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ORIGINAL RESEARCH article

Front. Immunol.
Sec. Viral Immunology
Volume 15 - 2024 | doi: 10.3389/fimmu.2024.1468258

CRISPR/Cas9-mediated knockout of STAT1 in porcine-derived cell lines to elucidate the role of STAT1 in autophagy following classical swine fever virus infection

Provisionally accepted
Li Yuan Zhang Li Yuan Zhang Dong Li Liang Dong Li Liang Kai Jun Min Kai Jun Min Jia Xin Liang Jia Xin Liang Yu Tian Yu Tian Cheng Liu Cheng Liu Ting Rong Luo Ting Rong Luo *Xiao Ning Li Xiao Ning Li
  • Guangxi University, Nanning, China

The final, formatted version of the article will be published soon.

    Signal transducer and activator of transcription 1 (STAT1) plays a critical role in immune response, human STAT1 as a transcriptional suppressor of autophagy genes and autophagic activity.Classical swine fever virus (CSFV)-infected induce autophagy, leading to immune evasion. However, there are limited reports on the function of porcine STAT1 in autophagy during CSFV infection.There is also lack of suitable in vitro models for studying porcine STAT1. The objective of this study was to establish porcine PK-15 STAT1-/-and 3D4/21 STAT1-/-cell lines using the CRISPR/Cas9 system to investigate the function of the STAT1 in autophagy. The PK-15 STAT1-/-and 3D4/21 STAT1-/-cell lines, featuring homozygous knockout of STAT1 gene were successfully constructed using the CRISPR/Cas9 editing system. The knockout efficiency determined to be 82.4% and 81.1%, respectively. Infection with CSFV in porcine PK-15 STAT1-/-and 3D4/21 STAT1-/-cells led to an observable increase in autophagosomes as evidenced by transmission electron microscope.Additionally, STAT1 knockout (STAT1 -/-) by the CRISPR/Cas9 system upregulated the expression of ULK1, Beclin1, and LC3 genes, thereby enhancing autophagy during CSFV infection. Conversely, overexpression of STAT1 downregulated the expression of ULK1, Beclin1, and LC3 genes, leading to inhibition of autophagy during CSFV infection.The application of an autophagy dual-fluorescent-Additionally, STAT1 knockout (STAT1 -/-) by the CRISPR/Cas9 system upregulated the expression of ULK1, Beclin1, and LC3 genes, thereby enhancing autophagy during CSFV infection. Conversely, overexpression of STAT1 downregulated the expression of ULK1, Beclin1, and LC3 genes, leading to inhibition of autophagy during CSFV infection.

    Keywords: STAT1 (Signal transducers and activators of transcription 1), CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems), gene knockout cell lines, Autophagy, CSFV (Classical swine fever virus), ULK1

    Received: 21 Jul 2024; Accepted: 10 Oct 2024.

    Copyright: © 2024 Zhang, Liang, Min, Liang, Tian, Liu, Luo and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Ting Rong Luo, Guangxi University, Nanning, China

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