Undergraduate Researcher University of California, Berkeley, United States
Introduction/Rationale: Oncolytic virotherapy is a promising immunotherapy strategy, but efficacy in solid tumors is limited by poor intratumoral viral spread. Vaccinia virus is a leading oncolytic platform due to its large capacity and cytoplasmic life cycle, yet it produces low levels of extracellular enveloped virus, the form responsible for tissue dissemination and immune evasion. Enhancing this phenotype requires interrogation of viral genes that are often essential, making knockout approaches unsuitable. We sought to develop a method to tune vaccinia gene expression without disrupting viral viability.
Methods: CRISPR interference and CRISPR activation systems were adapted for use in cytoplasm-replicating vaccinia virus. A catalytically inactive Cas9 was directed to viral promoters using engineered guide RNAs. CRISPR activation used MS2 hairpin–containing guides to recruit viral transcription factors, while CRISPR interference repressed transcription through steric blocking. Recombinant vaccinia viruses encoding a late promoter–driven GFP reporter were generated, and viral gene expression in infected HEK293T cells was quantified by flow cytometry.
Results: CRISPR interference targeting downstream of a late viral promoter produced a consistent and statistically significant reduction in GFP expression relative to non-target controls, demonstrating effective repression of late vaccinia transcription. In contrast, CRISPR activation targeting upstream promoter regions failed to increase expression, indicating that late viral promoters operate near transcriptional saturation. Thus, late-stage vaccinia transcription is resistant to activation but sensitive to graded repression.
Conclusion: This work establishes CRISPR interference as a robust tool for programmable gene regulation in vaccinia virus and defines constraints on late viral transcription. The platform enables functional analysis of essential viral genes that influence viral dissemination and supports the rational engineering of improved oncolytic immunotherapies.
