DNA methylation can alter CRISPR/Cas9 editing frequency and DNA repair outcome in a target-specific manner

Adéla Přibylová, Lukáš Fischer, Douglas Pyott, Andrew Bassett, Attila Molnar

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

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.

Original languageEnglish
Pages (from-to)2285-2299
Number of pages15
JournalNew Phytologist
Issue number6
Early online date6 May 2022
Publication statusPublished - 1 Sept 2022

Keywords / Materials (for Non-textual outputs)

  • Cas9
  • chromatin
  • DNA methylation
  • microhomology-medi ated
  • DNA repair
  • NGS
  • NHEJ
  • staggered-end


Dive into the research topics of 'DNA methylation can alter CRISPR/Cas9 editing frequency and DNA repair outcome in a target-specific manner'. Together they form a unique fingerprint.

Cite this