Over 100 novel repressor fusions were screened for their effectiveness in (CRISPRi).
Novel repressor fusions showed reduced dependence on guide RNA sequences.
These fusions resulted in better growth inhibition when knocking down essential genes.
The -ZIM3(KRAB)-MeCP2(t) platform demonstrated enhanced gene repression at both transcript and protein levels.
Improved gene repression was observed across several cell lines and in genome-wide screens.
The findings suggest potential for increased reproducibility and utility of CRISPRi in mammalian cells.
Simplified
BACKGROUND: (CRISPRi), the repurposing of the RNA-guided endonuclease 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.
RESULTS: Here, we explore the combination of novel repressor domains with strong Krüppel-associated box (KRAB) repressors, screening > 100 bipartite and tripartite fusion proteins for their ability to reduce gene expression as CRISPRi effectors. We show that these novel repressor fusions have reduced dependence on guide RNA sequences, better slow cell growth when used to knock down expression of essential genes, and function in either fusion or scaffold modalities. Furthermore, 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.
CONCLUSIONS: We posit that these novel repressor fusions can enhance the reproducibility and utility of CRISPRi in mammalian cells.
Key numbers
50%
Increase in Gene Efficiency
Average improvement across various cell lines compared to ().
100
Screened Repressor Variants
Number of bipartite and tripartite fusion proteins screened for gene repression.
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Declarations. Ethics approval and consent to participate: The authors declare that ethical approval was not required for the study. Competing interests: A.K. and J.B. have submitted patent applications related to this work. All other authors declare no conflicts of interest.
