CRISPR/Cas9-Mediated Chicken TBK1 Gene Knockout and Its Essential Role in STING-Mediated IFN-β Induction in Chicken Cells
CRISPR/Cas9-Mediated Chicken TBK1 Gene Knockout and Its Essential Role in STING-Mediated IFN-β Induction in Chicken Cells
复制标题
CRISPR/Cas9介导的鸡TBK1基因敲除及其在STING介导的鸡细胞IFN-β诱导中的重要作用
DOI：
10.3389/fimmu.2018.03010
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发表时间：
2019-01-04
影响因子：
7.3
通讯作者：
Sun, Jianhe
中科院分区：
文献类型：
--
作者：
Cheng, Yuqiang;Lun, Minxiang;Sun, Jianhe
TANK结合激酶1（TBK 1）参与先天免疫，促使I型干扰素的转录诱导应答病原性感染。许多研究都集中在哺乳动物，但TBK 1在鸡中的功能仍然不清楚。CRISPR/Cas9系统使基因敲除变得容易完成。尽管CRISPR/Cas9已用于鸡细胞，但较低的突变效率限制了其在鸡中的广泛应用。本研究基于鸡胚胎成纤维细胞DF-1的CRISPR/Cas9系统开发了一种有效的基因敲除系统。构建了两种CRISPR/Cas9质粒TBK 1-g1和TBK 1-g2，它们表达靶向鸡TBK 1基因的不同序列的gRNA。转染和富集嘌呤霉素筛选后，通过T7 E1测定评估的突变率分别为88.05%和89.55%，随后的序列分析显示突变效率为86.67%和93.33%。采用有限稀释法，获得了一株chTBK 1基因缺陷型单克隆细胞株，命名为DF-1-TBK 1-C3。与野生型细胞相比，DF-1-TBK 1-C3细胞表现出正常的形态并保持稳定的增殖能力。基因过表达系统和荧光素酶报告基因分析表明，在DF-1-TBK 1-C3细胞中，chSTING诱导的IFN-β诱导几乎完全被阻断。通过实时定量PCR，我们进一步证实了chTBK 1在chSTING介导的IFN-β诱导中的重要作用。最后，本研究证明chTBK 1基因敲除系统同样适用于原代鸡胚成纤维细胞（CEF）。本研究将一种有效的基因敲除系统应用于鸡，成功建立了TBK 1基因缺失的DF-1细胞系，并利用chTBK 1敲除细胞，揭示了chTBK 1在鸡细胞中STING介导的IFN-β激活中不可或缺。
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-beta 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-beta 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-beta activation in chicken cells.