Fig. 1.

CRISPR technologies to perturb gene functions in mammalian cells for pooled genetic screens. CRISPR loss-of-function technologies include A) CRISPR knockout (KO) and B) CRISPR interference (CRISPRi). A) Cas9-mediated DNA cleavage is directed to the coding region of a gene by a single guide RNA (sgRNA) and it results in error-prone repair by nonhomologous end joining pathways (NHEJ), and as a consequence of that gene function is disrupted (when indels and especially frame shifts are introduced). B) Catalytically dead Cas9 (dCas9) is fused to a transcriptional repressor domain (e.g. KRAB) and as that is recruited to the transcription start site (TSS) of a gene specified by an sgRNA, to repress its transcription. CRISPR gain-off-function technology is C) CRISPR activation (CRISRPa). C) dCas9 is fused with transcriptional activation domain(s) (e.g. VP64) and recruited to a given gene’s TSS, to activate its transcription.