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

Abstract

<jats:title>Abstract</jats:title><jats:p>Antiviral defense by type III CRISPR-Cas systems relies on two distinct activities of their effectors: the RNA-activated DNA cleavage and synthesis of cyclic oligoadenylate. Both activities are featured as indiscriminate nucleic acid cleavage and subjected to the spatiotemporal regulation. To yield further insights into the involved mechanisms, we reconstituted LdCsm, a lactobacilli III-A system in <jats:italic>Escherichia coli</jats:italic>. Upon activation by target RNA, this immune system mediates robust DNA degradation but lacks the synthesis of cyclic oligoadenylates. Mutagenesis of the Csm3 and Cas10 conserved residues revealed that Csm3 and multiple structural domains in Cas10 function in the allosteric regulation to yield an active enzyme. Target RNAs carrying various truncations in the 3′ anti-tag were designed and tested for their influence on DNA binding and DNA cleavage of LdCsm. Three distinct ternary LdCsm complexes were identified. In particular, binding of target RNAs carrying a single nucleotide in the 3′ anti-tag to LdCsm yielded an active LdCsm DNase regardless whether the nucleotide shows a mismatch, as in the cognate target RNA (CTR), or a match in the noncognate target RNAs (NTR), to the 5’ tag of crRNA. In addition, further increasing the number of 3′ anti-tag in CTR facilitated the substrate binding and enhanced the substrate degradation whereas doing the same as in NTR gradually decreased the substrate binding and eventually shut off the DNA cleavage by the enzyme. Together, these results provide the mechanistic insights into the allosteric activation and repression of LdCsm enzymes.</jats:p>

DNA targeting by subtype I-D CRISPR–Cas shows type I and type III features

“…To construct the plasmid for the purification of I-D backbone complex from Sulfolobus , the csc1 gene encoding a C-terminal His-tagged Csc1 ( csc1-his ) was amplified from S. islandicus LAL14/1 genome and cloned into pEXA2 ( 17 ) via NdeI and NheI, giving rise to pEXA2-Csc1. Artificial CRISPR arrays with 11 (spacer 5–6) or 9 (spacer 4–17) repeat spacer units were generated by PCR as described ( 18 ) and fused to an arabinose promoter by overlap PCR and cloned into the plasmid pEXA2-Csc1 via Nhe I and Not I, yieldding pEXA2-Csc1-S5–6 and pEXA2-Csc1-S4–17, respectively. The gene encoding Cas3′ with a C-terminal His-tag was amplified from LAL14/1 genome by PCR and inserted into the E. coli expression plasmid pMAL-TEV (derived from pMAL-c5X, NEB) by overlap PCR, generating pMAL-TEV-Cas3′.…”

Identification of a Type IV-A CRISPR-Cas System Located Exclusively on IncHI1B/IncFIB Plasmids in Enterobacteriaceae

“…CRISPR-Cas systems are confirmed, or expected, to provide immunity against viruses and other mobile genetic elements (MGEs), except for transposon-encoded CRISPR-Cas systems that lack the interference module and therefore are predicted to perform functions distinct from adaptive immunity ( Makarova et al, 2020 ). Most of the CRISPR types target DNA, some types specifically target RNA such as Type VI, while Type III CRISPR systems are unique because they exhibit both RNA interference and DNA interference in vivo to protect their microbial hosts ( Marraffini and Sontheimer, 2008 , 2010b ; Hale et al, 2009 ; Deng et al, 2013 ; Manica et al, 2013 ; Goldberg et al, 2014 ; Tamulaitis et al, 2014 ; Zebec et al, 2014 ; Peng et al, 2015 ; Samai et al, 2015 ; Elmore et al, 2016 ; Estrella et al, 2016 ; Kazlauskiene et al, 2016 ; Zhang and Ye, 2017 ; Ozcan et al, 2019 ; Lin et al, 2020 ).…”

Remodeling of a tripartite substrate-binding motif in the HD domain provides the mechanism for activation of CRISPR-Cas10 DNases

“…Cells were harvested by centrifugation at 5000 rpm for 5 min, and cell pellets were resuspended in 50 ml buffer A (20 mM Tris-HCl, 0.25 M NaCl, 20 mM imidazole, 10% glycerol, pH8.5). The resulting cell suspension was treated by French press at 4 o C and cell debris was removed by centrifugation at 10,000 rpm for 1 h at 4 o C. LdCsm complexes were purified in a two-step purification procedure as previously described 31 .…”

“…More recently, we found that the Lactobacillus delbrueckii subsp. bulgaricus III-A (LdCsm) system, a unique CRISPR-Cas10 system, mainly relies on the DNase activity to mediate immune responses 31 . Upon activation, the LdCsm effector cleaves transcription bubbles in vitro and the immune system indiscriminately cleaves accessible DNA substrates in the host cells, and mediating antiviral defense via abortive infection 32 .…”