8Y9M

Cas12h1(D465A)-crRNA-dsDNA ternary complex

Summary for 8Y9M

• Entry DOI: 10.2210/pdb8y9m/pdb
• EMDB information: 39083
• Descriptor: Cas12h1 (D465A) mutant, crRNA, DNA (29-mer), ... (4 entities in total)
• Functional Keywords: crispr-cas, type v cas effectors, immune system, immune system-rna-dna complex, immune system/rna/dna
• Biological source: unidentified; More
• Total number of polymer chains: 4
• Total formula weight: 137760.20

Authors

Zheng, W.W.,Liu, M.X. (deposition date: 2024-02-07, release date: 2025-02-12, Last modification date: 2025-03-19)

Primary citation

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

PubMed 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, 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.

PubMed: 39955288
DOI: 10.1038/s41392-025-02147-5

Experimental method

ELECTRON MICROSCOPY (2.76 Å)