Efficient Mutagenesis of Marek's Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System

Area of Science:

• Virology
• Molecular Biology
• Gene Editing

Background:

• Virus-encoded microRNAs (miRNAs) play key regulatory roles in herpesvirus biology, influencing replication, latency, pathogenesis, and tumorigenesis.
• The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is an effective tool for manipulating large DNA virus genomes.

Purpose of the Study:

• To establish a novel platform for mutagenesis of viral miRNAs in Marek's disease virus serotype 1 (MDV-1), an oncogenic alphaherpesvirus.
• To generate and characterize miRNA-knocked out (miR-KO) mutants of MDV-1 using CRISPR/Cas9 technology.

Main Methods:

• Utilized a CRISPR/Cas9 system with a double-guide RNA transfection/virus infection strategy.
• Generated several miR-KO mutants (RB-1B∆Meq-miRs, RB-1B∆M9-M2, RB-1B∆M4, RB-1B∆M9, RB-1B∆M11) from the vvMDV strain RB-1B virus.
• Focused on deletions of Meq- or mid-clustered miRNAs.

Main Results:

• Successfully generated a series of MDV-1 miR-KO mutants.
• Observed altered in vitro virus growth kinetics in the generated miRNA-mutagenized viruses.
• These findings align with previous in vivo proliferation data from mutants generated using BAC and Rec E/T homologous recombination.

Conclusions:

• CRISPR/Cas9-based gene editing provides a simple, efficient, and minimally disruptive method for manipulating small non-coding genes in herpesvirus genomes.
• This approach represents a significant advancement for future research in herpesvirus biology.
• The study highlights the utility of CRISPR/Cas9 for generating viral miRNA mutants to study their biological functions.