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CRISPR 激活作为一个平台，用于鉴定具有抗病毒功能的干扰素刺激基因。

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

机构信息

Research Centre for Infectious Diseases, The University of Adelaide, Adelaide, South Australia, Australia.

Discipline of Molecular and Biomedical Science, The University of Adelaide, Adelaide, South Australia, Australia.

出版信息

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

DOI:10.1177/17534259231225611
PMID:38258394
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11165661/
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 (WNV), 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.

摘要

干扰素刺激基因（ISG）的表达在控制病毒复制和产生强大的适应性反应中起着关键作用。了解病原体和宿主之间的这种动态关系对于我们理解病毒生命周期和开发潜在的新型抗病毒策略至关重要。传统上，基于质粒的外源性启动子驱动的 ISG 表达已被用于研究抗病毒 ISG 功能，但这种方法存在缺陷。为了克服这一问题，我们研究了 CRISPR 激活（CRISPRa）的实用性，它可以从其内源启动子靶向转录激活基因。我们使用 CRISPRa-SAM 系统诱导一组抗病毒 ISG 的靶向表达，显示出 mRNA 和蛋白质表达的强烈诱导。然后，我们在几种抗病毒筛选格式中使用我们的 CRISPRa-SAM ISG 面板来测试 ISG 防止病毒诱导的细胞病变效应（CPE）和登革热病毒（DENV）、寨卡病毒（ZIKV）、西尼罗河病毒 Kunjin（WNV）、甲型肝炎病毒（HAV）和人冠状病毒 229E（HCoV-229E）复制的能力。我们的 CRISPRa 方法证实了 IFI6、IFNβ 和 IFNλ2 等 ISG 的抗病毒活性，这些 ISG 可防止病毒诱导的 CPE，这得到了高内涵免疫荧光成像分析的支持。这项工作突出了 CRISPRa 作为一种快速、灵活和强大的方法，用于鉴定和表征 ISG 和病毒限制因子。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/81b6b4a86a8b/10.1177_17534259231225611-fig8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/761ad03f0272/10.1177_17534259231225611-fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/ec1b278713f8/10.1177_17534259231225611-fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/fd9b3bb795d7/10.1177_17534259231225611-fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/2932f0ed96b5/10.1177_17534259231225611-fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/84cb3f6dc05f/10.1177_17534259231225611-fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/b437777c2815/10.1177_17534259231225611-fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/fc505db3e602/10.1177_17534259231225611-fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/81b6b4a86a8b/10.1177_17534259231225611-fig8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/761ad03f0272/10.1177_17534259231225611-fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/ec1b278713f8/10.1177_17534259231225611-fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/fd9b3bb795d7/10.1177_17534259231225611-fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/2932f0ed96b5/10.1177_17534259231225611-fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/84cb3f6dc05f/10.1177_17534259231225611-fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/b437777c2815/10.1177_17534259231225611-fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/fc505db3e602/10.1177_17534259231225611-fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4de3/11165661/81b6b4a86a8b/10.1177_17534259231225611-fig8.jpg

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本文引用的文献

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High-content CRISPR screening.高内涵CRISPR筛选
Nat Rev Methods Primers. 2022;2(1). doi: 10.1038/s43586-022-00098-7. Epub 2022 Feb 10.
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Nonlytic cellular release of hepatitis A virus requires dual capsid recruitment of the ESCRT-associated Bro1 domain proteins HD-PTP and ALIX.非结构性细胞释放甲型肝炎病毒需要 ESCRT 相关 Bro1 结构域蛋白 HD-PTP 和 ALIX 的双重衣壳募集。
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Inducible CRISPR activation screen for interferon-stimulated genes identifies OAS1 as a SARS-CoV-2 restriction factor.
诱导型 CRISPR 激活筛选干扰素刺激基因，鉴定出 OAS1 是 SARS-CoV-2 的一种限制因子。
PLoS Pathog. 2022 Apr 14;18(4):e1010464. doi: 10.1371/journal.ppat.1010464. eCollection 2022 Apr.
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Increased Sensitivity of SARS-CoV-2 to Type III Interferon in Human Intestinal Epithelial Cells.人肠上皮细胞中 SARS-CoV-2 对 III 型干扰素的敏感性增加。
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A CRISPR Activation Screen Identifies an Atypical Rho GTPase That Enhances Zika Viral Entry.CRISPR 激活筛选鉴定出一种增强寨卡病毒进入的非典型 Rho GTPase。
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Genome-Wide CRISPR Screen Identifies RACK1 as a Critical Host Factor for Flavivirus Replication.全基因组 CRISPR 筛选鉴定 RACK1 为黄病毒复制的关键宿主因子。
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