Expression and Purification of the Cas10-Csm Complex from Staphylococci

Chou-Zheng, Lucy; Hatoum-Aslan, Asma

doi:10.21769/bioprotoc.2353

Cited by 10 publications

(6 citation statements)

References 25 publications

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“…Cas10-Csm expression and immobilized metal affinity chromatography were performed as reported by Chou-Zheng and Hatoum-Aslan (2017). Briefly, overnight cultures of S. epidermidis LM1680 carrying the plasmids pcrispr or pcrispr/csm3 D32A were used to inoculate 1 L of brain heart infusion media containing appropriate antibiotics.…”

Section: Purification Of Cas10-csmmentioning

confidence: 99%

Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex

Nasef

Muffly

Beckman

et al. 2019

RNA

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CRISPR-Cas systems are a class of adaptive immune systems in prokaryotes that use small CRISPR RNAs (crRNAs) in conjunction with CRISPR-associated (Cas) nucleases to recognize and degrade foreign nucleic acids. Recent studies have revealed that Type III CRISPR-Cas systems synthesize second messenger molecules previously unknown to exist in prokaryotes, cyclic oligoadenylates (cOA). These molecules activate the Csm6 nuclease to promote RNA degradation and may also coordinate additional cellular responses to foreign nucleic acids. Although cOA production has been reconstituted and characterized for a few bacterial and archaeal Type III systems, cOA generation and its regulation have not been explored for the Staphylococcus epidermidis Type III-A CRISPR-Cas system, a longstanding model for CRISPR-Cas function. Here, we demonstrate that this system performs Mg 2+-dependent synthesis of 3-6 nt cOA. We show that activation of cOA synthesis is perturbed by single nucleotide mismatches between the crRNA and target RNA at discrete positions, and that synthesis is antagonized by Csm3-mediated target RNA cleavage. Altogether, our results establish the requirements for cOA production in a model Type III CRISPR-Cas system and suggest a natural mechanism to dampen immunity once the foreign RNA is destroyed.

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Section: Purification Of Cas10-csmmentioning

confidence: 99%

Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex

Nasef

Muffly

Beckman

et al. 2019

RNA

Self Cite

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“…Cas10-Csm complexes containing a 6-His tag on the N-terminus of Csm2 or Csm5 in pcrispr-cas-based plasmids were overexpressed in S. epidermidis LM1680 cells, harvested, and stored exactly as described in Chou-Zheng and Hatoum-Aslan, 2017 . Final pellets were purified following the first affinity chromatography protocol (Ni 2+ -affinity chromatography) with slight modifications.…”

Section: Methodsmentioning

confidence: 99%

Critical roles for ‘housekeeping’ nucleases in type III CRISPR-Cas immunity

Chou-Zheng

Hatoum-Aslan

2022

eLife

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CRISPR-Cas systems are a family of adaptive immune systems that use small CRISPR RNAs (crRNAs) and CRISPR-associated (Cas) nucleases to protect prokaryotes from invading plasmids and viruses (i.e. phages). Type III systems launch a multi-layered immune response that relies upon both Cas and non-Cas cellular nucleases, and although the functions of Cas components have been well described, the identities and roles of non-Cas participants remain poorly understood. Previously, we showed that the Type III-A CRISPR-Cas system in Staphylococcus epidermidis employs two degradosome-associated nucleases, PNPase and RNase J2, to promote crRNA maturation and eliminate invading nucleic acids (Chou-Zheng and Hatoum-Aslan, 2019). Here, we identify RNase R as a third 'housekeeping' nuclease critical for immunity. We show that RNase R works in concert with PNPase to complete crRNA maturation, and identify specific interactions with Csm5, a member of the Type III effector complex, which facilitate nuclease recruitment/stimulation. Further, we demonstrate that RNase R and PNPase are required to maintain robust anti-plasmid immunity, particularly when targeted transcripts are sparse. Altogether, our findings expand the known repertoire of accessory nucleases required for Type III immunity and highlight the remarkable capacity of these systems to interface with diverse cellular pathways to ensure successful defense.

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“…Cas10-Csm expression and immobilized metal affinity chromatography (IMAC) were performed as previously described [ 36 ]. Briefly, overnight cultures of S. epidermidis LM1680, which carry the plasmid p crispr/csm3 D32A but lack a genomic crispr locus, were used to inoculate 1 L of Brain Heart Infusion media containing appropriate antibiotics.…”

Section: Methodsmentioning

confidence: 99%

The effect of crRNA–target mismatches on cOA-mediated interference by a type III-A CRISPR-Cas system

2022

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CRISPR systems elicit interference when a foreign nucleic acid is detected by its ability to base-pair to crRNA. Understanding what degree of complementarity between a foreign nucleic acid and crRNA is required for interference is a central question in the study of CRISPR systems. A clear description of which target–crRNA mismatches abrogate interference in type III, Cas10-containing, CRISPR systems has proved elusive due to the complexity of the system which utilizes three distinct interference activities. We characterized the effect of target–crRNA mismatches on in vitro cyclic oligoadenylate (cOA) synthesis and in vivo in an interference assay that depends on cOA synthesis. We found that sequence context affected whether a mismatched target was recognized by crRNA both in vitro and in vivo. We also investigated how the position of a mismatch within the target–crRNA duplex affected recognition by crRNA. Our data provide support for the hypothesis that a Cas10-activating region exists in the crRNA–target duplex, that the Cas10-proximal region of the duplex is the most critical in regulating cOA synthesis. Understanding the rules governing target recognition by type III CRISPR systems is critical: as one of the most prevalent CRISPR systems in nature, it plays an important role in the survival of many genera of bacteria. Recently, type III systems were re-purposed as a sensitive and accurate molecular diagnostic tool. Understanding the rules of target recognition in this system will be critical as it is engineered for biotechnology purposes.

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Expression and Purification of the Cas10-Csm Complex from Staphylococci

Cited by 10 publications

References 25 publications

Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex

Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex

Critical roles for ‘housekeeping’ nucleases in type III CRISPR-Cas immunity

The effect of crRNA–target mismatches on cOA-mediated interference by a type III-A CRISPR-Cas system

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