint patent poola development that medical and legal experts think could hasten the development of new human therapies. The Broad Institute of MIT and ...

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Crispr Patent-Holders Move Toward Easing Access to Gene-Editing Technology - Wall Street Journal (subscription)

After CRISPR, theres a new genetic technique with a tongue-in-cheek name in town: LASSO cloning.

Researchersfrom four institutions, including the US-based John Hopkins, Rutgers and Harvard, and the University of Trento in Italy, have developed a new technology tostudy large chunks of DNA and their function. The work behind it was recently published inNature Biomedical Engineering,and a patent was filed earlier this month.

This molecular tool is called long adapter single-stranded oligonucleotide, or LASSO for short. The lasso rope metaphor applies to the tools mechanism, which can capture and clone long sequences of DNA fragments. Fragment length had so far been the main challenge for cloning probes and the genome sequencing field at large. Next generation sequencing (NGS), which has gained a lot of attention in medical research, relies on sequencing short fragments that are then put together, like a puzzle, by bioinformatics tools. However, this method falls short for certain types of samples. Short reads capture only about 100 basepairs, or DNA letters, at a time, while LASSO can read more than1000 base pairs.

As a proof of concept, the researchers set out to test LASSO probes in biotechs favorite microorganism,E. coli. The tool managed to simultaneously clone over 3000 DNA fragments of the genome ofE. coli, capturing around 75% of the targets and leaving almost all of the non-targeted DNA alone, and the studys authors say theres still certainly room for improvement.

LASSO cloning should enable the scientific community to build libraries of a given organisms protein in a much faster and cheaper way, democratizing research that was so far only within the reach of big research consortia. The usefulness of such studies ranges from a better understanding of organisms to the ability to screen large libraries of natural enzymes and compounds that could be valuable leads in drug discovery,as it has been done before for some species likePenicilliumfungistrains, for example.

One of the organisms to be better studied is, of course, human beings. Researchers already tested LASSO cloning with human DNA, something has the potential to yield new biomarkers for a range of diseases. Another focus of interest is the human microbiome. As described in the same paper, LASSO was used to build the first protein library of the microbiome, and the research team hopes that it can improve precision medicine strategies that takeinto account the microbes living within us.

Images by DWilliam/Pixabay and Jennifer E. Fairman/John Hopkins University

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Better than CRISPR? LASSO Cloning ropes in Long-Read DNA - Labiotech.eu (blog)

How many mutations?

By Michael Le Page

As you were. In May, a study claimed that the revolutionary CRISPR gene editing technique can cause thousands of unwanted and potentially dangerous mutations. The authors called for regulators to reassess the safety of the technique.

But doubts were raised about these claims from the very beginning, not least because it was a tiny study involving just three mice. Some critics have called for the paper to be withdrawn. Now a paper posted online on 5 July has proposed a simple and more plausible explanation for the controversial results. If its right, the authors of the original study were wrong.

We strongly encourage the authors to restate the title and conclusions of their originalpaper or provide properly controlled experiments that can adequately support their claims, write the team behind the new study. Not doing so does a disservice to the field and leaves the misleading impression that the strong statements and recommendations found in their paper are adequately supported by the data presented.

The aim of gene editing is to make a precise change in a DNA sequence while leaving the rest of the genome untouched. Gene editing can be used to introduce desirable changes into plants and animals (and perhaps people too one day), and to treat a range of disorders in people.

Gene editing has been around for decades but it remained extremely difficult and expensive until the revolutionary CRISPR technique was discovered in 2013. CRISPR is so cheap and easy that it is already widely used by researchers around the world, and nearly 20 clinical trials in people are already getting underway. The rapid pace of development has been unprecedented.

But have doctors been rushing to use it too soon? When Stephen Tsang of Columbia University Medical Center and colleagues compared the entire genomes of two CRISPR-edited mice with a third one, they found thousands of shared mutations in the two edited mice.

Tsang and co attributed to these mutations to CRISPR, and issued a widely-covered press release that suggested CRISPR is far riskier than dozens of other studies had suggested.

It has always been clear that CRISPR, like other gene-editing techniques, can sometimes make alterations other than the intended one. These off-target changes are most likely to occur when the CRISPR machinery binds to DNA sequences very similar to the target one.

For this reason, studies on the safety of CRISPR have usually looked to see if any sequences resembling the target sequences have been altered. Most have found few unwanted changes, suggesting CRISPR is safe. And some teams have already tweaked the CRISPR machinery to reduce these off-targets effects even more.

Tsang and colleagues claimed that by sequencing the entire genome, they found off-target mutations missed by studies that only looked at sites resembling the target sequence. But there is a much simpler explanation, says the latest study: the two CRISPR-edited mice just happened to be more closely related and thus shared more mutations.

Tsang and colleagues assumed the three mice they studied were essentially genetically identical because their parents were very closely related, but the way the colony of mice was maintained means this was probably not the case, the team, which includes Luca Pinello of Harvard University, say.

This explanation makes sense for another reason, too. The shared mutations in the edited mice were nowhere near DNA sequences resembling the one were targeted for editing, Pinello and colleagues point out, so its far from clear why CRISPR would cause mutations in these same sites in two different mice.

I agree the two mice are indeed more likely to be closely related, says geneticist Gaetan Burgio of the Australian National University, one of the many critics of the original paper. He says its publication in a prominent journal was a failure of peer review.

Journal reference: bioRxiv, DOI: 10.1101/159707

Read more: CRISPR causes many unwanted mutations, small study suggests

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CRISPR gene editing technique is probably safe, study confirms ... - New Scientist

CRIPSR-Cas is one tool that could help keep pace with the growing demand for more sustainable agricultural solutions.

DuPont Pioneer

When Carmen and I were young, we made our home at the foot of the Sandia Mountains east of Albuquerque. Back then, for a weekend outing on a pleasant summers day, we would avoid Interstate 25 and travel the Turquoise Trail National Scenic Byway, the picturesque backroad that links Albuquerque with Santa Fe through the high country. The Byway is named for the semi-precious, blue-green mineral deposits of hydrated copper and aluminum phosphatesought after by Native Americans for more than a millenniumwhich are found near the northern stretches of the road. Once bustling mining towns, scattered along the way like nuggets of the wild west, may also be discovered and explored; ghost towns such as Golden and Madrid (pronounced MAD-rid, not Ma-DRID).

During one of these trips, we stopped at the Golden General Merchandise Store and struck up a conversation with the proprietors, Vera and Bill Henderson. We learned that in 1962, Bill and Vera purchased the store from her parents. Together they turned the failing store into a thriving arts and crafts trading post featuring Native American jewelry, rugs, Kachina dolls, and pottery for sale.

The Hendersons (both of these dear friends have passed away) were true and devoted connoisseurs and patrons of local and regional artisans. Vera especially wanted customers to share her passion in these handicrafts. If a potential patron showed lackadaisical interest in the artistic qualities of a piece or the artist who made it, she would send them on their way, telling them to take their business to Santa Fe!

I admired Veras spunk and forthrightness. She cared deeply, and she stood up for what she believed. She inspired me to learn the stories told about everyday Pueblo Indian life by silversmiths such as the Kewa Pueblo artist Vidal Aragon.

For example, a silver bracelet we purchased depicted a Pueblo village around which grew stalks of corn. As a kid growing up in the D.C. suburbs, about all I knew of corn was corn flakes. The Hendersons passion for Native American traditions encouraged me to deepen my cultural appreciation of food and agriculture.

The Keresan-speaking tribes of the American Southwest believed in a female corn goddess, whom they called Iyatiku. It is she who led the first people to the surface of the earth from Shipap, her underground realm. To provide for their sustenance and wellbeing, Iyatiku planted pieces of her heart in the fields surrounding their village. These tiny tokens of her body grew into lush fields of life-sustaining corn!

This worldviewthat seeds and plants are sacred gifts available to and shared by allis common among native peoples around the world. This ancient perspective stands in stark contrast to the modern view corporate ag-science offers us. To illustrate, I will use DuPont Pioneers CRISPR-Cas waxy corn as an example.

DuPont Pioneer has recently announced its intentions to commercialize waxy corn hybrids developed in the laboratory using a new and powerful gene editing technique called CRISPR-Cas: clustered regularly interspaced short palindromic repeats-CRISPR-associated system. (Thats not a typo. This two-part acronym incorporates its own acronym!)

Waxy corn produces a high amylopectin starch content, which is milled for a number of everyday consumer food and non-food uses including processed foods, adhesives and high-gloss paper. DuPont Pioneer created this hybrid using CRISPR-Cas advanced gene editing technology to program the plants molecular machinery to synthesize amylopectin starch in abnormally high levels.

According to DuPont Pioneers website and their September 2016 webinar, CRISPR-Cas is a more efficient and targeted plant breeding technology. In the past, genetic engineering of plants often has relied upon transgenic techniques, which modifies the host species by adding genetic material from different species (i.e., GMOs). CRISPR-Cas does not incorporate foreign DNA from other species. Its a continuation of what people have been doing since plants were first domesticatedselecting plants for their desired characteristics like higher yields, disease resistance, longer shelf life or better nutrition.

CRISPR-Cas gene editing technology is likened to word processing, by which scientist delete, edit, or search and replace specific portions of a plants genetic code. CRISPR-Cas uses molecular scissors to cut a specific section of DNA. After the DNA is cut, either a piece is removed and the loose ends are spliced back together eliminating an undesired trait; or a desired trait is inserted into the gap and the DNA is stitched together again.

DuPont Pioneer seeks to further the science and expand the adoption of CRISPR-Cas across all crops, including soybeans, rice, wheat, canola, sunflowers, fruits and vegetables. Agricultural products developed with CRISPR-Cas will be like the ones that we have been producing through conventional plant breeding for generations and will be subject to extensive testing prior to commercialization. As with every technology we apply, we hold ourselves accountable to our core values, to our customers and to consumers.

According to DuPont Pioneer, current Food and Drug Administration law will not require labelling of CRISPR-Cas waxy corn. Even under the newly enacted National Bioengineered Food Disclosure Law of 2016, their genetically edited waxy corn will not meet the definition of bioengineered as written in the law, and would therefore not require disclosure as a bioengineered food.

For much of human history, edible plants were cherished as gifts from the gods. Now, they are becoming high-tech, commercial products of industry. Once seeds were available to all. Now they are becoming proprietary, patented, intellectual property, licensed through end-user agreements.

Are these trends truly more sustainable agricultural solutions as industry claims they are? Are they empowering farmers? Or are corporate monopolies of engineered seeds leading to bondage and dependency for farmers who are getting trapped in debt to pay royalties in the words of Vandana Shiva, philosopher and eco-feminist.

On a personal level, I relish the smell of a freshly picked ear of corn. For me, summer would not be complete without a picnic lunch serving up hot corn-on-the-cob. I wonder, would my body and soul feel as nourished if I knew I was biting into kernels of CRISPR-Cas corn? If not, should I adapt my aesthetic sensibilities to these new agricultural realities by adopting the attitude CRISPR-Cas corn, and I dont care (sung to the antebellum minstrel tune Jimmy Crack Corn)?

J. Dirk Nies

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Science to Live By: CRISPR-Cas Corn - The Crozet Gazette

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The gene-editing tool CRISPR may cause thousands of unintended mutations, but critics say its way too soon to accuse it of infidelity1.

A two-page study, published 30 May in Nature Methods, put biologys hottest technique in the crosshairs, questioning its ability to rewrite DNA with the precision necessary for medicine. The papers findings quickly dominated social media conversations on biology. And the stocks of biotech companies with CRISPR-based projects dipped by as much as 15 percent.

But some researchers are loath to end their love affair with CRISPR. Reports of the methods infidelity, they say, are greatly exaggerated. One reason, they note, is that the study is small just two mice for CRISPR and a single control mouse.

I think its an important finding that we really need to follow up, but its really hard to judge why there are so many [mutations], says Guoping Feng, professor of brain and cognitive sciences at the Massachusetts Institute of Technology. He says another enzyme the team used may in fact have caused the errant mutations.

Two weeks after the papers publication, Nature Methods editors added a note to the paper, saying they are considering the criticism of the results and plan to respond soon.

