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

Isolation of genes whose knockout confer resistance to SubAB-Induced cell death A genome-wide CRISPR/CAS9 knockout screen in HeLa cells was performed to identify host factors involved in the regulation of glycan biosynthesis by exploiting SubAB 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 SubAB 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 SubAB-treated cells with passages several times. sgRNA profiles of control and SubAB-treated HeLa cell were generated by deep sequencing, in biological replicate 2, using Illumina MiSeq.