Hybrid Recombineering: CRISPR/Cas9 Mutagenesis of Murine Cytomegalovirus BAC

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that can asymptomatically infect immunocompetent hosts with little-to-no impact. Conversely, HCMV can trigger life-threatening disease in immunocompromised hosts such as neonates, organ transplant recipients, and people living with AIDS. HCMV is a unique pathogen in that it can hijack the immune system for its benefit and disseminates throughout the body using host immune cells. Previous studies suggest that innate immune cells are responsible for viral dissemination. However, adequate methods for investigating the spread of CMV within a murine model remain incomplete. We implicate the innate immune cell the neutrophil as a major cell responsible for HCMV dissemination due to their massive volume and quick response time. Unfortunately, the model system murine CMV (MCMV) does not induce a neutrophil specific immune response as HCMV does. In order to adequately study neutrophils as the culprit for CMV dissemination, we mutated our laboratory MCMV strain to induce a neutrophil specific response utilizing the novel CRISPR/Cas9 mutagenesis system in conjunction with standard recombineering mutagenesis practices. Following a series of hybrid recombineering strategies, we successfully mutated MCMV to encode the gene vCXCL-1, which is important for neutrophil recruitment. It was observed that the recombinant virus was able to infect cells in vitro at similar rates, as our wild type virus. This project lays the foundation for successfully generating mutant MCMV viruses and provides a basis to study the function of vCXCL-1 in vivo.