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Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors
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Multidimensional analysis of host-virus interactions using the virus-encoded CRISPR-based direct readout system

Anna Lilja1, Yaara Finkel1,2, Einav Aharon1

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

Nature Protocols
|September 22, 2025
PubMed

Insights

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.

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.

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