CRISPR/Cas9 knockout and activation screening of colorectal cancer allelic imbalance target genes

A 33-day long pooled CRISPR/Cas9 knockout and CRISPRa/Cas9d activation screens were done in three colorectal cancer cell lines, GP5d, COLO320DM and CaCO2, targeting genes located in allelic imbalance regions found in a set of 1699 colorectal tumors. These assays detected genes whose loss or overexpression increased or decreased growth of tumor cells in culture. In CRISPR knockout screen, total 24944 guide RNAs (gRNAs) were designed to target the exons of 1928 candidate target genes in allelic imbalance regions and 198 additional control genes. The oligos were designed to contain a six bases long random sequence label (RSL) before the gRNA sequence to facilitate monitoring of small subpopulations of edited cells. To facilitate activation of the candidate target genes, another set of 24944 guide RNAs were deigned to target gene promoters. In CRISPRa expreriments,the RSL was added downstream of the guide RNA so that it was not transcribed (RSL and sample barcode sequenced using index reads are added to the read name).