The Miniature CRISPR-Cas12m Effector Binds DNA To Block Transcription

CRISPR-Cas are prokaryotic adaptive immune systems. Cas nucleases generally use RNA guides to specifically bind and cleave DNA or RNA targets. We here describe the experimental characterization of a bacterial CRISPR effector protein Cas12m representing a novel subtype V-M. Despite being less than half the size of Cas12a, Cas12m processes a pre-crRNA to generate mature crRNA guides. Cas12m recognizes a 5’-TTN’ protospacer-adjacent motif (PAM) and stably binds double-stranded DNA (dsDNA). Cas12m lacks a typical RuvC nuclease catalytic site and accordingly fails to cleave nucleic acids either specifically or collaterally. Despite lacking target cleavage activity, the high binding affinity of Cas12m to dsDNA targets allows for interference as demonstrated by its ability to protect bacteria against invading plasmids by down-regulating invader replication. Based on these molecular characteristics, we repurposed Cas12m by fusing it to a cytidine deaminase that resulted in base editing within a unique window.