Pooled CRISPR Screening in Leukemia Cells

The GeCKO library in the lentiCRISPRv2 backbone, amplified as previously described [19 (link)], was a gift from Feng Zhang (Addgene plasmid #52961) [20 (link)]. Triplicate cultures of 2.56 × 10⁸ 697 cells were transduced with the packaged library at a MOI of 0.3. Genomic DNA extracted from 3.8 × 10⁷ cells (300-fold library coverage), immediately after puromycin selection (day 0), and weeks 2, 4, 6 and 8, was amplified with custom barcoded primers (Table S11) and sequenced using the Illumina NextSeq 550 platform (Genomics Core Facility, Newcastle University). Reads were aligned to the GeCKO reference sequence, quantified, normalised to non-target reference sequences and analysed statistically using MAGeCK [21 (link)] (version 0.5.7). The α-RRA method with 1000 rounds of permutation testing was implemented without removing zero-count sgRNA sequences, sgRNA variance was calculated using all samples, and results were normalised according to copy number status. Target genes were annotated using GRCh37 (hg19).