Browsing by Author "Walker, Forrest C."

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    Molecular determinants for CRISPR RNA maturation in the Cas10-Csm complex and roles for non-Cas nucleases
    (Oxford University Press, 2017) Walker, Forrest C.; Chou-Zheng, Lucy; Dunkle, Jack A.; Hatoum-Aslan, Asma; University of Alabama Tuscaloosa
    CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a prokaryotic immune system that destroys foreign nucleic acids in a sequence-specific manner using Cas nucleases guided by short RNAs (crRNAs). Staphylococcus epidermidis harbours a Type III-A CRISPR-Cas system that encodes the Cas10-Csm interference complex and crRNAs that are subjected to multiple processing steps. The final step, called maturation, involves a concerted effort between Csm3, a ruler protein in Cas10-Csm that measures six-nucleotide increments, and the activity of a nuclease(s) that remains unknown. Here, we elucidate the contributions of the Cas10-Csm complex toward maturation and explore roles of non-Cas nucleases in this process. Using genetic and biochemical approaches, we show that charged residues in Csm3 facilitate its self-assembly and dictate the extent of maturation cleavage. Additionally, acidic residues in Csm5 are required for efficient maturation, but recombinant Csm5 fails to cleave crRNAs in vitro. However, we detected cellular nucleases that co-purify with Cas10-Csm, and show that Csm5 regulates their activities through distinct mechanisms. Altogether, our results support roles for non-Cas nuclease(s) during crRNA maturation and establish a link between Type III-A CRISPR-Cas immunity and central nucleic acid metabolism.
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    Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex
    (Cold Spring Harbor Lab Press, 2019) Nasef, Mohamed; Muffly, Mary C.; Beckman, Andrew B.; Rowe, Sebastian J.; Walker, Forrest C.; Hatoum-Aslan, Asma; Dunkle, Jack A.; University of Alabama Tuscaloosa
    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 Mg2+-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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    Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10
    (American Chemical Society, 2017) Bari, S. M. Nayeemul; Walker, Forrest C.; Cater, Katie; Aslan, Barbaros; Hatoum-Aslan, Asma; University of Alabama Tuscaloosa
    Staphylococci are prevalent skin-dwelling bacteria that are also leading causes of antibiotic-resistant infections. Viruses that infect and lyse these organisms (virulent staphylococcal phages) can be used as alternatives to conventional antibiotics and represent promising tools to eliminate or manipulate specific species in the microbiome. However, since over half their genes have unknown functions, virulent staphylococcal phages carry inherent risk to cause unknown downstream side effects. Further, their swift and destructive reproductive cycle make them intractable by current genetic engineering techniques. CRISPR-Cas10 is an elaborate prokaryotic immune system that employs small RNAs and a multisubunit protein complex to detect and destroy phages and other foreign nucleic acids. Some staphylococci naturally possess CRISPR-Cas10 systems, thus providing an attractive tool already installed in the host chromosome to harness for phage genome engineering. However, the efficiency of CRISPR-Cas10 immunity against virulent staphylococcal phages and corresponding utility as a tool to facilitate their genome editing has not been explored. Here, we show that the CRISPR-Cas10 system native to Staphylococcus epidermidis exhibits robust immunity against diverse virulent staphylococcal phages. On the basis of this activity, a general two-step approach was developed to edit these phages that relies upon homologous recombination machinery encoded in the host. Variations of this approach to edit toxic phage genes and access phages that infect CRISPR-less staphylococci are also presented. This versatile set of genetic tools enables the systematic study of phage genes of unknown functions and the design of genetically defined phage-based antimicrobials that can eliminate or manipulate specific Staphylococcus species.