CRISPR Fitness Screening in Cancer

Cas9-containing cells were infected in two biological replicates with the Avana and GeCKO libraries in lentiGuide; unmodified, parental cells were infected in two biological replicates with the Avana library in lentiCRISPRv2. Small molecules were added to puromycin-selected cells 7 days post-infection. Cells either received a media change or were passaged every two or three days over the course of the screen in complete media supplemented with 1% penicillin/streptomycin. Vemurafenib (PLX-4032, Selleckchem, S1267) was screened at a final concentration of 2 μM. Selumetinib (AZD-6244, Selleckchem, S1008) was screened at a final concentration of 200 nM. Surviving cells were harvested after 14 days of small molecule treatment. For analysis, the log₂-fold-change of each sgRNA was determined relative to the starting plasmid DNA (pDNA) pool for each biological replicate, which we have previously validated as representative of an early time point in pooled screens^{6 (link)}.