A Next-Generation Platform for Highly Optimized CRISPR-Mediated Transcriptional Repression

CRISPR interference (CRISPRi) is a powerful genetic tool enabling site-specific suppression of gene expression; however, its performance remains inconsistent across individual cell lines and targeting loci. Using combinatorial domain fusion libraries and subdomain dissection of the MeCP2 repressor, we uncover a suite of novel, and highly potent, repressor fusions. Through rigorous validation across several cell lines, we establish that our strongest new repressor, dCas9-ZIM3-NID-MXD1-NLS, achieves superior gene silencing compared to existing CRISPRi platforms.