Engineering Novel CRISPRi Repressors for Highly Efficient Mammalian Gene Regulation

CRISPR interference (CRISPRi), the repurposing of the RNA-guided endonuclease dCas9 as a programmable transcriptional repressor, allows highly specific repression (knockdown) of gene expression. CRISPRi platforms can often have incomplete knockdown, performance variability across cell lines and gene targets, and inconsistencies dependent on the guide RNA sequence employed. Here, we explore the combination of novel repressor domains with strong Kruppel-associated box (KRAB) repressors, screening 100+ bipartite and tripartite fusion proteins for their ability to reduce gene expression as CRISPRi effectors. We isolate and characterize a particularly effective CRISPRi platform, dCas9-ZIM3(KRAB)-MeCP2(t), which shows improved gene repression of endogenous targets both at the transcript and protein level across several cell lines and when deployed in genome-wide screens. We posit that these novel repressor fusions can enhance the reproducibility and utility of CRISPRi in mammalian cells.