CRISPR is a molecular scalpel that cuts DNA. It can home in on a specific spot on the molecule using a piece of RNA as a guide. The tool holds significant medical promise because it could help modify or fix genes that cause medical conditions.

In 2016, Alexander Bassuks team reported injecting mouse embryos with CRISPR fused to its usual protein partner, CAS9, which binds to DNA. The researchers targeted a gene called PDE6B that is involved in vision. The experiment was designed to correct a mutation that causes blindness in the mice2. It did.

For the new study, Bassuk and his collaborators looked for off-target effects of the treatment. They sequenced the whole genomes of the two mice and the control mouse. They compared the results from the edited mice against a database that includes genomes of 36 mouse strains.

CRISPR-CAS9 introduced more than 100 unintended mutations and more than 1,600 one-letter swaps in the code of DNA, the researchers found.

None of these changes had any obvious consequences. As far as we can tell, it hasnt affected the mice, says Bassuk, professor of pediatric neurology at the University of Iowa in Iowa City. But the researchers tested only the mices vision and do not know if the mutations affected the animals behavior or perception.

This was not the first time that CRISPR had caused accidental mutations, though previous reports found far fewer mutations3.

Its always been a concern for everyone in the field that this is not a completely clean method, says Anis Contractor, associate professor of physiology at Northwestern University in Chicago. Contractor was not involved in the research but uses CRISPR to make mouse models. This is a big red flag.

Contractor and others say the findings may prompt a change in best practices when using the method. Scientists may need to sequence the genomes of their models an expensive task to uncover any unexpected mutations.

Others, however, are downplaying the results.

I dont think this paper has any merit for CRISPR research, says George Church, professor of genetics at Harvard University. I think its a negative example that we can use as a cautionary tale. Church is co-founder of Editas Medicine, a biotechnology company that is using the technique to develop gene-editing therapies. The company lost 12 percent of its value the day the study was published. (Its stock has since gone up, surpassing its previous value.)

Church and the companys other executives wrote to Nature Methods with a laundry list of concerns about the paper. Their biggest beef, says Church: The studys control mouse likely wasnt genetically identical to the ones that had been edited with CRISPR, so its impossible to know whether the mutations resulted from the method.

Instead, Church argues, its possible that the mutations represent natural genetic variance between the animals. Church stops short of saying the paper should be retracted but calls it a rushed job. Another gene-editing company, Intellia Therapeutics, voiced similar complaints.

The team also used an unusual version of the editing system, says Feng, who is using CRISPR to create animal models of autism. He says the use of extra nickase, an enzyme that can cause breaks in a strand of DNA, could have caused the mutations.

I couldnt figure out what reason youd need to do it this way, he says.

Bassuk points out that the team did the editing work in 2015, early in CRISPRs use.

We used what was available at the time, Bassuk says. Its obviously not what people are using in 2017. He says he is not sure whether the editing system made a difference in the results.

In the past three months, researchers have debuted the first two mouse models of autism created using CRISPR. No one has yet published work on using CRISPR to correct genes in animal models of the condition.

In the short term, the findings will definitely temper the enthusiasm for CRISPR models, says J. Tiago Gonalves, assistant professor of neuroscience at Albert Einstein College of Medicine in New York, who was not involved in the research. But in the end, Im confident the problems will be solved and well figure out whats happening.

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CRISPR goes wild, and scientists debate its fidelity | Spectrum ... - Spectrum

CRISPR-Cas3 is a subtype of the CRISPR-Cas system, a widely adopted molecular tool for precision gene editing in biomedical research. Aspects of its mechanism of action, however, particularly how it searches for its DNA targets, were unclear, and concerns about unintended off-target effects have raised questions about the safety of CRISPR-Cas for treating human diseases.

Harvard Medical School and Cornell University scientists have nowgenerated near-atomic resolution snapshots of CRISPR that reveal key steps in its mechanism of action. The findings, published in Cell on June 29, provide the structural data necessary for efforts to improve the efficiency and accuracy of CRISPR for biomedical applications.

Through cryo-electron microscopy, the researchers describe for the first time the exact chain of events as the CRISPR complex loads target DNA and prepares it for cutting by the Cas3 enzyme. These structures reveal a process with multiple layers of error detectiona molecular redundancy that prevents unintended genomic damage, the researchers say.

High-resolution details of these structures shed light on ways to ensure accuracy and avert off-target effects when using CRISPR for gene editing.

To solve problems of specificity, we need to understand every step of CRISPR complex formation, said Maofu Liao, assistant professor of cell biology at Harvard Medical School and co-senior author of the study. Our study now shows the precise mechanism for how invading DNA is captured by CRISPR, from initial recognition of target DNA and through a process of conformational changes that make DNA accessible for final cleavage by Cas3.

Discovered less than a decade ago, CRISPR-Cas is an adaptive defense mechanism that bacteria use to fend off viral invaders. This process involves bacteria capturing snippets of viral DNA, which are then integrated into its genome and which produce short RNA sequences known as crRNA (CRISPR RNA). These crRNA snippets are used to spot enemy presence.

Acting like a barcode, crRNA is loaded onto members of the CRISPR family of enzymes, which perform the function of sentries that roam the bacteria and monitor for foreign code. If these riboprotein complexes encounter genetic material that matches its crRNA, they chop up that DNA to render it harmless. CRISPR-Cas subtypes, notably Cas9, can be programmed with synthetic RNA in order to cut genomes at precise locations, allowing researchers to edit genes with unprecedented ease.

To better understand how CRISPR-Cas functions, Liao partnered with Ailong Ke of Cornell University. Their teams focused on type 1 CRISPR, the most common subtype in bacteria, which utilizes a riboprotein complex known as CRISPR Cascade for DNA capture and the enzyme Cas3 for cutting foreign DNA.

Through a combination of biochemical techniques and cryo-electron microscopy, they reconstituted stable Cascade in different functional states, and further generated snapshots of Cascade as it captured and processed DNA at a resolution of up to 3.3 angstromsor roughly three times the diameter of a carbon atom.

In CRISPR-Cas3, crRNA is loaded onto CRISPR Cascade, which searches for a very short DNA sequence known as PAM that indicates the presence of foreign viral DNA.

Liao, Ke and their colleagues discovered that as Cascade detects PAM, it bends DNA at a sharp angle, forcing a small portion of the DNA to unwind. This allows an 11-nucleotide stretch of crRNA to bind with one strand of target DNA, forming a seed bubble.

The seed bubble acts as a fail-safe mechanism to check whether the target DNA matches the crRNA. If they match correctly, the bubble is enlarged and the remainder of the crRNA binds with its corresponding target DNA, forming what is known as an R-loop structure.

Once the R-loop is completely formed, the CRISPR Cascade complex undergoes a conformational change that locks the DNA into place. It also creates a bulge in the second, non-target strand of DNA, which is run through a separate location on the Cascade complex.

Only when a full R-loop state is formed does the Cas3 enzyme bind and cut the DNA at the bulge created in the non-target DNA strand.

The findings reveal an elaborate redundancy to ensure precision and avoid mistakenly chopping up the bacterias own DNA.

To apply CRISPR in human medicine, we must be sure the system is accurate and that it does not target the wrong genes, said Ke, who is co-senior author of the study. Our argument is that the CRISPR-Cas3 subtype has evolved to be a precise system that carries the potential to be a more accurate system to use for gene editing. If there is mistargeting, we know how to manipulate the system because we know the steps involved and where we might need to intervene.

Structures of CRISPR Cascade without target DNA and in its post-R-loop conformational states have been described, but this study is the first to reveal the full sequence of events from seed bubble formation to R-loop formation at high resolution.

In contrast to the scalpel-like Cas9, CRISPR-Cas3 acts like a shredder that chews DNA up beyond repair. While CRISPR-Cas3 has, thus far, limited utility for precision gene editing, it is being developed as a tool to combat antibiotic-resistant strains of bacteria. A better understanding of its mechanisms may broaden the range of potential applications for CRISPR-Cas3.

In addition, all CRISPR-Cas subtypes utilize some version of an R-loop formation to detect and prepare target DNA for cleavage. The improved structural understanding of this process can now enable researchers to work toward modifying multiple types of CRISPR-Cas systems to improve their accuracy and reduce the chance of off-target effects in biomedical applications.

Scientists hypothesized that these states existed but they were lacking the visual proof of their existence, said co-first author Min Luo, postdoctoral fellow in the Liao lab at HMS. The main obstacles came from stable biochemical reconstitution of these states and high-resolution structural visualization. Now, seeing really is believing.

Weve found that these steps must occur in a precise order, Luo said. Evolutionarily, this mechanism is very stringent and has triple redundancy, to ensure that this complex degrades only invading DNA.

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Bringing CRISPR into Focus - Bioscience Technology

Recent debate over the safety of CRISPR/Cas9 genome editing following a study that suggested it can cause hundreds of unexpected mutations [1]left me puzzled. The research (see BioNews 903), published in Nature Methods and carried out in three living mice, could indeed be criticized for the lack of stringent controls and technical errors. In addition, a number of sloppy mistakes suggested a misinterpretation of the data and therefore incorrect conclusions [2]. It is hard to believe the assertion that CRISPR-Cas9 editing caused so many mutations. And it is also hard to believe how such sloppiness passed the scrutiny of one of the most highly-ranked scientific journals.

In defence of the authors, they did the right thing they used whole-genome sequencing to assess possible effects of CRISPR-Cas9-mediated genome editing in their experimental system. What they found - from their point of view - was quite alarming, and by publishing the study they wanted to make the data available to the public and warn the scientific community.

The puzzling part to me is the reaction of the companies Intellia Therapeutics and Editas Medicine and their calls for the paper to be retracted on the grounds of flawed design and interpretation (see BioNews 905). Intellia is working on permanently editing disease-associated genes in the human body with a single treatment course, whereas Editas Medicine is dedicated to treating patients with genetically defined diseases. The base technology used by both companies is CRISPR-Cas9.

Why did they get so upset? The notion that CRISPR-Cas9-mediated genome editing may not be flawless brought down the share values of the companies. If they are so cocksure that the technology is flawless, the companies must have proof of that from their own pipelines. Instead just launching an attack to put minds of the investors at peace, they could make available a few examples of whole-genome sequencing data sets before and after CRISPR-Cas9 gene editing from their own work. This would show (I assume) that the technology is indeed in their hands very precise and, therefore, safe.

Failure to do that, makes me question whether they do whole-genome sequencing before and after CRISPR-Cas9 gene editing. I cannot help but ask the question, what is their quality control?

How they can even think about developing clinical products without demonstrating that there are no unwanted genome alterations following genome editing? Regulatory bodies, concerned about patient safety, would never approve clinical studies without such proofs. Thus, naturally, the companies' shares have ended up plunging.

Such an approach is standard in other fields of emerging therapies. If you, for example, work in cellular therapy with pluripotent stem cells, either human embryonic stem cells (hESCs) or human iPSs (induced pluripotent stem) cells, you would have to demonstrate the efficiency of your differentiation protocol by showing that not a single cell remained in a pluripotent state afterwards (which can lead to teratoma formation) - even though the probability of this happening is very low. The field has been struggling for years with the challenge of validating a hESC/hiPSC-derived cell dose for the absence of pluripotent stem cells.

And whole-genome sequencing is a part of the quality control in hiPSC-based clinical trials due to the fear that the reprogramming of somatic cells into hiPSC might cause mutations - even when the method used to reprogram the cells in the first place does not involve integrating DNA into the genome. Hence the possibility of introducing mutations due to reprogramming is almost zero.[3]

With CRISPR-Cas9, we are dealing with a genome editing technology and a remote possibility of unwanted genome alterations cannot be neglected. The whole genome sequencing before and after the editing should be a paradigm of quality control for any serious research study, not only clinical work.

And cost should not be an issue. According to the US National Human Genome Research Institute, the price of generating a high-quality 'draft' whole human genome sequence had fallen below US$1,500 [4] by the end of 2015, whereas the cost of a whole-exome sequence was generally below US$1,000. With such low costs, using this method for quality control should be a no-brainer for any CRISPR-Cas9 gene editing research study, not only for clinical applications.

