Journal: Nature Communications

Article Title: Genome-wide CRISPR/Cas9 library screening identified PHGDH as a critical driver for Sorafenib resistance in HCC

doi: 10.1038/s41467-019-12606-7

Figure Lengend Snippet: CRISPR library screening identified PHGDH as a driver for Sorafenib resistance. a Schematic diagram illustrates the workflow of genome-wide CRISPR/Cas9 knockout library screening (CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats). Human genome-wide CRISPR/Cas9 knockout library (GeCKO v2A) containing 65,386 sgRNAs was packed into lentiviral particle and transduced into Cas9-overexpressing MHCC97L cells (MHCC97L-Cas9) at low multiplicity of infection (MOI). The sgRNA transduced cells were selected by puromycin to generate a mutant cell pool. Mutant cells were cultured in vehicle and Sorafenib for 7 days for genetic screening. Genomic DNA was extracted from the treated cells and the sgRNA fragment was amplified by PCR. Copy number of sgRNAs was determined by high-throughput sequencing and analyzed by MAGeCK v0.5.7 algorithm. b PHGDH (phosphoglycerate dehydrogenase) was identified as the most significant gene in the library screening as indicated by the red dot. The sgRNAs targeting PHGDH were consistently depleted in Sorafenib-treated cells. c Volcano plots revealed that PHGDH targeting sgRNAs were negatively selected during Sorafenib treatment, suggesting that PHGDH is an essential gene for HCC cells to survive from Sorafenib treatment. Source data are provided as a Source Data file

Article Snippet: The library construction and massive parallel sequencing was performed by Novogene Technology.

Techniques: CRISPR, Library Screening, Genome Wide, Knock-Out, Infection, Mutagenesis, Cell Culture, Amplification, Next-Generation Sequencing