Harnessing the LdCsm RNA Detection Platform for Efficient microRNA Detection.

In cancer diagnosis, diverse microRNAs (miRNAs) are used as biomarkers for carcinogenesis of distinctive human cancers. Thus, the detection of these miRNAs and their quantification are very important in prevention of cancer diseases in human beings. However, efficient RNA detection often requires RT-PCR, which is very complex for miRNAs. Recently, the development of CRISPR-based nucleic acid detection tools has brought new promises to efficient miRNA detection. Three CRISPR systems can be explored for miRNA detection, including type III, V, and VI, among which type III (CRISPR-Cas10) systems have a unique property as they recognize RNA directly and cleave DNA collaterally. In particular, a unique type III-A Csm system encoded by Lactobacillus delbrueckii subsp. bulgaricus (LdCsm) exhibits robust target RNA-activated DNase activity, which makes it a promising candidate for developing efficient miRNA diagnostic tools. Herein, LdCsm was tested for RNA detection using fluorescence-quenched DNA reporters. We found that the system is capable of specific detection of miR-155, a microRNA implicated in the carcinogenesis of human breast cancer. The RNA detection system was then improved by various approaches including assay conditions and modification of the 5'-repeat tag of LdCsm crRNAs. Due to its robustness, the resulting LdCsm detection platform has the potential to be further developed as a better point-of-care miRNA diagnostics relative to other CRISPR-based RNA detection tools.

Genetic Engineering of Therapeutic Phages Using Type III CRISPR-Cas Systems.

The functional characterization of "hypothetical" phage genes is a major bottleneck in basic and applied phage research. To compound this issue, the most suitable phages for therapeutic applications-the strictly lytic variety-are largely recalcitrant to classical genetic techniques due to low recombination rates and lack of selectable markers. Here we describe methods for fast and effective phage engineering that rely upon a Type III-A CRISPR-Cas system. In these methods, the CRISPR-Cas system is used as a powerful counterselection tool to isolate rare phage recombinants.

Critical roles for 'housekeeping' nucleases in type III CRISPR-Cas immunity.

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 multilayered 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. Furthermore, 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.

A type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity.

CRISPR-Cas systems provide sequence-specific immunity against phages and mobile genetic elements using CRISPR-associated nucleases guided by short CRISPR RNAs (crRNAs). Type III systems exhibit a robust immune response that can lead to the extinction of a phage population, a feat coordinated by a multi-subunit effector complex that destroys invading DNA and RNA. Here, we demonstrate that a model type III system in Staphylococcus epidermidis relies upon the activities of two degradosome-associated nucleases, PNPase and RNase J2, to mount a successful defense. Genetic, molecular, and biochemical analyses reveal that PNPase promotes crRNA maturation, and both nucleases are required for efficient clearance of phage-derived nucleic acids. Furthermore, functional assays show that RNase J2 is essential for immunity against diverse mobile genetic elements originating from plasmid and phage. Altogether, our observations reveal the evolution of a critical collaboration between two nucleic acid degrading machines which ensures cell survival when faced with phage attack.

CRISPR-Cas10 assisted editing of virulent staphylococcal phages.

Phages are the most abundant entities in the biosphere and profoundly impact the bacterial populations within and around us. They attach to a specific host, inject their DNA, hijack the host's cellular processes, and replicate exponentially while destroying the host. Historically, phages have been exploited as powerful antimicrobials, and phage-derived proteins have constituted the basis for numerous biotechnological applications. Only in recent years have metagenomic studies revealed that phage genomes harbor a rich reservoir of genetic diversity, which might afford further therapeutic and/or biotechnological value. Nevertheless, functions for the majority of phage genes remain unknown, and due to their swift and destructive replication cycle, many phages are intractable by current genetic engineering techniques. Whether to advance the basic understanding of phage biology or to tap into their potential applications, efficient methods for phage genetic engineering are needed. Recent reports have shown that CRISPR-Cas systems, a class of prokaryotic immune systems that protect against phage infection, can be harnessed to engineer diverse phages. In this chapter, we describe methods to genetically manipulate virulent phages using CRISPR-Cas10, a Type III-A CRISPR-Cas system native to Staphylococcus epidermidis. A method for engineering phages that infect a CRISPR-less Staphylococcus aureus host is also described. Both approaches have proved successful in isolating desired phage mutants with 100% efficiency, demonstrating that CRISPR-Cas10 constitutes a powerful tool for phage genetic engineering. The relatively widespread presence of Type III CRISPR-Cas systems in bacteria and archaea imply that similar strategies may be used to manipulate the genomes of diverse prokaryotic viruses.

Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10.

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.