GeCKO_NPC_mito - GFP_FNZ_CT + MITO_1_2

Cas9-NPCs cells were generated via lentivirus-mediated transduction of the Cas9 gene, followed by selection with 10 µg/ml blasticidin. Cas9-NPCs were further transduced with lentivirus particles containing a human sgRNA library at a MOI of 0.3 to achieve no greater than 1 sgRNA per cell, followed by selection with both 1 ug/ml puromycin and 10 μg/ml blasticidin for more than 9 days. After, 5 × 106 cells were collected for determining initial sgRNA abundance. Infected cells, expanded to 5 × 107 cells, were treated with FNZ for 3 days, followed by FACS sorting of GFP-positive cells. Genomic DNA was isolated from all samples, and the sgRNA sequences were amplified by PCR and sequenced on a HiSeq 2500.