A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction

Abstract

The CRISPR (clustered regularly interspaced short palindromic repeats) system protects archaea and bacteria by eliminating nucleic acid invaders in a crRNA-guided manner. The Sulfolobus islandicus type III-B Cmr–α system targets invading nucleic acid at both RNA and DNA levels and DNA targeting relies on the directional transcription of the protospacer in vivo. To gain further insight into the involved mechanism, we purified a native effector complex of III-B Cmr–α from S. islandicus and characterized it in vitro. Cmr–α cleaved RNAs complementary to crRNA present in the complex and its ssDNA destruction activity was activated by target RNA. The ssDNA cleavage required mismatches between the 5΄-tag of crRNA and the 3΄-flanking region of target RNA. An invader plasmid assay showed that mutation either in the histidine-aspartate acid (HD) domain (a quadruple mutation) or in the GGDD motif of the Cmr–2α protein resulted in attenuation of the DNA interference in vivo. However, double mutation of the HD motif only abolished the DNase activity in vitro. Furthermore, the activated Cmr–α binary complex functioned as a highly active DNase to destroy a large excess DNA substrate, which could provide a powerful means to rapidly degrade replicating viral DNA.

Conformational regulation of CRISPR-associated nucleases

“…RNA binding drives a conformational rearrangement that allosterically activates non-sequence-specific DNase activity in the Cas10 subunit [69–75]. The RNA target is cleaved in regular 6-nt intervals and structures of Type III complexes have identified residues in the backbone (Cas7-family) and belly subunits that are necessary for RNA cleavage [41,68,76].…”

“…Complementarity between the crRNA and the RNA target that extends beyond the guide and into the 5′-repeat of the crRNA inhibits DNase activation in all Type III systems [70–74,75,77], but requirements for DNase activation seem to vary between systems. In Thermotoga maritima , the DNase function is activated by protospacers lacking flanking sequence, suggesting complementation between the crRNA-guide and RNA target is the only signal necessary for DNase activation and base pairing with the 5′-repeat is inhibitory [71].…”

“…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].…”

Broad Targeting Specificity during Bacterial Type III CRISPR-Cas Immunity Constrains Viral Escape

“…The type III Cas10 effector complex uses the crRNA guide to interact with a complementary sequence in the target transcript, an interaction that triggers both the DNase and RNase activities of the complex (Elmore et al, 2016; Estrella et al, 2016; Kazlauskiene et al, 2016), and results in the degradation of both the viral target DNA and its transcripts (Samai et al, 2015). Analysis of the sequence requirements for type III targeting has shown that only mutations that create homology between flanking and repeat sequence upstream of the protospacer and spacer, respectively, can abrogate immunity (Han et al, 2017; Kazlauskiene et al, 2016; Marraffini and Sontheimer, 2010; Samai et al, 2015; Zebec et al, 2014). These results have argued against the presence of a PAM for type III targeting.…”

The CRISPR Spacer Space Is Dominated by Sequences from Species-Specific Mobilomes

“…The mature crRNA consists of a unique spacer of 25 to 65 bp in length (depending on the CRISPR-Cas type and subtype) flanked by portions of the adjacent repeats. In the third and final stage, interference, the effector Cas protein complex mediates recognition of the target DNA or RNA via base-pairing between the spacer and cognate protospacer, followed by cleavage of the target by Cas nucleases (19)(20)(21)(22)(23)(24).…”