Functionally diverse type V CRISPR-Cas systems

Abstract

Additional, diverse CRISPR systems CRISPR systems have been revolutionizing molecular biology. Mining the metagenomic database, Yan et al. systematically discovered additional subtypes of type V CRISPR-Cas systems. The additional Cas12 effectors displayed a range of activities, including target and collateral cleavage of single-stranded RNA and DNA, as well as double-stranded DNA nicking and cleavage. These diverse nuclease activities suggest how an ancient transposase may have evolved into various type V effectors and expand the nucleic acid detection and genome-editing toolbox. Science, this issue p. 88 Functionalities of newly identified type V CRISPR-Cas12 effectors include RNA cleavage and double-stranded DNA nicking. Type V CRISPR-Cas systems are distinguished by a single RNA-guided RuvC domain-containing effector, Cas12. Although effectors of subtypes V-A (Cas12a) and V-B (Cas12b) have been studied in detail, the distinct domain architectures and diverged RuvC sequences of uncharacterized Cas12 proteins suggest unexplored functional diversity. Here, we identify and characterize Cas12c, -g, -h, and -i. Cas12c, -h, and -i demonstrate RNA-guided double-stranded DNA (dsDNA) interference activity. Cas12i exhibits markedly different efficiencies of CRISPR RNA spacer complementary and noncomplementary strand cleavage resulting in predominant dsDNA nicking. Cas12g is an RNA-guided ribonuclease (RNase) with collateral RNase and single-strand DNase activities. Our study reveals the functional diversity emerging along different routes of type V CRISPR-Cas evolution and expands the CRISPR toolbox.