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| Title | Molecular insights and rational engineering of a compact CRISPR-Cas effector Cas12h1 with a broad-spectrum PAM. |
|---|---|
| Journal, issue, pages | Signal Transduct Target Ther, Vol. 10, Issue 1, Page 66, Year 2025 |
| Publish date | Feb 12, 2025 |
 Authors | Weiwei Zheng / Hongyu Li / Mengxi Liu / Yuhang Wei / Bo Liu / Zekai Li / Chenyang Xiong / Shiqing Huang / Chunyi Hu / Songying Ouyang /   |
| 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 ...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. |
 External links | Signal Transduct Target Ther / PubMed:39955288 / PubMed Central |
| Methods | EM (single particle) |
| Resolution | 2.76 - 3.0 Å |
| Structure data | EMDB-39082, PDB-8y9l: EMDB-39083, PDB-8y9m: EMDB-39084, PDB-8y9n: |
| Chemicals |  ChemComp-MG: |
| Source |
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 Keywords | IMMUNE SYSTEM/RNA / Cas effector / IMMUNE SYSTEM / IMMUNE SYSTEM-RNA complex / IMMUNE SYSTEM/RNA/DNA / CRISPR-Cas / type V Cas effectors / IMMUNE SYSTEM-RNA-DNA complex / type V Cas effector |
