The Hap1 cell line (8 (link)) was kindly provided by Dr. Thijn Brummelkamp, Netherlands Cancer Institute. The 293FT cell line used to generate high-titer lentiviruses was obtained from ThermoFisher Scientific; the A549, PC3, MDA-MB-361, HCC-1954 and NCI-H1650 cell lines were purchased from ATCC, where they were validated by STR profiling, and used at passage numbers <5. The KOPN8 cell line was a generous gift from Professor Michael Cleary, Stanford University. The Phoenix-Ampho cell line used for retrovirus production was purchased from Allele Biotechnology (San Diego, CA).
Null alleles for genes were constructed using the CRISPR/Cas9 system. For Hap1 cells the oligos encoding the guide RNAs were cloned into pSpCas9(BB)-2A-GFP (PX458, Addgene Plasmid #48138 from Dr. Feng Zhang) (9 (link)). Single cells were sorted using flow cytometry, expanded and clones bearing null alleles were identified by Sanger sequencing and immunoblotting. For gene disruption in cancer cell lines, the gRNAs validated in the Hap1 cells were introduced into LentiCRISPR v2 (Addgene Plasmid #52961 from Dr. Feng Zhang) (10 (link)) for lentiviral-mediated delivery. The oligo sequences for guide RNAs are provided in thesupplementary methods .
Null alleles for genes were constructed using the CRISPR/Cas9 system. For Hap1 cells the oligos encoding the guide RNAs were cloned into pSpCas9(BB)-2A-GFP (PX458, Addgene Plasmid #48138 from Dr. Feng Zhang) (9 (link)). Single cells were sorted using flow cytometry, expanded and clones bearing null alleles were identified by Sanger sequencing and immunoblotting. For gene disruption in cancer cell lines, the gRNAs validated in the Hap1 cells were introduced into LentiCRISPR v2 (Addgene Plasmid #52961 from Dr. Feng Zhang) (10 (link)) for lentiviral-mediated delivery. The oligo sequences for guide RNAs are provided in the
