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.

The structure of a Type III-A CRISPR-Cas effector complex reveals conserved and idiosyncratic contacts to target RNA and crRNA among Type III-A systems.

Type III CRISPR-Cas systems employ multiprotein effector complexes bound to small CRISPR RNAs (crRNAs) to detect foreign RNA transcripts and elicit a complex immune response that leads to the destruction of invading RNA and DNA. Type III systems are among the most widespread in nature, and emerging interest in harnessing these systems for biotechnology applications highlights the need for detailed structural analyses of representatives from diverse organisms. We performed cryo-EM reconstructions of the Type III-A Cas10-Csm effector complex from S. epidermidis bound to an intact, cognate target RNA and identified two oligomeric states, a 276 kDa complex and a 318 kDa complex. 3.1 Å density for the well-ordered 276 kDa complex allowed construction of atomic models for the Csm2, Csm3, Csm4 and Csm5 subunits within the complex along with the crRNA and target RNA. We also collected small-angle X-ray scattering data which was consistent with the 276 kDa Cas10-Csm architecture we identified. Detailed comparisons between the S. epidermidis Cas10-Csm structure and the well-resolved bacterial (S. thermophilus) and archaeal (T. onnurineus) Cas10-Csm structures reveal differences in how the complexes interact with target RNA and crRNA which are likely to have functional ramifications. These structural comparisons shed light on the unique features of Type III-A systems from diverse organisms and will assist in improving biotechnologies derived from Type III-A effector complexes.

Recurring and emerging themes in prokaryotic innate immunity.

A resurgence of interest in the pathways that bacteria use to protect against their viruses (i.e. phages) has led to the discovery of dozens of new antiphage defenses. Given the sheer abundance and diversity of phages - the ever-evolving targets of immunity - it is not surprising that these newly described defenses are also remarkably diverse. However, as their mechanisms slowly come into focus, some common strategies and themes are also beginning to emerge. This review highlights recurring and emerging themes in the mechanisms of innate immunity in bacteria and archaea, with an emphasis on recently described systems that have undergone more thorough mechanistic characterization.

Tandem mobilization of anti-phage defenses alongside SCCmec cassettes.

Bacterial viruses (phages) and the immune systems targeted against them significantly impact bacterial survival, evolution, and the emergence of pathogenic strains. While recent research has made spectacular strides towards discovering and validating new defenses in a few model organisms1-3, the inventory of immune systems in clinically-relevant bacteria remains underexplored, and little is known about the mechanisms by which these systems horizontally spread. Such pathways not only impact the evolutionary trajectory of bacterial pathogens, but also threaten to undermine the effectiveness of phage-based therapeutics. Here, we investigate the battery of defenses in staphylococci, opportunistic pathogens that constitute leading causes of antibiotic-resistant infections. We show that these organisms harbor a variety of anti-phage defenses encoded within/near the infamous SCC (staphylococcal cassette chromosome) mec cassettes, mobile genomic islands that confer methicillin resistance. Importantly, we demonstrate that SCCmec-encoded recombinases mobilize not only SCCmec, but also tandem cassettes enriched with diverse defenses. Further, we show that phage infection potentiates cassette mobilization. Taken together, our findings reveal that beyond spreading antibiotic resistance, SCCmec cassettes play a central role in disseminating anti-phage defenses. This work underscores the urgent need for developing adjunctive treatments that target this pathway to save the burgeoning phage therapeutics from suffering the same fate as conventional antibiotics.

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.

Microbial genome mining expedition unearths trove of antiviral defenses.

In this issue of Cell Host & Microbe, Millman et al. use bioinformatic and genetic approaches to discover 21 novel antiviral immune systems in prokaryotes. Remarkably, many of these systems bear homology to components of the human innate immune system, suggesting an evolutionary tie between prokaryotic and eukaryotic antiviral defenses.

Structure and host specificity of Staphylococcus epidermidis bacteriophage Andhra.

Staphylococcus epidermidis is an opportunistic pathogen of the human skin, often associated with infections of implanted medical devices. Staphylococcal picoviruses are a group of strictly lytic, short-tailed bacteriophages with compact genomes that are attractive candidates for therapeutic use. Here, we report the structure of the complete virion of S. epidermidis-infecting phage Andhra, determined using high-resolution cryo-electron microscopy, allowing atomic modeling of 11 capsid and tail proteins. The capsid is a T = 4 icosahedron containing a unique stabilizing capsid lining protein. The tail includes 12 trimers of a unique receptor binding protein (RBP), a lytic protein that also serves to anchor the RBPs to the tail stem, and a hexameric tail knob that acts as a gatekeeper for DNA ejection. Using structure prediction with AlphaFold, we identified the two proteins that comprise the tail tip heterooctamer. Our findings elucidate critical features for virion assembly, host recognition, and penetration.

