Repurposing Type-VI CRISPR Systems for Programmable mRNA Trans-Splicing

Abstract

The type VI-Cas13 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) enzymes of the bacterial adaptive immune system have been repurposed as programmable RNA guided, RNA targeting nucleases for eukaryotic RNA editing1–4. In these systems, the Cas ribonucleoprotein (RNP) is guided to its target RNA transcript by a single CRISPR RNA (crRNA), which it cleaves following satisfactory RNA base-pairing. The RNase domain of the Cas13 protein may be inactivated to serve as a RNA guided RNA binding protein with high affinity for its RNA target5–8. This has expanded the utility of Cas13 beyond programable RNA knockdown. The catalytically dead Cas13 (dCas13), now, can serve as a chassis upon which effector domains have been added to catalyze site-targeted single base edits, demethylation, RNA cleavage and alternative splicing with improved specificity and efficiency relative to existing antisense RNA technologies5,9,10. However, the ability to edit large stretches of mRNA transcripts remains a significant challenge.RNA splicing is well a conserved process in higher eukaryotes that joins the protein coding RNA regions (exons) along the same transcript via a dual trans-esterification reaction in cis11. The intergenic region (intron) between these two exons choreographs this reaction by recruiting the spliceosome to conserved molecular signatures. In this work, type VI CRISPR-Cas13 systems are leveraged to facilitate RNA rewriting by hijacking the cell’s RNA splicing machinery via an approach referred to herein as CRISPR Assisted RNA Fragment Trans-splicing (CRAFT). To achieve such, a Cas13 crRNA was linked with a sequence containing these intronic molecular signatures (hemi-intron) and one or more exons in a single recombinant trans-splicing RNA (rcRNA). This chimeric RNA was co-expressed with a catalytically inactive cognate dCas13 nuclease. This RNP employs CRISPR to target nascent pre-mRNA species, while the conserved molecular signals of the hemi-intron mediate incorporation of the linked exon(s) into the mature transcript in trans. Using split reporter-based assays, we evaluate orthogonal Cas13 systems, optimize guide RNA length and position for optimal trans-splicing 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. Additionally, we demonstrate that 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. We validate CRAFT across different transformed and primary cell lines, multiple endogenous transcripts, develop a novel barcoded approach for guide selection to facilitate mRNA trans-splicing and affirm that CRAFT can serve as a promising tool for gene therapy and studying RNA biology.