CRISPR-Cas technologies have provided programmable gene editing tools that have revolutionized research. The leading CRISPR-Cas9 and Cas12a enzymes are ideal for programmed genetic manipulation, however, they are limited for genome-scale interventions.Here, we utilize a Cas3-based system featuring a processive nuclease for genome engineering. This minimal Cascade-Cas3 system (Type I-C), programmed with a single crRNA, was optimized to generate deletions with near-100% efficiency, and used to rapidly generate large deletions ranging from 7 -424 kb in Pseudomonas aeruginosa. By comparison, Cas9 yielded small deletions and point mutations. Cas3-generated deletion boundaries were highly variable, but successfully specified by a homology-directed repair (HDR) template. HDR was much more efficient when lesions were generated by Cas3, compared to Cas9. The minimal Type I-C system

CRISPR-Cas adaptive immune systems protect bacteria and archaea against their invading genetic parasites, including bacteriophages/viruses and plasmids. In response to this immunity, many phages have anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas targeting. To date, anti-CRISPR genes have primarily been discovered in phage or prophage genomes. Here, we uncovered acr loci on plasmids and other conjugative elements present in Firmicutes , using the Listeria acrIIA1 gene as a marker. The four identified genes, found in Listeria, Enterococcus, Streptococcus, and Staphylococcus genomes, can inhibit Type II-A SpyCas9 or SauCas9, and are thus named acrIIA16 - 19 . In Enterococcus faecalis, conjugation of a Cas9-targeted plasmid was enhanced by anti-CRISPRs derived from Enterococcus conjugative elements, highlighting a role for Acrs in the dissemination of plasmids. Reciprocal co-immunoprecipitation showed that each Acr protein interacts with Cas9, and Cas9:Acr complexes were unable to cleave DNA. Northern blotting suggests that these anti-CRISPRs manipulate sgRNA loading or stability. Mirroring their activity in bacteria, AcrIIA16 and AcrIIA17 provide robust and highly potent broad-spectrum inhibition of distinct Cas9 proteins in human cells (e.g. SpyCas9, SauCas9, SthCas9, NmeCas9, CjeCas9). This work presents a focused analysis of non-phage Acr proteins, demonstrating a role in horizontal gene transfer bolstered by broad spectrum CRISPR-Cas9 inhibition.

Bacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy foreign DNA. Bacteriophages, in turn, have evolved diverse "anti-CRISPR" proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode 2-3 distinct anti-Cas9 proteins, with acrIIA1 always present; however, its mechanism is unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain and, in Listeria, triggers Cas9 degradation. AcrIIA1 displays broad-spectrum inhibition of Type II-A and II-C Cas9s, including an additional highly-diverged Listeria Cas9. During lytic infection, AcrIIA1 is insufficient for rapid Cas9 inactivation, thus phages require an additional "partner" Acr that rapidly blocks Cas9-DNA-binding. The AcrIIA1 N-terminal domain (AcrIIA1 NTD ) is dispensable for anti-CRISPR activity; instead it is required for optimal phage replication through direct transcriptional repression of the anti-CRISPR locus. AcrIIA1 NTD is widespread amongst Firmicutes, can repress anti-CRISPR deployment by other phages, and has been co-opted by hosts potentially as an "anti-anti-CRISPR." In summary, Listeria phages utilize narrow-spectrum inhibitors of DNA binding to rapidly inactivate Cas9 in lytic growth and the broad-spectrum AcrIIA1 to stimulate Cas9 degradation for protection of the Listeria genome in lysogeny.

Highlights d Listeria anti-CRISPR protein AcrIIA1 serves as an anti-CRISPR and a vital autorepressor d The strong early acr promoter must be repressed for maximal phage fitness d AcrIIA1 allows prophages to tune Acr expression to Cas9 levels d AcrIIA1 homologs have been co-opted by host bacteria as ''anti-anti-CRISPRs''

SUMMARYBacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy foreign DNA. Bacteriophages, in turn, have evolved diverse “anti-CRISPR” proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode 2-3 distinct anti-Cas9 proteins, with acrIIA1 always present; however, its mechanism is unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain and, in Listeria, triggers Cas9 degradation. AcrIIA1 displays broad-spectrum inhibition of Type II-A and II-C Cas9s, including an additional highly-diverged Listeria Cas9. During lytic infection, AcrIIA1 is insufficient for rapid Cas9 inactivation, thus phages require an additional “partner” Acr that rapidly blocks Cas9-DNA-binding. The AcrIIA1 N-terminal domain (AcrIIA1NTD) is dispensable for anti-CRISPR activity; instead it is required for optimal phage replication through direct transcriptional repression of the anti-CRISPR locus. AcrIIA1NTD is widespread amongst Firmicutes, can repress anti-CRISPR deployment by other phages, and has been co-opted by hosts potentially as an “anti-anti-CRISPR.” In summary, Listeria phages utilize narrow-spectrum inhibitors of DNA binding to rapidly inactivate Cas9 in lytic growth and the broad-spectrum AcrIIA1 to stimulate Cas9 degradation for protection of the Listeria genome in lysogeny.