CRISPR-based engineering of phages for in situ bacterial base editing.

Investigation of microbial gene function is essential to the elucidation of ecological roles and complex genetic interactions that take place in microbial communities. While microbiome studies have increased in prevalence, the lack of viable in situ editing strategies impedes experimental progress, rendering genetic knowledge and manipulation of microbial communities largely inaccessible. Here, we demonstrate the utility of phage-delivered CRISPR-Cas payloads to perform targeted genetic manipulation within a community context, deploying a fabricated ecosystem (EcoFAB) as an analog for the soil microbiome. First, we detail the engineering of two classical phages for community editing using recombination to replace nonessential genes through Cas9-based selection. We show efficient engineering of T7, then demonstrate the expression of antibiotic resistance and fluorescent genes from an engineered λ prophage within an Escherichia coli host. Next, we modify λ to express an APOBEC-1-based cytosine base editor (CBE), which we leverage to perform C-to-T point mutations guided by a modified Cas9 containing only a single active nucleolytic domain (nCas9). We strategically introduce these base substitutions to create premature stop codons in-frame, inactivating both chromosomal (lacZ) and plasmid-encoded genes (mCherry and ampicillin resistance) without perturbation of the surrounding genomic regions. Furthermore, using a multigenera synthetic soil community, we employ phage-assisted base editing to induce host-specific phenotypic alterations in a community context both in vitro and within the EcoFAB, observing editing efficiencies from 10 to 28% across the bacterial population. The concurrent use of a synthetic microbial community, soil matrix, and EcoFAB device provides a controlled and reproducible model to more closely approximate in situ editing of the soil microbiome.

Phage Milagro: a platform for engineering a broad host range virulent phage for Burkholderia.

IMPORTANCE: Burkholderia infections are a significant concern in people with CF and other immunocompromising disorders, and are difficult to treat with conventional antibiotics due to their inherent drug resistance. Bacteriophages, or bacterial viruses, are now seen as a potential alternative therapy for these infections, but most of the naturally occurring phages are temperate and have narrow host ranges, which limit their utility as therapeutics. Here we describe the temperate Burkholderia phage Milagro and our efforts to engineer this phage into a potential therapeutic by expanding the phage host range and selecting for phage mutants that are strictly virulent. This approach may be used to generate new therapeutic agents for treating intractable infections in CF patients.

Making the leap from technique to treatment - genetic engineering is paving the way for more efficient phage therapy.

Bacteriophages (phages) are viruses specific to bacteria that target them with great efficiency and specificity. Phages were first studied for their antibacterial potential in the early twentieth century; however, their use was largely eclipsed by the popularity of antibiotics. Given the surge of antimicrobial-resistant strains worldwide, there has been a renaissance in harnessing phages as therapeutics once more. One of the key advantages of phages is their amenability to modification, allowing the generation of numerous derivatives optimised for specific functions depending on the modification. These enhanced derivatives could display higher infectivity, expanded host range or greater affinity to human tissues, where some bacterial species exert their pathogenesis. Despite this, there has been a noticeable discrepancy between the generation of derivatives in vitro and their clinical application in vivo. In most instances, phage therapy is only used on a compassionate-use basis, where all other treatment options have been exhausted. A lack of clinical trials and numerous regulatory hurdles hamper the progress of phage therapy and in turn, the engineered variants, in becoming widely used in the clinic. In this review, we outline the various types of modifications enacted upon phages and how these modifications contribute to their enhanced bactericidal function compared with wild-type phages. We also discuss the nascent progress of genetically modified phages in clinical trials along with the current issues these are confronted with, to validate it as a therapy in the clinic.

Phage-based delivery systems: engineering, applications, and challenges in nanomedicines.

