Cargando…

CRISPR/Cas9-Mediated Chicken TBK1 Gene Knockout and Its Essential Role in STING-Mediated IFN-β Induction in Chicken Cells

TANK-binding kinase 1 (TBK1) is involved in innate immunity, prompting transcriptional induction of type I interferons in response to pathogenic infection. Many studies have focused on mammals but the function of TBK1 in chickens remains poorly defined. CRISPR/Cas9 system has made gene-knockout easy...

Descripción completa

Detalles Bibliográficos
Autores principales: Cheng, Yuqiang, Lun, Minxiang, Liu, Yunxia, Wang, Hengan, Yan, Yaxian, Sun, Jianhe
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328437/
https://www.ncbi.nlm.nih.gov/pubmed/30662438
http://dx.doi.org/10.3389/fimmu.2018.03010
_version_ 1783386639599403008
author Cheng, Yuqiang
Lun, Minxiang
Liu, Yunxia
Wang, Hengan
Yan, Yaxian
Sun, Jianhe
author_facet Cheng, Yuqiang
Lun, Minxiang
Liu, Yunxia
Wang, Hengan
Yan, Yaxian
Sun, Jianhe
author_sort Cheng, Yuqiang
collection PubMed
description TANK-binding kinase 1 (TBK1) is involved in innate immunity, prompting transcriptional induction of type I interferons in response to pathogenic infection. Many studies have focused on mammals but the function of TBK1 in chickens remains poorly defined. CRISPR/Cas9 system has made gene-knockout easy to accomplish. Although CRISPR/Cas9 has been used in chicken cells, low mutation efficiency limits its wide application in chickens. In this study, an effective gene-knockout system was developed based on the CRISPR/Cas9 system in chicken embryonic fibroblast DF-1. Two CRISPR/Cas9 plasmids were constructed, TBK1-g1 and TBK1-g2, which express gRNAs targeting different sequences of the chicken TBK1 gene. After transfection and enrichment with puromycin screening, the mutation rates as assessed via T7E1 assay were 88.05 and 89.55%, respectively, and subsequent sequence analysis showed mutation efficiencies of 86.67 and 93.33%. With the limiting-dilution method, a chTBK1 gene-deficiency monoclonal cell line was obtained and was named DF-1-TBK1-C3. The DF-1-TBK1-C3 cells exhibited normal morphology and maintained stable proliferation ability compared to wild-type cells. The gene-overexpression system and luciferase reporter assay showed that IFN-β induction induced by chSTING was almost completely blocked in DF-1-TBK1-C3 cells. With quantitative real-time PCR, we further confirmed the essential role of chTBK1 in the chSTING-mediated IFN-β induction. At last, the study demonstrated that the chTBK1 knockout system is also applicable in primary chick embryo fibroblasts (CEFs). In this study, an effective gene-knockout system was applied in chickens, a TBK1 gene-deleted DF-1 cell line was successfully created using this system, and with the chTBK1 knockout cells, chTBK1 was revealed to be indispensable in STING-mediated IFN-β activation in chicken cells.
format Online
Article
Text
id pubmed-6328437
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-63284372019-01-18 CRISPR/Cas9-Mediated Chicken TBK1 Gene Knockout and Its Essential Role in STING-Mediated IFN-β Induction in Chicken Cells Cheng, Yuqiang Lun, Minxiang Liu, Yunxia Wang, Hengan Yan, Yaxian Sun, Jianhe Front Immunol Immunology TANK-binding kinase 1 (TBK1) is involved in innate immunity, prompting transcriptional induction of type I interferons in response to pathogenic infection. Many studies have focused on mammals but the function of TBK1 in chickens remains poorly defined. CRISPR/Cas9 system has made gene-knockout easy to accomplish. Although CRISPR/Cas9 has been used in chicken cells, low mutation efficiency limits its wide application in chickens. In this study, an effective