Selection of Genetically Modified Bacteriophages Using the CRISPR-Cas System.

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• Bacteriophages, like T7, are crucial tools in molecular biology and biotechnology.
• Efficient genetic manipulation of bacteriophages is essential for advancing their applications.
• Existing methods for bacteriophage genome editing can be inefficient or complex.

Purpose of the Study:

• To develop a novel and efficient CRISPR-Cas based technique for targeted gene deletion in the T7 bacteriophage genome.
• To establish a protocol for seamless genetic modification of T7 phage.
• To provide a foundation for broader applications in phage engineering and genetic manipulation.

Main Methods:

• A plasmid was constructed containing homologous arms flanking the target gene for deletion.
• T7 bacteriophage was propagated in *Escherichia coli* strains carrying the engineered plasmid.
• The CRISPR-Cas system was employed to selectively eliminate non-edited phage genomes.
• Homologous recombination between the phage genome and the plasmid facilitated targeted gene deletion.

Main Results:

• Successfully demonstrated the deletion of target genes from the T7 bacteriophage genome.
• The CRISPR-Cas system effectively selected for recombinant phages with deleted genes.
• The protocol allows for seamless and precise gene deletion in T7 phage.

Conclusions:

• The presented CRISPR-Cas based technique offers an efficient method for targeted gene deletion in T7 bacteriophage.
• This approach simplifies genetic manipulation of T7 phage.
• The methodology can be extended to other bacteriophages and diverse genetic modifications.