Functional characterization of the CRISPR-Cas Immune Systems of Pyrococcus furiosus and Thermococcus kodakarensis
Elmore, Joshua Ryan
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There are many CRISPR-Cas immune systems that use diverse mechanisms to silence invasive genetic elements in prokaryotes. CRISPR-Cas effector complexes are guided by CRISPR (cr)RNAs, containing sequences captured from invader nucleic acids, to silence invaders via nucleic acid degradation by immune system-specific Cas proteins. The work presented here demonstrates that the CRISPR-Cas system of the hyperthermophilic archaeon Thermococcus kodakarensis is active, silences invader DNA and can be programmed to target user-designated “invader” sequences. This work also indicates that three distinct CRISPR-Cas immune systems - Type I-A, Type I-G, and Type III-B, in the hyperthermophilic archaeon Pyrococcus furiosus function independently, and utilize distinct mechanisms to silence invader DNA. Previously, we identified the components of the three effector complexes in P. furiosus; however, the mechanisms these effector complexes use to effect invader silencing were unknown. Using genetically engineered single effector strains, we characterized the invader silencing functionality of all three systems in vivo. Plasmid infection assays revealed that the Type I-A and Type I-G systems require a crRNA target-adjacent sequence (PAM) to effect invader DNA silencing. Furthermore, silencing by each Type I system specifically requires its cognate Cas3 protein. The Type III-B system was previously demonstrated to cleave target RNAs. Here, our results demonstrate that the Type III-B system also silences plasmids by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Furthermore, we found that the target RNA induces DNA cleavage by the Cmr complex in vitro. The DNA cleavage activity of the Cmr complex does not depend on cleavage of the target RNA, but does require a PAM sequence within the activating target RNA. The activated complex does not require a target sequence in the DNA substrate. Unlike other studied Type III systems, plasmid elimination by the Pfu Cmr system does not require the Csx1/CARF family protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2/Cas10 protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a protospacer adjacent motif (rPAM).