Rewriting endogenous human transcripts with trans-splicing

Splicing bridges the gap between static DNA sequence and the diverse and dynamic set of protein products that execute a genes biological functions. While exon skipping technologies enable influence over splice site selection, many desired perturbations to the transcriptome require replacement or addition of exogenous exons to target mRNAs: for example, to replace disease-causing exons, repair truncated proteins, or engineer protein fusions. Here, we report the development of RNA-guided trans-splicing with Cas editor (RESPLICE), inspired by the rare, natural process of trans-splicing that joins exons from two distinct primary transcripts. RESPLICE uses two orthogonal RNA-targeting CRISPR effectors to co-localize a trans-splicing pre-mRNA and to inhibit the cis-splicing reaction, respectively. We demonstrate efficient, specific, and programmable trans-splicing of multi-kilobase RNA cargo into nine endogenous transcripts across two human cell types, achieving up to 45% trans-splicing efficiency in bulk, or 90% when sorting for high effector expression. Our results present RESPLICE as a new mode of RNA editing for fine-tuned and transient control of cellular programs without permanent alterations to the genetic code.