Genome-Wide CRISPR Screening in PIGS-HRD1-DKO Cells

Approximately, 5 × 10⁷ unsorted and 2 × 10⁷ twice-sorted PIGS-HRD1-DKO cells were extracted for genomic DNA using a Wizard Genomic DNA Purification Kit (Promega). The gRNAs were amplified from genomic DNA of unsorted and twice-sorted cells. PCR (25 cycles) was performed to amplify the gRNAs using KOD FX Neo Polymerase (TOYOBO LIFE SCIENCE), making up a total of 65 tubes for unsorted cells and 12 tubes for twice-sorted cells (oligos for amplification of gRNAs are shown in Data S1). PCR products for unsorted cells (1,050 μl) and for twice-sorted cells (250 μl) were applied to a 2% agarose gel for purification. The PCR products were concentrated, mixed (unsorted to sorted 4.2:1), and analyzed by paired-end sequencing with a NovaSeq 6000 system (Illumina). Deep sequencing raw data were processed for gRNA counting using Python scripts. The high-throughput sequencing reads were demultiplexed using the 5-bp adapter by cutadapt version v1.18 (Martin, 2011 (link)). By using MAGeCK workflow version 0.5.8, the adapters of the demultiplexed reads were trimmed to obtain 20-bp gRNA sequences, and the single-guide RNA (sgRNA) sequences were mapped to the sequences of the Human GeCKO v2 sgRNA library to determine the total number of gRNA counts. The robust rank aggregation values and P values were determined using the MAGeCK algorithm (Li et al., 2014 (link)).