(236f) Engineering Next-Generation Crispri Effectors for Targeted Mammalian Gene Regulation

The ability to systematically control gene expression is vital for performing robust whole-genome screens, discovering non-coding transcriptional regulatory motifs, and analyzing complex cellular processes such as differentiation. CRISPR interference (CRISPRi) has emerged as a powerful tool enabling site-specific transcriptional repression for these applications. The CRISPRi platform employs a deactivated bacterial endonuclease, dCas9, for recruiting native transcriptional repressor proteins to target genes. Almost all CRISPRi platforms implement direct fusions of dCas9 with the Krüppel-associated box (KRAB) domain from the human protein KOX1. This domain is traditionally known as KRAB, but here is called KOX1-KRAB to prevent ambiguity. Previous studies demonstrated improved CRISPRi activity by combining KOX1-KRAB with additional repressor domains or by using KRAB domains from other human proteins. Despite this progress, CRISPRi still suffers from incomplete repression, high performance variability across cell lines, and general overreliance on the KOX1-KRAB effector. In addition, precise epigenetic alterations and temporal dynamics resulting from CRISPRi-mediated gene knockdown also remain largely unexplored.

To address these limitations, we developed novel CRISPRi systems permitting tunable and highly efficacious gene knockdown across mammalian cell models. We first identified putative transcriptional repressor domains with diverse mechanisms of action and characterized their individual gene knockdown efficiencies using a fluorescent reporter assay in HEK293T cells. Eleven unique, non-KRAB repressor domains exhibited compatibility with the CRISPRi system, and one achieved greater silencing than KOX1-KRAB. Next, we assessed whether combining domains with diverse modalities can synergistically improve CRISPRi activity. To test this, we generated a library of bipartite repressors combining top-performing KRAB domains with the novel domains from our initial studies. Screening in HEK293T cells revealed four unique variants each achieving 20-30% improved gene knockdown efficiency compared to current gold standards. In addition, we assembled and screened tripartite repressor libraries and discovered one best-in-class repressor fusion achieving ~50% improved knockdown efficiency over established CRISPRi systems. We also demonstrate that our novel CRISPRi effectors exhibit superior performance in targeting endogenous genes and display minimal performance variability across diverse target sites. To build on our findings, we are assessing the generalizability of our top-performing repressor fusions across a large panel of mammalian cell lines. We envision these efforts will mark a significant step forward in making CRISPRi technology more robust and universally available for applications requiring precise gene control in mammalian cells.