Programmable multi-kilobase RNA editing using CRISPR-mediated trans-splicing.

bioRxiv : the preprint server for biology
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Abstract

Current gene editing approaches in eukaryotic cells are limited to single base edits or small DNA insertions and deletions, and remain encumbered by unintended permanent effects and significant challenges in the delivery of large DNA cargo. Here we describe Splice Editing, a generalizable platform to correct gene transcripts by programmable insertion or replacement of large RNA segments. By combining CRISPR-mediated RNA targeting with endogenous cellular RNA-splicing machinery, Splice Editing enables efficient, precise, and programmable large-scale editing of gene targets without DNA cleavage or mutagenesis. RNA sequencing and measurement of spliced protein products confirm that Splice Editing achieves efficient and specific targeted RNA and protein correction. We show that Splice Editors based on novel miniature RNA-targeting CRISPR-Cas systems discovered and characterized in this work can be packaged for effective delivery to human cells and affect different types of edits across multiple targets and cell lines. By editing thousands of bases simultaneously in a single reversible step, Splice Editing could expand the treatable disease population for monogenic diseases with large allelic diversity without the permanent unintended effects of DNA editing.

Year of Publication
2023
Journal
bioRxiv : the preprint server for biology
Date Published
08/2023
DOI
10.1101/2023.08.18.553620
PubMed ID
37645763
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