Molecular basis for methylation-sensitive editing by Cas9

CpC), however, significantly inhibits ThermoCas9 activity. Cryo-electron microscopy structures of ThermoCas9 in pre-cleavage and post-cleavage states at 2.8 Å and 2.2 Å resolution, respectively, reveal the molecular basis for the stringent requirement of the unmethylated cytosine in PAM binding and provide guidance for further enzyme engineering. We demonstrate methylation-sensitive editing by ThermoCas9 in human cell lines with distinct DNA methylation landscapes. Moreover, we demonstrate that a catalytically enhanced ThermoCas9 efficiently targets luminal expression signature genes that are consistently hypomethylated in patients with breast cancer. Owing to its sensitivity to DNA methylation, ThermoCas9 can specifically target cells with disease-related hypomethylation, which adds another layer of precision to genome-editing technologies.

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Publication Details

Journal
Nature
Published
2026-04-15
DOI
https://doi.org/10.1038/s41586-026-10384-z
Primary Topic
CRISPR and Genetic Engineering
Type
article
Field-Weighted Citation Impact
0.00
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article

Molecular basis for methylation-sensitive editing by Cas9

John van der Oost, Despoina Trasanidou, Mitchell O. Roth, Renee D. Hoffman et al.
Nature
CRISPR and Genetic Engineering
article

Molecular basis for methylation-sensitive editing by Cas9

John van der Oost, Despoina Trasanidou, Mitchell O. Roth, Renee D. Hoffman, Eugenios Bouzetos, Anuska Das, Nikolaos Trasanidis, Jay Rai, Michael Zawrotny, Christian Südfeld, Yuerong Shu, Megan Medina, Bing Wang, Hong Li, Yu Zhao, Mary K. Gelasco, Hemant N. Goswami
article en

Abstract

CpC), however, significantly inhibits ThermoCas9 activity. Cryo-electron microscopy structures of ThermoCas9 in pre-cleavage and post-cleavage states at 2.8 Å and 2.2 Å resolution, respectively, reveal the molecular basis for the stringent requirement of the unmethylated cytosine in PAM binding and provide guidance for further enzyme engineering. We demonstrate methylation-sensitive editing by ThermoCas9 in human cell lines with distinct DNA methylation landscapes. Moreover, we demonstrate that a catalytically enhanced ThermoCas9 efficiently targets luminal expression signature genes that are consistently hypomethylated in patients with breast cancer. Owing to its sensitivity to DNA methylation, ThermoCas9 can specifically target cells with disease-related hypomethylation, which adds another layer of precision to genome-editing technologies.

Nature
Florida State University (US), St. Jude Children's Research Hospital (US), Van Andel Institute (US), Sanquin (NL), Thermo Fisher Scientific (United States) (US), Centre for Inflammation Research (GB), Wageningen University & Research (NL)
Openalex Percentile: Top 17%
CRISPR and Genetic Engineering
DOIOpen Access PDFOpenAlex
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Molecular basis for methylation-sensitive editing by Cas9 — John van der Oost, Despoina Trasanidou, et al. · Nature (2026) | TGRS Research Map | TGRS