ArticleACS synthetic biology2017
Engineering of Bacteriophage T4 Genome Using CRISPR-Cas9.
Article in ACS synthetic biology, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 83 papers.
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Who cites it
83 citing papers in PubMed, 133 citations in OpenAlex.
- Scarless one-tube genome assemblyRSC chemical biology · 2026Article
- Defect-complementation homologous recombination: A novel strategy for precise genome engineering of virulent phages.Synthetic and systems biotechnology · 2026Article
- Bacteriophages as Therapeutic Agents for Pulmonary Infections: From Biological Principles to Clinical Applications.Pharmaceutics · 2026Review
- Periplasmic gatekeeping of phage DNA entry by an rSAM enzyme matured effector with HxS repeats.Nature communications · 2026Article
- An efficient CRISPR-Cas12a tool for iterative genome editing, streamlined minimization, and payload engineering ofSynthetic and systems biotechnology · 2026Article
- Cryo-EM structures of bacteriophage T4 portal-neck assembly intermediates reveal a viral genome retention mechanism.Nature communications · 2026Article
- Bacteriophage T4 genome packaging: mechanism and application.EcoSal Plus · 2025Review
- Rapid detection ofActa biochimica et biophysica Sinica · 2025Article
- CRISPR-Cas9 enables efficient genome engineering of the strictly lytic, broad-host-range staphylococcal bacteriophage K.Applied and environmental microbiology · 2025Article
- CRISPR-Cas10-Assisted Structural Modification of StaphylococcalACS synthetic biology · 2025Article
- Systematic, high-throughput characterization of bacteriophage gene essentiality on diverse hosts.Cell host & microbe · 2025Article
- Synthetic and Functional Engineering of Bacteriophages: Approaches for Tailored Bactericidal, Diagnostic, and Delivery Platforms.Molecules (Basel, Switzerland) · 2025Review
- A Modular Bacteriophage T4 Nanoparticle Platform Enables Rapid Design of Dual COVID-19-Flu Mucosal Vaccines.Small science · 2025Article
- Engineering Phages to Fight Multidrug-Resistant Bacteria.Chemical reviews · 2025Review
- Precision targeting of genetic variations in mixed bacterial cultures using CRISPR-Cas12a-programmed λ phages.Frontiers in microbiology · 2025Article
- Tevenvirinae phages encode a family of LSm-like fold proteins.Computational and structural biotechnology journal · 2025Article
- Utilizing Golden Gate Assembly to Streamline CRISPR-Cas/NgTET-Based Phage Mutagenesis.Methods in molecular biology (Clifton, N.J.) · 2025Article
- CRISPR/Cas9-Mediated Genome Editing of T4 Bacteriophage for High-Throughput Antimicrobial Susceptibility Testing.Analytical chemistry · 2024Article
- Article
- Bacteriophage T4 as a Protein-Based, Adjuvant- and Needle-Free, Mucosal Pandemic Vaccine Design Platform.Annual review of virology · 2024Review
23 more citing papers are in PubMed but not listed here.
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
Abstract
Bacteriophages likely constitute the largest biomass on Earth. However, very few phage genomes have been well-characterized, the tailed phage T4 genome being one of them. Even in T4, much of the genome remained uncharacterized. The classical genetic strategies are tedious, compounded by genome modifications such as cytosine hydroxylmethylation and glucosylation which makes T4 DNA resistant to most restriction endonucleases. Here, using the type-II CRISPR-Cas9 system, we report the editing of both modified (ghm-Cytosine) and unmodified (Cytosine) T4 genomes. The modified genome, however, is less susceptible to Cas9 nuclease attack when compared to the unmodified genome. The efficiency of restriction of modified phage infection varied greatly in a spacer-dependent manner, which explains some of the previous contradictory results. We developed a genome editing strategy by codelivering into E. coli a CRISPR-Cas9 plasmid and a donor plasmid containing the desired mutation(s). Single and multiple point mutations, insertions and deletions were introduced into both modified and unmodified genomes. As short as 50-bp homologous flanking arms were sufficient to generate recombinants that can be selected under the pressure of CRISPR-Cas9 nuclease. A 294-bp deletion in RNA ligase gene rnlB produced viable plaques, demonstrating the usefulness of this editing strategy to determine the essentiality of a given gene. These results provide the first demonstration of phage T4 genome editing that might be extended to other phage genomes in nature to create useful recombinants for phage therapy applications.
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