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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...

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Autores principales: Feng, Yi-Li, Liu, Si-Cheng, Chen, Ruo-Dan, Sun, Xiu-Na, Xiao, Jing-Jing, Xiang, Ji-Feng, Xie, An-Yong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2023
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.
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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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