How about CRISPR-Cas9-mediated genome editing in human embryos? The UK's HFEA (Human Fertilisation and Embryology Authority) last year granted a research licence for this (see BioNews 837) and, therefore, the same rules should apply as for any other research study. The complexity of the system and number of cells available would determine quality control. How investigators who have the licence granted will tackle the issue, remains to be seen.

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Safety matters can we be sure that CRISPR-Cas9 is not producing unwanted genetic alterations? - BioNews

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DuPont Pioneer announced it has secured exclusive rights to CRISPR-cas technology for all agricultural uses and applications in plants. CRISPR is one of the newest ways to edit biological genomes.

DuPont Pioneer Vice-President Neal Gutterson says,We see CRISPR-Cas technology as an advancement in plant breeding which can enable a new era in crop improvement. This licensing agreement with ERS is a piece of DuPont Pioneers strategy to position our business as a leader in the application of CRISPR-Cas in agriculture."

The licensing agreement is with ERS Genomics and already DuPont has 60 patents or patent applications for CRISPR bacteria identification and immunization, as well as gene editing technology.

Pioneer says it wants to use CRISPR to develop better environmental resiliency, productivity, and sustainability.

In another statement DuPont promises open and transparent communications and appropriate, science-based regulatory oversight for its work with CRISPR.

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Seed Company Secures CRISPR Rights | whotv.com - whotv.com

China's State Intellectual Property Office (SIPO) has granted the University of California a patent on CRISPR/Cas9 genome editing technology in the country.

The SIPO patent granted to University of California Berkeley, University of Vienna and researcher Professor Emanuelle Charpentier, will allow them to license CRISPR technologies to firms and researchers in China. It will also allow Professor Charpentier's company CRISPR Therapeutics and that of Professor Jennifer Doudna at Berkeley - Intellia Therapeutics, to market any CRISPR-based therapies they develop in future in the country.

'SIPO's decision further expands our IP portfolio, and is further global recognition that Jennifer Doudna, Emmanuelle Charpentier and their team are the pioneers in the application of CRISPR/Cas9 in all cell types,' said Intellia Therapeutics Chief Executive Officer and President, Dr Nessan Bermingham.

CRISPR intellectual property rights are the subject of an ongoing dispute in the USA between the University of California who were first to file and hold a general patent, and the Broad Institute, whose use of CRISPR in eukaryotic cells was determined to be separately patentable. The University of California argue that Professor Doudna and Professor Charpentier's team were the first to invent the technique, and has since filed an appeal for patent rights to uses of CRISPR in all cell types.

'China is following the lead of the EU and UK in saying that Doudna and Charpentier were the first to invent the CRISPR-Cas9 gene-editing technology,' said University of California Berkeley spokesperson, Robert Sanders, according to The Daily Californian. 'We are arguing that the US Patent and Trademark Office should also recognize Doudna and Charpentier were the first to invent the technology.'

The Broad Institute may yet be granted its own patents in China, as patent applications from the Broad Institute are still being considered by SIPO. 'In China, patents are subject to invalidation proceedings after they are issued,' Lee McGuire, the chief communications officer for the Broad Institute told The Daily Californian.

Continued here:

China sides with Berkeley on CRISPR patent - BioNews

With CRISPR being one of the most significant discoveries in genome engineering history, many scientists are now turning towards it as a basic foundation of their research. In response, companies are developing new technologies and products designed to help these very scientists become more effective in their research. One such company is Silicon Valley entity Synthego.

Founded by engineers, Paul and Michael Dabrowski, Synthego has risen from an unknown startup to one of the leading providers of genome engineering solutions today. Having both worked at Elon Musks SpaceX, the Dabrowski brothers seemed like an unlikely pair to create a biotech company focused on CRISPR genome engineering. However, it is their knowledge and experience of precision engineering and automation that has become a key differentiator.

In 2015, Synthego brought on biotech industry veteran Ted Tisch as Chief Operating Officer. In the following years, Ted helped build Synthegos product development, manufacturing and commercial operations, and formally launch the company in 2016.

Eight of the worlds 10 largest biopharma companies are already working with Synthego. Out of the top 25 biology universities in the world, 24 are using its products, and even CRISPR pioneer Jennifer Doudnas interest was sparked she is now an investor in the company.

We caught up with Tisch to talk about how CRISPR is revolutionizing the biotech field and the role Synthego is playing in all of this.

With genetic engineering as a key scientific research area, more and more researchers are using it as a foundation of their studies (source: Shutterstock)

PCR and next-generation sequencing (NGS) have been the two breakthrough technologies of the past 25 years. I call them core platform technologies, because they have been built up and expanded over time. These technologies took 10 to 15 years to reach wide adoption because researchers had to adjust to a technology that was very expensive. This consequently limited the adoption rate.

The cool thing about CRISPR is that it requires only a few pieces of equipment for it to work, so anyone can do it. I have not observed a technology lift like this in the market and this is why I believe that CRISPR will be the third platform technology in life science. Synthego aims to revolutionize CRISPR in two ways: Firstly, it automates the laboratory workflow, and secondly, it focuses on simplifying the workflow.

Biological workflows are extremely complex, as they include many complicated steps, which can lead to errors and wrong assumptions. This is a downward spiral, as many researchers rely on the work of others and subsequently, mistakes might be replicated in other labs. One of the biggest challenges with fast moving technologies is that people start publishing quickly with poor results; Synthego is trying to keep the industry safe from the dissemination of poor results by providing high-quality effective products and ultimately performing the basic research steps themselves.

Synthego is working to revolutionize genetic engineering by offering high-quality synthetic gRNA and a next-generation Design Tool (Source: Shutterstock)

Synthego produces synthetic guide RNA (gRNA) and a next-generation Design Tool. Imagine synthetic gRNA like a shuttle that escorts the enzyme Cas9 to a specific target gene where the Cas9 can cut the DNA. Poor quality gRNA products can lead to off-target events, meaning that the enzyme cuts the wrong part of the DNA strand. Synthegos gRNA has a high on-target efficiency, allowing researchers to work more quickly. Subsequently, there is an increased percentage of edited cells, making research experiments much easier.

The other product is a CRISPR Design Tool, which was launched in May. Synthego developed a software tool that identifies the target gRNA sequence with the highest on-target percentage while reducing off-target events. Researchers can go online, look up over 100,000 genomes and within seconds the tool will present the sequence of the gRNA. Its like shopping on Amazon: users can order it directly online and a receive their product a couple of days later.

The Design Tool coupled with the synthesized gRNA really simplifies the workflow in the lab.

Synthegos main goal is to automate and simplify the laboratory workflow, in order to minimize errors and maximize research success (source: Shutterstock)

There has been a great resonance from the industry. Worldwide, academic institutions and commercial organizations in about 33 countries are working with Synthego. To date, 24 of the 25 top life science universities in the world, and 8 of the worlds 10 largest biopharmaceutical companies are using our technologies.

Following the US, Europe represents one of our largest areas of interest. We have customers in most of the major Western European countries, including the United Kingdom, Germany, France, Spain and the Netherlands. For example, Synthego is working closely with the University of Oxford in the UK, and in Germany, the University of Freiburg.

Well, the first but good problem were facing is that Synthego has a huge customer demand. Helped by the fact that we received series B funding in January, we are now focusing on upscaling our capacities and on the development of new products and solutions.

An automated workflow brings many advantages with it. In the case of CRISPR, off-target events are minimized, whilst on-target events increase dramatically (source: Shutterstock)

One major challenge is to get people to move away from outdated processes. Scientists are notoriously dogmatic and careful about adapting new technologies. However, many researchers are exploring new ways of using genome editing in their processes, whether its therapies, improving the performance of their cell lines, or the development of their drug products. As our products help to deliver good and quick results, we have a high adoption rate.

As a small company that is trying to make noise in a big industry, we have to focus on quality, innovation and quick execution. Our competition are the large multimillion dollar life sciences companies. But these large life science suppliers have tried to address genome editing with old technologies or processes, making their products more expensive, slow to be delivered, and often unclean.

In the near term, we see further simplifying and improving the research workflow by providing easy-to-use kits and tools.

Our long-term strategy is to offer biology as a service. Imagine a scientist who wants to run thousands of experiments, but is lacking the time, budget, or infrastructure. As Synthego continues to develop its platform, any scientist will be able to insert his experimental parameters online via a computer, and Synthego will run the experiment and deliver the results.

Our goal is to make biology research accessible for all scientists worldwide.

Images vianoeldelmar, TarikVision, ibreakstock, science photo, Bloomicon/Shutterstock.com

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Rockets to DNA - How Two SpaceX Engineers became CRISPR ... - Labiotech.eu (blog)

The gene-editing tool known as CRISPR is fast becoming known for its potential to treat disease by snipping genetic mutations from DNA.

But genomic tools like CRISPR also have other possible capabilities, such as the ability to screen people for the presence of viruses, like dengue and Zika, as well as debilitating diseases like Parkinson's.

"I think the public perception of CRISPR is very focused on the idea of using gene editing clinically to cure disease. This is no doubt an exciting possibility, but this is only one small piece," said Neville Sanjana, of the New York Genome Center and an assistant professor of biology, neuroscience and physiology at New York University. [10 Amazing Things Scientists Just Did with CRISPR]

"With CRISPR, I think you'll see many applications in synthetic biology," like sensors for pathogens, Sanjana told Live Science.

At its core, CRISPR is a natural defense system that evolved in single-celled microorganisms to fight against invading viruses. The fight is an all-out war. Scientist estimate that for every cell on Earth, there are about 10 viruses, all launching relentless missions to replicate themselves by inserting their DNA into the machinery in cells.

Bacteria use an arsenal to fight back, including CRISPR, which is an array of short, repeated sequences of DNA that are separated by spacers that have unique sequences.Bacteria use it when they are infected with a virus. As the virus's genetic bits replicate inside the bacteria, CRISPR steps in, guiding the bacterial defenses toward the foreign material.

The protein in CRISPR cuts up the intruder, but also collects a short DNA sequence from the invader, which the protein inserts it into the bacteria's CRISPR as a spacer. Each time a virus invades and is destroyed, a new spacer gets added to the CRISPR.

In a sense, the spacers in CRISPR are an account of the bacteria's battlefield wins, like kill marks in the stock of a rifle barrel. But the spacers provide another function.

When a virus that was previously defeated tries to invade, the bacteria recognizes it and sets about chopping the invader up into tiny bits. And when the bacteria itself multiplies, it passes it's defense system on to its daughter cells.

"It turns out you can actually leverage these properties to potentially develop a very sensitive diagnostic device" that could detect small amounts of molecules from viruses in human blood, such as Zika virus, said biochemist and CRISPR expert Sam Sternberg, the group leader of Technology Development at Berkeley, California-based Caribou Biosciences Inc., which is advancing new applications for CRISPR-based technologies. [5 Amazing Technologies That Are Revolutionizing Biotech]

One of the most recent CRISPR advances in this area is a tool called SHERLOCK (which stands for Specific High Sensitivity Enzymatic Reporter UnLOCKing). In April 2017, a team of researchers led by bioengineer James Collins and CRISPR pioneer Feng Zhang of the Broad Institute of MIT and Harvard reported in Science that they had programmed a CRISPR molecule to seek out strains of Zika and dengue viruses in blood serum, urine and saliva and cut them up.

The researchers programmed the CRISPR molecules to release a fluorescent signal when they were chopping away at the viruses, so that the presence of the virus could be detected. SHERLOCK was so sensitive, it was able to distinguish the American strain of Zika from the African strain and differentiate one strain of dengue from another one.

Collins and his team were able to see the presence of the viruses even in extremely low concentrations, as low as two molecules in a quintillion.

In a separate test, SHERLOCK was able to detect two different strains of the antibiotic-resistant superbug Klebsiella pneumoniae. [6 Superbugs to Watch Out For]

Then, in June 2017, a team at the University of Central Florida reported in the journal Scientific Reports that they had used a CRISPR system to detect the presence of Parkinson's disease. This disorder of the central nervous system causes malfunction and death of nerve cells in the brain, and gets worse over time, causing tremors and problems with movement. The disease affects about 1 million people in the United States, according to the Parkinson's Disease Foundation.

