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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...

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Autores principales: Kosicki, Michael, Allen, Felicity, Steward, Frances, Tomberg, Kärt, Pan, Yangyang, Bradley, Allan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
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.
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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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