Bacteriophages (phages) represent a unique category of viruses with a remarkable ability to selectively infect host bacteria, characterized by their assembly from proteins and nucleic acids. Leveraging their exceptional biological properties and modifiable characteristics, phages emerge as innovative, safe, and efficient delivery vectors. The potential drawbacks associated with conventional nanocarriers in the realms of drug and gene delivery include a lack of cell-specific targeting, cytotoxicity, and diminished in vivo transfection efficiency. In contrast, engineered phages, when employed as cargo delivery vectors, hold the promise to surmount these limitations and attain enhanced delivery efficacy. This review comprehensively outlines current strategies for the engineering of phages, delineates the principal types of phages utilized as nanocarriers in drug and gene delivery, and explores the application of phage-based delivery systems in disease therapy. Additionally, an incisive analysis is provided, critically examining the challenges confronted by phage-based delivery systems within the domain of nanotechnology. The primary objective of this article is to furnish a theoretical reference that contributes to the reasoned design and development of potent phage-based delivery systems.

Bacteriophage genome engineering for phage therapy to combat bacterial antimicrobial resistance as an alternative to antibiotics.

Bacteriophages (phages) are viruses that mainly infect bacteria and are ubiquitously distributed in nature, especially to their host. Phage engineering involves nucleic acids manipulation of phage genome for antimicrobial activity directed against pathogens through the applications of molecular biology techniques such as synthetic biology methods, homologous recombination, CRISPY-BRED and CRISPY-BRIP recombineering, rebooting phage-based engineering, and targeted nucleases including CRISPR/Cas9, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Management of bacteria is widely achieved using antibiotics whose mechanism of action has been shown to target both the genetic dogma and the metabolism of pathogens. However, the overuse of antibiotics has caused the emergence of multidrug-resistant (MDR) bacteria which account for nearly 5 million deaths as of 2019 thereby posing threats to the public health sector, particularly by 2050. Lytic phages have drawn attention as a strong alternative to antibiotics owing to the promising efficacy and safety of phage therapy in various models in vivo and human studies. Therefore, harnessing phage genome engineering methods, particularly CRISPR/Cas9 to overcome the limitations such as phage narrow host range, phage resistance or any potential eukaryotic immune response for phage-based enzymes/proteins therapy may designate phage therapy as a strong alternative to antibiotics for combatting bacterial antimicrobial resistance (AMR). Here, the current trends and progress in phage genome engineering techniques and phage therapy are reviewed.

Cross-Genus "Boot-Up" of Synthetic Bacteriophage in Staphylococcus aureus by Using a New and Efficient DNA Transformation Method.

Staphylococcus aureus is an opportunistic pathogen that causes a wide range of infections and food poisoning in humans with antibiotic resistance, specifically to methicillin, compounding the problem. Bacteriophages (phages) provide an alternative treatment strategy, but these only infect a limited number of circulating strains and may quickly become ineffective due to bacterial resistance. To overcome these obstacles, engineered phages have been proposed, but new methods are needed for the efficient transformation of large DNA molecules into S. aureus to "boot-up" (i.e., rescue) infectious phages. We presented a new, efficient, and reproducible DNA transformation method, NEST (non-electroporation Staphylococcus transformation), for S. aureus to boot-up purified phage genomic DNA (at least 150 kb in length) and whole yeast-assembled synthetic phage genomes. This method was a powerful new tool for the transformation of DNA in S. aureus and will enable the rapid development of engineered therapeutic phages and phage cocktails against Gram-positive pathogens. IMPORTANCE The continued emergence of antibiotic-resistant bacterial pathogens has heightened the urgency for alternative antibacterial strategies. Phages provide an alternative treatment strategy but are difficult to optimize. Synthetic biology approaches have been successfully used to construct and rescue genomes of model phages but only in a limited number of highly transformable host species. In this study, we used a new, reproducible, and efficient transformation method to reconstitute a functional nonmodel Siphophage from a constructed synthetic genome. This method will facilitate the engineering of Staphylococcus and Enterococcus phages for therapeutic applications and the engineering of Staphylococcus strains by enabling transformation of higher molecular weight DNA to introduce more complex modifications.

Engineered Reporter Phages for Rapid Bioluminescence-Based Detection and Differentiation of Viable Listeria Cells.

