CRISPR/Cas9 screening targeting the deregulated metabolic genes in Huh 7 cells

The exact functional roles of most of the dysregulated metabolic genes in tumorigenicity are still unclear. We report the application of CRISPR/Cas9 knockout screen technology in hepatocellular carcinoma cells. By an in vivo CRISPR/Cas9 knockout screen that targets 1,121 differentially expressed metabolic genes in HCC, we identified 67 metabolic genes as oncogenic candidates for HCC. Overall design: A pooled sgRNA library, which contained 6,726 sgRNAs targeting 1,121 metabolic genes, 12 sgRNAs targeting two validated oncogenes and 500 control sgRNAs, was constructed and transduced into HUH7 cells stably expressing Cas9-GFP-Luc at a multiplicity of infection (MOI) of 0.3. The transduced cells were selected with puromycin, propagated and subcutaneously transplanted into NOD/SCID mice. After we harvested genomic DNA from the primary tumors at 4 weeks after transplantation and HUH7 cells, the sgRNA sequences were PCR-amplified and measured by next-generation sequencing to determine the enrichment in day 28 (T28) cells relative to day 0 (T0) cells.