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Updated: Aug 25, 2025
Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy
Published on: April 3, 2018
A Genome-Wide CRISPR-Cas9 Loss-of-Function Screening to Identify Host Restriction Factors Modulating Oncolytic
Michela Muscolini1, John Hiscott1, Evelyne Tassone2
1Istituto Pasteur Italia - Fondazione Cenci Bolognetti, Rome, Italy.
Abstract:
Oncolytic virotherapy represents an efficient immunotherapeutic approach for cancer treatment. Oncolytic viruses (OVs) promote antitumor responses through tumor-selective cell lysis and immune system activation. However, some tumor cell lines and primary tumors display resistance to therapy. Here we describe a protocol to identify novel host factors responsible for tumor resistance to oncolysis using an unbiased genome-wide CRISPR-Cas9 loss-of-function screening. Cas9-expressing tumor cells are transduced with a library of pooled single-guide RNA (sgRNA)-expressing lentiviruses that target all human genes to obtain a cell population where each cell is knocked out for a single gene. Upon OV infection, resistant cells survive, while sensitive cells die. The relative abundance of each genome-integrated sgRNA is measured by next-generation sequencing (NGS) in resistant and control cells. This protocol is amenable to uncover host factors involved in the resistance to different OVs in multiple tumor models.
Insights
This study introduces a genome-wide CRISPR-Cas9 screening method to discover host factors that cause resistance to oncolytic viruses (OVs) in cancer. This approach helps identify new therapeutic targets for improving cancer immunotherapy.
Area of Science:
- Oncology
- Immunotherapy
- Genetics
Background:
- Oncolytic virotherapy uses oncolytic viruses (OVs) to treat cancer by selectively killing tumor cells and activating immune responses.
- Tumor resistance to oncolytic virotherapy limits its clinical efficacy.
- Identifying host factors contributing to this resistance is crucial for enhancing treatment outcomes.
Purpose of the Study:
- To develop and present a protocol for identifying novel host factors responsible for tumor resistance to oncolytic virotherapy.
- To enable unbiased, genome-wide screening for resistance mechanisms.
Main Methods:
- Utilized a genome-wide CRISPR-Cas9 loss-of-function screen in Cas9-expressing tumor cells.
- Transduced cells with a pooled single-guide RNA (sgRNA) lentiviral library targeting all human genes.
- Infected cells with oncolytic viruses (OVs) and selected for resistant survivors.
- Quantified sgRNA abundance in resistant versus control cells using next-generation sequencing (NGS).
Main Results:
- The protocol successfully generated a population of tumor cells with individual gene knockouts.
- Differential sequencing identified specific genes associated with resistance or sensitivity to OV infection.
- The screening method is adaptable to various tumor models and OVs.
Conclusions:
- The described CRISPR-Cas9 screening protocol is effective for discovering host factors that mediate resistance to oncolytic virotherapy.
- This method provides a powerful tool for uncovering new therapeutic targets to overcome treatment resistance in cancer.
