Rewriting endogenous human transcripts with dual CRISPR-guided 3' trans-splicing

Unlike genome editing, RNA editing offers the ability to transiently alter cells with minimal risk from off-target effects. While exon-skipping technologies can influence splice site selection, many desired perturbations to the transcriptome require replacement or addition of exogenous exons to target mRNAs, such as replacing disease-causing exons, repairing truncated proteins, or engineering protein fusions. Here, we report the development of RNA-guided trans-splicing with Cas editor (RESPLICE). 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 RNA cargo (up to 2.1 kb) into 11 endogenous transcripts across 3 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 mode of RNA editing that could provide fine-tuned and transient control of cellular programs.