Genome-wide essential gene identification for Shiga toxin-induced cell death using CRISPR/CAS9 screening system

Isolation of genes whose knockout conferring resistance to STx-Induced cell death A genome-wide CRISPR/CAS9 knockout screen in HeLa cells was performed to identify host factors involved in the regulation of Gb3 biosynthesis by exploiting STx sensitivity. Lentivirus-based GeCKO v2 pooled library, which is delivered as two half-libraries (A and B), was used. Two independent sgRNAs-expressing cell libraries (A-1, A-2, B-1, B-2) were prepared by transducing the lentivirus libraries, and cells were then treated with STx1 to assess toxicity. The sgRNAs integrated into the cellular genomes of the surviving cells were amplified by PCR and analyzed with high-throughput sequencing. For untreated controls, cells in each cell library were cultured for the same period as STx1-treated cells with passages several times. sgRNA profiles of control and STx1-treated HeLa cell were generated by deep sequencing, in biological replicate 2, using Illumina MiSeq.