Cas10 is the signature gene for type-III CRISPR-Cas surveillance complexes. Unlike type-I and type-II systems, type-III systems do not require a PAM, and target nascent RNA associated with transcriptionally active DNA. Further, target RNA recognition activates the cyclase domain of Cas10, resulting in the synthesis of cyclic oligoadenylate (cOA) second messengers. These second messengers are recognized by ancillary Cas proteins harboring CARF domains, and regulate the activities of these proteins in response to invading nucleic acid. Csx3 is a distant member of the CARF domain superfamily previously characterized as a Mn2+ dependent deadenylation exoribonuclease. However, its specific role in CRISPR-Cas defense remains to be determined. Here we show Csx3 is strongly associated with type-III systems and that Csx3 binds cyclic tetra-adenylate (cA4) second messenger with high affinity. Further, Csx3 harbors cyclic oligonucleotide phosphodiesterase activity that quickly degrades this cA4 signal.  In addition, structural analysis identifies core elements that define the CARF domain fold, and the mechanistic basis for ring nuclease activity are discussed. Overall, the work suggests Csx3 functions within CRISPR-Cas as a counter balance to Cas10 to regulate the duration and amplitude of the cA4 signal, providing an off-ramp from the programmed cell death pathway in cells that successfully cure viral infection.

Csa3 family transcription factors are ancillary CRISPR-associated proteins composed of N-terminal CARF domains and C-terminal winged helix-turn-helix domains. The activity of Csa3 transcription factors is thought to be controlled by cyclic oligoadenyate (cOA) second messengers produced by type III CRISPR-Cas surveillance complexes. Here we show that Saccharolobus solfataricus Csa3a recognizes cyclic tetra-adenylate (cA4) and that Csa3a lacks self-regulating “ring nuclease” activity present in some other CARF domain proteins. The crystal structure of the Csa3a/cA4 complex was also determined and the structural and thermodynamic basis for cA4 recognition are described, as are conformational changes in Csa3a associated with cA4 binding. We also characterized the effect of cA4 on recognition of putative DNA binding sites. Csa3a binds to putative promoter sequences in a nonspecific, cooperative and cA4-independent manner, suggesting a more complex mode of transcriptional regulation. We conclude the Csa3a/cA4 interaction represents a nexus between the type I and type III CRISPR-Cas systems present in S. solfataricus, and discuss the role of the Csa3/cA4 interaction in coordinating different arms of this integrated class 1 immune system to mount a synergistic, highly orchestrated immune response.

While there is extreme interest in CRISPR‐Cas gene editing technology, nature did not invent CRISPR‐Cas for this purpose. Rather, CRISPR‐Cas naturally serves as an adaptive immune system in single celled prokaryotic organisms, where it is found in more than 40% of Bacteria and 85% of Archaea. These systems are incredibly diverse, and classified by cas gene content into 2 major classes, six types and more than 20 different subtypes. In each case, however, they operate in the same three stage process; i) spacer acquisition, ii) crRNA expression and maturation and iii) target interference(Annu Rev Biochem, 82:237). The well‐known CRISPR‐Cas9 systems are class 2, type II systems and represent less than 10% of identified CRISPR‐Cas systems. Much more common are the Class 1 systems, especially type I and type III. Like class II systems, type I systems also target dsDNA in a PAM dependent process. Type III systems, however, do not require a PAM, and instead recognize nascent mRNAs emanating from transcriptionally active DNA. And upon recognition, they degrade both the RNA and DNA. Further, while complexed with the target RNA, the cyclase domain of the Cas10 subunit synthesizes cyclic oligo‐adenylate (cAn) signals (Science 357:605, Nature 548:543), which are in turn degraded by ring nucleases (Nature, 562:277). Sulfolobus solfataricus is a model archaeon utilizing both Type‐IA and Type‐IIIB CRISPR‐Cas systems. Structural studies suggest Csa3 is a transcription factor under the allosteric control of a 4‐base cyclic RNA signal that regulates expression of CRISPR‐Cas (J Mol Biol, 405:939, RNA Biol, 13:254). Here we show that Csa3 specifically recognizes a palindromic sequence present in the promoters for stage I acquisition genes (Cas1, Cas2, Csa1) and three CRISPR loci, that promoter recognition is enhanced by cyclic tetra‐adenylate (cA4), that over‐expression of Csa3 in S. solfataricus activates spacer acquisition, and that Csa3 lacks ring nuclease activity, suggesting long‐term potentiation of the cA4 signal. Further, we present the structure of Csa3 in complex with cA4, describe recognition of cA4 by Csa3 in atomic detail, and cA4 induced conformational changes that enhance promoter recognition. In all, this provides a molecular understanding for feedback activation of spacer acquisition and crRNA expression in cells struggling to clear viral infections. This feedback system is dependent on both type I Csa3 and type III Cas10, and thus represents a coordinated response by two different arms of CRISPR‐Cas. In this light, type I and type III CRISPR‐Cas in S. solfataricus are not independent systems, but instead represent two complementary arms of a coordinated anti‐viral response. Support or Funding Information Supported by National Science Foundation grant MCB‐1413534.

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