Cell culture

MT4CCR5 cells are a human CD4+ T cell-line modified to stably express CCR5 co-receptor after being transduced with a lentiviral vector expressing human CCR5 under the control of SFFV promoter. These cells are susceptible to both R5-and X4-tropic HIV-1. The MT4CCR5 cells were kindly provided by Dr. Koki Morizono (UCLA, Los Angeles). These cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 2mM L-glutamine, 100 units penicillin, and 100g/ml streptomycin (Gibco). The cell cultures were maintained in a humidified incubator at 37C and 5% CO2. HEK293T cell line (Clontech) was used for viral production and lentiviral titer. The cells were cultured in Dulbeccos Modification of Eagles Medium (Hyclone) with the same supplements as MT4CCR5 cells.

The third-generation lentiviral vector, pLVX-AcGFP1-N1 (Clontech, Cat# 632154), was used as the backbone vector to transfer the C46 HIV-1 fusion inhibitor gene into the target cells. The C46 C-peptide fusion inhibitor sequence obtained from the publication of Felix G. Hermann et al., 200950 were gene synthesized and cloned into pUC57 plasmid (Integrated DNA Technologies, Inc). The C46 HIV-1 fusion inhibitor sequence was designed to flank XhoI and EcoRI restriction sites to clone in the lentiviral vector, resulting pLVX-C46-AcGFP1. The pLVX-C46 plasmid was cloned by amplifying C46 HIV-1 fusion inhibitor gene without the expression of AcGFP1 from the synthesized pUC57 plasmid with the forward primer having the XhoI restriction site and the reverse primer adding a stop codon before the EcoRI restriction site. All construct lentiviral plasmids were confirmed to contain the C46 HIV-1 fusion inhibitor by restriction enzyme digestion and DNA sequencing.

VSVG-pseudotyped lentiviral vector particles were produced in HEK293T cells, using four separate plasmids and the calcium phosphate transfection method, as previously described51,52,53. HEK293T cells were seeded on 10-cm cell culture dishes, and co-transfected with the construct lentiviral vector with the third-generation packaging plasmid system (Addgene plasmid #12251 (pMDLg/pRRE), #12253 (pRSVREV), #12259 (pMD2.G). Vector particles were harvested from the culture supernatant collected at 24 and 48h. The viral vector titers, determined by infection of HEK293T cells with serial dilution of the samples, were expressed as the percentage of AcGFP1+positive cells evaluated by flow cytometry.

MT4CCR5 cells and MT4CCR5 CRISPR/Cas9 knockout CCR5 cells (1106) were transduced with lentiviral vectors at a multiplicity of infection (MOI) of 0.1 with 8g/ml polybrene for 24h. The transduced cells were seeded in a 6-well plate and incubated at 37C for 3days before determining the percentage of transduced cells by flow cytometry. The transduced cells were enriched for those containing the lentiviral vectors by treating them with 1g/ml puromycin (SigmaAldrich) in culture medium and refreshing the medium every 34days.

3xNLS-SpCas9 plasmid (Addgene plasmid#114365), was transformed into ClearColi BL21(DE3) E.coli cells for protein expression. The recombinant protein was expressed and purified as previously published54. In detail, the cells were cultured in LuriaBertani medium supplemented with 100g/ml of ampicillin at 37C until the cell density reached 0.6 at OD600 nm. Subsequently, protein expression was induced by adding 0.4mM isopropyl--D-thiogalactopyranoside (IPTG), and the culture was maintained at 18C in a shaking incubator for 24h. After induction, cells were centrifuged, and the cell pellets were stored at20C. To purify the Cas9 protein, the cell pellets were lysed in a buffer containing 20mM TrisHCl pH 7.5, 200mM NaCl, 0.5mg/ml lysozyme, and 1mM DTT by incubating on ice for 10min. The cells were then physically lysed by sonication and centrifuged at 15,000rpm for 1h at 4C. The supernatant was applied onto the first 1ml of HisTrap FF prepacked column (Cytiva). After extensive washing with binding buffer (20mM TrisHCl pH 7.5, 200mM NaCl) and binding buffer containing 20mM Imidazole for 5ml, the partially purified Cas9 protein was eluted with a buffer containing 200mM imidazole. For the second purification step, hydrophobic interaction chromatography (HIC) column (phenyl sepharose HP) was utilized. The HIC column was pre-equilibrated with 20mM TrisHCl pH 7.5, 200mM NaCl, and 2M NaCl buffer. The elution sample from the first column was also supplemented with salt up to 2M to promote protein binding. After applying the sample onto the column, Cas9 protein was eluted with a buffer containing 20mM TrisHCl pH 7.5, 200mM NaCl, and 1M NaCl. Finally, the Cas9 protein was further purified using gel filtration chromatography (superdex 200 increase 10/300 GL) as a third purification step. The purity of the final protein sample was evaluated by SDS-PAGE. The protein concentration was determined by the Bradford method using BSA as protein standard.

