Nuclease-deactivated Cas9 fused to the KRAB repressor domain (dCas9-KRAB)
Transcriptional repressor made by fusing nuclease-deactivated Cas9 (dCas9) to the Krüppel-associated box (KRAB) repressor domain of human ZNF10. A guide RNA directs the fusion to a promoter, where KRAB silences the endogenous gene without cutting DNA. This approach is called CRISPR interference (CRISPRi).
Length: 4443 bp (1481 aa)
Function: repression
Base protein: dCas9 Catalytically Dead Nuclease
Origin: dCas9 fused to the KRAB repressor domain
Characteristics
dCas9 carries the D10A and H840A substitutions that inactivate both nuclease domains, so the fusion binds DNA at the guide RNA target without cutting it. The KRAB domain silences the bound gene. Coupling dCas9 to a repressor domain silences several endogenous genes in human cells, and RNA-seq showed CRISPRi repression to be highly specific. Across 49 genes tiled with sgRNAs in K562 cells, CRISPRi activity was strongest when dCas9-KRAB was targeted from -50 to +300 bp relative to the transcription start site, with a maximum at +50 to +100 bp.
Applications: Silencing of endogenous genes in human and yeast cells with a coexpressed guide RNA, and genome-scale CRISPRi screens in K562 cells. Silencing is guide RNA dependent, so several genes can be repressed at once with several guide RNAs.
Limitations: Repression is strongest only when the guide RNA targets a narrow window around the transcription start site, from -50 to +300 bp, so sites further away silence poorly. dCas9 derives from SpCas9, so target sites need an NGG PAM, and its coding sequence of about 4.1 kb leaves little room for other elements in a single AAV vector.
Architecture: Catalytically dead Cas9 (D10A/H840A) fused to the KRAB repressor domain of human ZNF10 (KOX1). KRAB recruits the KAP1 corepressor complex.
