Targeting monogenetic liver diseases by CRISPR/Cas9

Class 2 type V CRISPR-Cas systems are characterized by a single effector nuclease which requires guide RNA for activity, making them attractive genome-editing tools. A distinct group of type V effector variants, termed Cms1, has recently been identified in metagenomic datasets. Their size and domain architecture set them apart from other known type V nucleases, suggesting likely functional differences. Many of the more than 40 predicted putative Cms1 nucleases exhibit editing activity in plants. However, catalytic properties and biotechnological potential of these effectors remain largely unexplored. In this study we characterized the SuCms1 nuclease identified in a metagenomic sequence of an uncultured chemolithoautotrophic epsilonproteobacterium, Sulfuricurvum spp., from the Frasassi Cave, Italy. We carried out our assessment of the fundamental properties of SuCms1 in Escherichia coli BL21-AI heterologous host. SuCms1 exhibited two nuclease activities, mediating CRISPR-RNA biogenesis and catalyzing RNA-dependent interference by cleaving dsDNA targets. This cleavage was dependent on the three RuvC domains, similar to Cas12a. However, unlike any know type V nuclease, SuCms1 exhibited a broad PAM preference, recognizing approximately half of the nucleotide combinations in a 5N library. Furthermore, the role of the -5 position, which is not part of the Cas12a consensus PAM, was context dependent in the case of SuCms1. Finally, we showed that known type V anti-CRISPR (AcrVA) proteins did not inhibit SuCms1. Particularly notable is the lack of inhibition by AcrVA5 which inactivates Cas12a by acetylating a conserved PAM recognition lysine residue also found in SuCms1. These data suggest that the PAM recognition mechanism of SuCms1 is entirely different from that of Cas12a, potentially relying on a combination of sequence identities and associated structural DNA features. This work helps establish SuCms1 as a unique variant among type V CRISPR-nucleases and informs efforts of adopting Cms1 effectors for biotechnological applications.