Engineering of Bacteriophage T4 Genome Using CRISPR-Cas9

Area of Science:

• Microbiology
• Genetics
• Molecular Biology

Background:

• Bacteriophages, like T4 phage, are abundant but poorly characterized.
• T4 phage DNA modifications hinder genetic analysis and DNA manipulation.
• Classical genetic methods for T4 phage are laborious and inefficient.

Purpose of the Study:

• To develop and demonstrate a CRISPR-Cas9 based genome editing system for T4 phage.
• To enable precise genetic modifications in both native and engineered T4 phage genomes.
• To explore the potential of phage genome editing for biological research and therapeutic applications.

Main Methods:

• Utilized the type-II CRISPR-Cas9 system for targeted DNA cleavage in E. coli.
• Co-delivered CRISPR-Cas9 and donor plasmids for homology-directed repair.
• Introduced single/multiple point mutations, insertions, and deletions using short homologous arms (50 bp).
• Assessed editing efficiency on both modified (ghm-Cytosine) and unmodified T4 genomes.

Main Results:

• Successfully edited both modified and unmodified T4 phage genomes.
• Demonstrated that modified T4 DNA is less susceptible to Cas9 cleavage than unmodified DNA.
• Achieved efficient genome editing with short homologous arms, generating viable recombinants.
• Confirmed gene essentiality by creating a viable deletion in the RNA ligase gene (rnlB).

Conclusions:

• Established the first CRISPR-Cas9 based genome editing strategy for T4 phage.
• The method is effective for various genetic modifications in T4 phage.
• This approach can be extended to other phage genomes for research and phage therapy development.