Simultaneous deletion of virulence genes and heterologous gene insertions into the viruses belonging to alphaherpesvirinae subfamily using CRISPR/Cas9 system

CRISPR-mediated precision genome editing is considered as a revolutionary technology, which was discovered as a result of intensive studies on prokaryotic host-bacteriophage interactions. Along with other branches in biology science, CRISPR comes forward a highly efficient, versatile tool frequently implementing in virology and antiviral therapy, especially in gene knock-out and knock-in studies. The main goal of this thesis study was to modify two members of alphaherpesvirinae subfamily, Bovine alphaherpesvirus 1 (BHV-1) and Gallid alphaherpesvirus 1 (GaHV-1). For this purpose, thymidine kinase (tk) and dUTPase genes were targeted for BHV-1 and GaHV-1, respectively. A red fluorescent protein (rfp) gene, DsRed was aimed to insert in tk position of BHV- 1, while DsRed and fusion (F) gene of Newcastle disease virus were chosen for the insertion of GaHV-1. A donor plasmid construction carrying desired gene sequences was excised via CRISPR bait sequences to release linear expression cassettes. As a result, both DsRed and F genes were successfully inserted into desired position of GaHV-1 genome. Next, DsRed reporter gene was erased from the viral genome with using Cre-Lox recombination system, and the F gene expression was proven to remain intact and functional. Finally, obtained recombinant virus possessed insignificant features in the aspects of plaque forming capacity and growth properties than tk deleted and double heterologous gene