Although the cause is unknown, the amount of a protein called alpha-synuclein, normally found in the brain, rises in people who develop the disease. The researchers used CRISPR to edit the gene that makes the alpha-synuclein protein so that the protein would fluoresce. The larger the amount of the protein, the stronger the fluorescent signal.

The scientists said they think they could use this technique to test out new drugs to treat Parkinson's disease.

"If we take one of these modified cells and treat it with a particular drug, if it doesn't produce light anymore, then this means the drug is a potential treatment for this disease," study co-author Sambuddha Basu, a postdoctoral researcher at Central Florida,said in a statement.

It's still the very early days for these and other CRISPR-related biological tools, and because of the diversity of the immune systems in bacteria, it's quite possible that other tools remain to be discovered, Sternberg said.

"I think it's a really nice example of yet another basic science discovery that has led to a potential breakthrough technology," he said.

Originally published on Live Science.

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Could CRISPR Sniff Out Viruses? - Live Science

The 15 kilometer cross-country ski race at the 1964 Winter Olympics should have been a close race. But in fact Finlands Eero Mntyranta streaked over the line fully 40 seconds ahead of his closest competitor.

His secret? He was born with a mutation of the EPOR gene, which regulates the production of the erythropoietin protein. In turn this determines the production of red blood cells, which are crucial for physical endurance.

Disgraced cyclist Lance Armstrong achieved the same outcome by taking erythropoietin as a drug. But in future if indeed it has not already started athletes may go straight to the root of the matter and mutate their own EPOR gene, to give themselves the same advantage that Mntyranta was born with. How could this possibly be detected?

That athletes will turn to gene editing is, in my view, a certainty. We have the tools to manipulate genes and we know at least some of the genes that code for the sort of physical attributes that athletes crave. Here is a picture of the Belgian bull.

This double muscled specimen has a natural mutation of the gene that codes for the protein myostatin and the result is uninhibited muscle growth. For farmers that means lots of lean meat. But for weight lifters it could be a short cut to an Olympic medal.

A crack in creation

This is just one of the ethical dilemmas unleashed by our newfound ability to edit the genome. As I described last week Jennifer Doudna of UC Berkeley is credited with the discovery of CRISPR, the technique that has made gene editing considerably faster, cheaper, more accurate and accessible, and today I want to tell you a little more about her new book A Crack in Creation.

Much of it covers the voyage of the discovery of CRISPR the hypotheses, the laboratory tests, the international conferences, the chance meetings between researchers and the occasional flashes of inspiration. The book also tells us exactly how CRISPR works.

You may have to read this several times before getting your head around it but essentially CRISPR was adapted from the method that bacteria use to identify and cut the DNA of viruses that are trying to attack them a pair of designer molecular scissors that homes in on a specific twenty-letter DNA sequence and cuts apart both strands of the double helix..

Today we are able to use CRISPR to disable target genes and to add new stretches of DNA. One of the impressive aspects of Doudnas book is her confidence in these techniques.

Scientists tend to be circumspect, but not here. Scientists have succeeded in bringing this primordial process (of the evolution of life) fully under human control. Using powerful biotechnology tools to tinker with DNA inside living cells, scientists can now manipulate and rationally modify the genetic code that defines every species on the planet. And using CRISPR an organisms entire DNA content has become almost as editable as a piece of text.

That is not to say that CRISPR can achieve anything. Many traits are the result of numerous genetic interactions and may be too complicated to affect. And CRISPR is not perfect. DNA does not always get altered as desired, and there are off-target effects that hit other areas of the genome.

Then there is the challenge of delivering the CRISPR mechanism into cells. This is hard enough when the cells are in the laboratory but even harder when the cells are still inside the human body. We are finding ways of making gene editing more accurate, but a certain amount of inaccuracy may not matter anyway.

Most medical treatments have some unforeseen consequences and the judgement is not whether they are perfect but whether the advantages outweigh the disadvantages.

We must though draw an important distinction between the editing of germline cells and of somatic cells. Germline cells contain DNA that is handed down to the next generation. Somatic cells (sometimes called adult cells) are all the others.

Suppose that I have muscular dystrophy, a disease that can be tracked down to specific genetic mutations. If CRISPR is used to correct these mutations then there may be off-target effects that could affect the function of my cells and body in unwelcome ways. But this is my problem alone.

However if in trying to correct a specific gene in germline cells there are off-target effects then these will be passed on. This, it seems to me, is highly problematical.

And yet Doudna believes that germline editing is something that we should accept, arguing that since our genes are constantly and randomly changing as our cells divide and copy, a few extra CRISPR-induced changes wont make much difference.

What this tech can do

What can this new power over the living world do for mankind? First, it can enable us to understand it. The best way to find the function of a gene is to disable it and see the result.

Once we discover the genetic mutation responsible for, say, Huntingdons or cancer we can create cells or laboratory mice with this same mutation as use them as models upon which to test potential therapies. Already we have numerous examples of the potential of gene editing.

In human medicine CRISPR has already been used to develop potential cures for diseases including cystic fibrosis, sickle cell disease, muscular dystrophy, HIV/AIDS and even Alzheimers. Tests have been conducted on laboratory cells and animals and, in some pioneering cases, in humans.

CRISPR is critical to cancer immunotherapy, which sees immune cells engineered to recognize cancer cells. Elsewhere we have made barley that is resistant to powdery mildew, tomatoes that do not rot as soon as they are picked, mosquitoes that are unable to transmit malaria, ultra-muscular police dogs and cows with no horns. On the horizon are pigs that can serve as donors of human organs and even woolly mammoths and unicorns.

This all sounds great and yet Doudna is worried. The availability of CRISPR means that it could fall into villainous hands. She is worried that the same ignorant outcry that has hampered the progress of GM crops could impede the progress of gene editing. And she concedes that CRISPR is forcing us to confront difficult, perhaps unanswerable questions, many of which boil down to conundrums about the relationship between humans and nature.

But mankind has for ever tried to conquer nature and Doudna clearly believes that gene editing is in many ways superior to techniques we have used in the past. In the realm of agriculture historic practice has been to bombard plants with chemicals or radiation in order to cause random genetic changes.

When this has thrown up a plant with superior traits then we have bred from it. Is it not better to identify the desired trait, work out its genetic cause and then deliberately engineer the gene? And if we have hunted the great auk to extinction, should we not use genetic engineering to bring it back?

The most pressing debate concerns germline editing. When allied to established practices like In Vitro Fertilization and pre-implantation testing it is highly likely that we will be able to ensure that babies do not carry the genetic mutations that in some cases virtually guarantee a life of suffering.

Of course these same techniques could be used to create designer babies and yet Doudna favours their approval. I dont believe theres an ethical defence for banning germline modification outright, nor do I think we can justifiably prevent parents from using CRISPR to improve their chances of having a healthy, genetically related child, so long as the methods are safe and offered in an equitable manner.

If we have the tools to prevent suffering, surely we should use them. Doudna argues that some existing practices like PGD (pre-implantation genetic diagnosis) that allow parents to choose the sex of their baby or abort those with Downs syndrome are already facilitating forms of designer baby and that ultimately matters of conception are best left to parental choice.

Finally Doudna dismisses the argument that germline editing should be banned because it unnatural. As she points out natural evolution has not been entirely benign and, in the world of medicine the line between natural and unnatural blurs to the point of disappearing.In my mind the distinction between natural and unnatural is a false dichotomy, and if it prevents us from alleviating human suffering, its also a dangerous one.

So this book is both a description of the extraordinary possibilities of gene editing but also, if indeed the horse has not already bolted, a plea for its acceptance. We should, Doudna believes, be bold and brave. Few technologies are inherently good or bad; what matters is how we use them and in the case of CRISPR it has incredible potential to improve our world.

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CRISPR 'it Has Incredible Potential To Improve Our World - The Daily Reckoning

In BriefScientists have developed a tool that can test an entiregenome against a CRISPR molecule to predict potential errors andinteractions. This will allow doctors to ensure treatments aresafer and more effective. Editing The Editor

The CRISPR gene-editing tool is already in use by scientists all over the world who are racing to cure deadly diseases by editing the genomes of patients. However, as human trials for various treatments are slated to begin, we still face the hurdle of ensuring that any errors in CRISPR edits wont causing problems. Scientists from The University of Texas at Austin may have come up with a possible solution. Theyve developedsomething that works like a predictive editor for CRISPR: a method for anticipating and catching the tools mistakes as it works, thereby allowing for the editing ofdisease-causing errors out of genomes.Click to View Full Infographic

Scientists have already learned how to use CRISPR to edit errors in almost any genome and its these errors that can cause a wide range of diseases. Many forms of cancer, Huntingtons disease, and even HIV can be targeted usingCRISPR. That being said, itsnot a perfect solution. Just as the autocorrect on your smartphone can cause you to send an unintentional and embarrassing text message, CRISPR can correct something that was actually right the consequences of which can make it adangerous mistake. One that actually causes a disease as opposed to an embarrassing social gaffe.

The researchers developed a method for quickly testing a CRISPR molecule against a persons entire genome, rather than onlythe target area,in order topredict other segments of DNA the tool might accidentally interact with. This new technique functions like an early warning system, giving doctors a chance to more closely tailor gene therapies to specific patients, while ensuring they are effective andsafe.

If were going to use CRISPR to improve peoples health, we need to make sure we minimize collateral damage, and this work shows a way to do that,Stephen Jones, UT Austin postdoctoral researcher and co-lead author of the study, told the UT News.

This research will also allow scientists to improve their own predictive skills when it comes to CRISPR molecule behaviors even without genome testing. This is because the work is actually revealing the rule book CRISPR molecules follow when they choose targets.

One CRISPR molecule the team tested, Cascade, targets DNA sequences but pays less attention to every third letterin the sequence. So if it were looking for the word shirt and instead found the word short, it might be fine with that, Jones said, explaining the significance of the quirk to the UT News.

As researchers master these rules, they will be able to develop better predictive models for CRISPR therapies. This will make the technique faster and cheaper, which will in turn render personalized gene therapies more accessible to more patients. Most important of all, it will also help make theentire process far safer.

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This New Gene-Editing Technique Can Spot CRISPR's Mistakes - Futurism

CRISPR-Cas3 is a subtype of the CRISPR-Cas system, a widely adopted molecular tool for precision gene editing in biomedical research. Aspects of its mechanism of action, however, particularly how it searches for its DNA targets, were unclear, and concerns about unintended off-target effects have raised questions about the safety of CRISPR-Cas for treating human diseases.

Harvard Medical School and Cornell University scientists have now generated near-atomic resolution snapshots of CRISPR that reveal key steps in its mechanism of action. The findings, published in Cell on June 29, provide the structural data necessary for efforts to improve the efficiency and accuracy of CRISPR for biomedical applications.

Through cryo-electron microscopy, the researchers describe for the first time the exact chain of events as the CRISPR complex loads target DNA and prepares it for cutting by the Cas3 enzyme. These structures reveal a process with multiple layers of error detectiona molecular redundancy that prevents unintended genomic damage, the researchers say.

High-resolution details of these structures shed light on ways to ensure accuracy and avert off-target effects when using CRISPR for gene editing.

To solve problems of specificity, we need to understand every step of CRISPR complex formation, said Maofu Liao, assistant professor of cell biology at Harvard Medical School and co-senior author of the study. Our study now shows the precise mechanism for how invading DNA is captured by CRISPR, from initial recognition of target DNA and through a process of conformational changes that make DNA accessible for final cleavage by Cas3.

Target search

Discovered less than a decade ago, CRISPR-Cas is an adaptive defense mechanism that bacteria use to fend off viral invaders. This process involves bacteria capturing snippets of viral DNA, which are then integrated into its genome and which produce short RNA sequences known as crRNA (CRISPR RNA). These crRNA snippets are used to spot enemy presence.

Acting like a barcode, crRNA is loaded onto members of the CRISPR family of enzymes, which perform the function of sentries that roam the bacteria and monitor for foreign code. If these riboprotein complexes encounter genetic material that matches its crRNA, they chop up that DNA to render it harmless. CRISPR-Cas subtypes, notably Cas9, can be programmed with synthetic RNA in order to cut genomes at precise locations, allowing researchers to edit genes with unprecedented ease.

