Multidimensional analysis of host-virus interactions using the virus-encoded CRISPR-based direct readout system (VECOS).

CRISPR-Cas9 technology has transformed the study of gene function, enabling the systematic investigation of host-virus interactions. However, most CRISPR-based screens in the context of viral infections rely on cell survival as a readout, which limits their sensitivity and biases results toward early infection stages. To address these challenges, we developed the virus-encoded CRISPR-based direct readout system (VECOS), a virus-centric approach in which human cytomegalovirus is engineered to express single-guide RNA (sgRNA) libraries directly from its genome. This system allows sgRNA abundance, embedded in the viral genome, to serve as a direct and quantitative readout of gene-perturbation effects on viral propagation. By tracking sgRNA levels at distinct stages of the viral infection cycle, VECOS enables a detailed, multidimensional analysis of virus-host interactions. Here we present a modular detailed Protocol for (1) constructing and reconstituting complex sgRNA libraries in double-stranded DNA viruses using bacterial artificial chromosomes, (2) performing multipassage screens to investigate perturbation effects on various stages of viral infection and (3) analyzing the multipassage and multistage sgRNA abundance measurements utilizing a comprehensive framework for data analysis. Successful implementation of this full Protocol takes 14-22 weeks and requires proficiency in molecular biology, as well as basic familiarity with Unix-based computing and programming in R for data processing. This Protocol offers researchers a robust tool for uncovering the molecular mechanisms that drive viral propagation and host-virus interactions.