CRISPR/Cas9 stable knockout cell lines

HEK-293T cells were transfected with the 3 plasmid lentiCRISPR v2 system. In 12-well plates, 4 µl of lipofectamine 2000 (Invitrogen^TM) was combined with 1 µg lentiCRISPR V2 plasmid expressing the guide RNA (gRNA) targeting the protein of interest, 0.65 µg of pVSV.G and 0.65 µg psPAX2. Two days post transfection the viral supernatant was harvested and filtered (0.45 µm pore, Merck Millipore) before transduction of TREx BCBL1-Rta cells in the presence of 8 µg/mL of polybrene (Merck Millipore). Virus supernatant was removed 6 h post transduction and fresh media added. Cells were maintained for 48 h before 2 µg/µl puromycin (Sigma-Aldrich) selection. Stable mixed population cell lines were maintained until confluency before single cell selection. Single cell populations were generated through serial dilution of ~50 cells distributed across a 96 well plate. Successfully cultured wells were maintained for 3–5 weeks with regular exchange of fresh media before transferal into 6 well plates. Upon confluency, single cell clones were harvested and DNA extracted using Monarch Genomic DNA Purification Kit (New England Biolabs) as described in manufacturer’s protocol. Extracted genomic DNA was subsequently sequenced (Source Bioscience, Sanger sequencing service) with ORF11 site specific primers. Editing efficacy and indels generated were assessed using TIDE (http://shinyapps.datacurators.nl/tide)⁵².