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

Anna Lilja¹, Yaara Finkel^1,2, Einav Aharon¹

¹Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

Nature Protocols

|September 22, 2025

Summary

Researchers developed a virus-encoded CRISPR-based direct readout system (VECOS) to overcome limitations in studying host-virus interactions. This novel method uses viral genome-integrated sgRNA libraries for sensitive, stage-specific analysis of viral propagation.

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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.