Abstact

The type I-FHNH CRISPR-Cas system is a non-canonical Class 1 effector complex distinguished by the replacement of the Cas3 recruitment domain with a catalytic HNH domain in Cas8, enabling autonomous DNA cleavage without accessory nucleases. Using cryo-EM, we determined high-resolution structures of the effector complex in three catalytic states-precatalytic, NTS-cleaved, and post-catalytic-revealing a dynamic trajectory of the HNH domain through inward, middle, and outward conformations. Biochemical assays demonstrated that the complex cleaves the nontarget strand (NTS) prior to the target strand (TS), consistent with a sequential cleavage mechanism similar to Cas12 effectors but notably lacking trans-cleavage activity on single-stranded DNA. Structural comparisons confirmed a minimal PAM requirement (5'-CN) and a constrained HNH catalytic site poised for precise strand scission. We engineered a ΔLinker variant of Cas8 that repositions the HNH domain, selectively abolishing TS cleavage and converting the system into a programmable NTS-specific nickase. Importantly, we validated the functionality of both wild-type and mutant complexes in human cells. While the wild-type system induced indels and base substitutions, the ΔLinker variant triggered targeted single-strand nicks without double-stranded breaks. Together, our work establishes type I-FHNH as a compact and precise genome editing platform with in vivo efficacy.