A unique mode of nucleic acid immunity performed by a multifunctional bacterial enzyme.

The perpetual arms race between bacteria and their viruses (phages) has given rise to diverse immune systems, including restriction-modification and CRISPR-Cas, which sense and degrade phage-derived nucleic acids. These complex systems rely upon production and maintenance of multiple components to achieve antiphage defense. However, the prevalence and effectiveness of minimal, single-component systems that cleave DNA remain unknown. Here, we describe a unique mode of nucleic acid immunity mediated by a single enzyme with nuclease and helicase activities, herein referred to as Nhi (nuclease-helicase immunity). This enzyme provides robust protection against diverse staphylococcal phages and prevents phage DNA accumulation in cells stripped of all other known defenses. Our observations support a model in which Nhi targets and degrades phage-specific replication intermediates. Importantly, Nhi homologs are distributed in diverse bacteria and exhibit functional conservation, highlighting the versatility of such compact weapons as major players in antiphage defense.

Prophages self-destruct to eliminate competitors.

Bacteria have evolved many immune systems to combat their viral parasites (i.e., phages). In this issue of Cell Host & Microbe, Owen et al. discover a mechanism of anti-phage immunity that is mediated by a phage-encoded protein, and thus provide an example of how inter-phage conflict can promote survival of the bacterial population.

The phages of staphylococci: critical catalysts in health and disease.

The phages that infect Staphylococcus species are dominant residents of the skin microbiome that play critical roles in health and disease. While temperate phages, which can integrate into the host genome, have the potential to promote staphylococcal pathogenesis, the strictly lytic variety are powerful antimicrobials that are being exploited for therapeutic applications. This article reviews recent insights into the diversity of staphylococcal phages and newly described mechanisms by which they influence host pathogenicity. The latest efforts to harness these viruses to eradicate staphylococcal infections are also highlighted. Decades of research has focused on the temperate phages of Staphylococcus aureus as model systems, thus underscoring the need to broaden basic research efforts to include diverse phages that infect other clinically relevant Staphylococcus species.

Complete Genome Sequence of Staphylococcus aureus Siphophage Lorac.

Staphylococcus aureus is a leading cause of a wide range of clinical infections. Here, we announce the complete genome sequence of S. aureus siphophage Lorac, a phiETA-like temperate phage that is similar at the nucleotide level to the previously described S. aureus prophage phiNM2.

Complete Genome Sequences of Staphylococcus epidermidis Myophages Quidividi, Terranova, and Twillingate.

Staphylococcus epidermidis is an opportunistic pathogen that commonly colonizes human skin and mucous membranes. We report here the complete genome sequences of three S. epidermidis phages, Quidividi, Terranova, and Twillingate, which are members of the Twort-like group of large myophages infecting Gram-positive hosts.

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

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.

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.

Draft Genome Sequences of Staphylococcus Podophages JBug18, Pike, Pontiff, and Pabna.

We report here the draft genome sequences of Staphylococcus bacteriophages JBug18, Pike, Pontiff, and Pabna, which infect and lyse S. epidermidis and S. aureus strains. All bacteriophages belong to the morphological family Podoviridae and constitute attractive candidates for use as whole-phage therapeutics due to their compact genomes and lytic lifestyles.

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.

Phage Genetic Engineering Using CRISPR⁻Cas Systems.

Since their discovery over a decade ago, the class of prokaryotic immune systems known as CRISPR⁻Cas have afforded a suite of genetic tools that have revolutionized research in model organisms spanning all domains of life. CRISPR-mediated tools have also emerged for the natural targets of CRISPR⁻Cas immunity, the viruses that specifically infect bacteria, or phages. Despite their status as the most abundant biological entities on the planet, the majority of phage genes have unassigned functions. This reality underscores the need for robust genetic tools to study them. Recent reports have demonstrated that CRISPR⁻Cas systems, specifically the three major types (I, II, and III), can be harnessed to genetically engineer phages that infect diverse hosts. Here, the mechanisms of each of these systems, specific strategies used, and phage editing efficacies will be reviewed. Due to the relatively wide distribution of CRISPR⁻Cas systems across bacteria and archaea, it is anticipated that these immune systems will provide generally applicable tools that will advance the mechanistic understanding of prokaryotic viruses and accelerate the development of novel technologies based on these ubiquitous organisms.