gene-knockout system was developed based on the CRISPR/Cas9 system in chicken embryonic fibroblast DF-1. Two CRISPR/Cas9 plasmids were constructed, TBK1-g1 and TBK1-g2, which express gRNAs targeting different sequences of the chicken TBK1 gene. After transfection and enrichment with puromycin screening, the mutation rates as assessed via T7E1 assay were 88.05 and 89.55%, respectively, and subsequent sequence analysis showed mutation efficiencies of 86.67 and 93.33%. With the limiting-dilution method, a chTBK1 gene-deficiency monoclonal cell line was obtained and was named DF-1-TBK1-C3. The DF-1-TBK1-C3 cells exhibited normal morphology and maintained stable proliferation ability compared to wild-type cells. The gene-overexpression system and luciferase reporter assay showed that IFN-β induction induced by chSTING was almost completely blocked in DF-1-TBK1-C3 cells. With quantitative real-time PCR, we further confirmed the essential role of chTBK1 in the chSTING-mediated IFN-β induction. At last, the study demonstrated that the chTBK1 knockout system is also applicable in primary chick embryo fibroblasts (CEFs). In this study, an effective gene-knockout system was applied in chickens, a TBK1 gene-deleted DF-1 cell line was successfully created using this system, and with the chTBK1 knockout cells, chTBK1 was revealed to be indispensable in STING-mediated IFN-β activation in chicken cells. Frontiers Media S.A. 2019-01-04 /pmc/articles/PMC6328437/ /pubmed/30662438 http://dx.doi.org/10.3389/fimmu.2018.03010 Text en Copyright © 2019 Cheng, Lun, Liu, Wang, Yan and Sun. http://creativecommons.org/licenses/by/4.0/ 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) and the copyright owner(s) 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.
spellingShingle Immunology
Cheng, Yuqiang
Lun, Minxiang
Liu, Yunxia
Wang, Hengan
Yan, Yaxian
Sun, Jianhe
CRISPR/Cas9-Mediated Chicken TBK1 Gene Knockout and Its Essential Role in STING-Mediated IFN-β Induction in Chicken Cells
title CRISPR/Cas9-Mediated Chicken TBK1 Gene Knockout and Its Essential Role in STING-Mediated IFN-β Induction in Chicken Cells
title_full CRISPR/Cas9-Mediated Chicken TBK1 Gene Knockout and Its Essential Role in STING-Mediated IFN-β Induction in Chicken Cells
title_fullStr CRISPR/Cas9-Mediated Chicken TBK1 Gene Knockout and Its Essential Role in STING-Mediated IFN-β Induction in Chicken Cells
title_full_unstemmed CRISPR/Cas9-Mediated Chicken TBK1 Gene Knockout and Its Essential Role in STING-Mediated IFN-β Induction in Chicken Cells
title_short CRISPR/Cas9-Mediated Chicken TBK1 Gene Knockout and Its Essential Role in STING-Mediated IFN-β Induction in Chicken Cells
title_sort crispr/cas9-mediated chicken tbk1 gene knockout and its essential role in sting-mediated ifn-β induction in chicken cells
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328437/
https://www.ncbi.nlm.nih.gov/pubmed/30662438
http://dx.doi.org/10.3389/fimmu.2018.03010
work_keys_str_mv AT chengyuqiang crisprcas9mediatedchickentbk1geneknockoutanditsessentialroleinstingmediatedifnbinductioninchickencells
AT lunminxiang crisprcas9mediatedchickentbk1geneknockoutanditsessentialroleinstingmediatedifnbinductioninchickencells
AT liuyunxia crisprcas9mediatedchickentbk1geneknockoutanditsessentialroleinstingmediatedifnbinductioninchickencells
AT wanghengan crisprcas9mediatedchickentbk1geneknockoutanditsessentialroleinstingmediatedifnbinductioninchickencells
AT yanyaxian crisprcas9mediatedchickentbk1geneknockoutanditsessentialroleinstingmediatedifnbinductioninchickencells
AT sunjianhe crisprcas9mediatedchickentbk1geneknockoutanditsessentialroleinstingmediatedifnbinductioninchickencells