Establishment of a CRISPR/Cas9 knockout library for screening type I interferon-inducible antiviral effectors in pig cells

Abstract

Diseases caused by emerging swine viruses had a great economic impact, constituting a new challenge for researchers and practicing veterinarians. Innate immune control of viral pathogen invasion is mediated by interferons (IFNs), resulting in transcriptional elevation of hundreds of IFN-stimulated genes (ISGs). However, the ISG family is vast and species-specific, and despite remarkable advancements in uncovering the breadth of IFN-induced gene expression in mouse and human, it is less characterized with respect to the repertoire of porcine ISGs and their functional annotation. Herein, with the application of RNA-sequencing (RNA-Seq) gene profiling, the breadth of IFN-induced gene expression in the context of type I IFN stimulation was explored by using IBRS-2 cell, a commonly used high-efficient cultivation system for porcine picornaviruses. By establishing inclusion criteria, a total of 359 ISGs were selected. Aiming to identify key effectors mediating type I IFN inhibition of swine viruses, a CRISPR/Cas9 knockout library of 1908 sgRNAs targeting 5’ constitutive exons of 359 ISGs with an average of 5 to 6 sgRNAs per gene was constructed. Using VSV-eGFP (vesicular stomatitis virus, fused with GFP) as a model virus, a subset of highest-ranking candidates were identified, including previously validated anti-VSV genes IRF9, IFITM3, LOC100519082 and REC8, as well as several novel hits. This approach attains a high level of feasibility and reliability, and a high rate of hit identification, providing a forward-looking platform to systematically profile the effectors of type I IFN antiviral response against porcine viruses.

Interferon inhibits a model RNA virus via a limited set of inducible effector genes

“…Recently, CRISPR‐Cas9 gene knockout screens have begun to address some of these limitations. By genetically inactivating individual genes in the context of a functional IFN response, CRISPR screens have the potential to identify those ISGs that are necessary for IFN‐mediated protective effects (OhAinle et al , 2018 ; Richardson et al , 2018 ; Dang et al , 2022 ; Mac Kain et al , 2022 ). A second question of interest in the field is “what are the mechanisms of ISG effectors?” Mechanistic studies include determining which stage in the viral replication cycle an ISG effector acts and the molecular details of how the effector achieves viral inhibition.…”

“…Recent CRISPR‐based ISG discovery screens have focused on yellow fever virus (Richardson et al , 2018 ), HIV‐1 (OhAinle et al , 2018 ), vesicular stomatitis virus (Dang et al , 2022 ), and SARS‐CoV‐2 (Mac Kain et al , 2022 ). We sought to use similar approaches to identify potentially novel ISGs that inhibit alphaviruses.…”

Interferon inhibits a model RNA virus via a limited set of inducible effector genes

“…Recently, CRISPR-Cas9 gene silencing screens have begun to address some of these limitations. By genetically silencing individual genes in the context of a functional IFN response, CRISPR screens have the potential to identify those ISGs that are necessary for IFN-mediated protective effects (Dang et al , 2022; Mac Kain et al , 2022; OhAinle et al , 2018; Richardson et al , 2018). A second question of interest in the field is “what are the mechanisms of ISG effectors?” Mechanistic studies include determining which stage in the viral replication cycle an ISG effector acts and the molecular details of how the effector achieves viral inhibition.…”