Abstract

Efficient targeted control of splicing is a major goal of functional genomics and therapeutic applications. Guide (g)RNA-directed, deactivated (d)Cas CRISPR enzymes fused to splicing effectors represent a promising strategy due to the flexibility of these systems. However, efficient, specific, and generalizable activation of endogenous exons using this approach has not been previously reported. By screening over 300 dCasRx-splicing factor fusion proteins tethered to splicing reporters, we identify dCasRx-RBM25 as a potent activator of exons. Moreover, dCasRx-RBM25 efficiently activates the splicing of ∼90% of targeted endogenous alternative exons and displays high on-target specificity. Using gRNA arrays for combinatorial targeting, we demonstrate that dCasRx-RBM25 enables multiplexed activation and repression of exons. Using this feature, the targeting of neural-regulated exons in Ptpb1 and Puf60 in embryonic stem cells reveals combinatorial effects on downstream alternative splicing events controlled by these factors. Collectively, our results enable versatile, combinatorial exon-resolution functional assays and splicing-directed therapeutic applications.