Figure 2.

Several CRISPR systems are applied for drug target screening. (A) The schematic diagram of CRISPR-Cas9 system, CRISPRi, CRISPRa and their variants. The CRISPRi and CRISPRa distinguish CRISPR/Cas9 system with the dCas9 rather than Cas9. Cas9 combined with sgRNA perform shearing function to specific site on target DNA, causing DNA double-strand breaks. The gene repair approaches include the nonhomologous end joining and homology-directed repair. For achieving higher efficiency, dCas9 often fuses with repressed proteins such as KRAB and DNMT3A in CRISPRi, while in CRISPRa it often fuses with activated protein VP64. (B) The flow diagram of cell-based high-throughput screening using pooled sgRNA library synthesis. The synthesized sgRNAs are cloned into plasmid for amplifying by lentivirus to establish sgRNA library. Cells which are expressed Cas9 or dCas9 undergo drug treatment to select against sgRNA library according to phenotype changes, following the drug target genes are analyzed by the next generation sequencing (NGS).