Bacterial abortive-infection systems limit the spread of foreign invaders by shutting down or killing infected cells before the invaders can replicate1,2. Several RNA-targeting CRISPR–Cas systems (that is, types III and VI) cause abortive-infection phenotypes by activating indiscriminate nucleases3–5. However, a CRISPR-mediated abortive mechanism that leverages indiscriminate DNase activity of an RNA-guided single-effector nuclease has yet to be observed. Here we report that RNA targeting by the type V single-effector nuclease Cas12a2 drives abortive infection through non-specific cleavage of double-stranded DNA (dsDNA). After recognizing an RNA target with an activating protospacer-flanking sequence, Cas12a2 efficiently degrades single-stranded RNA (ssRNA), single-stranded DNA (ssDNA) and dsDNA. Within cells, the activation of Cas12a2 induces an SOS DNA-damage response and impairs growth, preventing the dissemination of the invader. Finally, we harnessed the collateral activity of Cas12a2 for direct RNA detection, demonstrating that Cas12a2 can be repurposed as an RNA-guided RNA-targeting tool. These findings expand the known defensive abilities of CRISPR–Cas systems and create additional opportunities for CRISPR technologies.

CRISPR-associated DinG protein (CasDinG) is essential to type IV-A CRISPR function. However, the enzymatic activities of CasDinG are unknown. Here we demonstrate that CasDinG from Pseudomonas aeruginosa strain 83 is an ATP- and metal-dependent 5′-3′ DNA helicase. The crystal structure of CasDinG reveals a helicase core of two RecA-like domains with three accessory domains (N terminal, arch, and vestigial FeS). To examine the in vivo function of these CasDinG domains, we first identified the preferred PAM sequence (5′-GNAWN-3′ on the 5′-side of the target) with a plasmid library containing all combinations of the five nucleotides upstream of the target sequence. Plasmid clearance assays (using a 5′-GGAAA-3′ PAM) with CasDinG domain mutants demonstrated the vFeS and arch accessory domains are both essential for type IV immunity. These results provide a needed structural and biochemical framework for understanding the type IV-A CRISPR system.

Bacterial abortive infection systems limit the spread of foreign invaders by shutting down or killing infected cells before the invaders can replicate. Several RNA-targeting CRISPR-Cas systems (e.g., types III and VI) cause Abi phenotypes by activating indiscriminate RNases. However, a CRISPR-mediated abortive mechanism that relies on indiscriminate DNase activity has yet to be observed. Here we report that RNA targeting by the type V Cas12a2 nuclease drives abortive infection through non-specific cleavage of double-stranded (ds)DNA. Upon recognition of an RNA target with an activating protospacer-flanking sequence, Cas12a2 efficiently degrades single–stranded (ss)RNA, ssDNA, and dsDNA. Within cells, the dsDNase activity induces an SOS response and impairs growth, stemming the infection. Finally, we harnessed the collateral activity of Cas12a2 for direct RNA detection, demonstrating that Cas12a2 can be repurposed as an RNA-guided, RNA-targeting tool. These findings expand the known defensive capabilities of CRISPR-Cas systems and create additional opportunities for CRISPR technologies.

TereSa lorenz, uSFS Pacific northwest research Station, 3625 93rd ave. SW, olympia, Washington 98512PHil FiSCHer, 22620 u.S. Highway 12, naches, Washington 98937 aBSTraCT: Woodpeckers provide important ecological services by excavating nesting cavities that are used by many forest birds and other animals. Demand for nesting cavities by secondary cavity nesters can lead to intense competition for this limited resource. The Western Bluebird (Sialia mexicana) is known to usurp nests from its own and other species. However, the process by which bluebirds take over nests from woodpeckers larger than themselves has not been well documented. in order to understand this process, we analyzed 112 hours of video footage of nests of a Black-backed (Picoides arcticus) and a Hairy Woodpecker (P. villosus) located in the okanogan-Wenatchee national Forest in Washington. usurpation first involves a short period of physical confrontation followed by a prolonged period of constant presence around the nest. The male and female bluebirds cooperate by taking turns harassing the woodpecker and guarding the nest. This may be of concern to managers as the Black-backed Woodpecker is considered a species at risk in certain locations.