Cas12a2 elicits abortive infection through RNA-triggered destruction of dsDNA

Abstract

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.

RNA targeting unleashes indiscriminate nuclease activity of CRISPR–Cas12a2

“…In Cas12a, this region is highly sensitive to mismatches because it initiates R-loop formation following recognition of the protospacer adjacent motif (PAM) and is considered a seed region 16 . By contrast, Cas12a2 is insensitive to single mismatches within the entirety of the crRNA but has reduced in vivo activity when truncated on the 3′ end 1 . Our structure suggests that Cas12a2-crRNA-target strand (TS) duplex formation may initiate and propagate from the 3′ end of the crRNA, enabling Cas12a2 to target phage that have escaped surveillance by Cas12a through mutagenesis of the PAM or 5′ seed regions.…”

“…Plasmid curing assays were conducted as described previously 1 . In short, immune system plasmids were prepared with Cas12a2 and a 3× CRISPR repeat and transformed into BL21 AI cells by heat-shock transformation.…”

Inactive long prokaryotic Argonaute systems employ various effectors to confer immunity via abortive infection

“…Genomic DNA degradation are performed by some defense systems as an immune strategy [29][30][31] , and potentially by more systems that employ nuclease effectors. In particular, a few type III CRISPR-Cas systems with NucC nucleases as effectors and type V-A2 CRISPR-Cas systems cleave genomic DNA after crRNA-directed target RNA recognition 29,30 .…”

“…Genomic DNA degradation are performed by some defense systems as an immune strategy [29][30][31] , and potentially by more systems that employ nuclease effectors. In particular, a few type III CRISPR-Cas systems with NucC nucleases as effectors and type V-A2 CRISPR-Cas systems cleave genomic DNA after crRNA-directed target RNA recognition 29,30 . This suggests that a subgroup of pAgo and CRISPR-Cas, the two nucleic acid-directed defense systems, have convergently evolved to adopt the genomic DNA degradation Abi strategy.…”

“…Relying on these associated effectors, the long-B pAgos mediate Abi responses to provide immunoprotection against invading plasmid. In particular, the long-B pAgo-nuclease system performs RNA-guided unspeci c DNA degradation following recognition of a speci c DNA target, indicating that both CRISPR-Cas systems and pAgo systems can utilize the target recognition-activated collateral DNA degradation as a defense strategy [29][30][31][32] . Our ndings also provide novel opportunities to develop pAgo-based technologies.…”