To better understand how CRISPR-Cas functions, Liao partnered with Ailong Ke of Cornell University. Their teams focused on type 1 CRISPR, the most common subtype in bacteria, which utilizes a riboprotein complex known as CRISPR Cascade for DNA capture and the enzyme Cas3 for cutting foreign DNA.

Through a combination of biochemical techniques and cryo-electron microscopy, they reconstituted stable Cascade in different functional states, and further generated snapshots of Cascade as it captured and processed DNA at a resolution of up to 3.3 angstromsor roughly three times the diameter of a carbon atom.

Seeing is believing

In CRISPR-Cas3, crRNA is loaded onto CRISPR Cascade, which searches for a very short DNA sequence known as PAM that indicates the presence of foreign viral DNA.

Liao, Ke and their colleagues discovered that as Cascade detects PAM, it bends DNA at a sharp angle, forcing a small portion of the DNA to unwind. This allows an 11-nucleotide stretch of crRNA to bind with one strand of target DNA, forming a seed bubble.

The seed bubble acts as a fail-safe mechanism to check whether the target DNA matches the crRNA. If they match correctly, the bubble is enlarged and the remainder of the crRNA binds with its corresponding target DNA, forming what is known as an R-loop structure.

Once the R-loop is completely formed, the CRISPR Cascade complex undergoes a conformational change that locks the DNA into place. It also creates a bulge in the second, non-target strand of DNA, which is run through a separate location on the Cascade complex.

Only when a full R-loop state is formed does the Cas3 enzyme bind and cut the DNA at the bulge created in the non-target DNA strand.

The findings reveal an elaborate redundancy to ensure precision and avoid mistakenly chopping up the bacterias own DNA.

To apply CRISPR in human medicine, we must be sure the system is accurate and that it does not target the wrong genes, said Ke, who is co-senior author of the study. Our argument is that the CRISPR-Cas3 subtype has evolved to be a precise system that carries the potential to be a more accurate system to use for gene editing. If there is mistargeting, we know how to manipulate the system because we know the steps involved and where we might need to intervene.

Setting the sights

Structures of CRISPR Cascade without target DNA and in its post-R-loop conformational states have been described, but this study is the first to reveal the full sequence of events from seed bubble formation to R-loop formation at high resolution.

In contrast to the scalpel-like Cas9, CRISPR-Cas3 acts like a shredder that chews DNA up beyond repair. While CRISPR-Cas3 has, thus far, limited utility for precision gene editing, it is being developed as a tool to combat antibiotic-resistant strains of bacteria. A better understanding of its mechanisms may broaden the range of potential applications for CRISPR-Cas3.

In addition, all CRISPR-Cas subtypes utilize some version of an R-loop formation to detect and prepare target DNA for cleavage. The improved structural understanding of this process can now enable researchers to work toward modifying multiple types of CRISPR-Cas systems to improve their accuracy and reduce the chance of off-target effects in biomedical applications.

Scientists hypothesized that these states existed but they were lacking the visual proof of their existence, said co-first author Min Luo, postdoctoral fellow in the Liao lab at HMS. The main obstacles came from stable biochemical reconstitution of these states and high-resolution structural visualization. Now, seeing really is believing.

Weve found that these steps must occur in a precise order, Luo said. Evolutionarily, this mechanism is very stringent and has triple redundancy, to ensure that this complex degrades only invading DNA.

This article has been republished frommaterialsprovided by Harvard Medical School. Note: material may have been edited for length and content. For further information, please contact the cited source.

Reference

Xiao, Y., Luo, M., Hayes, R. P., Kim, J., Ng, S., Ding, F., . . . Ke, A. (2017). Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System. Cell, 170(1). doi:10.1016/j.cell.2017.06.012

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CRISPR Mechanism of Action Imaged Near-atomic Resolution - Technology Networks

Jennifer Doudna: Experiments fail. To have people around that get along with each other is super important. Photograph: Bryan Derballa/Kintzing.com

Jennifer Doudna, 53, is an American biochemist based at the University of California, Berkeley. Together with the French microbiologist Emmanuelle Charpentier, she led the discovery of the revolutionary gene-editing tool, Crispr. The technology has the potential to eradicate previously incurable diseases, but also poses ethical questions about the possible unintended consequences of overwriting the human genome.

Were you nerdy as a child? What got youhooked on science? Yes, I was nerdy. My father was a professor of American literature in Hawaii and he loved books. One day I came home from school and he haddropped a copy of The Double Helixon the bed, by Jim Watson. Onerainy afternoon I read it and Iwasjust stunned. I was blown awaythat you could do experiments about what a molecule looks like. I was probably 12 or 13. I think that wasthebeginning ofstarting to think,Wow, that could be an amazingthing to work on.

Youve spent most of your career uncovering the structure of RNA and never set out to create a tool to copy andpaste human genes. How did you endup working on Crispr? I think you can put scientists into two buckets. One is the type who dives very deeply into one topic for their whole career and they know it better than anybody else in the world. Then theresthe other bucket, where I wouldput myself, where its like youre at a buffet table and you see an interesting thing here and do it for a while, and that connects you to another interesting thing and you take a bit of that. Thats how I came to be working on Crispr it was a total side-project.

But when you first started your collaboration with Emmanuelle Charpentier, did you have a hunch youwere on to something special? We met at a conference in San Juan, Puerto Rico, and took a walk around the old town together. She was so passionate, her excitement was very infectious. I still remember walking down this street with her and she said: Well Im really glad you want to work with us on the mysterious [Cas9 the enzyme that snips DNA at the chosen location in the editing process]. It was this kind of electrifying moment. Even then I just had this gut feeling that this was something really interesting.

I would have loved to continue working with Emmanuelle. Im not blaming her: she had her reasons and I respect her

How important is personal chemistry inscience collaborations? Its essential. Working in a lab is analogous to being in a high-school play: youre rehearsing long hours, itscrowded, there are stressful things that come up. Its the same thing in science. Things never work as you think they will, experiments fail and so to have people around that really get along with each other is super important. Many collaborations dont work out, usually just because peoples interests arent aligned or people dont really like working together.

The real frenzy around your work started in 2012, when you showed that Crispr-Cas9 could be used to slice up DNA at any site [of the DNA molecule] you wanted. Did you realise this was abig deal gradually orimmediately? It wasnt a gradual realisation, it was one of those OMG moments where you look at each other and say holy moly. This was something we hadnt thought about before, but now we could see how it worked, we could see it would be such a fantastic way to do gene editing.

After you demonstrated Crispr could edit bacterial DNA, two rival labs (Harvard and the Broad Institute) got there first in human cells. How come they beat you to it? They were absolutely set up to do that kind of experiment. They had all the tools, the cells growing, everything was there. For us, they were hard experiments to do because its not thekind of science we do. What speaksto the ease of the system was that a lab like mine could even do it.

The Broad Institute won the latest round of an ongoing legal battle over patent rights they claim that it wasnt obvious that Crispr could be used to edit human cells too. Where do you stand? People have asked me over and over again: Did you know it was going to work? But until you do an experiment you dont know thats science. Ive been lambasted for this in the media, but I have to be true to who I am as a scientist. We certainly had a hypothesisand it certainly seemed likea very good guess that it would.

Theres the patent dispute and you and Emmanuelle Charpentier also ended up pursuing rival projects to commercialise the technology. Are you all still friends? If theres a sadness to me about all of this and a lot of its been wonderful and really exciting its that I wouldve loved to continue working with Emmanuelle, scientifically. For multiple reasons that wasnt desirable to her. Im not blaming her at all she had her reasons and I respect her a lot.

The media loves to drive wedges, but we are very cordial. I was just with her in Spain and she was telling me about the challenges [of building her new lab in Berlin]. I hope on her side, certainly on my side, we respect each others work and in the end were all init together.

In your book you describe a nightmare youhad involving Hitler wearing a pig mask, asking to learn more about your amazing technology. Do you still have anxiety dreams about where Crispr mightleave the human race? I had the Hitler dream and Ive had a couple of other very scary dreams, almost like nightmares, which is quite unusual for an adult. Not so much lately, but in the first couple of years after I published my work, the field was moving so fast. I had this incredible feeling that the science was getting out way ahead of any considerations about ethics, societal implications and whether we should be worrying about random people in various parts of the world using this for nefarious purposes.

In 2015, you called for a moratorium on the clinical use of gene editing. Where do you stand on using Crispr to edit embryos these days? It shouldnt be used clinically today, but in the future possibly. Thats a big change for me. At first, I just thought why would you ever do it? Then I started to hear from people with genetic diseases in their family this is now happening every day for me. Alot of them send me pictures of their children. There was one that Icant stop thinking about, just sent to me in the last 10 days or so. A mother who told me that her infant son was diagnosed with a neurodegenerative disease, caused by a sporadic rare mutation. She sent me a picture of thislittle boy. He was this adorable little baby, he was bald, in his little carrier and so cute. I have a son and myheart just broke.

What would you do as a mother? You see your child and hes beautiful, hes perfect and you know hes going to suffer from this horrible disease and theres nothing you can do about it. Its horrible. Getting exposed to that, getting to know some of these people, its not abstract any more, its very personal. And you think, if there were away to help these people, we should do it. It would be wrong not to.

Are people going to start saying I want a child thats 6ft 5in with blue eyes and so on? Do we really want to go there?

What about the spectre of designerbabies? A lot of it will come down to whether the technology is safe and effective, are there alternatives that would be equally effective that we should consider, and what are the broader societal implications of allowing gene editing? Are people going to start saying I want a child thats 6ft 5in and has blue eyes and so on? Do we really want to go there? Would you do things that are not medically necessary but are just nice-to-haves, for some people?Its a hard question. There area lot of grey areas.

Are you worried about cuts to science funding, including to the National Institutes of Health (NIH) budget? I am very concerned. Science funding is not a political football but in fact a down payment on discovery, the seed money to fund a critical step toward ending Alzheimers or curing cancer.

Researchers currently working on projects aimed at improving numerous aspects of our agriculture, environment and health may be forced to abandon their work. The outcome is that people will not receive the medical treatments they need, our struggle to feed our exploding population will deepen, and our efforts to manage climate change will collapse.

Over the long term, the very role of fundamental science as a means to better our society may come into question. History and all evidence points to the fact that when we inspire and support our scientific community we advance our way of life and thrive.

Were you disturbed when Trump tweeted, If U.C. Berkeley does not allow free speech and practices violence on innocent people with a different point of view NO FEDERAL FUNDS? in response to a planned alt-right speaker being cancelled due to violent protests on campus? Yes. It was a confusing tweet since the university was clearly committed to ensuring that the event would proceed safely and first amendment rights were supported. Few expected the awful actions of a few to be met with a willingness from the highest office to deprive more than 38,000 students access to an education.

Youve spoken at Davos, shared the $3m2015 Breakthrough prize, been listedamong the 100 most influential people in the world by Time magazine. Areyou still motivated about heading intothe lab these days? Yesterday I was getting ready to go to a fancy dinner. I was in a cocktail gown and had my makeup on and my hair done, but I wanted to talk to a postdoc in my lab about an experiment he was doing, so I texted him saying can we Skype? It was 8am in California, I was over here [in the UK] in my full evening gown, talking abouttheexperiment.Thats how nerdy I am.

A Crack in Creation: The New Power to Control Evolution by Jennifer Doudna and Sam Sternberg is published by The Bodley Head (20). To order a copy for 17 go to bookshop.theguardian.com or call 0330 333 6846. Free UK p&p over 10, online orders only. Phone orders min p&p of 1.99

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Jennifer Doudna: 'I have to be true to who I am as a scientist ... - The Guardian

I set a high bar for writing about mouse studies. I dont include them in my textbooks or news articles, and only rarely blog about them. But when experiments in mice shine a glimmer of hope on a horrific illness with a long history of failed treatments, I pay attention. That happened last week for a report on editing out of mice the human version of the mutant Htt gene that causes Huntington disease (HD), published in the Journal of Clinical Investigation.

