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首页> 外文期刊>Journal of Molecular Biology >CRISPR/Cas9-based Knockout Strategy Elucidates Components Essential for Type 1 Interferon Signaling in Human HeLa Cells
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CRISPR/Cas9-based Knockout Strategy Elucidates Components Essential for Type 1 Interferon Signaling in Human HeLa Cells

机译：基于CRISPR / CAS9的敲除策略阐明了人Hela细胞中1型干扰素信号所必需的组分

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摘要

Type I interferons (IFNs) have a central role in innate and adaptive immunities, proliferation, and cancer surveillance. How IFN binding to its specific receptor, the IFN alpha and beta receptor (IFNAR), can drive such variety of processes is an open question. Here, to systematically and thoroughly investigate the molecular mechanism of IFN signaling, we used a CRISPR/Cas9-based approach in a human cell line (HeLa) to generate knockouts (KOs) of the genes participating in the type 1 IFN signaling cascade. We show that both IFNAR chains (IFNAR1 and IFNAR2) are absolutely required for any IFN-induced signaling. Deletion of either signal transducer and activator of transcription 1 (STAT1) or STAT2 had only a partial effect on IFN-induced antiviral activity or gene induction. However, the deletion of both genes completely abrogated any IFN-induced activity. So did a double STAT2 IFN regulatory factor 1 (IRF1) KO and, to a large extent, a STAT1 KO together with IRF9 knockdown. KO of any of the STATs had no effect on the phosphorylation of other STATs, indicating that they bound IFNAR independently. STAT3 and STAT6 phosphorylations were fully induced by type 1 IFN in the STAT1 STAT2 KO, but did not promote gene induction. Moreover, STAT3 KO did not affect type 1 IFN-induced gene or protein expression. Type 1 IFN also did not activate p38, AKT, or ERK kinase. We conclude that type 1 IFN-induced activities in HeLa cells are mediated by STAT1/STAT2/IRF9, STAT1/STAT1, or STAT2/IRF9 complexes and do not require alternative pathways. (C) 2019 Elsevier Ltd. All rights reserved.
机译：I型干扰素（IFNS）在先天和适应症免疫，增殖和癌症监测中具有核心作用。如何与其特异性受体结合，IFNα和β受体（IFNAR），可以驱动这些各种过程是一个打开的问题。这里，为了系统地和彻底地研究IFN信号传导的分子机制，我们在人体细胞系（HELA）中使用了基于CRISPR / CAS9的方法，以产生参与IFN信号传导级联的基因的敲除（KOS）。我们表明，任何IFN诱导的信令都需要IFNAR链（IFNAR1和IFNAR2）。转录1（STAT1）或Stat2的信号传感器和活化剂的缺失仅对IFN诱导的抗病毒活性或基因诱导具有部分效果。然而，缺失两个基因完全消除了任何IFN诱导的活性。因此，DACE STAT2 IFN调节因子1（IRF1）KO和在很大程度上与IRF9敲低一起。任何统计数据的KO对其他统计数据的磷酸化没有影响，表明它们独立地联系了IFNAR。 STAT3和STAT6磷酸化通过IFN 1 IFN诱导STAT1 Stat2 KO，但未促进基因诱导。此外，Stat3 Ko不影响1型IFN诱导的基因或蛋白质表达。类型1 IFN也没有激活P38，AKT或ERK激酶。我们得出结论，1型IFN诱导的HELA细胞的活动由Stat1 / Stat2 / IRF9，Stat1 / Stat1或STAT2 / IRF9复合物介导，并且不需要替代途径。 （c）2019 Elsevier Ltd.保留所有权利。

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