Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing

David N. Fiflis, Nicolas A. Rey, Harshitha Venugopal-Lavanya, Beatrice Sewell, Aaron Mitchell-Dick, Katie N. Clements, Sydney Milo, Abigail R. Benkert, Alan Rosales, Sophia Fergione and Aravind Asokan ()
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David N. Fiflis: Duke University
Nicolas A. Rey: Duke University School of Medicine
Harshitha Venugopal-Lavanya: Duke University
Beatrice Sewell: Duke University School of Medicine
Aaron Mitchell-Dick: Duke University School of Medicine
Katie N. Clements: Duke University School of Medicine
Sydney Milo: Duke University
Abigail R. Benkert: Duke University School of Medicine
Alan Rosales: Duke University
Sophia Fergione: Duke University School of Medicine
Aravind Asokan: Duke University

Nature Communications, 2024, vol. 15, issue 1, 1-14

Abstract: Abstract Type VI CRISPR enzymes have been developed as programmable RNA-guided Cas proteins for eukaryotic RNA editing. Notably, Cas13 has been utilized for site-targeted single base edits, demethylation, RNA cleavage or knockdown and alternative splicing. However, the ability to edit large stretches of mRNA transcripts remains a significant challenge. Here, we demonstrate that CRISPR-Cas13 systems can be repurposed to assist trans-splicing of exogenous RNA fragments into an endogenous pre-mRNA transcript, a method termed CRISPR Assisted mRNA Fragment Trans-splicing (CRAFT). Using split reporter-based assays, we evaluate orthogonal Cas13 systems, optimize guide RNA length and screen for optimal trans-splicing site(s) across a range of intronic targets. We achieve markedly improved editing of large 5’ and 3’ segments in different endogenous mRNAs across various mammalian cell types compared to other spliceosome-mediated trans-splicing methods. CRAFT can serve as a versatile platform for attachment of protein tags, studying the impact of multiple mutations/single nucleotide polymorphisms, modification of untranslated regions (UTRs) or replacing large segments of mRNA transcripts.

Date: 2024
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DOI: 10.1038/s41467-024-46172-4

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