Viral genome editing methods and applications in the CRISPR era

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• CRISPR-Cas systems revolutionize genetic manipulation of large DNA viruses.
• Traditional methods like BAC recombination have limitations.

Purpose of the Study:

• Provide a practical framework for applying CRISPR technology to herpesviruses and other large DNA viruses.
• Serve as an alternative and complement to traditional BAC recombination.

Main Methods:

• Nuclease selection and sgRNA design for efficient editing.
• Optimized donor template configuration and homology arm length.
• Synchronized delivery of Cas complexes and donor DNA, alongside HDR enhancement strategies.

Main Results:

• Demonstrated reproducible, scarless knock-ins and conditional gene manipulation in herpes simplex virus type 1 and human cytomegalovirus.
• Achieved direct editing of clinical isolates previously inaccessible with BAC methods.

Conclusions:

• CRISPR technology provides a flexible and scalable platform for viral functional genomics.
• Enables advancements in antiviral target discovery and translational virology.