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Cas9-induced large deletions and small indels are controlled in a convergent fashion
Repair of Cas9-induced double-stranded breaks results primarily in formation of small insertions and deletions (indels), but can also cause potentially harmful large deletions. While mechanisms leading to the creation of small indels are relatively well understood, very little is known about the ori...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9197861/ https://www.ncbi.nlm.nih.gov/pubmed/35701408 http://dx.doi.org/10.1038/s41467-022-30480-8 |
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author | Kosicki, Michael Allen, Felicity Steward, Frances Tomberg, Kärt Pan, Yangyang Bradley, Allan |
author_facet | Kosicki, Michael Allen, Felicity Steward, Frances Tomberg, Kärt Pan, Yangyang Bradley, Allan |
author_sort | Kosicki, Michael |
collection | PubMed |
description | Repair of Cas9-induced double-stranded breaks results primarily in formation of small insertions and deletions (indels), but can also cause potentially harmful large deletions. While mechanisms leading to the creation of small indels are relatively well understood, very little is known about the origins of large deletions. Using a library of clonal NGS-validated mouse embryonic stem cells deficient for 32 DNA repair genes, we have shown that large deletion frequency increases in cells impaired for non-homologous end joining and decreases in cells deficient for the central resection gene Nbn and the microhomology-mediated end joining gene Polq. Across deficient clones, increase in large deletion frequency was closely correlated with the increase in the extent of microhomology and the size of small indels, implying a continuity of repair processes across different genomic scales. Furthermore, by targeting diverse genomic sites, we identified examples of repair processes that were highly locus-specific, discovering a role for exonuclease Trex1. Finally, we present evidence that indel sizes increase with the overall efficiency of Cas9 mutagenesis. These findings may have impact on both basic research and clinical use of CRISPR-Cas9, in particular in conjunction with repair pathway modulation. |
format | Online Article Text |
id | pubmed-9197861 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-91978612022-06-16 Cas9-induced large deletions and small indels are controlled in a convergent fashion Kosicki, Michael Allen, Felicity Steward, Frances Tomberg, Kärt Pan, Yangyang Bradley, Allan Nat Commun Article Repair of Cas9-induced double-stranded breaks results primarily in formation of small insertions and deletions (indels), but can also cause potentially harmful large deletions. While mechanisms leading to the creation of small indels are relatively well understood, very little is known about the origins of large deletions. Using a library of clonal NGS-validated mouse embryonic stem cells deficient for 32 DNA repair genes, we have shown that large deletion frequency increases in cells impaired for non-homologous end joining and decreases in cells deficient for the central resection gene Nbn and the microhomology-mediated end joining gene Polq. Across deficient clones, increase in large deletion frequency was closely correlated with the increase in the extent of microhomology and the size of small indels, implying a continuity of repair processes across different genomic scales. Furthermore, by targeting diverse genomic sites, we identified examples of repair processes that were highly locus-specific, discovering a role for exonuclease Trex1. Finally, we present evidence that indel sizes increase with the overall efficiency of Cas9 mutagenesis. These findings may have impact on both basic research and clinical use of CRISPR-Cas9, in particular in conjunction with repair pathway modulation. Nature Publishing Group UK 2022-06-14 /pmc/articles/PMC9197861/ /pubmed/35701408 http://dx.doi.org/10.1038/s41467-022-30480-8 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Kosicki, Michael Allen, Felicity Steward, Frances Tomberg, Kärt Pan, Yangyang Bradley, Allan Cas9-induced large deletions and small indels are controlled in a convergent fashion |
title | Cas9-induced large deletions and small indels are controlled in a convergent fashion |
title_full | Cas9-induced large deletions and small indels are controlled in a convergent fashion |
title_fullStr | Cas9-induced large deletions and small indels are controlled in a convergent fashion |
title_full_unstemmed | Cas9-induced large deletions and small indels are controlled in a convergent fashion |
title_short | Cas9-induced large deletions and small indels are controlled in a convergent fashion |
title_sort | cas9-induced large deletions and small indels are controlled in a convergent fashion |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9197861/ https://www.ncbi.nlm.nih.gov/pubmed/35701408 http://dx.doi.org/10.1038/s41467-022-30480-8 |
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