Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing
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 stretche...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2024-03-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-024-46172-4 |
| _version_ | 1797259208423374848 |
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| author | 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 Aravind Asokan |
| author_facet | 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 Aravind Asokan |
| author_sort | David N. Fiflis |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-24T23:05:46Z |
| format | Article |
| id | doaj.art-4a9e799fbf1b4f619012e055594df9cf |
| institution | Directory Open Access Journal |
| issn | 2041-1723 |
| language | English |
| last_indexed | 2024-04-24T23:05:46Z |
| publishDate | 2024-03-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj.art-4a9e799fbf1b4f619012e055594df9cf2024-03-17T12:30:12ZengNature PortfolioNature Communications2041-17232024-03-0115111410.1038/s41467-024-46172-4Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicingDavid N. Fiflis0Nicolas A. Rey1Harshitha Venugopal-Lavanya2Beatrice Sewell3Aaron Mitchell-Dick4Katie N. Clements5Sydney Milo6Abigail R. Benkert7Alan Rosales8Sophia Fergione9Aravind Asokan10Department of Biomedical Engineering, Duke UniversityDepartment of Surgery, Duke University School of MedicineDepartment of Biomedical Engineering, Duke UniversityDepartment of Surgery, Duke University School of MedicineDepartment of Surgery, Duke University School of MedicineDepartment of Surgery, Duke University School of MedicineDepartment of Biomedical Engineering, Duke UniversityDepartment of Surgery, Duke University School of MedicineDepartment of Biomedical Engineering, Duke UniversityDepartment of Surgery, Duke University School of MedicineDepartment of Biomedical Engineering, Duke UniversityAbstract 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.https://doi.org/10.1038/s41467-024-46172-4 |
| spellingShingle | 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 Aravind Asokan Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing Nature Communications |
| title | Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing |
| title_full | Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing |
| title_fullStr | Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing |
| title_full_unstemmed | Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing |
| title_short | Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing |
| title_sort | repurposing crispr cas13 systems for robust mrna trans splicing |
| url | https://doi.org/10.1038/s41467-024-46172-4 |
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