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bioRxiv · 10.1101/2024.02.06.579165

Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing

Fiflis, D. N.·Rey, N.·Lavanya, H. V.·Sewell, B.·Mitchell-Dick, A.·Milo, S.·Clements, K.·Benkert, A.·Rosales, A.·Fergione, S.·Asokan, A.
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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.

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Fiflis, D. N., Rey, N., Lavanya, H. V., Sewell, B., Mitchell-Dick, A., Milo, S., Clements, K., Benkert, A., Rosales, A., Fergione, S., Asokan, A.. 2024-02-08. Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing. https://doi.org/10.1101/2024.02.06.579165

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