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Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining
In CRISPR/Cas9 genome editing, the tight and persistent target binding of Cas9 provides an opportunity for efficient genetic and epigenetic modification on genome. In particular, technologies based on catalytically dead Cas9 (dCas9) have been developed to enable genomic regulation and live imaging i...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10085678/ https://www.ncbi.nlm.nih.gov/pubmed/36864759 http://dx.doi.org/10.1093/nar/gkad116 |
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author | Feng, Yi-Li Liu, Si-Cheng Chen, Ruo-Dan Sun, Xiu-Na Xiao, Jing-Jing Xiang, Ji-Feng Xie, An-Yong |
author_facet | Feng, Yi-Li Liu, Si-Cheng Chen, Ruo-Dan Sun, Xiu-Na Xiao, Jing-Jing Xiang, Ji-Feng Xie, An-Yong |
author_sort | Feng, Yi-Li |
collection | PubMed |
description | In CRISPR/Cas9 genome editing, the tight and persistent target binding of Cas9 provides an opportunity for efficient genetic and epigenetic modification on genome. In particular, technologies based on catalytically dead Cas9 (dCas9) have been developed to enable genomic regulation and live imaging in a site-specific manner. While post-cleavage target residence of CRISPR/Cas9 could alter the pathway choice in repair of Cas9-induced DNA double strand breaks (DSBs), it is possible that dCas9 residing adjacent to a break may also determine the repair pathway for this DSB, providing an opportunity to control genome editing. Here, we found that loading dCas9 onto a DSB-adjacent site stimulated homology-directed repair (HDR) of this DSB by locally blocking recruitment of classical non-homologous end-joining (c-NHEJ) factors and suppressing c-NHEJ in mammalian cells. We further repurposed dCas9 proximal binding to increase HDR-mediated CRISPR genome editing by up to 4-fold while avoiding exacerbation of off-target effects. This dCas9-based local inhibitor provided a novel strategy of c-NHEJ inhibition in CRISPR genome editing in place of small molecule c-NHEJ inhibitors, which are often used to increase HDR-mediated genome editing but undesirably exacerbate off-target effects. |
format | Online Article Text |
id | pubmed-10085678 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-100856782023-04-11 Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining Feng, Yi-Li Liu, Si-Cheng Chen, Ruo-Dan Sun, Xiu-Na Xiao, Jing-Jing Xiang, Ji-Feng Xie, An-Yong Nucleic Acids Res Genome Integrity, Repair and Replication In CRISPR/Cas9 genome editing, the tight and persistent target binding of Cas9 provides an opportunity for efficient genetic and epigenetic modification on genome. In particular, technologies based on catalytically dead Cas9 (dCas9) have been developed to enable genomic regulation and live imaging in a site-specific manner. While post-cleavage target residence of CRISPR/Cas9 could alter the pathway choice in repair of Cas9-induced DNA double strand breaks (DSBs), it is possible that dCas9 residing adjacent to a break may also determine the repair pathway for this DSB, providing an opportunity to control genome editing. Here, we found that loading dCas9 onto a DSB-adjacent site stimulated homology-directed repair (HDR) of this DSB by locally blocking recruitment of classical non-homologous end-joining (c-NHEJ) factors and suppressing c-NHEJ in mammalian cells. We further repurposed dCas9 proximal binding to increase HDR-mediated CRISPR genome editing by up to 4-fold while avoiding exacerbation of off-target effects. This dCas9-based local inhibitor provided a novel strategy of c-NHEJ inhibition in CRISPR genome editing in place of small molecule c-NHEJ inhibitors, which are often used to increase HDR-mediated genome editing but undesirably exacerbate off-target effects. Oxford University Press 2023-03-02 /pmc/articles/PMC10085678/ /pubmed/36864759 http://dx.doi.org/10.1093/nar/gkad116 Text en © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Genome Integrity, Repair and Replication Feng, Yi-Li Liu, Si-Cheng Chen, Ruo-Dan Sun, Xiu-Na Xiao, Jing-Jing Xiang, Ji-Feng Xie, An-Yong Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining |
title | Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining |
title_full | Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining |
title_fullStr | Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining |
title_full_unstemmed | Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining |
title_short | Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining |
title_sort | proximal binding of dcas9 at a dna double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining |
topic | Genome Integrity, Repair and Replication |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10085678/ https://www.ncbi.nlm.nih.gov/pubmed/36864759 http://dx.doi.org/10.1093/nar/gkad116 |
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