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

Isolation of genes whose knockout conferring resistance to lysenin-Induced cell death Overall design: A genome-wide CRISPR/CAS9 knockout screen in HeLa cells was performed to identify factors involved in the regulation of sphingomyelin biosynthesis, delivery, or distribution by exploiting lysenin 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 lysenin three times. 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 kept subcultured for the same period as lysenin-treated cells. sgRNA profiles of control and lysenin-treated HeLa cell were generated by next-generation sequencing, using Illumina MiniSeq, in biological duplicates.