The pathogen Listeria monocytogenes causes listeriosis, a severe foodborne disease associated with high mortality. Rapid and sensitive methods are required for specific detection of this pathogen during food production. Bioluminescence-based reporter bacteriophages are genetically engineered viruses that infect their host cells with high specificity and transduce a heterologous luciferase gene whose activity can be detected with high sensitivity to indicate the presence of viable target cells. Here, we use synthetic biology for de novo genome assembly and activation as well as CRISPR-Cas-assisted phage engineering to construct a set of reporter phages for the detection and differentiation of viable Listeria cells. Based on a single phage backbone, we compare the performance of four reporter phages that encode different crustacean, cnidarian, and bacterial luciferases. From this panel of reporter proteins, nanoluciferase (NLuc) was identified as a superior enzyme and was subsequently introduced into the genomes of a broad host range phage (A511) and two serovar 1/2- and serovar 4b/6a-specific Listeria phages (A006 and A500, respectively). The broad-range NLuc-based phage A511::nlucCPS detects one CFU of L. monocytogenes in 25 g of artificially contaminated milk, cold cuts, and lettuce within less than 24 h. In addition, this reporter phage successfully detected Listeria spp. in potentially contaminated natural food samples without producing false-positive or false-negative results. Finally, A006::nluc and A500::nluc enable serovar-specific Listeria diagnostics. In conclusion, these NLuc-based reporter phages enable rapid, ultrasensitive detection and differentiation of viable Listeria cells using a simple protocol that is 72 h faster than culture-dependent approaches.IMPORTANCE Culture-dependent methods are the gold standard for sensitive and specific detection of pathogenic bacteria within the food production chain. In contrast to molecular approaches, these methods detect viable cells, which is a key advantage for foods generated from heat-inactivated source material. However, culture-based diagnostics are typically much slower than molecular or proteomic strategies. Reporter phage assays combine the best of both worlds and allow for near online assessment of microbial safety because phage replication is extremely fast, highly target specific, and restricted to metabolically active host cells. In addition, reporter phage assays are inexpensive and do not require highly trained personnel, facilitating their on-site implementation. The reporter phages presented in this study not only allow for rapid detection but also enable an early estimation of the potential virulence of Listeria isolates from food production and processing sites.

Engineering microbes for targeted strikes against human pathogens.

Lack of pathogen specificity in antimicrobial therapy causes non-discriminant microbial cell killing that disrupts the microflora present. As a result, potentially helpful microbial cells are killed along with the pathogen, altering the biodiversity and dynamic interactions within the population. Moreover, the unwarranted exposure of antibiotics to microbes increases the likelihood of developing resistance and perpetuates the emergence of multidrug resistance. Synthetic biology offers an alternative solution where specificity can be conferred to reduce the non-specific, non-targeted activity of currently available antibiotics, and instead provides targeted therapy against specific pathogens and minimising collateral damage to the host's inherent microbiota. With a greater understanding of the microbiome and the available genetic engineering tools for microbial cells, it is possible to devise antimicrobial strategies for novel antimicrobial therapy that are able to precisely and selectively remove infectious pathogens. Herein, we review the strategies developed by unlocking some of the natural mechanisms used by the microbes and how these may be utilised in targeted antimicrobial therapy, with the promise of reducing the current global bane of multidrug antimicrobial resistance.

Current status of clinical trials for phage therapy.

Recently, bacteriophages have been considered alternatives to antibacterial treatments. Infectious diseases continue to plague the world because bacteria can adapt and develop defence mechanisms against antibiotics. The growing incidence of antibiotic-resistant bacterial infections necessitated the development of new techniques for treating bacterial infections worldwide. Clinical trials have shown efficiency against antibiotic-resistant bacteria. However, scientists in future clinical trials should scrutinize phage resistance implications, assess combination strategies with antimicrobial agents and address challenges in phage therapy delivery for effective implementation.

Engineered bacteriophages: A panacea against pathogenic and drug resistant bacteria.

