CRISPR/Cas9-Based Genome Editing of HSV

Area of Science:

• Molecular Biology
• Virology
• Gene Editing

Background:

• The CRISPR/Cas9 system offers powerful genome engineering capabilities across diverse organisms.
• Large double-stranded DNA (dsDNA) viruses, such as HSV-1, present unique challenges for genetic manipulation.
• Efficient and flexible editing tools are crucial for studying viral genomes and developing therapeutic strategies.

Purpose of the Study:

• To develop and present an efficient methodology for CRISPR/Cas9-mediated genome editing of the Herpes Simplex Virus type 1 (HSV-1).
• To enable both marker-based and marker-free genetic modifications of the HSV-1 genome.
• To demonstrate the versatility of the developed method for various types of genomic alterations.

Main Methods:

• Co-transfection of plasmids encoding Cas9, guide RNAs, and a homology-directed repair template into host cells.
• Introduction of the HSV-1 genome into the edited cells via viral infection.
• Application of the CRISPR/Cas9 system for precise nucleotide alterations, insertions, and deletions within the HSV-1 genome.

Main Results:

• Successful implementation of an efficient methodology for CRISPR/Cas9 gene editing of the HSV-1 genome.
• Demonstration of both marker-based and marker-free editing capabilities.
• Facilitation of a wide range of genomic modifications, from single nucleotide changes to large insertions and deletions.

Conclusions:

• The presented methodology provides a flexible and efficient approach for engineering the HSV-1 genome.
• This tool significantly advances the study of HSV-1 biology and opens avenues for therapeutic development.
• CRISPR/Cas9 gene editing is a powerful strategy for manipulating large dsDNA viral genomes.