摘要
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,Cas12h1hf,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.
基金
supported by the National Key Research and Development Program of China(2021YFC2301403)
National Natural Science Foundation of China grants(82225028 and 82172287)
Natural Science Foundation of Fujian Province of China(2022J01638).
