NGS analysis of Cas9-gRNA CRISPR edited herpesviruses

Description: Herpesvirus such as EBV and KSHV are prevalent in the human population and are the etiological agent of several neoplastic malignancies. To investigate the therapeutic potential of Cas9-gRNA CRISPR system to suppress and eliminate virus-driven cancers, several Cas9-gRNA targeting sequences of murine gammaherpesvirus 68 (MHV68), a natural pathogen of mice with extensive homology to KSHV were evaluated. Murine NIH3T3 fibroblast cells (3T3) stably express Cas9 and the gRNAs were infected de novo with MHV68. DNA fragments encompassing the gRNA targeting sequences were PCR amplified using high fidelity polymerases and subject to next generation sequencing (NGS). In addition, A20-HE B-cell line that is latently infected with reactivation-competent MHV68 was nucleofected to stably express Cas9 and the gRNAs against MHV68. Viral reactivation was induced through stimulation with TPA. DNA fragments were prepared and sequenced similarly as those after virus infected NIH3T3 cells. In both analyses, no targeting gRNAs NT1-3 were used as controls. Amplicons were named according to the ORFs or virus element that the gRNAs targeted.