CRISPR-Cas9-induced double-strand breaks disrupt maintenance of epigenetic information

Dec 2, 2025Genome biology

CRISPR-Cas9 DNA cuts may disrupt the preservation of gene regulation signals

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Abstract

Cas9-induced DNA cause significant changes in patterns in human embryonic stem cells.

Changes in DNA methylation occur at target sites due to mechanisms like homologous recombination and large structural variations.
These epigenetic alterations can happen with or without accompanying genetic changes.
Cas9-induced double-strand breaks also disrupt DNA methylation patterns in colorectal cancer cells and in hypermethylated regions of human embryonic stem cells.
Abnormal methylation changes are stable during in vitro passaging of cells.
Methylation alterations are observed around endogenous deletions in unedited genomic regions, indicating a broader impact of double-strand break repair.

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BACKGROUND: CRISPR-Cas9 genome editing enables precise genetic modifications by introducing targeted DNA (DSBs). While Cas9-induced DSBs are known to cause unintended on-target mutations, their impact on the epigenetic landscape remains unexplored.

RESULTS: Here, we investigate how Cas9-induced DSBs affect patterns in human embryonic stem cells (hESCs). We induce DSBs at differentially methylated regions of imprinted genomic loci and perform high-coverage, long-read native DNA sequencing to simultaneously obtain genetic variant and base-resolution methylation data in a haplotype-resolved manner. Our findings reveal that DSBs cause significant changes in DNA methylation at target sites through mechanisms including homologous recombination, large structural variations, or defective methylation maintenance during DNA repair. Notably, these epigenetic changes can occur either together with or independently of genetic alterations. Beyond imprinted loci, Cas9-induced DSBs significantly disrupt DNA methylation patterns of the MLH1 epimutation alleles in colorectal cancer cells, and hypermethylated heterochromatin loci in hESCs. Clonal analysis indicates that the aberrant methylation changes are stable during in vitro passaging. Intriguingly, significant changes in DNA methylation levels are also detected around endogenous deletions in unedited genomic regions, suggesting that methylation alterations are not unique to Cas9 nuclease activity but represent a general outcome of DSB repair in human cells.

CONCLUSIONS: This study underscores the importance of assessing and mitigating unintended epigenetic consequences in genome editing applications, as such changes can profoundly affect gene regulation and cellular function.

Key numbers

5mCpG 84.90%

Efficiency Change

efficiency at the SNRPN locus in Target-SNRPN sample vs. Non-target sample.

3,045×

Enrichment Fold

Enrichment efficiency achieved for the SNRPN locus.

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CRISPR edits cause unintended epigenetic changes

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CRISPR-Cas9-induced double-strand breaks disrupt maintenance of epigenetic information

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