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

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• CRISPR-Cas9 technology facilitates gene function studies, including host-virus interactions.
• Existing CRISPR screens for viral infections often use cell survival, limiting sensitivity and focusing on early stages.
• A more sensitive and comprehensive method is needed to analyze virus-host interactions across the entire infection cycle.

Purpose of the Study:

• To develop and present a protocol for the virus-encoded CRISPR-based direct readout system (VECOS).
• To enable sensitive, quantitative, and stage-specific analysis of host-virus interactions during viral infections.
• To provide a robust tool for uncovering molecular mechanisms of viral propagation.

Main Methods:

• Engineered human cytomegalovirus to express single-guide RNA (sgRNA) libraries directly from its genome (VECOS).
• Constructed complex sgRNA libraries in double-stranded DNA viruses using bacterial artificial chromosomes.
• Performed multipassage screens and analyzed sgRNA abundance across distinct viral infection stages.

Main Results:

• VECOS allows sgRNA abundance within the viral genome to serve as a direct readout of gene-perturbation effects.
• The system enables multidimensional analysis of virus-host interactions by tracking sgRNA levels at different infection stages.
• A comprehensive data analysis framework was developed for multipassage and multistage measurements.

Conclusions:

• VECOS offers a robust and sensitive approach to study host-virus interactions, overcoming limitations of previous methods.
• This system provides a detailed, quantitative understanding of viral propagation and host responses throughout the infection cycle.
• The protocol facilitates the discovery of molecular mechanisms driving viral infections and host-pathogen dynamics.