The Card1 nuclease provides defence during type III CRISPR immunity

Abstract

During the prokaryotic type III CRISPR-Cas immune response, infection triggers the production of cyclic oligoadenylates, which bind and activate CARF domain-containing proteins 1 , 2 . Many type III loci are associated with proteins in which the CARF domain is fused to an endonuclease-like domain 3 , 4 ; however, with the exception of the well-characterized Csm6/Csx1 RNases 5 , 6 , whether and how these inducible effectors provide defense is not known. Here we investigated one of such type III CRISPR accessory proteins, Card1. Card1 forms a symmetrical dimer with a large central cavity between its CARF and restriction endonuclease (REase) domains that binds cA 4 . Ligand binding results in a conformational change where individual monomers rotate relative to each other to form a more compact dimeric scaffold wherein a Mn cation coordinates to the catalytic residues and activates the cleavage of single, but not double, stranded nucleic acids (DNA and RNA). In vivo , Card1 activation induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used by CRISPR systems to provide immunity.

Accelerated RNA detection using tandem CRISPR nucleases

“…Furthermore, we show that this assay can be implemented on a compact detector and thus would be amenable for use in point-of-care diagnostics. FIND-IT demonstrates the value of stabilizing Csm6 nuclease activation and deploying it in tandem with RNA-cleaving CRISPR-Cas proteins for fast, sensitive RNA detection in a simple, amplification-free format [33][34][35][36] . This technology could enable practical on-site detection of viral or human RNA in clinical samples or plant, fungal or microbial RNA in environmental samples.…”

Molecular mechanisms of the CdnG-Cap5 antiphage defense system employing 3’,2’-cGAMP as the second messenger

“…Structures of effectors in complex with some of these signaling molecules are available. They include eukaryotic and bacterial STING 10,26 , bacterial endonuclease 17 , and CRISPR-associated RNA and DNA nucleases [27][28][29][30] . All of them employ one surface where the ligand-binding pocket locates to recognize the signaling molecules.…”

Type III CRISPR-Cas Systems: Deciphering the Most Complex Prokaryotic Immune System

“…This cOA-activated Card1 protein from the Type III-A locus of Treponema succinifaciens , exhibits nuclease activity towards both single-stranded DNA and RNA in vitro . However, Card1 activation in S. aureus cells did not produce detectable changes in the transcriptome [ 50 ], suggesting that its RNase activity is either inhibited in vivo or is highly specific and does not cause massive transcript degradation. Expression of Card1 compensates for the lack of the csm6 gene in the Type III-A system of S. epidermidis and restores the ability of the cells to resist phage infection.…”

“…Expression of Card1 compensates for the lack of the csm6 gene in the Type III-A system of S. epidermidis and restores the ability of the cells to resist phage infection. However, cells harboring Cas10 gene with mutated catalytic residues of the HD domain could not clear the infection by a phage even in the presence of Card1 [ 50 ], suggesting that its single-strand DNase activity is not sufficient for interference. The clearance of the targeted plasmid from the cells required the activities of both Cas10 HD and Card1, which suggests that the Cas10 HD nuclease activity is specific towards the protospacer-containing DNA [ 50 ].…”