Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clones

hPSCs culture

Wild-type hiPSCs PC056c2 were derived by Phenocell (Grasse, France) from human primary fibroblasts using Sendai vectors [2] and cultured with StemMACS iPS-Brew XF medium (Miltenyi Biotec) in vitronectin (Gibco-coated dishes). Culture medium was changed three times a week. Cells were passaged following gentle harvesting with EDTA 0.25 mM (Invitrogen every 5–7 days). For CRISPR/Cas9 editing experiments, hiPSCs were pretreated with a rock inhibitor (Y-27632; Tocris) at least one-hour prior transfection and harvested with an enzymatic treatment (Stempro Accutase cell dissociation reagent; Life technologies). Following amplification of single cell-derived clones, cells were banked using CryoStor CS10 (Sigma-Aldrich).

CRISPR/Cas9 gene editing

For p.Glu192fs mutation, guide RNA (gRNA) was selected based on CRISPOR.org predictions [8]. Twenty possible gRNAs were proposed of which 18 gRNAs with a required MIT specificity score superior to 50. Of these 18 gRNAs, we selected one with both the higher Lindel score (prediction probability of frameshift) and the higher Doench’16 score (predicted cleavage efficiency). One hundred thirty-seven off-targets were predicted with up to four mismatches with the original guide sequence. Of these, the top 15 off-targets were not localized into an exon. Integrated DNA Technologies (Iowa, USA) synthesized selected gRNA. hiPSCs were plated in 24-well plates at 80–120,000 cells per well. Lipid-based transfection was performed the next day with 15 µM of ribonucleoprotein (RNP) complexes (gRNA, SpCas9 protein), according to the manufacturer’s protocol (Lipofectamine Stem Transfection Reagent, ThermoFisher Scientific). SpCas9 purified protein is a gift from Jean-Paul Concordet (MNHN-CNRS UMR 7196/INSERM U1154).

After enzymatic dissociation with Stempro Accutase (Life technologies), 150,000 hiPSCs in 7.5 µL of resuspension buffer (Neon Transfection Kit, Invitrogen) were mixed with 7.5 µL of RNP complexes. The mix was then electroporated at 1200 V/20 ms/2 pulses using 10 µL Neon Tips. For limiting dilution cloning experiments, the cell suspension was plated in several 100-mm culture dishes at different concentrations (1000/2000/5000/10,000 cells/dish). For semi-automated cell picking, cells were seeded in 6-well plates or directly seeded following electroporation in a vitronectin-coated nanowell plate at a density of 3000 cells per well.

For the base editing strategy, different pathogenic nonsense mutations were identified within ALMS1 using the ClinVar database (NCBI). Among the identified mutations, we selected the p.Gln3613Ter mutation as it consists in a C → T nonsense mutation. A gRNA targeting this position was then designed using CRISPR RGEN Tools and synthesized by Integrated DNA Technologies (Iowa, USA).

After enzymatic dissociation with Stempro Accutase (Life technologies), 150,000 hiPSCs in 10 µL of resuspension buffer (Neon Transfection Kit, Invitrogen) were mixed with 10 µL of RNA AncBE4max (pCMV_AncBE4max_P2A_GFP was a gift from David Liu (Addgene plasmid # 112100; http://n2t.net/addgene:112100; RRID: Addgene_112100)) and 2.5 pmol gRNA. The mix was then electroporated using the same protocol. For semi-automated cell picking, cells were seeded in a vitronectin-coated nanowell plate at a density of 3,000 cells per well.

Sequence analysis

Genomic DNA was extracted with QuickExtract DNA extraction solution (Lucigen) according to the manufacturer’s instructions. PCR amplification corresponding to the edited DNA sequence (p.Glu192fs: forward primer: TATACACTGGCAGCGGCG; reverse primer: TCTGTAGAGACCTACTCAGAGGG; p.Gln3613Ter: forward primer: CCCGTACACCTACCAAGTGA; reverse primer: CATCATCGAGCTGGGAGGTC) was subjected to Sanger sequencing (Genewiz, Germany). To evaluate edition rates, decomposition of quantitative sequence trace data was based on TIDE [4]. Sequence analysis was performed using Snapgene software (Insightful Science).

