RNA activation‐independent DNA targeting of the Type III CRISPR‐Cas system by a Csm complex

Abstract

The CRISPR-Cas system is an adaptive and heritable immune response that destroys invading foreign nucleic acids. The effector complex of the Type III CRISPR-Cas system targets RNA and DNA in a transcription-coupled manner, but the exact mechanism of DNA targeting by this complex remains elusive. In this study, an effector Csm holocomplex derived from is reconstituted with a minimalistic combination of Csm12345, and shows RNA targeting and RNA-activated single-stranded DNA (ssDNA) targeting activities. Unexpectedly, in the absence of an RNA transcript, it cleaves ssDNA containing a sequence complementary to the bound crRNA guide region in a manner dependent on the HD domain of the Csm1 subunit. This nuclease activity is blocked by a repeat tag found in the host CRISPR loci. The specific cleavage of ssDNA without a target RNA suggests a novel ssDNA targeting mechanism of the Type III system, which could facilitate the efficient and complete degradation of foreign nucleic acids.

Structure Studies of the CRISPR-Csm Complex Reveal Mechanism of Co-transcriptional Interference

“…Importantly, the stoichiometry of both Csm and Cmr complexes agrees with several high or medium resolution EM structures, including TtCmr complexes (Spilman et al, 2013;Staals et al, 2013;Taylor et al, 2015) and the Pyrococcus furiosus Cmr (PfCmr) complex (Ramia et al, 2014;Spilman et al, 2013), as well as the Cmr1-deficient chimeric Cmr-ssDNA complex (Osawa et al, 2015). In contrast, our stoichiometry does not agree with those obtained from low-resolution Csm complexes, including TthCsm complex (Staals et al, 2014), Sulfolobus solfataricus Csm (Rouillon et al, 2013), and Thermococcus onnurineus Csm (Park et al, 2017). Also, our stoichiometry differs from that for previously reported SthCsm, for which a stoichiometry was calculated on the basis of mass spectrometric data (Kazlauskiene et al, 2016).…”

Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase

“…2c, only CTR activated the LdCsm DNase, indicating that mismatches between the 3ʹ anti-tag of the target RNA and the corresponding 5ʹ tag of crRNA are essential for the activation. These results are in good agreement with those reported for other characterized type III CRISPR-Cas systems 15,17,19,20,31,34,35 . We further tested the minimal concentration of CTR is required for the ssDNase activity of LdCsm.…”

“…Biochemical and structural analyses of different type III CRISPR-Cas systems have revealed that the HD domain is responsible for ssDNA cleavage 15,19,20,31,34,35 , Palm1 and Palm2 domains function in cOA synthesis [38][39][40][41]43 whereas the Linker domain plays a regulatory role in both target RNA-activated DNA cleavage and cOA synthesis 54 . However, it remains unclear whether a concerted action of different Cas10 domains would be required to yield Csm DNase with optimal activity.…”

“…Type III CRISPR systems are unique because they exhibit both RNA interference and DNA interference in vivo to protect their microbial hosts against invading nucleic acids 4,[12][13][14][15][16][17][18][19][20][21][22][23] . Three activities are associated with these Type III immune systems, including target RNA cleavage 18,[24][25][26][27][28][29][30] , target RNAactivated indiscriminate single-stranded (ss) DNA cleavage, a secondary DNase activity 15,19,20,[31][32][33][34][35][36][37] and synthesis of cyclic oligoadenylate (cOA), a second messenger that activates RNases of Csm6/Csx1 families, mediating cell dormancy or cell death [38][39][40][41][42][43][44][45] . Activation of the immunity requires mismatches between the 3ʹ anti-tag of a cognate target RNA (CTR) and the 5ʹ tag of crRNA whereas the full match between the 3ʹ anti-tag of non-cognate target RNA (NTR) and the 5ʹ tag of crRNA completely represses the immune response (see reviews [46][47][48][49].…”

Reprogramming the endogenous type III-A CRISPR-Cas system for genome editing, RNA interference and CRISPRi screening inMycobacterium tuberculosis

“…The type III CRISPR-Cas system possess transcription dependent RNA and DNA cleavage capabilities, hence efficiently providing immunity against invading genomic elements in Staphylococcus epidermidis (Koonin and Makarova, 2013; Samai et al, 2015; Guo et al, 2019). The RNA cleavage is mediated by the Csm3/cmr4 or csm6/csx1 complex, while the DNA cleavage is catalyzed by Cas10 (Csm1) (Grüschow et al, 2019a; Park et al, 2017; Samai et al, 2015; Tamulaitis et al, 2017). ).…”