Antimicrobial resistance (AMR) is a major global concern; antibiotics and other regular treatment methods have failed to overcome the increasing number of infectious diseases. Bacteriophages (phages) are viruses that specifically target/kill bacterial hosts without affecting other human microbiome. Phage therapy provides optimism in the current global healthcare scenario with a long history of its applications in humans that has now reached various clinical trials. Phages in clinical trials have specific requirements of being exclusively lytic, free from toxic genes with an enhanced host range that adds an advantage to this requisite. This review explains in detail the various phage engineering methods and their potential applications in therapy. To make phages more efficient, engineering has been attempted using techniques like conventional homologous recombination, Bacteriophage Recombineering of Electroporated DNA (BRED), clustered regularly interspaced short palindromic repeats (CRISPR)-Cas, CRISPY-BRED/Bacteriophage Recombineering with Infectious Particles (BRIP), chemically accelerated viral evolution (CAVE), and phage genome rebooting. Phages are administered in cocktail form in combination with antibiotics, vaccines, and purified proteins, such as endolysins. Thus, phage therapy is proving to be a better alternative for treating life-threatening infections, with more specificity and fewer detrimental consequences.

Phages to the rescue: in situ editing of the gut microbiota.

The gut microbiome contains numerous bacteria tied to our health. However, genetically modifying this community remains a major challenge. Brödel et al. take a critical step by engineering bacteriophages to efficiently deliver gene editors without propagation of the genetic cargo, efficiently introducing edits to bacteria residing in the mouse gut.

Engineered phage with cell-penetrating peptides for intracellular bacterial infections.

IMPORTANCE: Salmonella infection is a significant threat to global public health, and the increasing prevalence of antibiotic resistance exacerbates the situation. Therefore, finding new and effective ways to combat this pathogen is essential. Phages are natural predators of bacteria and can be used as an alternative to antibiotics to kill specific bacteria, including drug-resistant strains. One significant limitation of using phages as antimicrobial agents is their low cellular uptake, which limits their effectiveness against intracellular bacterial infections. Therefore, finding ways to enhance phage uptake is crucial. Our study provides a straightforward strategy for displaying cell-penetrating peptides on non-model phages, offering a promising novel and effective therapeutic approach for treating intracellular and drug-resistant bacteria. This approach has the potential to address the global challenge of antibiotic resistance and improve public health outcomes.

The Biotechnological Application of Bacteriophages: What to Do and Where to Go in the Middle of the Post-Antibiotic Era.

Amid the escalating challenges of antibiotic resistance, bacterial infections have emerged as a global threat. Bacteriophages (phages), viral entities capable of selectively infecting bacteria, are gaining momentum as promising alternatives to traditional antibiotics. Their distinctive attributes, including host specificity, inherent self-amplification, and potential synergy with antibiotics, render them compelling candidates. Phage engineering, a burgeoning discipline, involves the strategic modification of bacteriophages to enhance their therapeutic potential and broaden their applications. The integration of CRISPR-Cas systems facilitates precise genetic modifications, enabling phages to serve as carriers of functional genes/proteins, thereby enhancing diagnostics, drug delivery, and therapy. Phage engineering holds promise in transforming precision medicine, addressing antibiotic resistance, and advancing diverse applications. Emphasizing the profound therapeutic potential of phages, this review underscores their pivotal role in combatting bacterial diseases and highlights their significance in the post-antibiotic era.

Approaches for bacteriophage genome engineering.

In recent years, bacteriophage research has been boosted by a rising interest in using phage therapy to treat antibiotic-resistant bacterial infections. In addition, there is a desire to use phages and their unique proteins for specific biocontrol applications and diagnostics. However, the ability to manipulate phage genomes to understand and control gene functions, or alter phage properties such as host range, has remained challenging due to a lack of universal selectable markers. Here, we discuss the state-of-the-art techniques to engineer and select desired phage genomes using advances in cell-free methodologies and clustered regularly interspaced short palindromic repeats-CRISPR associated protein (CRISPR-Cas) counter-selection approaches.