The sgRNA1# sequences, 5-ACTATGCTGCCGCCCAGT-3; the sgRNA2# sequence 5-CAGAAGGGGACAGTAAG-314. The sgRNAs targeting to CCR5 gene were synthesized with chemically modified nucleotides at the terminal positions at both the 5 and 3 ends were purchased from Integrated DNA Technologies, Inc. Ribonucleoprotein (RNP) complex was made by incubating 6g (36.81pmol) or 10g (61.35pmol) of the in-house purified Cas9 protein, 2g (61.86pmol) or 4g (123.72pmol) of sgRNA#1 or sgRNA#2 at room temperature for 20min. Resuspended 1106 cells in 20l of SF cell line nucleofector solution, mixed with the RNP complex, and transferred into the Nucleocuvette (Lonza), program DC100. The nucleotransfection protocol was followed according to the manufacturers recommendations (Amaxa 4D-Nucleofector, Lonza).

Cells were stained with monoclonal antibodies against human CCR5 (2D7: PECy7 labeled, eBioscience, Cat#557752), human CXCR4 (12G5: APC labeled, Biolegend, Cat#306510), human CD4 (OKT4: PE labeled, Biolegend, Cat#317410), according to the manufacturers instructions. C46 HIV-1 fusion inhibitor expression was measured by staining with 0.2g of anti-HIV-1 gp41 Monoclonal (2F5) (NIH AIDS Reagent Program, Cat#1475) followed by 50l of 1:500 of the secondary antibody (PE-anti-human Fc, Jackson Immunoresearch, cat#109-115-098). Isotype control antibodies were included with mouse IgG2b control-PE (ImmunoTools, Cat#21275534), mouse IgG1 control-APC (ImmunoTool, Cat#2185016), and mouse IgG1 control-PECy7 (Biolegand, Cat#400126). CCR5, CXCR4, CD4, C46 HIV-1 fusion inhibitor, and AcGFP1 expression were measured by flow cytometry using the CytoFLEX Flow Cytometer (Beckman Coulter). A live cell population defined by 7AAD- staining (Biolegend, cat#420404) was subjected to single-cell population analysis for each antibody staining. Additionally, a positive control for dead cells was established by exposing MT4CCR5 cells to heat at 56C for 30min before staining and determined by the 7AAD+ population. Data analysis for gene expression was performed with FlowJo version 10 software (BD Biosciences).

The genomic DNA was isolated from cells using Invitrogen Purelink Genomic DNA Mini Kit (Invitrogen, cat#K182001) according to the manufacturers instructions. 25ng of genomic DNA per reaction was validated with real-time PCR reactions set up in duplicate in 25l reaction volumes using Agilent Brilliant II master mix (Invitrogen) in a 96-well plate on CFX Connect Thermal Cycler platform (Bio-Rad). Taqman primer and probe sequences for this assay were as follows: C46 probe: 5 6-FAM/CA CTC CAC G/ZEN/C AGC ACT TCC GCT CG/IABkFQ 3, C46 forward primer: 5 CAC AGC CTG ATC GAG GAG AG 3, C46 reverse primer: 5 GTC CTG CCA CTG GTG GTG 3, -globin probe: 5 HEX/CT CCT GAG GAG AAG TCT GCC GTT ACT GCC /BHQ-2 3, -Globin forward primer: 5 CAA CCT CAA ACA GAC ACC ATG G 3, -Globin reverse primer: 5 TCC ACG TTC ACC TTG CCC 3. Thermocycling conditions were 50C 2min, 95C 10min, 40(95C 15s; 60C 1min)18.

