Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox
Abstract Pre- and post-transcriptional mechanisms, including alternative promoters, termination signals, and splicing, play essential roles in diversifying protein output by generating distinct RNA and protein isoforms. Two major challenges in characterizing the cellular function of alternative isof...
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| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-07-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-62066-5 |
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| author | Megan D. Schertzer Andrew Stirn Keren Isaev Laura Pereira Stella H. Park Anjali Das Aline Réal Erin D. Jeffery Claire Harbison Hans-Hermann Wessels Gloria M. Sheynkman Neville E. Sanjana David A. Knowles |
| author_facet | Megan D. Schertzer Andrew Stirn Keren Isaev Laura Pereira Stella H. Park Anjali Das Aline Réal Erin D. Jeffery Claire Harbison Hans-Hermann Wessels Gloria M. Sheynkman Neville E. Sanjana David A. Knowles |
| author_sort | Megan D. Schertzer |
| collection | DOAJ |
| description | Abstract Pre- and post-transcriptional mechanisms, including alternative promoters, termination signals, and splicing, play essential roles in diversifying protein output by generating distinct RNA and protein isoforms. Two major challenges in characterizing the cellular function of alternative isoforms are the lack of experimental methods to specifically and efficiently modulate isoform expression and computational tools for complex experimental design and analysis. To address these gaps, we develop and methodically test an isoform-specific knockdown strategy which pairs the RNA-targeting CRISPR/Cas13d system with guide RNAs that span exon-exon junctions. In parallel, we provide computational tools for experimental design and analysis. In this study, we demonstrate that junction-targeting achieves robust and isoform-specific RNA knockdown across diverse alternative isoform events, genes, and cell types. |
| format | Article |
| id | doaj-art-73cd1e5da5e84c9b9e8b22cd094789dc |
| institution | DOAJ |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-73cd1e5da5e84c9b9e8b22cd094789dc2025-08-20T03:05:14ZengNature PortfolioNature Communications2041-17232025-07-0116111910.1038/s41467-025-62066-5Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolboxMegan D. Schertzer0Andrew Stirn1Keren Isaev2Laura Pereira3Stella H. Park4Anjali Das5Aline Réal6Erin D. Jeffery7Claire Harbison8Hans-Hermann Wessels9Gloria M. Sheynkman10Neville E. Sanjana11David A. Knowles12New York Genome CenterNew York Genome CenterNew York Genome CenterNew York Genome CenterNew York Genome CenterNew York Genome CenterNew York Genome CenterDepartment of Molecular Physiology and Biological Physics, University of VirginiaNew York Genome CenterNew York Genome CenterDepartment of Molecular Physiology and Biological Physics, University of VirginiaNew York Genome CenterNew York Genome CenterAbstract Pre- and post-transcriptional mechanisms, including alternative promoters, termination signals, and splicing, play essential roles in diversifying protein output by generating distinct RNA and protein isoforms. Two major challenges in characterizing the cellular function of alternative isoforms are the lack of experimental methods to specifically and efficiently modulate isoform expression and computational tools for complex experimental design and analysis. To address these gaps, we develop and methodically test an isoform-specific knockdown strategy which pairs the RNA-targeting CRISPR/Cas13d system with guide RNAs that span exon-exon junctions. In parallel, we provide computational tools for experimental design and analysis. In this study, we demonstrate that junction-targeting achieves robust and isoform-specific RNA knockdown across diverse alternative isoform events, genes, and cell types.https://doi.org/10.1038/s41467-025-62066-5 |
| spellingShingle | Megan D. Schertzer Andrew Stirn Keren Isaev Laura Pereira Stella H. Park Anjali Das Aline Réal Erin D. Jeffery Claire Harbison Hans-Hermann Wessels Gloria M. Sheynkman Neville E. Sanjana David A. Knowles Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox Nature Communications |
| title | Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox |
| title_full | Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox |
| title_fullStr | Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox |
| title_full_unstemmed | Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox |
| title_short | Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox |
| title_sort | cas13d mediated isoform specific rna knockdown with a unified computational and experimental toolbox |
| url | https://doi.org/10.1038/s41467-025-62066-5 |
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