HD affects about 30,000 people in the US, and more than 200,000 family members are at-risk, possibly having inherited the mutation. The disease arises from a repeat of the DNA triplet CAG beyond the 35 or fewer copies that most of us have, at the start of the gene that encodes the protein huntingtin. CAG specifies the amino acid glutamine, and the extra stretch of it clogs certain neurons in the striatum in the brain, affecting movement, cognition, and behavior.

Symptoms typically begin in adulthood, but 10 percent of cases are juvenile. Karli Mukka developed symptoms at age 5, and died within weeks of her father Karl, she just 14 years old, he 43, in 2010. Karlis huntingtin gene did a loop-de-loop upon itself, giving her 99 CAG repeats to her fathers 47. DNA Science told her story here.

ONLY ONE TREATMENT, FOR ONE SYMPTOM

An expanding triplet repeat presents a thorny drug-targeting challenge. Countering it isnt as simple as supplying a missing enzyme, depleting a biochemical buildup, unfolding and refolding an errant protein, or even introducing a functional gene with gene therapy.

Unlike other genes in which mutations remove a normal function, abnormal huntingtin protein confers a toxic gain of function. Having two mutations is no worse than having just one, which means that lacking the normal (wild type) allele has no effect, at least after birth thats important.

The only FDA-approved treatment for HD is tetrabenazine, a repurposed schizophrenia drug used in other nations for decades before its FDA approval to treat the movement (chorea) part of HD in 2008. An altered version (deutetrabenazine) became available in April 2017: heavy hydrogen atoms (deuterium) keep the drug circulating longer.

Researchers have for years thrownevery tool imaginableat the formidable expanded Htt gene:

Deploying small molecules to target RNA loops or metabolites. Manipulating growth factor levels. Implanting stem cells to replace neurons. Dampening expression of the mutant gene using RNAi or antisense nucleic acids.

New biomarkers, prediction studies, scans, and induced pluripotent stem cells track the onset of the disease, with the hope of eventual early intervention.

I was once a fly-on-the-wall at private meetings of HD researchers, writing reports for a funding organization. I learned a great deal about the mouse models, the treatment modalities, and ways of detecting the disease early and tracking its progression. Progress was slow. That gig ended in 2010 before the debut of CRISPR/Cas9 gene editing. And now its beginning to sound like a whole different ballgame.

CRISPR EDITING OUT MUTANT HTT

The peculiarities of HD make gene editing, which can add, replace, or remove a gene, the most logical therapeutic strategy. HD requires DNA to be jettisoned, not augmented.

While RNAi and antisense oligonucleotides can dampen expression of the extended gene, the effect isnt permanent in the way that snipping out the repeat or even the entire gene would be. And a one-time or few-times editing out is preferable to a regular need for treatment, especially given the unsettling healthcare situationin the US.

Xiao-Jiang Li, MD, PhD, distinguished professor of human genetics at the Emory University School of Medicine, with colleagues there and at the Chinese Academy of Sciences, used CRISPR/Cas9 gene editing on mice that have the first exon (protein-encoding part) from the human Htt gene, including 140 CAG repeats its called an HD140Q knockin (Q stands for polyglutamine). Specks of the toxic protein appear when the mice are 4 to 6 months old, aggregating by 9-10 months. The timetable is like that in people, because mice live about 2 years.

Technical details (jargon alert): CRISPR/Cas9 was delivered to the striata of two dozen 9-month-old mice in two batches of adeno-associated viruses: guide RNAs targeting exon 1 and the Cas9 enzyme that cleaves both DNA strands, removing the gene and triggering repair of the breaks. The guide RNA part included instructions for red fluorescent protein, and both batcheswere under different promoter (control) sequences, so that the researchers could compare delivery of the dual intervention to either alone. Both are required: find the target and cut it out.

For 3 months the mice were tested on the rotarod, the balance beam, and a device to assess grip strength. Although the sample was small, the findings were clear and compelling.

In the brain parts given both components of the treatment, levels of toxic protein fell, in lockstep with improving performance on the rotating rods, balance beams, and grippers. Weight loss slowed and astrocytes (another type of brain cell) became less reactive. The red marker glowed from the right places the medium spiny neurons of the striatum while protein markers for other neurological diseases as well as for cell death (apoptosis) and degrading used parts (autophagy) werent altered. The experiment was elegantly controlled.

Because the mice had two Htt mutations unlike patients who typically have one and removing both copies didnt do harm, yanking both copies of the gene in people might work. That might mean a one-size-fits-all approach is feasible, rather than tweaking treatment to a specific repeat number. Normal Htt might be required in stem cells, though.

A major concern about CRISPR/Cas9 gene editing is off-target effects. This is relevant to tackling HD because expanded CAG repeats lie behind 7 other neurodegenerative diseases. What would ripping away the CAGs in these genes do? So far, the gene editing was restricted to Htt the researchers sequenced the genomes of the mice to check.

Perhaps most importantly, the treated mice were middle-aged! So older neurons can still throw out their garbage if appropriately stimulated. That may mean, someday, that HD patients flinging themselves helplessly on the floor or bashing their heads might find relief from a possibly one-time treatment that trims the repeats.

THE BIG PICTURE

Of course its a long journey from rodents to patients. But when the only options for families with HD are tetrabenazine to dampen movements and embryo selection to avoid transmitting the mutation, a potential treatment for those in the throes of the illness is good news indeed.

The enormous potential of gene editing to treat intractable diseases is why the anti-CRISPR backlash troubles me. I fear that negative depictions and predictions about the technology could obstruct the quest to develop one-time treatments for genetic diseases with concernsthat a lunatic will someday gene-edit an enhanced master race.

I wonder how many of the more than 6.5 million people who have clicked like toGenetic Engineering Will Change Everything Forever CRISPRhave read the media reports on the Journal of Clinical Investigation article? People love to exaggerate and panic, to imagine the worst, especially when the details of a new technology are unfamiliar or require a base of scientific knowledge.

Once again, politics cant be ignored. Gene editing could help to counter the deadly combination of cutting funding of basic biomedical research while threatening to take away healthcare for millions, simultaneously dismantling the pipeline for new treatments while dooming patients. Meanwhile, the mouse experiments provide something priceless to the HD community: hope. Sums up Jane Mervar, who lost her daughter Karli to juvenile HD and is now caring for two other daughters, CRISPR is my dream.

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Can CRISPR Conquer Huntington's? - PLoS Blogs (blog)

There may be new hope for those suffering from a fatal brain disorder called Huntingtons Disease. Recent research at Emory University, using a groundbreaking gene editing tool called CRISPR-Cas9, has provided new insight into how the disease works, and possible ways to reverse its cruelest effects.

Huntingtons Disease is a genetically inherited condition that leads to nerve cell destruction in the brain. Symptoms, which usually appear in mid-life, include uncontrolled muscle movement, balance issues, mood swings and cognitive decline. Though rates and recording vary from country to country, approximately 30,000 in the U.S. currently suffer from the disease.

While there is no known cure for Huntingtons, a recent study by Chinese scientists at Emory University in Atlanta, Georgia is showing promise. Early results suggest possible treatments for the disease and a path to preventing its occurrence in the first place.

The research is part of an ongoing medical collaboration between the U.S. National Institute of Health (NIH) and the National Natural Science Foundation of China. Under this program, both the U.S. and China contribute funds and scientists for research in both countries.

Emory School of Medicine

Using the revolutionary gene editing technique known as CRISPR-Cas9, researchers at Emory were able to reverse the effects of Huntingtons in test mice.The mice had been genetically modified to carry a human version of the hungtingtin gene that causes the disease. While considered essential for nervous system development in early life, a mutated huntingtin gene can also produce toxic proteins that cause neural generation.

After nine months, when the mice developed the animal version of Huntingtons Disease, researchers used CRISPR-Cas9 to replace the mutant gene with a normal one and then reintroduce the repaired DNA into mice.

Weeks after treatment, the brain-damaging proteins had almost disappeared and motor functions of the mice dramatically improvedthough not to the same level in healthy control mice in which Huntingtons hadnt been induced.

While the results show promise for future human trials involving humans, clinical trials remain a long way off. The long term effectiveness and safety of CRISPR-Cas9 is still under review.

The studys senior author Dr. Xiao-Jiang Li, PhD is optimistic. The findings open up an avenue for treating Huntingtons as well as other inherited neurodegenerative diseases, although more testing of safety and long-term effects is needed, said Xiao-Jiang.

In addition to developing a treatment for victims of Huntingtons, the Sino-U.S. research group hopes to develop ways to reduce the risk for people who are genetically predisposed to developing Huntingtons.

Last year, the same group of Emory researchers had shown they could delete the huntingtin gene in mice older than four months without any known adverse effects. Younger mice without this gene developed fatal pancreatitis. The findings suggest it may someday be possible to safely shut off the gene in adult humans, as well.

Full results of the groups research was published June 19, 2017 in the Journal of Clinical Investigation.

MORE ON CRISPR:

For the second time in a year, doctors in China have used the CRISPR-Cas9 gene editing technique for the treatment of cancer. It is also only the second time CRISPR-Cas9 has been used in human tria

CRISPR-Cas9 is a recently developed gene editing technique that has received worldwide attention because of its relative technical simplicity and wide applications. It is being used in research throughout the world in areas including agriculture, creating new and effective drugs, as well as treating a wide array of genetic disorders.

Defective genes can cause disease. Researchers can use CRISPR Cas-9 like a surgeon uses a scalpelslicing out bad DNA from a damaged genome. Molecular biologists can also transplant normal genes into cellsreplacing damaged or mutated DNA with a new sequence assembled in a lab.

A visualization from the McGovern Institute at MIT explains the science and approach of CRISPR-Cas9.

As the technology advances, scientists hope to someday use CRISPR-Cas9 to create gene therapies that can prevent other inherited diseases, including sickle-cell anemia, Parkinsons disease and cancers that appear to have a genetic component like colon cancer.

Continued here:

CRISPR gene-editing reverses Huntington's Disease in mice - CGTN America (blog)

Jennifer Doudna remembers a moment when she realized how important CRIPSRthe gene-editing technique that she co-discoveredwas going to be. It was in 2014, and a Silicon Valley entrepreneur had contacted Sam Sternberg, a biochemist who was then working in Doudnas lab. Sternberg met with the entrepreneur in a Berkeley cafe, and she told him, with what he later described to Doudna as a very bright look in her eye that was also a little scary, that she wanted to start applying CRISPR to humans. She wanted to be the mother of the first baby whose genome had been edited with the technique. And she wanted to establish a business that would offer a menu of such edits to parents.

Nothing of the kind could currently happen in the U.S., where editing the genomes of human embryos is still verboten. But the entrepreneur apparently had connections that would allow her to offer such services in other countries. Thats a true story, Doudna told a crowd at the Aspen Ideas Festival, which is co-hosted by the Aspen Institute and The Atlantic. That blew my mind. It was a heads-up that people were already thinking about thisthat at some point, someone might announce that they had the first CRISPR baby.

The possibility had always been there. Bacteria have been using CRISPR for billions of years to slice apart the genetic material of viruses that invade their cells. In 2012, Doudna and others showed how this system could be used to deliberately engineer the genomes of bacteria, cutting their DNA with exceptional precision. In quick succession, researchers found that they could do the same in mammalian cells, mice, plants, andin early 2014monkeys. I had all of this at the back of my mind, Doudna told me after her panel. But Sternbergs story about his meeting was the moment where I said I needed to get involved in this conversation. Im not going to feel good about myself if I dont talk about it publicly.

That has not been an easy journey. Doudna built her career on molecules and microbes. As few as five years ago, she was, by her own admission, working head-down in an ivory tower, with no plans of milking practical applications from her discoveries, and little engagement with the broader social impact of her work.

But CRISPR forcefully yanked Doudna out of that closeted environment, and dumped her into the midst of intense ethical debates about whether its ever okay to change the DNA of human embryos, whether eradicating mosquitoes is a good idea, and whether fixing the genes behind inherited diseases is a blow to disabled communities. Now, shes a spokesperson for a field, and an influencer of policy. She regularly makes appearances at conferences and panel discussions, which she often shares with not just scientists but also philosophers, ethicists, and policy-makers. With Sternberg, she is the author of a new book called A Crack in Creation, describing her role in the CRISPR story.

