CRISPR screen with FANCA–/– CRISPRi cells

In total, 1 × 106 cells were electroporated with 400 pmol SpCas9 nuclear localization sequence (NLS), 480 pmol L2 gRNA targeting BFP and 500 pmol BFP-to-GFP ssODN template using CM-189 and SF solution (Lonza, 4D electroporator) per replicate. Cells were further expanded in culture before sorting. 

RNP electroporation for BFP-to-GFP reporter assay and genomic loci targeting
RNP electroporation was performed as described (https://doi.org/ 10.17504/protocols.io.dm649d)9. In brief, 36 pmol sgRNA and 30 pmol SpCas9-NLS were mixed in Cas9 buffer (20 mM HEPES at pH 7.5, 150 mM KCl, 1 mM MgCl2, 10% glycerol and 1 mM tris (2-carboxyethyl) phosphine (TCEP) reducing agent). The mixture was incubated at room temperature for 20 min. Then, 1 × 10^5 to 2 × 10^5 cells were collected and spun down at 300g for 5 min. The cell pellets were resuspended in 15 µl of nucleofection buffer (Lonza). Then, 5 µl of RNP mixture was added to the cell suspension with 0.3 µl of 100 µM (30 pmol) ssODN (BFP-to-GFP template) template. Five days after electroporation, cells were collected and subjected to flow cytometry with an Attune Flow Cytometer (Thermo Fisher Scientific). Downstream analysis was performed using FlowJo version 10.8.2 software. Mouse cells were electroporated following the same protocol as other cells (see above) using the supplemented SE buffer provided by Lonza and using the CM-150 program. For endogenous locus targeting, 100 pmol SpCas9-NLS was mixed with 120 pmol gRNA in Cas9 buffer, and the mixture was incubated for 20–30 min at room temperature or 37 °C. In total, 1 × 105 to 2 × 10^5 cells were collected and resuspended in 15 µl of nucleofection buffer (Lonza). For each reaction, 100 pmol ssODN was then added before nucleofections. Electroporations were performed in the strip format, with 20-µl volume of cells and RNP mix. The following kit and program for each cell type was selected: K-562 (SF kit/FF-120), RPE-1 (P3 kit/EA-104), U2OS (SE kit/CM-130), MDA-MB-231 (SE kit/CM-130), HeLa (SE kit/CM-130) and Jurkat (SE kit/CL-120). After electroporation, pre-warmed 80 µl of DMEM or RPMI medium was added into strips. Cells were incubated in the hood for 10 min and then transferred to the plates and returned to 37 °C. 

For the primary cell nucleofection, the protocol described above was used with the following changes. For HSPCs, cells were electroporated with P3 kit, ER-100 or DS-130 program. Activated T cells were electroporated with P3 kit and CN-114 program, and iPSCs were electroporated with P3 kit and CB-150 program. After electroporation,
cells were returned to their corresponding media, as indicated above. 

Imaging of TREX1-GFP and H2B-iRFP during gene editing
In total, 700,000 RPE hTERT GFP-TREX1 H2B-iRFP cells were nucleofected with Cas9 RNP (final concentrations: sgNT (L2 sgRNA) or CXCR sgRNA 0.72 µM, Cas9 enzyme 20 µM) with or without ssODN templates (BFP ssODN or CXCR4 template ssODN, final 5 µM). The nucleofected cells were plated on poly-lysine-coated chambered coverslips (ibidi, 80807) and imaged 24 h after nucleofection.