文章：

Cas9甲基化敏感编辑的分子基础

Molecular basis for methylation-sensitive editing by Cas9

原文发布日期：2026-04-15

DOI: 10.1038/s41586-026-10384-z

类型: Article

开放获取: 是

英文摘要：

The bacterial CRISPR–Cas9 (Cas9) nuclease has become a powerful genome manipulation tool for a wide range of organisms1,2,3. However, it has yet to fully leverage the pervasive presence of DNA methylation in genomes4,5,6,7,8,9,10. Here, to fill this gap, we report biochemical, structural and human genome-editing characterizations of a methylation-sensitive Cas9 (ThermoCas9). ThermoCas9 efficiently binds to and cleaves DNA upstream of its protospacer adjacent motif (PAM) 5′-NNNNCGA-3′ or 5′-NNNNCCA-3′ in vitro. Methylation of the fifth cytosine in either PAM sequence (5mCpG or 5mCpC), 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.

摘要翻译： 

细菌CRISPR–Cas9（Cas9）核酸酶已成为广泛应用于多种生物体的强大基因组操作工具[1,2,3]。然而，它尚未充分利用基因组中普遍存在的DNA甲基化修饰[4,5,6,7,8,9,10]。为填补这一空白，我们报道了一种甲基化敏感的Cas9（ThermoCas9）的生化、结构及人类基因组编辑特性。ThermoCas9在体外能高效结合并切割其前间隔序列邻近基序（PAM）5′-NNNNCGA-3′或5′-NNNNCCA-3′上游的DNA。然而，任一PAM序列中第五位胞嘧啶的甲基化（5mCpG或5mCpC）会显著抑制ThermoCas9的活性。通过冷冻电镜分别以2.8 Å和2.2 Å分辨率解析ThermoCas9在切割前和切割后的结构，揭示了PAM结合中严格依赖未甲基化胞嘧啶的分子基础，并为后续酶工程改造提供了指导。我们在具有不同DNA甲基化谱的人类细胞系中验证了ThermoCas9的甲基化敏感编辑能力。此外，我们证明催化增强型ThermoCas9能高效靶向乳腺癌患者中持续低甲基化的管腔表达特征基因。由于对DNA甲基化的敏感性，ThermoCas9可特异性靶向具有疾病相关低甲基化状态的细胞，为基因组编辑技术增添了另一重精准性。

原文链接：

Molecular basis for methylation-sensitive editing by Cas9