All of this work consumes up to half of her time, taking her away from her lab of 25 people. I find myself really struggling to maintain that balance, she says. But those are the cards Ive been dealt and I feel an obligation to being involved in [the debates around CRISPR]. There arent that many people who know the technology deeply and willing to talk publicly about the societal and ethical issues. I have many science colleagues who dont want to get involved. Yet it has to be done.

Her upbringing prepared her well for this newfound role. Her father was a professor of American literature at the University of Hawaii, who was fiercely intellectual and politically conservative but never dogmatic. Her family dinner table was a place where opposing views were shared openly and debated open-mindedly. It still is: Many of Doudnas in-laws staunchly oppose any form of genetic modification, so her work is a point of contention, even among close family. I spend a lot of time talking to people like me, and its a big challenge is to reach out those who arent, she says. Its a paradigm for the challenges in our country right now.

With her increasing slate of talks, many of those unfamiliar opinions now seek her out. After a recent panel, a fellow speaker told her that her sister was born with a rare mutation that left her intellectually disabled and led to her dying in her 20s. I want you to know, the speaker said, that if it were possible to use gene-editing to get rid of that mutation permanently, I would have no hesitation. On the flipside, Doudna was recently interviewed by a journalist whose son has Downs syndrome. I want you to know, the journalist said, that I would never use CRISPR on him because hes perfect just the way he is.

Im very respectful of both those points of view, she tells me. And Ive learned a lot about myself in these last five years.

Much of the rhetoric around CRISPR is overblown. It is unlikely, for example, that CRISPR could ever be used to design babies to be smarter, taller, or free of conditions like obesity or schizophrenia, because such traits are the work of hundreds of genes, each with small effects. The threat of the technique can also be exaggerated in equal measure to its promise. One of Doudnas colleagues recently attended a meeting at the Department of Energy, and was asked by a member of the Trump administration: What about CRISPR? Thats dangerous. We need to get rid of it.

Well you cant, Doudna says plainly. Were in the system were in, and we have to deal with the technology in that context. Ive been encouraging an international discussion because the worst thing we could do is to ignore it, and for scientists not to get involved.

Read more here:

How CRISPR Yanked Jennifer Doudna Out of the Ivory Tower - The Atlantic

On the eve of publishing her new book, Jennifer Doudna, a pioneer in the field of CRISPR-Cas9 biology and genome engineering, spoke with Fast Company about the potential for this new technology to be used for good or evil.

The worst thing that could happen would be for [CRISPR] technology to be speeding ahead in laboratories, Doudna tells Fast Company. Meanwhile, people are unaware of the impact thats coming down the road. Thats why Doudna and her colleagues have been raising awareness of the following issues.

Related:CRISPR Pioneer Jennifer Doudna On Gene Editings Potential For Good And Evil

Editing sperm cells or eggsknown as germline manipulationwould introduce inheritable genetic changes at inception. This could be used to eliminate genetic diseases, but it could also be a way to ensure that your offspring have blue eyes, say, and a high IQ. As a result, several scientific organizations and the National Institutes of Health have called for a moratorium on such experimentation. But, writes Doudna, its almost certain that germline editing will eventually be safe enough to use in the clinic.

Using a CRISPR-related technique known as gene drive, bioengineers can encode DNA with a selected-for trait, which propagates to future generationsand across entire populationswith unnatural speed. This could give mosquitoes resistance to a parasite responsible for malaria or encode them with a gene for female sterilitythus breeding the pests themselves out of existence. But theres also the risk of spreading unwanted mutations and crossbreeding the change into another species. There could be real dangers to releasing organisms into the environment that are out of control at some level genetically, Doudna writes, where theres some trait thats being driven through a population before we understand what the implications of that really are.

Woolly mammoths roaming the earth once again? Its far from easy to do, but scientists are working on just such a Jurassic Park scenario. Ever since I first heard about experiments like these, Ive struggled to decide whether theyre admirable, deplorable, or something in between, writes Doudna. They could enhance our planets biodiversity, but bringing back certain species could also open the door to dangerous pathogens or upset ecosystems.

Since CRISPRs discovery, scientists around the world have been finding new ways to apply gene editing to plants and animals. Here are some of the developments Doudna tracks in A Crack in Creation.

Citrus fruit [Illustration: Alex J. Walker]1. Citrus Fruit:Researchers at South Carolinas Clemson University are employing CRISPR to create citrus trees that are resistant to a disease known as Huanglongbing, or citrus greening, which has devastated the countrys industry over the past decade.

Soybeans [Illustration: Alex J. Walker]2. Soybeans:Using a gene-editing tool called TALEN, Minneapolis-based Calyxt has developed soybeans with an overall fat profile similar to that of olive oil, Doudna writes. The company plans to launch commercial soybean oil next year.

3. Pigs:The University of Missouri has bred pigs that are resistant to porcine reproductive and respiratory syndrome. The virus costs U.S. pork producers more than $500 million annually, Doudna writes, and reduces production by 15%.

Goats. [Illustration: Alex J. Walker]4. Goats:Chinese scientists have applied CRISPR to suppress the gene that controls hair growth in Shanbei goats, prized for their cashmere wool. The enhanced goats produce a third more fur than their counterparts.

5. Monkeys:Researchers in China are harnessing CRISPR to create monkeys that mimic human conditions and diseases, from muscular dystrophy to cancer, which would allow scientists to hunt for disease cures without endangering human lives, Doudna writes.

Chickens. [Illustration: Alex J. Walker]6. Chickens:A team in Australia is exploring ways to rewrite the chicken genome to eliminate the proteins that cause egg allergies in humans. The new eggs could be used in foods and vaccines.

I'm the executive editor of Fast Company and Co.Design.

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The Ethics Of CRISPR - Fast Company

T

he ruckus over the CRISPR gene-editing system hides a dark reality: its high cost may make it unaffordable and questions remain whether most insurance companies will pay for it.

As CRISPR begins to move forward in clinical trials, there are some signals about how it may or may not be received commercially. Other types of gene therapies carry a price tag that is likely to induce sticker shock. If adopted, these therapies will add striking new cost burdens to our health care system.

The cost isnt coming down, said Mark Trusheim, director of the Massachusetts Institute of Technologys NEW Drug Development Paradigms, a think tank working on the problem of how we will pay for expensive new drugs. Companies will say, We are developing these medicines, just pay us; insurers will say, We cant afford it.'

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A few years ago, Dutch drug company uniQure set up a plant in Lexington, Mass., to make a gene therapy called Glybera, at the time the most expensive drug in the world. It used viruses to slip copies of a gene into human cells to restore an enzyme needed to break down fats. The cost? $1.4 million per patient. The company eventually abandoned its bid to bring Glybera to the U.S. and, after having sold it just once in Germany, recently withdrew it from European markets, rendering it a commercial failure.

Spark Therapeutics of Philadelphia is vying to bring the first gene therapy to market in the U.S. to treat a rare genetic eye disease called Leber congenital amaurosis 2. Analysts said it could cost a half-million dollars per eye. Like Glybera, Sparks treatment is a form of traditional gene therapy, which makes use of viruses to get bits of restorative code into our cells.

Do CRISPR enthusiasts have their head in the sand about the safety of gene editing?

CRISPR will allow us to alter our existing genes. But it often relies on using viruses to shuttle the molecular gene-editing systems into our cells, and can be as expensive as other gene therapies.

Editas Medicine plans to use CRISPR-Cas9 to treat various diseases, including Leber congenital amaurosis. Enthusiasm is great for interventions in the eye, New York University bioethicist Arthur Caplan told me. They permit trying one eye at a time and it is easy to tell if anything positive happens. Safety is much easier to ensure. But in its annual report, Editas noted significant uncertainty on whether payers would cover the treatment. In fact, a handful of insurance companies (VantageBlue from Blue Cross Blue Shield of Rhode Island, Select Health, and VIVA Health) have issued policy documents that exclude gene therapy from coverage, a move that experts say establishes policy against paying for CRISPR-based therapeutics.

The Institute for Clinical and Economic Review released a report in March stating there are 12 to 14 gene therapy candidates that are expected to be among the first for commercial approval. With payer budgets already stretched, and reining in the costs high on the agenda, both public and private payers will likely balk at the cost of some of these gene-based treatments, the American Journal of Managed Care wrote in a reflection on the report. Europe has the lead in approved gene therapies, and the first such drug to be approved had a launch price of $1.4 million. Can the US health care system absorb the cumulative impact of such prices, considering that 10 percent of the population has a rare condition linked to a genetic defect?

Five major gene therapy companies went publiclast year, suggesting that investors are ready to bet on the commercial prospects. Editas signed a deal with Juno Therapeutics that could be worth up to $737 million. The companies would combine CRISPR with other tactics to trick the immune systems T cells to fight cancer. Those tactics could include disabling genes in T cells that prevent cancer cells from shutting down a T cell response, and adding bits of genetic code to engineer new receptors into T cells to let them attach to abnormal proteins in cancer cells called neoantigens.

Gene and cell therapies that run into the six figures and beyond are poised to heighten the cost of cancer treatments, which not everyone may be able to afford. In fact, oncologist Dr. Siddhartha Mukherjee, author of the bestselling Emperor of All Maladies, gave a speech this month at the annual American Society of Clinical Oncology meeting that warned about dividing the world into the rich who can afford personalized cancer treatment and the poor who cannot.

Tania Bubela, a law and policy expert, and Chris McCabe, a health economist, both at the University of Alberta, will be holding a workshop in late June in Banff, Canada, to explore how to enable access to high-priced technologies. According to Bubela, gene-editing systems such as CRISPR-Cas9 promise to heighten the tension around health care policy. One idea for easing the tension is for regulators to permit drug makers to get reimbursed from insurers before their gene therapy gets FDA approval, while requiring drug makers to collect more data before charging full price a kind of price control.

Companies will charge whatever the market will bear, Bubela told me. Im not even sure that many of these gene therapies will work, and not all medicine is worth the price. But if these technologies become broadly used, especially in altering T cells for cancer, payers wont meet the demands of steep prices, and Bubela predicts that the system implodes under its own weight.

I believe that part of the problem lies in financial dealings. The Broad Institute, for instance, holds patents to gene editing tools such as CRISPR-Cas9 and CRISPR-Cpf1 and has issued exclusive licenses to Editas to use these tools for medical purposes. It could issue more-affordable CRISPR licenses one gene at a time, say directly to Juno Therapeutics, which now accesses them through its multimillion dollar deals with Editas. But that would cut Editas investors out of the loop. Such deals tend to inflate drug prices, since venture and public investors in Editas demand a cut on each CRISPR application. As investors engage in layers of transactional deals along the top of the food chain, the costs of gene therapies go up while the financiers may shift blame for a lack of patient coverage to insurance companies.

Dr. Stuart Orkin, a pediatric oncologist at Boston Childrens Center, and Dr. Philip Reilly, a partner at Third Rock Ventures, an Editas funder, coauthored a paper in Science magazine saying that sticker shock shouldnt halt commercialization. It can cost $300,000 a year to treat a single hemophilia patient with existing standard treatments and $25,000 to treat a single sickle-cell patient. Given costs like those, one-time gene therapy treatments running into the six figures may be comparatively affordable if an insurer makes payments to a drug-maker over a decade that are tied to the drugs continued performance. In fact, the idea of spreading payments over years as annuities originated with corporate-friendly FDA commissioner Scott Gottlieb in a 2014 paper he co-authored for the American Enterprise Institute.

Other performance-based models are being tested. GlaxoSmithKline, for example, is trying to bring a $665,000 gene therapy to the U.S. to treat an immune system disorder. The company said it will tie the cost of the drug to its performance in patients with a money-back guarantee. The reality is its very tough, and it doesnt come easy, said Jonathan Appleby, a chief scientific officer for the companys rare disease unit.

