Kristof Andrew, Karunakaran Krithika, Allen Christopher, Mizote Paula, Briggs Sophie, Jian Zixin, Nash Patrick, Blazeck John
School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, 30332, USA.
Genome Biol. 2025 Jun 12;26(1):164. doi: 10.1186/s13059-025-03640-4.
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 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.
We posit that these novel repressor fusions can enhance the reproducibility and utility of CRISPRi in mammalian cells.
CRISPR干扰（CRISPRi）是将RNA引导的核酸内切酶dCas9重新用作可编程转录阻遏物，可实现基因表达的高度特异性抑制（敲低）。CRISPRi平台往往存在敲低不完全、不同细胞系和基因靶点间性能差异以及依赖所使用的引导RNA序列而产生的不一致性等问题。
在此，我们探索了新型阻遏结构域与强Krüppel相关盒（KRAB）阻遏物的组合，筛选了100多种二分体和三分体融合蛋白作为CRISPRi效应物降低基因表达的能力。我们表明，这些新型阻遏物融合对引导RNA序列的依赖性降低，用于敲低必需基因表达时能更好地减缓细胞生长，且在融合或支架模式下均能发挥作用。此外，我们分离并表征了一个特别有效的CRISPRi平台dCas9-ZIM3（KRAB）-MeCP2（t），它在多个细胞系中以及在全基因组筛选中使用时，在内源靶点的转录本和蛋白质水平上均显示出改善的基因抑制效果。
我们认为这些新型阻遏物融合可提高CRISPRi在哺乳动物细胞中的可重复性和实用性。
