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. 2024 Feb;30(2-4):40-54.
doi: 10.1177/17534259231225611. Epub 2024 Jan 23.

CRISPR activation as a platform to identify interferon stimulated genes with anti-viral function

Emily N Kirby  1   2 , Xavier B Montin  1   2 , Timothy P Allen  2 , Jaslan Densumite  3 , Brooke N Trowbridge  1   2 , Michael R Beard  1   2
Affiliations

CRISPR activation as a platform to identify interferon stimulated genes with anti-viral function

Emily N Kirby et al. Innate Immun. 2024 Feb.

Abstract

Interferon Stimulated Gene (ISG) expression plays a key role in the control of viral replication and development of a robust adaptive response. Understanding this dynamic relationship between the pathogen and host is critical to our understanding of viral life-cycles and development of potential novel anti-viral strategies. Traditionally, plasmid based exogenous prompter driven expression of ISGs has been used to investigate anti-viral ISG function, however there are deficiencies in this approach. To overcome this, we investigated the utility of CRISPR activation (CRISPRa), which allows for targeted transcriptional activation of a gene from its endogenous promoter. Using the CRISPRa-SAM system to induce targeted expression of a panel of anti-viral ISGs we showed robust induction of mRNA and protein expression. We then employed our CRISPRa-SAM ISG panel in several antiviral screen formats to test for the ability of ISGs to prevent viral induced cytopathic cell death (CPE) and replication of Dengue Virus (DENV), Zika Virus (ZIKV), West Nile Virus Kunjin (WNVKUN), Hepatitis A Virus (HAV) and Human Coronavirus 229E (HCoV-229E). Our CRISPRa approach confirmed the anti-viral activity of ISGs like IFI6, IFNβ and IFNλ2 that prevented viral induced CPE, which was supported by high-content immunofluorescence imaging analysis. This work highlights CRISPRa as a rapid, agile, and powerful methodology to identify and characterise ISGs and viral restriction factors.

Keywords: Antiviral; CRISPR activation; interferon stimulated genes; virus.

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Conflict of interest statement

Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
CRISPRa-SAM and CRISPRa-SunTag methods of inducing transcriptional activation
Figure 2.
Figure 2.
CRISPRa-SAM and SunTag induce transcriptional activation of target ISGs overtime
Figure 3.
Figure 3.
CRISPRa-SAM and SunTag transcriptional activation of ISGs is not significantly different between cell types
Figure 4.
Figure 4.
Stable integration of CRISPRa-SAM componentry induces transcriptional activation of target genes
Figure 5.
Figure 5.
CRISPRa mediated transcriptional activation of select ISGs results in increased protein expression
Figure 6.
Figure 6.
Protection from virally induced cytopathic cell death is ISG dependent
Figure 7.
Figure 7.
CRISPRa ISG screening identifies inhibitors of HAV-NLuc replication
Figure 8.
Figure 8.
High-content imaging of CRISPRa ISG cells identifies inhibitors of flavivirus replication
See this image and copyright information in PMC

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