Broad Institute prevails in heated dispute over CRISPR patents

Orkin and Reilly also like the idea of using U.S. government funds from the Orphan Drug Act, established in 1983, to pay gene therapy companies for their commercial products. Another idea for keeping gene therapy, including CRISPR-based therapies, affordable is that investors could ask insurance companies to buy in bulk. MITs Ernst Berndt, inspired in part by volume purchases of vaccines in Africa, has proposed advanced market commitments through which insurance groups commit to buying a bunch of expensive drugs. That model that could be applied to gene therapies, but the insurers may not go for it without a bit more give.

In 2009, the Biologics Price Competition and Innovation Act created a pathway for approving generic biologics, also known as biosimilars. It may apply to CRISPR-based biosimilars, but generic gene-editing and thus competition to drive down prices is unlikely to appear for decades. Cathryn Donaldson, a spokesperson for Americans Health Insurance Plans, noted that a lack of generic forms of CRISPR means drug makers may charge whatever they want for their branded medication.

In 1968, Garrett Hardin argued in his now-classic essay, The Tragedy of the Commons, that a shared-resource system will tend to be depleted by self-interested individuals. He also argued against exponential growth to which we could add today the growth of biotech valuation.

Health care is a limited shared resource, and expensive new technologies could add pressures resulting in unequal access, especially to cancer therapies. Given the aggressive drive for money, and without new approaches in thinking, we are headed for disaster. One of two things will happen: either we will embrace a national health care system with broad access but that severely limits expensive new drugs, gene therapies, and CRISPR-based biologics; or these treatments will be available to only the wealthiest among us who can pay for them, a dystopian vision which is perverse but perhaps more realistic considering the pressures for a return on investment.

Writer Jim Kozubek is the author of Modern Prometheus: Editing the Human Genome with Crispr-Cas9, published by the Cambridge University Press.

Jim Kozubek can be reached at jimkozubek@gmail.com

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Who will pay for CRISPR? - STAT News - STAT

Bill Lundberg, CSO, CRISPR

CRISPR/Cas9 tech is still at a very early stage of development. But one of the top biotechs looking to make a breakthrough in the clinic got a chance today to explain why one of its preclinical studies helps demonstrate gene editings promise in developing a radically new kind of therapy.

The company is CRISPR Therapeutics $CRSP and its preclinical program focuses on a different kind of approach in treating sickle cell disease as well as beta-thalassemia two diseases triggered by a genetic mutation that slashes the natural production of hemoglobin.

Some people, though, have a genetic mutation that allows their bodies to continue to produce fetal hemoglobin. Its a benign condition that is typically only found by chance. But creating this condition in these patients is a potential cure, and CRISPR Therapeutics made it their showcase program.

Fetal hemoglobin can fully replace adult hemoglobin, CRISPR Therapeutics CSO Bill Lundberg tells me. There are some patients in whom that switch fails to turn off.

Using its gene editing tech, investigators took CD34-positive progenitor cells from patient samples and created this condition in a therapeutic batch. Their abstract presented at the EHA meeting in Madrid on Friday concludes:

Using CRISPR/Cas9 we successfully created gene edits that upregulate HbF in both healthy donor and patient samples. We have also dissected the genotype-phenotype relationship for specific genetic modifications, identifying the editing strategies which are most promising for re-expressing HbF. We have optimized the conditions for modifying HSPCs, including at clinical scale in a GMP-compliant setting, and demonstrated potential safety with no detectable off-target editing. These experiments support the further development of specific CRISPR/Cas9 editing strategies of HSPCs to treat SCD and -Thal patients.

One of the great things about this program, adds the CSO, is that we know what effects it should lead to. We would look for that, look for fetal hemoglobin two to three months in, after patients receive it.

Its worth emphasizing again that this is a preclinical study and all such early lab experiments can at best just set the stage for what has to be tested in humans for an extensive period before any biotech can take a drug to regulators.

That is a long and risky journey at every stage, with plenty of twists expected along the way. And there are several rivals in the field, including Editas $EDIT and Intellia $NTLA. But for CRISPR Therapeutics, it also represents another goal post on the crucial lead-up to the clinic as they start to visualize getting to an application 3 or 4 or 5 years down the road.

Birthing any new technology isnt easy. These kinds of potential revolutions never come cheap or easy, which is why its good for CRISPR Therapeutics to have $290 million in cash. But it will be studied at every step.

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CRISPR crew's lab test spotlights lead program in sickle cell disease, beta-thalassemia - Endpoints News

CRISPR-Powered Viruses

Earlier this month, the annualCRISPR 2017 conferencewas held at Montana State University. Attendees were the first to hear about successes companies have had using CRISPR to engineer viruses to kill bacteria. One of the most exciting potential application for these viruses, called bacteriophages, would be killingbacteria that have becomeresistant to antibiotics. At least two of the companies aim to start clinical trials of these engineered viruses within 18 to 24 months.

The use of bacteriophages isnt new. In the past, they have been isolated in the wild and purified for use.Although bacteriophages are regarded as being safe and effective for use in humans, because they are found in the wild, research on them has been sluggish. New discoveries cant be patented, and furthermore, these discoveries can also betransient, because bacteria can, and often do, rapidly evolve.

However, usingCRISPR to engineer them is definitely innovative. It renders viruses uniquely lethal to the most dangerous bacteria in the world, and initial tests saved the lives of mice who were infected withantibiotic-resistant infections that would have ultimately killed them, explained conference speaker Rodolphe Barrangou, chief scientific officer of Locus Biosciences.

This ability has lead researchers from at least two companies to useCRISPR in an attempt to turnthe tables on antibiotic-resistant bacteria. Both companies cite treating bacterial infections linked to serious diseases as their primary goal. Eventually, they intend to engineer viruses that would allow them to do much more by taking a precision approach to the human microbiome as a whole. The idea would be to selectively remove any bacteria that occur naturally andhave been associatedwith various health conditions. This could be anything from autism to obesity and possibly even some forms of cancer.

Onecompany, Locus, is using CRISPR to send DNA that will create modified guide RNAs tofind pieces of the antibiotic-resistance gene. After the virus infects the bacterium and the guide RNA connects with the resistance gene, the bacterium produces a phage-killing enzyme called Cas3. This is the bacteriums usual response, only in this instance,it destroys its own antibiotic-resisting genetic sequence. Over time Cas3 destroys all of the DNA, and the bacterium dies.

Another company, Eligo Bioscience, is taking a slightly different approach. The team chose to insertthe DNA that creates guide RNAs (this time with the bacterial enzyme Cas9), which removes all genetic replication instructions. Cas9 then severs the DNA of the bacterium at a specific place, and that cut triggers the self-destruct mechanism in the bacterium.

The third approach, by Synthetic Genomics,involves creating supercharged phages thatcontain dozens of enzymes. Each enzyme offers its own unique set of benefits, including the ability to camouflage the phages from the human immune system by breaking down proteins or biofilms.

Despite these promising results thus far, there will be challenges to bringing successfulengineered phages to market. For example, there is a risk that phages could actually spread genes for antibiotic-resistance to non-resistant bacteria. Another potential issue is that it might take a very large number of phages to treat an infection, which in turn could trigger immune reactions that would sabotage the treatment.

Ideally, though, if clinical trials go well, engineered phages could provide humans with a powerful weapon in the fight against superbugs.A fight that has, thus far, included a variety of strategies. Whenever it happens, it wouldnt be soon enough:this past January, the Centers for Disease Control (CDC) reported that a patient died from a superbug that was resistant to all 26 antibiotics available in the US.

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Scientists Modify Viruses with CRISPR to Kill Antibiotic-Resistant Bacteria - Futurism

We've seen how CRISPR/Cas9 can be used to tackle HIV and cancer, and now the revolutionary gene editing technique has Huntington's disease in its sights, as scientists have used it to reverse signs of the condition in mice.

That suggests CRISPR/Cas9 might one day be able to do the same in humans, so we can push Huntington's higher up the list of priorities for future research.

Huntington's disease is a fatal, inherited condition where brain cells die off due to a toxic protein released by a mutant version of the Huntingtin gene (mHTT). Symptom onset is typically in early middle age, making it a devastating illness at a time when victims are often parents of young children.

It's that mutant mHTT gene that CRISPR/Cas9 could fix, according to the researchers from Emory University.

The new approach could "efficiently and permanently eliminate" the poisoning of the brain that leads to Huntington's, the researchers write.

If you're completely new to CRISPR, or clustered regularly interspaced short palindromic repeats, it enables scientists to "cut and paste" DNA data with great accuracy. Cas9 refers to one particular way of using CRISPR that's currently being explored. Other recent research usesCas3 to attack antibiotic-resistant superbugs.

Potentially, bad genetic code responsible for diseases could be cut out, and healthy genetic code could be pasted in instead. We haven't got that far yet in humans, but it's a goal researchers are working towards.

With mice engineered to have the same mutant Huntington's-causing gene as humans, the scientists used CRISPR/Cas9 to snip out the gene and remove the flow of the toxic protein that eventually leads to problems with motor control and mood swings.

After three weeks, almost all traces of the damaging protein had disappeared.

What's more, the treated mice showed "significant improvements" in their motor control, balance, and grip strength, though they didn't get back up to the same levels of mobility and dexterity shown by the control mice used in the experiments.

That suggests the nerve cells were able to heal themselves to some extent after the troublesome gene had been snipped out by CRISPR/Cas9.

The team says the technique would not have to be customised to each patient's genome, making it easier to apply, although they do stress that a lot more testing and research is required before we'll be sure this is healthy for humans to try.

Clinical trials are already underway for gene-silencing drugs that would block off the protein that causes Huntington's, but the advantage of CRISPR/Cas9 is that it could provide a one-time fix for the disease, with no further treatment required.

"Given that CRISPR/Cas9 can permanently eliminate the expression of targeted genes, using CRISPR/Cas9 should more efficiently deplete the expression of mHTT than has been possible with previous therapeutic approaches, which require continuous administration," the team saysin its paper.

Neurodegenerative diseases in humans have not yet been tackled by CRISPR/Cas9, as the complexity and delicacy of the brain means any kind of tinkering could have catastrophic consequences.

Scientists want to be sure they're on the right track first, which is where this latest research can help, showing one approach that might be effective.

The research has been published in the Journal of Clinical Investigation.

Continue reading here:

CRISPR could point to a cure for Huntington's disease, suggests ... - ScienceAlert

Dive Brief:

The fields of synthetic lethality (a combination of DNA defects that work together to destroy a cancer cell) and DNA repair, fueled by gene editing technologies such as CRISPR/Cas9, have only been around a couple of decades but they are creating a solid foothold in basic research and are now moving into active development.

Repare Therapeutics is using its high-throughput, genomic synthetic lethal screening platform, which includes CRISPR/Cas9 genome editing, to find and exploit DNA damage repair (DDR) defects found across virtually all cancers.

"Versants commitment to and confidence in Repares distinct science has enabled the company to build the team, operations and initial programs away from the spotlight,"said Repare CEO Lloyd M. Segal. "With the added leadership of MPM and this syndicate, we are financed to achieve our goal of testing our multiple new, precision oncology therapeutics in a clinical setting."

CRISPR/Cas9 is becoming a busy field, with a number of companies and groups edging towards the clinic. The first in human study of CRISPR/Cas9 was approved by a Federal panel of the National Institutes of Health in mid-2016. The University of Pennsylvania study will target myeloma, melanoma and sarcoma. This was beaten to the clinic by Chinese scientists, who gave a patient with aggressive lung cancer cells edited using CRISPR/Cas9 in October 2016.

Editas Medicine expected to begin clinical trials by the end of 2017 for Leber congenital amaurosis type 10, a rare eye disorder, but these have now been delayed until mid-2018 because of issues with a third-party manufacturer. This is in development in collaboration with Allergan.

CRISPR Therapeutics expects to file a European clinical trial authorization for beta-thalassemia by the end of 2017, using CRISPR techniques to create variants that artificially induce hereditary persistence of fetal hemoglobin (HPFH), an asymptomatic and naturally-occurring condition that has been linked with better outcomes in people with beta-thalassemia and sickle cell disease.

Intellia Therapeutics, CRISPR Therapeutics and Editas Medicine all floated in 2016, though share prices for Intellia and Editas slumped towards the end of the year.

Continued here:

Celgene-backed CRISPR company emerges with $68M Series A - BioPharma Dive