{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"omics_type":["Unknown"],"volume":["15(1)"],"submitter":["Fiflis DN"],"pubmed_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."],"journal":["Nature communications"],"pagination":["2325"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC10940283"],"repository":["biostudies-literature"],"pubmed_title":["Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing."],"pmcid":["PMC10940283"],"pubmed_authors":["Fiflis DN","Clements KN","Rosales A","Mitchell-Dick A","Venugopal-Lavanya H","Asokan A","Rey NA","Benkert AR","Sewell B","Milo S","Fergione S"],"additional_accession":[]},"is_claimable":false,"name":"Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing.","description":"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.","dates":{"release":"2024-01-01T00:00:00Z","publication":"2024 Mar","modification":"2025-04-05T12:06:03.631Z","creation":"2025-04-05T12:06:03.631Z"},"accession":"S-EPMC10940283","cross_references":{"pubmed":["38485709"],"doi":["10.1038/s41467-024-46172-4"]}}