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Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases

CRISPR-Cas9 introduces targeted DNA breaks that engage competing DNA repair pathways, producing a spectrum of imprecise insertion/deletion mutations (indels) and precise templated mutations (precise edits). The relative frequencies of these pathways are thought to primarily depend on genomic sequenc...

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Detalles Bibliográficos
Autores principales: Chauhan, Vikash P., Sharp, Phillip A., Langer, Robert
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10242711/
https://www.ncbi.nlm.nih.gov/pubmed/36881621
http://dx.doi.org/10.1073/pnas.2300605120
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author Chauhan, Vikash P.
Sharp, Phillip A.
Langer, Robert
author_facet Chauhan, Vikash P.
Sharp, Phillip A.
Langer, Robert
author_sort Chauhan, Vikash P.
collection PubMed
description CRISPR-Cas9 introduces targeted DNA breaks that engage competing DNA repair pathways, producing a spectrum of imprecise insertion/deletion mutations (indels) and precise templated mutations (precise edits). The relative frequencies of these pathways are thought to primarily depend on genomic sequence and cell state contexts, limiting control over mutational outcomes. Here, we report that engineered Cas9 nucleases that create different DNA break structures engage competing repair pathways at dramatically altered frequencies. We accordingly designed a Cas9 variant (vCas9) that produces breaks which suppress otherwise dominant nonhomologous end-joining (NHEJ) repair. Instead, breaks created by vCas9 are predominantly repaired by pathways utilizing homologous sequences, specifically microhomology-mediated end-joining (MMEJ) and homology-directed repair (HDR). Consequently, vCas9 enables efficient precise editing through HDR or MMEJ while suppressing indels caused by NHEJ in dividing and nondividing cells. These findings establish a paradigm of targeted nucleases custom-designed for specific mutational applications.
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spelling pubmed-102427112023-09-07 Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases Chauhan, Vikash P. Sharp, Phillip A. Langer, Robert Proc Natl Acad Sci U S A Physical Sciences CRISPR-Cas9 introduces targeted DNA breaks that engage competing DNA repair pathways, producing a spectrum of imprecise insertion/deletion mutations (indels) and precise templated mutations (precise edits). The relative frequencies of these pathways are thought to primarily depend on genomic sequence and cell state contexts, limiting control over mutational outcomes. Here, we report that engineered Cas9 nucleases that create different DNA break structures engage competing repair pathways at dramatically altered frequencies. We accordingly designed a Cas9 variant (vCas9) that produces breaks which suppress otherwise dominant nonhomologous end-joining (NHEJ) repair. Instead, breaks created by vCas9 are predominantly repaired by pathways utilizing homologous sequences, specifically microhomology-mediated end-joining (MMEJ) and homology-directed repair (HDR). Consequently, vCas9 enables efficient precise editing through HDR or MMEJ while suppressing indels caused by NHEJ in dividing and nondividing cells. These findings establish a paradigm of targeted nucleases custom-designed for specific mutational applications. National Academy of Sciences 2023-03-07 2023-03-14 /pmc/articles/PMC10242711/ /pubmed/36881621 http://dx.doi.org/10.1073/pnas.2300605120 Text en Copyright © 2023 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by-nc-nd/4.0/This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) .
spellingShingle Physical Sciences
Chauhan, Vikash P.
Sharp, Phillip A.
Langer, Robert
Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases
title Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases
title_full Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases
title_fullStr Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases
title_full_unstemmed Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases
title_short Altered DNA repair pathway engagement by engineered CRISPR-Cas9 nucleases
title_sort altered dna repair pathway engagement by engineered crispr-cas9 nucleases
topic Physical Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10242711/
https://www.ncbi.nlm.nih.gov/pubmed/36881621
http://dx.doi.org/10.1073/pnas.2300605120
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