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.

A Genome-Wide CRISPR-Cas9 Loss-of-Function Screening to Identify Host Restriction Factors Modulating Oncolytic Virotherapy / Muscolini, Michela; Hiscott, John; Tassone, Evelyne. - (2023), pp. 379-399. - METHODS IN MOLECULAR BIOLOGY. [10.1007/978-1-0716-2788-4_25].

A Genome-Wide CRISPR-Cas9 Loss-of-Function Screening to Identify Host Restriction Factors Modulating Oncolytic Virotherapy

Muscolini, Michela;Tassone, Evelyne
Ultimo
2023

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.
2023
HDAC/HAT Function Assessment and Inhibitor Development: Methods and Protocols
978-1-0716-2787-7
978-1-0716-2788-4
CRISPR-Cas9; Genome-wide; Host restriction Factors; Oncolytic virus; Pooled screening; Resistance
02 Pubblicazione su volume::02a Capitolo o Articolo
A Genome-Wide CRISPR-Cas9 Loss-of-Function Screening to Identify Host Restriction Factors Modulating Oncolytic Virotherapy / Muscolini, Michela; Hiscott, John; Tassone, Evelyne. - (2023), pp. 379-399. - METHODS IN MOLECULAR BIOLOGY. [10.1007/978-1-0716-2788-4_25].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/1669538
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