DNA methylation can alter CRISPR/Cas9 editing frequency and DNA repair outcome in a target-specific manner.
Cas9
DNA methylation
DNA repair
chromatin
microhomology-mediated
next-generation sequencing
nonhomologous end joining
staggered end
Journal
The New phytologist
ISSN: 1469-8137
Titre abrégé: New Phytol
Pays: England
ID NLM: 9882884
Informations de publication
Date de publication:
09 2022
09 2022
Historique:
received:
11
03
2022
accepted:
02
05
2022
pubmed:
8
5
2022
medline:
20
8
2022
entrez:
7
5
2022
Statut:
ppublish
Résumé
The impact of epigenetic modifications on the efficacy of CRISPR/Cas9-mediated double-stranded DNA breaks and subsequent DNA repair is poorly understood, especially in plants. In this study, we investigated the effect of the level of cytosine methylation on the outcome of CRISPR/Cas9-induced mutations at multiple Cas9 target sites in Nicotiana benthamiana leaf cells using next-generation sequencing. We found that high levels of promoter methylation, but not gene-body methylation, decreased the frequency of Cas9-mediated mutations. DNA methylation also influenced the ratio of insertions and deletions and potentially the type of Cas9 cleavage in a target-specific manner. In addition, we detected an over-representation of deletion events governed by a single 5'-terminal nucleotide at Cas9-induced DNA breaks. Our findings suggest that DNA methylation can indirectly impair Cas9 activity and subsequent DNA repair, probably through changes in the local chromatin structure. In addition to the well described Cas9-induced blunt-end double-stranded DNA breaks, we provide evidence for Cas9-mediated staggered DNA cuts in plant cells. Both types of cut may direct microhomology-mediated DNA repair by a novel, as yet undescribed, mechanism.
Identifiants
pubmed: 35524464
doi: 10.1111/nph.18212
pmc: PMC9545110
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
2285-2299Subventions
Organisme : Wellcome Trust
ID : 206194
Pays : United Kingdom
Commentaires et corrections
Type : CommentIn
Informations de copyright
© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.
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