CellCelector clonal isolation

24-well nanowell plates (ALS, Germany) were coated with vitronectin (Gibco) and centrifugated for 5 min at 1258 g (to eliminate micro-bubbles) prior to incubation at 37 °C for 1 h. Approximately 3000 cells were seeded per well (4300 square-shaped nanowells in total in a well of a 24-well plate). Seeded cells are randomly distributed in nanowells following Poisson distribution resulting in ~ 30% of nanowells occupied with single cells.

After cell seeding, plates were centrifuged again for 3 min at 100 g to allow all cells to be captured in nanowells. Nanowells with seeded cells were automatically scanned on the CellCelector system (ALS, Germany) on D0 just after cell seeding. Identification of single cell nanowells on D0 was performed using a specific image analysis algorithm using CellCelector software (v3.1) including gating with single cell size and sphericity. At time of picking on D3-5, the nanowell plate was scanned again and the system automatically selected single cell-derived colonies based on their outgrowth. Selected single cell-derived colonies were detached with Stempro Accutase (Life technologies) just prior to picking run. Single-use CellCelector glass capillaries (ALS, Germany) with internal diameter of 80 µm with aspiration volume of 0.5 µL were used for picking. The tip of the capillary was calibrated at the height of approximately 80 µm from the bottom of nanowells. Colonies were deposited in a vitronectin-coated 96-well plate with 200 µL of StemMACS iPS-Brew XF medium (Miltenyi Biotec) containing rock inhibitor. Cell growth was followed using INCUCYTE S3 (Sartorius).

Flow cytometry

Cells were dissociated with TrypLE (ThermoFisher) and stained with TRA1-81-AF647 (Biolegend) or SSEA-4-PE (Miltenyi Biotec) and corresponding isotypes from the same suppliers. For intracellular markers, cells were fixed with 4% PFA following dissociation. Cells were treated with PBS 0.1% tritonX100 and then incubated overnight with OCT3/4 (Santa Cruz, sc-5279, 1:100) and LIN28A (R&D SYSTEMS, AF3757, 1:200). The day after, cells were washed and incubated with corresponding secondary antibodies (Invitrogen). Acquisitions were done with a MACSQuant analyzer (Miltenyi Biotec) and data were analyzed using the FlowJo software.

Immunofluorescence

After fixation with 4% PFA for 15 min at room temperature, cells were incubated overnight at 4 °C with the following primary antibodies: OCT3/4 (Santa Cruz, sc-5279, 1:100), LIN28A (R&D SYSTEMS, AF3757, 1:200), Pericentrin (Abcam, ab28144, 1:200) and ALMS1 (Abcam, ab84892, 1:200). Appropriate Alexa fluorescent secondary antibodies (Invitrogen) and DAPI were added for 1 h. Image acquisitions were performed on a Zeiss LSM880-Airyscan Confocal Microscope driven by the Zeiss Zen black software.

Single-nucleotide polymorphism (SNP) analysis

High-quality genomic DNA was obtained with Nucleospin Tissue kit (Macherey Nagel) according to manufacturer instruction. gDNA hybridization was achieved on Infinium Core-24v1-2 BeadChip (Illumina). Data were analyzed with GenomeStudio v2.0.5 software (Illumina).

Multiplex fluorescence in situ hybridization (mFISH) karyotype analysis

Cells were blocked in metaphase with colchicine (Eurobio) for 90 min and then they were detached with trypsin. After centrifugation, the supernatant was removed and cell pellet was suspended and warmed in a hypotonic solution for several minutes and then fixed with a Carnoy fixative. Drops of the cell suspension were spread on glass slides and let to dry. mFISH 24Xcite probe (MetaSystems) and ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) were used for mFISH staining. Seventy metaphases were acquired with Metafer MetaSystems software coupled to an AxioImager Zeiss Z2 microscope furnished with a camera cool cube and 10X and 63X objectives. Images were analyzed with Isis software (MetaSystems).

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