A highly efficient KRAB domain for CRISPRi applications

CRISPR interference (CRISPRi), based the fusion of enzymatically inactive Cas9 (dCas9) to the KRAB repressor domain, has emerged as a powerful platform to silence gene expression and decipher the logic of transcriptional regulatory elements, non-coding RNAs, and genetic interactions. However, a major challenge has been the sensitivity of CRISPRi to guide RNA selection and incomplete silencing of target gene expression. We assayed 57 KRAB domains fused to dCas9 for their ability to repress transcription in human cells. We show that, surprisingly, the currently used KRAB domain from KOX1 is not a particularly strong KRAB-based repressor. We characterize the KRAB domain from ZIM3 as a highly potent repressor and establish that ZIM3 KRAB-dCas9 fusion silences gene expression more efficiently in multiple contexts and is less sensitive to gRNA selection than current CRISPRi platforms, including the recently described KOX1 KRAB-MeCP2 fusion.