Engineering large viral DNA genomes using the CRISPR-Cas9 system

Area of Science:

• Molecular Biology
• Virology
• Genetic Engineering

Background:

• Viral genome engineering is crucial for understanding viral functions and developing virus-based therapies.
• Traditional methods like homologous recombination and bacterial artificial chromosome (BAC) systems have limitations, including low efficiency, labor intensity, and potential interference with viral function.

Purpose of the Study:

• To develop a highly efficient method for engineering herpes simplex virus (HSV) genomes.
• To create a system that avoids the insertion of artificial genes into the viral genome, preventing potential functional alterations.

Main Methods:

• Utilized the clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system for precise viral genome editing.
• Applied phenotype-based selection to enhance the isolation efficiency of desired viral clones.

Main Results:

• Successfully engineered HSV genomes with high efficiency using the CRISPR-Cas9 system.
• Generated both gene-ablated (knockout) and gene-inserted (knock-in) HSV mutants without incorporating artificial DNA sequences.
• Demonstrated that phenotype-based selection significantly improves the isolation of correctly edited viral clones.

Conclusions:

• The CRISPR-Cas9 system offers a powerful and efficient tool for engineering HSV genomes, overcoming limitations of previous methods.
• This versatile system is applicable to a broad range of DNA viruses, including Epstein-Barr virus, cytomegaloviruses, vaccinia virus, and baculovirus.
• The developed method holds significant promise for advancing viral research, genetic studies, and the development of novel viral-based therapeutics, including oncotherapy.