Recombinant Reporter Phage rTUN1::nLuc Enables Rapid Detection and Real-Time Antibiotic Susceptibility Testing of Klebsiella pneumoniae K64 Strains.

The emergence of multi-drug-resistant Klebsiella pneumoniae (Kp) strains constitutes an enormous threat to global health as multi-drug resistance-associated treatment failure causes high mortality rates in nosocomial infections. Rapid pathogen detection and antibiotic resistance screening are therefore crucial for successful therapy and thus patient survival. Reporter phage-based diagnostics offer a way to speed up pathogen identification and resistance testing as integration of reporter genes into highly specific phages allows real-time detection of phage replication and thus living host cells. Kp-specific phages use the host's capsule, a major virulence factor of Kp, as a receptor for adsorption. To date, 80 different Kp capsule types (K-serotypes) have been described with predominant capsule types varying between different countries and continents. Therefore, reporter phages need to be customized according to the locally prevailing variants. Recently, we described the autographivirus vB_KpP_TUN1 (TUN1), which specifically infects Kp K64 strains, the most predominant capsule type at the military hospital in Tunis (MHT) that is also associated with high mortality rates. In this work, we developed the highly specific recombinant reporter phage rTUN1::nLuc, which produces nanoluciferase (nLuc) upon host infection and thus enables rapid detection of Kp K64 cells in clinical matrices such as blood and urine. At the same time, rTUN1::nLuc allows for rapid antibiotic susceptibility testing and therefore identification of suitable antibiotic treatment in less than 3 h.

The long and sinuous road to phage-based therapy of Clostridioides difficile infections.

With the antibiotic crisis and the rise in antimicrobial resistance worldwide, new therapeutic alternatives are urgently needed. Phage therapy represents one of the most promising alternatives but for some pathogens, such as Clostridioides difficile, important challenges are being faced. The perspective of phage therapy to treat C. difficile infections is complicated by the fact that no strictly lytic phages have been identified so far, and current temperate phages generally have a narrow host range. C. difficile also harbors multiple antiphage mechanisms, and the bacterial genome is often a host of one or multiple prophages that can interfere with lytic phage infection. Nevertheless, due to recent advances in phage host receptor recognition and improvements in genetic tools to manipulate phage genomes, it is now conceivable to genetically engineer C. difficile phages to make them suitable for phage therapy. Other phage-based alternatives such as phage endolysins and phage tail-like bacteriocins (avidocins) are also being investigated but these approaches also have their own limitations and challenges. Last but not least, C. difficile produces spores that are resistant to phage attacks and all current antibiotics, and this complicates therapeutic interventions. This mini-review gives a brief historical overview of phage work that has been carried out in C. difficile, presents recent advances in the field, and addresses the most important challenges that are being faced, with potential solutions.

Engineering bacteriophages for enhanced host range and efficacy: insights from bacteriophage-bacteria interactions.

Bacteriophages, the most abundant organisms on earth, have the potential to address the rise of multidrug-resistant bacteria resulting from the overuse of antibiotics. However, their high specificity and limited host range can hinder their effectiveness. Phage engineering, through the use of gene editing techniques, offers a means to enhance the host range of bacteria, improve phage efficacy, and facilitate efficient cell-free production of phage drugs. To engineer phages effectively, it is necessary to understand the interaction between phages and host bacteria. Understanding the interaction between the receptor recognition protein of bacteriophages and host receptors can serve as a valuable guide for modifying or replacing these proteins, thereby altering the receptor range of the bacteriophage. Research and development focused on the CRISPR-Cas bacterial immune system against bacteriophage nucleic acids can provide the necessary tools to promote recombination and counter-selection in engineered bacteriophage programs. Additionally, studying the transcription and assembly functions of bacteriophages in host bacteria can facilitate the engineered assembly of bacteriophage genomes in non-host environments. This review highlights a comprehensive summary of phage engineering methods, including in-host and out-of-host engineering, and the use of high-throughput methods to understand their role. The main aim of these techniques is to harness the intricate interactions between bacteriophages and hosts to inform and guide the engineering of bacteriophages, particularly in the context of studying and manipulating the host range of bacteriophages. By employing advanced high-throughput methods to identify specific bacteriophage receptor recognition genes, and subsequently introducing modifications or performing gene swapping through in-host recombination or out-of-host synthesis, it becomes possible to strategically alter the host range of bacteriophages. This capability holds immense significance for leveraging bacteriophages as a promising therapeutic approach against antibiotic-resistant bacteria.

