Structural basis for target DNA cleavage and guide RNA processing by CRISPR-Casλ2

RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Among the diverse CRISPR-Cas effectors, CRISPR-Casλ, a recently identified miniature type V effector encoded in phage genomes, has emerged as a promising candidate for genome editing due to its nuclease activity in mammalian and plant cells. However, the detailed molecular mechanisms of Casλ family of enzymes remain poorly understood. In this study, we report the identification and detailed biochemical and structural characterizations of CRISPR-Casλ2. The cryo-electron microscopy structures of Casλ2 in five different functional states unveiled the dynamic domain rearrangements during its activation. The structures revealed that, unlike other type V CRISPR-Cas effectors, the REC2 domain directly interacts with the substrate DNA within the RuvC active site to facilitate the target DNA cleavage. Our biochemical analyses indicated that Casλ2 processes its precursor crRNA to a mature crRNA using the RuvC active site through a unique ruler mechanism, in which Casλ2 defines the spacer length of the mature crRNA. Furthermore, structural comparisons of Casλ2 with Casλ1 and CasΦ highlighted the diversity and conservation of phage-encoded type V CRISPR-Cas enzymes. Collectively, our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and establish a framework for rational engineering of the CRISPR-Casλ-based genome-editing platform.