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.

Bacterial abortive infection systems limit the spread of foreign invaders by shutting down or killing infected cells before the invaders can replicate[1][1], [2][2]. Several RNA-targeting CRISPR-Cas systems (e.g., types III and VI) cause Abi phenotypes by activating indiscriminate RNases[3][3]–[5][4]. 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.### Competing Interest StatementBenson Hill, O.D., R.N.J., and C.L.B. have filed provisional patent applications on the related concepts. G.C.N., M.B.B., and B.N.G. are employees of Benson Hill. C.L.B. is a co-founder of Locus Biosciences and is a scientific advisory board member of Benson Hill. The other authors declare no competing financial interests. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-5

CRISPR-Cas systems comprise diverse adaptive immune systems in prokaryotes whose RNA-directed nucleases have been co-opted for various technologies. Recent efforts have focused on expanding the number of known CRISPR-Cas subtypes to identify nucleases with novel properties. However, the functional diversity of nucleases within each subtype remains poorly explored. Here, we used cell-free transcription-translation systems and human cells to characterize six Cas12a single-effector nucleases from the V-A subtype, including nucleases sharing high sequence identity. While these nucleases readily utilized each other's guide RNAs, they exhibited distinct PAM profiles and apparent targeting activities that did not track based on phylogeny. In particular, two Cas12a nucleases encoded by Prevotella ihumii (PiCas12a) and Prevotella disiens (PdCas12a) shared over 95% amino-acid identity yet recognized distinct PAM profiles, with PiCas12a but not PdCas12a accommodating multiple G's in PAM positions -2 through -4 and T in position -1. Mutational analyses transitioning PiCas12a to PdCas12a resulted in PAM profiles distinct from either nuclease, allowing more flexible editing in human cells. Cas12a nucleases therefore can exhibit widely varying properties between otherwise related orthologs, suggesting selective pressure to diversify PAM recognition and supporting expansion of the CRISPR toolbox through ortholog mining and PAM engineering.

CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression-all without protein purification or live cells. We used TXTL to measure the dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength in E. coli, determine the specificities of 24 diverse anti-CRISPR proteins, and develop a fast and scalable screen for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses.

CRISPR-based genome editing is an enabling technology with potential to dramatically transform multiple industries. Identification of additional editing tools will be imperative for broad adoption and application of this technology. A novel Type V, Class 2 CRISPR nuclease system was identified from Microgenomates and Smithella bacterial species ( C RISPR from M icrogenomates and S mithella , Cms1). This system was shown to efficiently generate indel mutations in the major crop plant rice ( Oryza sativa ). Cms1 are distinct from other Type V nucleases, are smaller than most other CRISPR nucleases, do not require a tracrRNA, and have an AT-rich protospacer-adjacent motif site requirement. A total of four novel Cms1 nucleases across multiple bacterial species were shown to be functional in a eukaryotic system. This is a major expansion of the Type V CRISPR effector protein toolbox and increases the diversity of options available to researchers.

Extremely broad claims surrounding Cas9 nucleases have the potential to stifle innovation in the field of genome editing.

Precise genome editing of plants has the potential to reshape global agriculture through the targeted engineering of endogenous pathways or the introduction of new traits. To develop a CRISPR nuclease-based platform that would enable higher efficiencies of precise gene insertion or replacement, we screened the Cpf1 nucleases from Francisella novicida and Lachnospiraceae bacterium ND2006 for their capability to induce targeted gene insertion via homology directed repair. Both nucleases, in the presence of a guide RNA and repairing DNA template flanked by homology DNA fragments to the target site, were demonstrated to generate precise gene insertions as well as indel mutations at the target site in the rice genome. The frequency of targeted insertion for these Cpf1 nucleases, up to 8%, is higher than most other genome editing nucleases, indicative of its effective enzymatic chemistry. Further refinements and broad adoption of the Cpf1 genome editing technology have the potential to make a dramatic impact on plant biotechnology.