2 Materials and methods
CD4+ T cells cultivation and editing: CD4+ T cells and PBMCs were isolated from leukocyte reduction system (LRS) chambers obtained from the Blood Donation Center at the Medical Center - University of Freiburg after informed consent. CD4+ T cells were isolated using CD4 MicroBeads (Miltenyi, Germany), cultivated in X-Vivo15 (Lonza, Switzerland) supplemented with 20 U/ml IL-2 (Miltenyi, Germany), and activated with the T Cell Activation/Expansion Kit (Miltenyi, Germany). After 3 days, beads were removed and cells reactivated every 7 days. Electroporation was performed combining 5x106 cells with 10 µg of each TALEN mRNA or 5 µg of chemically modified GFP mRNA [20]. Cells were electroporated in 100 µl electroporation buffer using P3 Primary Cell 4D-Nucleofector™ kit (Lonza, Switzerland) and program EO-115, or in 200µl of BTXpress Cytoporation medium T (BTX, Harvard Bioscience USA) (1 pulse 1200 V, 0.1 ms, 0.2 ms interval, 1 pulse 1200 V, 0.1 ms, 100 ms interval and 4 pulses 130 V, 0.2 ms, 2 ms intervals). After harvesting, genomic DNA was extracted with DNeasy Blood & Tissue Kits (Qiagen, Netherlands). NGS or T7E1 assay was performed and analyzed as previously described [21].
mRNA production : For in vitro RNA transcription, 10 µg of TALEN encoding plasmid DNA containing a T7 promoter and a poly(A) were linearized with HindIII-HF (NEB, Germany). The templates were purified using the QIAquick® Gel Extraction Kit (Qiagen, Netherlands) and RNA transcribed with mMESSAGE mMACHINE® T7 Ultra kit (Thermo Fisher Scientific, USA). The RNA pellets were resuspended in buffer EB (Qiagen, Netherlands) and stored at -80°C.
Lentiviral transduction : For production [22], HEK293T cells (ATCC) were grown in DMEM GlutaMAX I (Gibco, Life Technologies, USA) supplemented with 10% FCS (GE Healthcare, UK), 10.000 U/ml penicillin (GE Healthcare, UK), 10 mg/ml streptomycin (GE Healthcare, UK) and 100 mM sodium pyruvate (PAA Laboratories, Austria). 15x106cells were seeded per 15 cm plate 24 h before transfection. Prior to transfection medium was supplemented with chloroquine 1:1000 (Sigma-Aldrich, Germany) and polyethyleneimine (PEI) transfection performed with 15 µg pMDLg/pRRE (Addgene), 3 µg pRSV-Rev (Addgene), 15 µg of the lentivirus encoding plasmid, and 5.1 µg of the envelope encoding plasmid (Bal_gp120, kind gift of Dr. Boris Fehse, or pMD2.G, Addgene). After 12 h, transfection mix was exchanged with medium containing 10 mM sodium butyrate (Sigma-Aldrich, Germany). Supernatants were harvested 32 h and 56 h post-transfection, centrifuged at 20000xg at 4°C for 2 h on a 20% sucrose cushion using a swingout rotor. Pellets were resuspended in 50 µl of PBS and stored at -80°C. Lentiviral EGFP vectors were titrated on PM1 cells (NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH [23]) that were cultivated in RPMI 1640 medium containing L-Glutamine (Gibco Life Technologies, USA), 10% FCS (GE Healthcare, UK), 10.000 U/ml penicillin (GE Healthcare, UK) and 10 mg/ml streptomycin (GE Healthcare). 5x104 PM1 cells in medium supplemented with 4 µg/ml polybrene were transduced with serial dilutions of the vectors by spinoculation for 1 h at 200xg at 32°C. For transduction, 5x104 CD4+ T cells were cultured in X-Vivo15 medium (Lonza, Switzerland) containing 4 μg/ml polybrene with 6 transducing units/cell by spinoculation for 1 h at 200xg at 32°C.
