CRISPR/Cas9 cloning, lentiviral production, and infection

Single-guide RNAs (sgRNAs) (sequences in Table S1) were selected by inputting gene sequences into the Broad Institute GPP sgRNA CRISPR KO Designer. Two high-score sgRNAs were selected for each gene. sgRNA oligos were cloned into the lentiCRISPR V2 (Addgene) lentiviral plasmid according to depositor instructions.

Lentivirus was prepared in HEK293T cells. Cells were transfected at 50%–60% confluency with lentiCRISPR V2, psPAX2 (lentiviral packaging), and pMD2.G (lentiviral envelope) (Addgene) using polyjet (SignaGen). 72 h post-transfection the supernatant was collected, mixed with 8 μg/ml of polybrene and 1% PEG, and added to SH-SY5Y, 786-O, or HEK293T cells that were spinfected at 1,000 rpm for 1 h at room temperature. Cells were selected for 2 weeks in media containing either 300 μg/ml hygromycin B (Invitrogen; rtTA doxycycline-inducible element), 5 μg/ml blasticidin (Invivogen; inducible RIG-I), or 5 μg/ml puromycin (Sigma; lenti-CRISPR V2 knockouts).