Figure 2.

A schematic diagram illustrates the workflow of high-throughput CRISPR-Cas9 screening for novel regulators of drug sensitivity. CRISPR-Cas9 sgRNA libraries are packed into lentiviral vectors and transfected into Cas9- or dCas9-expressing cancer cells. In the case of loss-of-function screening, CRISPR-Cas9-mediated genome editing leads to gene knockout (or transcriptional inhibition) in individual cells, which are subsequently selection by various drugs. The residual drug-resistant cells are collected. The abundance of cells with different sgRNAs is determined in the drug-treated and control pool. Cells with sgRNAs targeting genes that cause drug resistance upon knockout (or transcriptional inhibition) will be enriched while those resulting in enhanced sensitivity to the drug will be depleted in the final pool. For gain-of-function screening, activation of gene expression by dCas9-mediated recruitment of transcriptional activation domains to transcriptional start site. Other procedures are similar to loss-of-function screening. The unique sgRNA sequence in the genome serves as a genetic barcode for high-throughput phenotyping by next-generation sequencing. Essential genes for drug sensitivity are identified for further validation. dCas9, nuclease-dead Cas9. sgRNA, single guide RNA.