RESUMO
The generation of cyclic oligoadenylates and subsequent allosteric activation of proteins that carry sensory domains is a distinctive feature of type III CRISPR-Cas systems. In this work, we characterize a set of associated genes of a type III-B system from Haliangium ochraceum that contains two caspase-like proteases, SAVED-CHAT and PCaspase (prokaryotic caspase), co-opted from a cyclic oligonucleotide-based antiphage signaling system (CBASS). Cyclic tri-adenosine monophosphate (AMP)-induced oligomerization of SAVED-CHAT activates proteolytic activity of the CHAT domains, which specifically cleave and activate PCaspase. Subsequently, activated PCaspase cleaves a multitude of proteins, which results in a strong interference phenotype in vivo in Escherichia coli. Taken together, our findings reveal how a CRISPR-Cas-based detection of a target RNA triggers a cascade of caspase-associated proteolytic activities.
Assuntos
Proteínas de Bactérias , Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Caspases , Myxococcales , Proteólise , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Caspases/química , Caspases/genética , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , RNA/metabolismo , Myxococcales/enzimologia , Myxococcales/genética , Domínios ProteicosRESUMO
CRISPR-Cas are prokaryotic adaptive immune systems. Cas nucleases generally use CRISPR-derived RNA guides to specifically bind and cleave DNA or RNA targets. Here, we describe the experimental characterization of a bacterial CRISPR effector protein Cas12m representing subtype V-M. Despite being less than half the size of Cas12a, Cas12m catalyzes auto-processing of a crRNA guide, recognizes a 5'-TTN' protospacer-adjacent motif (PAM), and stably binds a guide-complementary double-stranded DNA (dsDNA). Cas12m has a RuvC domain with a non-canonical catalytic site and accordingly is incapable of guide-dependent cleavage of target nucleic acids. Despite lacking target cleavage activity, the high binding affinity of Cas12m to dsDNA targets allows for interference as demonstrated by its ability to protect bacteria against invading plasmids through silencing invader transcription and/or replication. Based on these molecular features, we repurposed Cas12m by fusing it to a cytidine deaminase that resulted in base editing within a distinct window.
Assuntos
Proteínas Associadas a CRISPR , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA/genética , Plasmídeos , RNA , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3' end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5' end of the crRNA. Moreover, we uncover that the variation in the size and composition of type III complexes within a single host results in variable seed regions. This may prevent escape by invading genetic elements, while controlling cOA production tightly to prevent unnecessary damage to the host. Lastly, we use these findings to develop a new diagnostic tool, SCOPE, for the specific detection of SARS-CoV-2 from human nasal swab samples, revealing sensitivities in the atto-molar range.