Molecular basis for DNA cleavage by the mini-RNA-guided endonuclease Casδ−1

CRISPR-Cas adaptive immune systems are powerful and versatile tools for genome engineering. CRISPR-Casδ−1 is a recently discovered, evolutionarily distinct endonuclease that enables versatile cross-kingdom genome editing under miniature RNA-guiding. In this study, we present a comprehensive biochemical and structural characterization of CRISPR-Casδ−1. Biochemical analysis not only verifies that Casδ−1 exhibits robust double-stranded DNA cleavage activity and target-dependent trans-cleavage activity, but also explores in depth its precise cleavage sites and substrate saturation. Furthermore, we determine a 3.04 Å resolution cryo-EM structure of the Casδ−1-crRNA-dsDNA ternary complex, which reveals the molecular basis of protospacer-adjacent motif (PAM) recognition, crRNA-DNA heteroduplex assembly, and RuvC domain-mediated DNA cleavage. Collectively, our study reveals the structural basis and evolutionary insights into Casδ−1, advances the mechanistic understanding of type V CRISPR-Cas effectors, and offers opportunities for the rational design of CRISPR-Casδ−1 as a versatile genome-editing tool. Casδ−1 is a recently discovered type V CRISPR endonuclease enabling miniature RNA-guided genome editing. In this study, the authors reveal its structural basis and evolutionary insights, advancing understanding of compact CRISPR systems.