RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System

Abstract

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus) Type III-A Csm complex (TthCsm) with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA) by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (5′ tag) of the crRNA and the 3′ flanking region of the ssRNA. Mutation of the histidine-aspartate (HD) nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA) during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryo-electron microscopy and x-ray crystallography.

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].…”

Conformational regulation of CRISPR-associated nucleases

“…However, in several other systems, hybridization with the protospacer sequence alone is not sufficient for DNase activation. In these systems, the DNase remains inactive unless a non-complementary sequence extends from the protospacer into the 5′-repeat [72,73,75], and in some systems (e.g. Type III-B Cmr system in P. furiosus ) this 3′ non-complementary RNA sequence must meet specific sequence requirements [70].…”

A type III CRISPR ancillary ribonuclease degrades its cyclic oligoadenylate activator

“…Thus far, ring nucleases have only been identified in the crenarchaea, and, as highlighted by Mo and Marraffini [14], the enzyme(s) responsible for cOA degradation in bacteria remain unknown. The type III CRISPR system of the bacterium Thermus thermophilus HB8 has been investigated [15,16], and its CRISPR ancillary ribonuclease TTHB152 was among the first shown to be activated by cA4 [10]. The type III CRISPR locus of T. thermophilus also encodes an uncharacterised CARF domain-containing protein, TTHB144, which was reported to be Csm6-like [17].…”