The diverse arsenal of type III CRISPR-Cas-associated CARF and SAVED effectors

Abstract

Type III CRISPR-Cas systems make use of a multi-subunit effector complex to target foreign (m)RNA transcripts complementary to the guide/CRISPR RNA (crRNA). Base-pairing of the target RNA with specialized regions in the crRNA not only triggers target RNA cleavage, but also activates the characteristic Cas10 subunit and sets in motion a variety of catalytic activities that starts with the production of cyclic oligoadenylate (cOA) second messenger molecules. These messenger molecules can activate an extensive arsenal of ancillary effector proteins carrying the appropriate sensory domain. Notably, the CARF and SAVED effector proteins have been responsible for renewed interest in type III CRISPR-Cas due to the extraordinary diversity of defenses against invading genetic elements. Whereas only a handful of CARF and SAVED proteins have been studied so far, many of them seem to provoke abortive infection, aimed to kill the host and provide population-wide immunity. A defining feature of these effector proteins is the variety of in silico-predicted catalytic domains they are fused to. In this mini-review, we discuss all currently characterized type III-associated CARF and SAVED effector proteins, highlight a few examples of predicted CARF and SAVED proteins with interesting predicted catalytic activities, and speculate how they could contribute to type III immunity.

Keywords: CRISPR; abortive infection; antiviral defense; cOA; nuclease; signaling.

Figure 1.. Mechanism of type III CRISPR-Cas immunity.

(A) Overview of type III effector complex formation. Expression of the cas genes and processing of the CRISPR array. Repeats and spacers are indicated by blue diamonds and gray rectangles, respectively. Following endonucleolytic cleavages of Cas6, a variable 3′ end processing step of the crRNAs leads to a heterogeneous complex size population. (B) Biological context of type III CRISPR–Cas systems. A transcription bubble is formed when MGE-derived dsDNA is transcribed by an RNA polymerase into (m)RNA, which is subsequently targeted by type III CRISPR–Cas. (C) Target RNA requirements for the various activities of type III. Note that RNA cleavage only relies on complementarity in the seed region, whereas ssDNase and cyclase (cOA production) activity requires additional strict base-pairing in the CAR and no base-pairing interactions with the 5′ handle.

Figure 2.. Schematic illustration of the activities of characterized type III-associated, cOA-activatable effectors.

(A) Csm6 and Csx1 homodimers bind cOA, stabilizing them into an active form where the HEPN domains catalyze ssRNA degradation. (B) Can1 occurs as monomers with two CARF domains, a nuclease-like and nuclease domain. Upon cOA binding, the nuclease-like and nuclease domain form a composite active site that catalyzes dsDNA nicking. (C) Can2 homodimers bind cOA and shift to an active form where the nuclease domains form a composite active site that catalyzes dsDNA nicking, ssRNA and ssDNA degradation. (D) Lon-SAVED is initially bound to CRISPR-T, and upon cOA binding, cleaves CRISPR-T into CRISPR-T₂₃ and CRISPR-T₁₀. CRISPR-T₂₃ then proceeds to degrade a yet unknown nucleic acid target. (E) TIR-SAVED forms superhelical structures upon cOA₃ binding, forming multiple composite NADase active sites for NAD⁺ degradation. (F) NucC homotrimers bind cOA₃, causing conformational changes that promote homohexamer formation, and forming dsDNA cleavage sites across the two homotrimers.

Figure 3.. Schematic illustration of the anticipated activities of bioinformatically-predicted type III-associated, cOA-activatable effectors.

(A) CARF or SAVED proteins with promiscuous nuclease activity, cleaving both self- and non-self nucleic acids. (B) CARF or SAVED proteins with DNA binding domains could enhance or repress downstream effector genes. (C) CARF or SAVED proteins with transmembrane domains could form pores that depolarize the membrane, depriving the cell of energy. Alternative strategies to disrupt the membrane could be employed too. (D) CARF proteins with predicted adenosine deaminase domains converting ATP into Inosine triphosphate (ITP), depleting cellular ATP levels. (E) CARF or SAVED proteins with a fused Lon protease domain liberating a toxin that kills the cell.