Bacteriophage T4 as a nanovehicle for delivery of genes and therapeutics into human cells.

The ability to deliver therapeutic genes and biomolecules into a human cell and restore a defective function has been the holy grail of medicine. Adeno-associated viruses and lentiviruses have been extensively used as delivery vehicles, but their capacity is limited to one (or two) gene(s). Bacteriophages are emerging as novel vehicles for gene therapy. The large 120 × 86-nm T4 capsid allows engineering of both its surface and its interior to incorporate combinations of DNAs, RNAs, proteins, and their complexes. In vitro assembly using purified components allows customization for various applications and for individualized therapies. Its large capacity, cell-targeting capability, safety, and inexpensive manufacturing could open unprecedented new possibilities for gene, cancer, and stem cell therapies. However, efficient entry into primary human cells and intracellular trafficking are significant barriers that must be overcome by gene engineering and evolution in order to translate phage-delivery technology from bench to bedside.

Directed Evolution of Replication-Competent Double-Stranded DNA Bacteriophage toward New Host Specificity.

In the fight against antimicrobial resistance, bacteriophages are a promising alternative to antibiotics. However, due to their narrow spectra, phage therapy requires the careful matching between the host and bacteriophage to be effective. Despite our best efforts, nature remains as the only source of novel phage specificity. Directed evolution can potentially open an avenue for engineering phage specificity and improving qualities of phages that are not strongly selected for in their natural environments but are important for therapeutic applications. In this work, we present a strategy that generates large libraries of replication-competent phage variants directly from synthetic DNA fragments, with no restriction on their host specificity. Using the T7 bacteriophage as a proof-of-concept, we created a large library of tail fiber mutants with at least 107 unique variants. From this library, we identified mutants that have broadened specificity as evidenced by their novel lytic activity against Yersinia enterocolitica, a strain that the wild-type T7 was unable to lyse. Using the same concept, mutants with improved lytic efficiency and characteristics, such as lytic condition tolerance and resistance suppression, were also identified. However, the observed limitations in altering host specificity by tail fiber mutagenesis suggest that other bottlenecks could be of equal or even greater importance.

In Vivo Capsid Engineering of Bacteriophages for Oriented Surface Conjugation.

The current state-of-the-art in bacteriophage (phage) immobilization onto magnetic particles is limited to techniques that are less expensive and/or facile but nonspecific or those that are more expensive and/or complicated but ensure capsid-down orientation of the phages, as necessary to preserve infectivity and performance in subsequent applications (e.g., therapeutics, detection). These cost, complexity, and effectiveness limitations constitute the major hurdles that limit the scale-up of phage-based strategies and thus their accessibility in low-resource settings. Here, we report a plasmid-based technique that incorporates a silica-binding protein, L2, into the T7 phage capsid, during viral assembly, with and without inclusion of a flexible linker peptide, allowing for targeted binding of the phage capsid to silica without requiring the direct modification of the phage genome. L2-tagged phages were then immobilized onto silica-coated magnetic nanoparticles. Inclusion of the flexible linker between the phage capsid protein and the L2 protein improved immobilization density compared to both wild type T7 phages and L2-tagged phages without the flexible linker. Taken together, this work demonstrates phage capsid modification without engineering the phage genome, which provides an important step toward reducing the cost and increasing the specificity/directionality of phage immobilization methods and could be more broadly applied in the future for other phages for a range of other capsid tags and nanomaterials.