Flow cytometry : GFP expression after lentiviral transduction and/or cell viability (7AAD, AppliChem, Germany) were assessed using BD Accuri C6 Flow Cytometer (BD Biosciences, USA). CCR5 expression was detected by labelling 1x105 cells in 50 µl of PBS with 2µl of APC-labelled mouse anti-human CD195 antibody (BD Biosciences, USA) for 20 min at room temperature. Cells were analyzed on BD FACS Canto-II (BD Biosciences, USA).
HIV-1 infection: Full-length HIV-1 provirus encoding plasmids were obtained from NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH [24, 25]. Virus stocks HIV-1JR-FL (CCR5-tropic) and HIV-1NL4-3(CXCR4-tropic) were generated and titrated as previously described [26]. For infection cells were activated as described above, and 2x105 CD4+ T cells were infected with either HIV-1JR-FL or HIV-1NL4-3 at MOIs of 0.01 and 0.001. At indicated time points 50 µl of cell culture supernatant were harvested and used to determine the amount of p24 by ELISA [27].
Cytokine release : 200 µl of supernatants were harvested from 1x106 cells per sample at indicated timepoints. Cytokine concentrations were determined using Cytometric Bead Array plus beads and standards for each analyte: IFN-γ, IL-2 or TNF-α (BD Biosciences, USA). Cells were stimulated with CD3/CD28/CD2 beads (Miltenyi Biotec, Germany), 1 µg/ml ionomycin (Merck Millipore, Germany) or 10 ng/ml PMA (Sigma-Aldrich, Germany.
Off-target analyses : Potential off-target sites were predicted with PROGNOS web tool (http://bao.rice.edu/cgi-bin/prognos/prognos.cgi) using the TALEN v2.0 algorithm [28]. Five mismatches per half sites were allowed. Loci were PCR amplified using the primers listed in Table S1. Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (NEB, Germany) and quantified using ddPCR Library Quantification Kit for Illumina TruSeq (Biorad, Germany). Samples were sequenced on an Illumina MiSeq platform using MiSeq Reagent Kit v2, 500-cycles (Illumina, USA), and data analyzed with Crispresso2 to establish significance and indel types [29] . p values were adjusted with the Benjamini & Hochberg [30] method. For oligonucleotide capture assay (OCA), human PBMCs (ALLCELLS, USA) were plated at a density of 1x106 cells/ml in X-vivo-15 media (Lonza, Switzerland) supplemented with 5% human AB serum (Gemini, USA) and 20 ng/ml of IL-2 (Miltenyi, Germany). The next day, PBMCs were activated using human T activator CD3/CD28 (Thermo Fisher Scientific, USA). Four days later, 5x106 T cells in cytoporation medium T were electroporated with 20 µg of TALEN mRNA (10 µg each of left and right subunit) and 10 µl of pre-annealed oligodeoxynucleotides (dsODN, 100 µM) using an AgilePulse MAX system (Harvard Apparatus, USA) and a 0.4 cm cuvette. Genomic DNA was extracted 3 days later, randomly sheared to 300 bp fragments by sonication (Covaris LE220-plus), fragments end-repaired/A-tailed (NEBNext® Ultra™ End Repair/dA-Tailing Module, USA), and next-generation sequencing Y-adapters (TruSeq Annealed Adapter) were added. Two rounds of anchored PCR using dsODN-specific and adapter-specific primers were performed. Adapter-specific (P5_1) and dsODN-specific primers were used in the first PCR. Adapter-specific (P5_2) primers, dsODN-specific primers P7 and primers adding the barcode and P7 sequence to the ends of the PCR product were used in the second PCR. PCR products were pooled and sequenced using Illumina NextSeq (2×150 bp). The resulting sequences reads were mapped to the human genome to identify potential off-target sites.
Statistical analyses : A time series analysis [31] was performed to compare the samples in both the HIV-1 challenge and growth curves experiments. Briefly, two cubic regression models were fitted to each growth curve. The full-model captures specific time course for each separate treatment whereas the reduced-model specifies a single set of parameters for two tested treatments. Finally, the goodness of the fit between the full and the reduced models was tested by Anova whereP -value indicates the probability that the two models are the same. P -values are indicated with * ≤0,05; ** ≤0,001; ***≤0,0001. For all other analyses, the Student’s T-test was applied.