The New phytologistMay 7, 2022

DNA methylation can change how often CRISPR edits DNA and how the DNA is repaired, depending on the target

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Jan 13, 2026

Abstract

High levels of methylation decreased the frequency of Cas9-mediated mutations in Nicotiana benthamiana leaf cells.

Cytosine methylation levels impact the efficacy of in plants.
Only promoter methylation, not gene-body methylation, was associated with reduced mutation frequency.
influenced the ratio of insertions and deletions following Cas9 cuts.
Evidence for staggered DNA cuts by Cas9 in plant cells was observed, in addition to the known blunt-end breaks.
A novel mechanism for microhomology-mediated DNA repair may be involved in the repair process.

Simplified

The impact of epigenetic modifications on the efficacy of -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 methylation, but not gene-body methylation, decreased the frequency of Cas9-mediated mutations. 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.

Key numbers

2.3×

Decrease

Comparing mutation frequencies between high and low methylation contexts.

61%

/ Ratio Shift

Analyzing the distribution of mutation types at various target sites.

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DNA methylation can change how often CRISPR edits DNA and how the DNA is repaired, depending on the target

Abstract

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You found one interesting study. We’ll send the next 7.

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