SNAP annotations: PDB entry 8y9l

PDB-id

8y9l; DSSR-derived features in text and JSON formats; DNAproDB

Class

immune system-RNA

Method

cryo-EM (3.0 Å)

Summary

Cas12h1-crrna binary complex

Reference

Zheng W, Li H, Liu M, Wei Y, Liu B, Li Z, Xiong C, Huang S, Hu C, Ouyang S (2025): "Molecular insights and rational engineering of a compact CRISPR-Cas effector Cas12h1 with a broad-spectrum PAM." Signal Transduct Target Ther, 10, 66. doi: 10.1038/s41392-025-02147-5.

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.