Molecular insights and rational engineering of a compact CRISPR-Cas effector Cas12h1 with a broad-spectrum PAM

作者：Weiwei Zheng, Hongyu Li, Mengxi Liu, Yuhang Wei, Bo Liu, Zekai Li, Chenyang Xiong, Shiqing Huang, Chunyi Hu, Songying Ouyang · 发表于：Signal Transduction and Targeted Therapy · 年份：2025 · DOI：10.1038/s41392-025-02147-5 · 被引用次数：6 · 研究领域：CRISPR and Genetic Engineering、Plant Virus Research Studies、Insect symbiosis and bacterial influences

Abstract Cas12h1 is a compact CRISPR-associated nuclease from functionally diverse type V CRISPR-Cas effectors and recognizes a purine-rich protospacer adjacent motif (PAM) distinct from that of other type V Cas effectors. Here, we report the nickase preference of Cas12h1, which predominantly cleaves the nontarget strand (NTS) of a double-stranded DNA (dsDNA) substrate. In addition, Cas12h1 acts as a nickase in human cells. We further determined the cryo-EM structures of Cas12h1 in the surveillance, R-loop formation, and interference states, revealing the molecular mechanisms involved in the crRNA maturation, target recognition, R-loop formation, nuclease activation and target degradation. Cas12h1 notably recognizes a broad 5’-DHR-3’ PAM (D is A, G, or T; H is A, C, or T; R is A or G) both in vitro and in human cells. In addition, Cas12h1 utilizes a distinct activation mechanism that the lid motif undergoes a “flexible to stable” transition to expose the catalytic site to the substrate. A high-fidelity nucleic acid detector, Cas12h1 hf , was developed through rational engineering, which distinguishes single-base mismatches and retains comparable on-target activities. Our results shed light on the molecular mechanisms underlying Cas12h1 nickase, improve the understanding of type V Cas effectors, and expand the CRISPR toolbox for genome editing and molecular diagnosis.