CRISPR-Mediated Viral Gene Knock-In for Studying Viral-Host Interactions

CRISPR-mediated gene knock-in is a powerful tool for studying virus-host interactions. In this protocol, we describe the methodology for the knock-in of a red fluorescent protein, mCherry, fused to the viral protein VP26, a component of the herpesvirus capsid. By integrating the mCherry gene into the HSV-1 genome, we enable detailed studies of viral protein dynamics, viral replication, and virus-host interactions in living cells. In particular, this approach allows real-time monitoring of the subcellular localization of the viral capsid. The protocol provides a step-by-step guide for designing the gene knock-in vectors, transfection, selection, and validation of the mCherry fusion to the VP26 protein in the HSV-1 genome.