Bacteriophage genome engineering with CRISPR–Cas13a

Guan, Jingwen; Oromí-Bosch, Agnès; Mendoza, Senén D.; Karambelkar, Shweta; Berry, Joel D.; Bondy‐Denomy, Joseph

doi:10.1038/s41564-022-01243-4

Cited by 57 publications

(49 citation statements)

References 53 publications

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“…Our engineering workflow was validated through the construction of K::nluc, a reporter gene-coding phage K variant for viable S. aureus detection. The bioluminescent nanoluciferase, NanoLuc ® , has been utilized in prior reporter phage development studies [35][36][37]59] [42,60,[77][78][79]. Unlike antimicrobial payloads that disrupt key metabolic pathways and alter bacterium-phage dynamics, intracellular expression of NanoLuc ® is less likely to impose significant fitness costs on the phage or host bacterium.…”

Section: Discussionmentioning

confidence: 99%

“…However, like all synthetic methods, engineering becomes challenging when dealing with larger phage genomes that require assembly from numerous long DNA fragments. We therefore chose a homologous recombination (HR)-based approach, which has been successfully used for engineering larger phage genomes [19,[31][32][33][34][35][36] and included a downstream CRISPR-Cas-assisted counterselection (CS) step, which allowed us to rapidly obtain recombinant phages. To demonstrate the functionality and applicability of this approach, we engineered a phage K-based reporter phage that enables detection of viable S. aureus cells.…”

Section: Introductionmentioning

confidence: 99%

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CRISPR-Cas9 enables efficient genome engineering of the strictly lytic, broad host-range staphylococcal bacteriophage K

Fernbach,

Baggenstos,

Riedo

et al. 2024

Preprint

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Staphylococcus aureus is an important opportunistic pathogen, responsible for a range of diseases that often prove challenging to treat due to resistance to methicillin, vancomycin, and other antimicrobials. Bacteriophages present a promising alternative to target such pathogens, particularly when conventional drugs are ineffective. The antimicrobial efficacy of phage therapeutics can be further improved through genetic engineering. Among S. aureus phages, members of the Twortvirinae subfamily, characterized by their strictly lytic nature and broad host range, are considered the most promising therapeutic candidates. However, their large genome sizes make them notoriously difficult to engineer. In this study, we utilized Twortvirus K as a model to develop an efficient phage engineering platform, leveraging homologous recombination and CRISPR-Cas9-assisted counterselection. As proof of principle, this platform was utilized to construct a nanoluciferase (nluc)-encoding reporter phage (K::nluc) and tested as a preliminary, bioluminescence-based approach for identifying viable Staphylococcus cells. Independent of their phage-resistance profile, 100% of tested clinical S. aureus isolates emitted bioluminescence upon K::nluc challenge. This diagnostic assay was further adapted to complex matrices such as human whole blood and bovine raw milk, simulating S. aureus detection scenarios in bacteremia and bovine mastitis. Beyond reporter phage-based diagnostics, our engineering technology opens avenues for the design and engineering of therapeutic Twortvirinae phages to combat drug-resistant S. aureus strains.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

CRISPR-Cas9 enables efficient genome engineering of the strictly lytic, broad host-range staphylococcal bacteriophage K

Fernbach,

Baggenstos,

Riedo

et al. 2024

Preprint

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“…ϕ KZ particles packaged with virion proteins bearing a C-terminal 3xFLAG-tag were generated by adapting a protocol used to generate ϕ KZ particles packaged with mNeonGreen-tagged inner body proteins[18, 55]. PAO1 cells transformed with the appropriate pHERD30T − (PHIKZxxx) − 3xFLAG construct were grown overnight in 3 mL LB supplemented with gentamicin (50 µ g/ml) at 37°C with aeration at 175 rpm.…”

Section: Methodsmentioning

confidence: 99%

Next-generation interaction proteomics for quantitative Jumbophage-bacteria interaction mapping

Fossati

Mozumdar

Kokontis

et al. 2023

Preprint

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Host-pathogen interactions (HPIs) are pivotal in regulating establishment, progression, and outcome of an infection. Affinity-purification mass spectrometry has become instrumental for the characterization of HPIs, however the targeted nature of exogenously expressing individual viral proteins has limited its utility to the analysis of relatively small pathogens. Here we present the use of co-fractionation mass spectrometry (SEC-MS) for the high-throughput analysis of HPIs from native viral infections of two jumbophages (phiKZ and phiPA3) in Pseudomonas aeruginosa. This enabled the detection >6000 unique host-pathogen and >200 pathogen-pathogen interactions for each phage, encompassing more then 50% of the phage proteome. Interactome-wide comparison across phages showed similar perturbed protein interactions suggesting fundamentally conserved mechanisms of phage predation within the KZ-like phage family. Prediction of novel ORFs revealed a phiPA3 complex showing strong structural and sequence similarity to phiKZ nvRNAp, suggesting phiPA3 also possesses two RNA polymerases acting at different stages of the infection cycle. We further expanded our understanding on the molecular organization of the injected phage proteome by providing 23 novel virion components and 5 novel injected proteins, as well as providing the first evidence for phage manipulation of the host ribosome. To enable accessibility to this data, we developed PhageMAP, an online resource for network query, visualization, and interaction prediction \url{http://phagemap.ucsf.edu/}. We anticipate this study will lay the foundation for the application of co-fractionation mass spectrometry for the scalable profiling of host-pathogen interactomes and protein complex dynamics upon infection.

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“…This leads to degradation of both phage and host cellular RNA, cell dormancy, and phage restriction (36) (Fig 3A). Cas13a produces a strong selection against the wildtype gene so only phage with mutations in the targeted region can replicate (35,37).…”

Section: Mutations In the Conserved Shell-associated Protein Pica Alt...mentioning

confidence: 99%

An essential and highly selective protein import pathway encoded by nucleus-forming phage

Morgan,

Enustun,

Armbruster

et al. 2024

Preprint

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Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here we identify two components of this novel protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA, that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together our results allow us to propose a multistep model for the Protein Import Chimallivirus (PIC) pathway, where proteins are targeted to PicA by amino acids on their surface, and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely-related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.

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Bacteriophage genome engineering with CRISPR–Cas13a

Cited by 57 publications

References 53 publications

CRISPR-Cas9 enables efficient genome engineering of the strictly lytic, broad host-range staphylococcal bacteriophage K

CRISPR-Cas9 enables efficient genome engineering of the strictly lytic, broad host-range staphylococcal bacteriophage K

Next-generation interaction proteomics for quantitative Jumbophage-bacteria interaction mapping

An essential and highly selective protein import pathway encoded by nucleus-forming phage

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