Vol. 12, Special Issue 9 (2023)

Author(s):
Sushmita Nautiyal, Pallavi Deol, S Nandi, Sonalika Mahajan, Kirtika Sharma, Ragini Mishra and GK Sharma

Abstract:
Genome editing using CRISPR/Cas9 technology offers several advantages over conventional methods of gene-editing. The method is cost-effective, time saving and highly efficient. The technology has been successfully applied for genome editing of various human and animal DNA viruses for understanding viral replication, pathogenesis, host-virus interaction and generation of viral mutants. In present study we applied this technology for editing of bovine herpesvirus-1(BoHV-1) genome. Plasmid based expression system was used for the delivery of single guide RNA (sgRNA) and targeting of the non-essential gene (US8 coding for glycoprotein E) of the virus. In order to trace the mutant virus, homology directed repair (HDR) method was used. A reporter gene (DsRed) producing fluorescence was inserted at the site of mutation. The fluorescence was detected after third passage of the mutant virus in MDBK cells. The method appears to be promising for tracing of virus mutant from wild-type virus population which is a challenging step in genome editing using CRISPR/Cas9 technology.