A naturally DNase-free CRISPR-Cas12c enzyme silences gene expression

Used widely for genome editing in human cells, plants and animals, CRISPR-Cas enzymes including Cas9 and Cas12 provide RNA-guided immunity to microbes by targeting foreign DNA sequences for cleavage. We show here that the native activity of CRISPR-Cas12c protects bacteria from phage infection by binding to DNA targets without cleaving them, revealing that antiviral interference can be accomplished without chemical attack on the invader or general metabolic disruption in the host. Biochemical experiments demonstrate that Cas12c is a site-specific ribonuclease capable of generating mature CRISPR RNAs (crRNAs) from precursor transcripts. Furthermore, we find that crRNA maturation is essential for Cas12c-mediated DNA targeting. Surprisingly, however, these crRNAs direct double-stranded DNA binding by Cas12c using a mechanism that precludes DNA cutting. Cas12c’s RNA-guided DNA binding activity enables robust transcriptional repression of fluorescent reporter proteins in cells. Furthermore, this naturally DNase-free Cas12c enzyme can protect bacteria from lytic bacteriophage infection when targeting an essential phage gene. Together these results show that Cas12c employs targeted DNA binding to provide anti-viral immunity in bacteria, providing a native DNase-free pathway for transient antiviral immunity. ### Competing Interest Statement J.A.D. is a cofounder of Caribou Biosciences, Editas Medicine, Scribe Therapeutics, Intellia Therapeutics and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Vertex, Caribou Biosciences, Intellia Therapeutics, Scribe Therapeutics, Mammoth Biosciences, Algen Biotechnologies, Felix Biosciences, The Column Group and Inari. J.A.D. is a Director at Johnson & Johnson and Tempus and has research projects sponsored by Biogen, AppleTree Partners, and Roche.