Cells were washed with ice-cold PBS and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo-Fisher Scientific). The total protein concentration in the cell lysates was measured using the bicinchoninic acid assay (Thermo-Fisher Scientific). Subsequently, 30g of total protein was separated by SDS-PAGE and transferred onto a PVDF membrane (Thermo-Fisher Scientific). Blocking was carried out using 5% skim milk (Sigma) in 0.05% PBST for 24h, followed by overnight incubation with the primary antibody (anti-CCR5 antibody; CUSABIO TECHNOLOGY, cat#CSB-PA006994). After washing the blots five times with 0.05% PBST, they were probed with the secondary antibody (Goat pAb to Rb IgG (HRP); AbCam, cat#ab205718) for 1h and subsequently detected using ECL Western Blotting Substrate (Bio-Rad, cat#1705060) and scanned using the Odyssey InfraRed Imaging System (LI-COR BioSciences, Lincoln, NE). The blots were then stripped with mild stripping buffer (Thermo-Fisher Scientific), re-blocked, and re-probed with anti--Actin antibody (Abcam, cat#ab8227).

Genomic DNA was extracted from cells using Invitrogen Purelink Genomic DNA Mini Kit (Invitrogen, cat#K182001) according to the manufacturers instructions. The target sequence was amplified using the Phusion high-fidelity DNA polymerase (Thermo Scientific, cat#F-530XL) and XhoI-CCR5 forward primers 5-TGGACAGGGAAGCTAGCAGCAAA-3 and EcoRI-CCR5 reverse primer 5-TCACCACCCCAAAGG TGACCG-3. The PCR products were annealed after purification. Next, 2l of hybridized DNA were subjected to digestion with 0.5L T7EI (New England Biolabs) in NE Buffer 2 for 30min at 37C. Subsequently, the samples were loaded onto a 2% agarose gel electrophoresis with an equal amount of PCR product controls from non-edited samples.

R5-tropic HIV-1BaL virus and X4-tropic HIV-1NL4-3 virus were produced by transient transfection of pWT/BaL plasmid (NIH AIDS Reagent Program, cat#11414) or pNL4-3 plasmid (NIH AIDS Reagent Program, cat#114) into HEK293T cells. Monolayers of HEK293T cells (5106 cells per 10-cm dishes) were transfected with 10g of each plasmid using a calcium phosphate transfection method51,52,53. After 8h, the transfection mixture was withdrawn, replaced by 10ml of 10% FBS in DMEM medium. The transfected cells were incubated for 24h. HIV-1 viruses were harvested from the culture supernatants and filtered through sterile syringe filters with a 0.45-m pore size (Merck Millipore). HIV-1 samples were aliquoted and kept at80C. The virus titer was determined for the HIV viral load using the COBAS AMPLICOR HIV-1 Monitortest (version 1.5; Roche Molecular Systems, Branchburg, NJ).

The 1106 cells were incubated with HIV-1 at MOI of 1 and 10 for 16h. The cells were then washed three times with serum-free medium and resuspended in fresh growth medium. The infected cells were split into half at 3-day intervals, to maintain a cell density of approximately 106cells/ml. HIV-1 replication was monitored in culture supernatants, using HIV-1 p24 Simple Step ELISA kit (Abcam, cat#ab218268) and viral load assay, as described above. The cell pellets were kept determining cell viability by 7AAD staining (Biolegend, cat#420404) and flow cytometry.

Data was obtained from triplicate experiments (n=3). The data were analyzed, and standard deviations (SD) were calculated using the Prism 7 (GraphPad) statistical software program. Unpaired t-tests with Welchs correction were used to calculate p values, and p<0.05 was considered statistically significant.

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CRISPR/Cas9 genome editing of CCR5 combined with C46 HIV-1 fusion inhibitor for cellular resistant to R5 and X4 ... - Nature.com

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