基于CRISPR/Cas9技术构建与鉴定敲除ACE2基因的Huh7肝癌细胞株

doi:10.13560/j.cnki.biotech.bull.1985.2022-1251

摘要/Abstract

摘要：

利用CRISPR-Cas9基因编辑技术敲除人Huh7肝癌细胞的血管紧张素转化酶2（ACE2）基因，构建ACE2基因敲除细胞株，为研究ACE2在肝细胞癌的作用提供细胞模型。首先，对ACE2结构域进行鉴定，利用在线网站设计两条破坏所有结构域、靶向作用于ACE2外显子的sgRNA。其次，构建重组载体并转染肝癌细胞Huh7，嘌呤霉素筛选出单克隆细胞株。最后，免疫印迹鉴定敲除效果。结构域鉴定结果显示，在340-520号氨基酸位置存在Zn结合位点和激活位点，根据sgRNA靶点设计原则，采用片段敲除的方式，针对ACE2的第9、第10外显子设计两对sgRNA，并成功构建PX459-ACE2-sgRNA重组质粒。嘌呤霉素成功筛选出单克隆细胞株，并测序证实了发生片段敲除，ACE2蛋白在敲除细胞株中不表达；成功构建ACE2敲除的Huh7细胞株，为日后研究ACE2在肝细胞癌的发生机制奠定基础。

关键词: 血管紧张素转化酶2, 肝细胞癌, CRISPR-Cas9

Abstract:

The experiment is aimed to establish ACE2（angiotensin converting enzyme 2）-knock out Huh7 liver cancer cells using CRISPR-Cas9, which could be used as cell model to study the role of ACE2 in HCC（hepatocellular carcinoma）. Firstly, the ACE2 domain was identified, and two sgRNAs targeting ACE2 exons were designed using an online website that destroyed all the domains. Secondly, the recombinant vector was constructed and transfected into HCC cell Huh7, and the monoclonal cell line was screened by puromycin. Finally, the knockout effect was identified by immunoblotting. The domain identification results showed that there were Zn-binding sites and activation sites at amino acids 340-520. According to the sgRNA target design principle, two pairs of sgRNA were designed for the ninth and tenth exons of ACE2 by fragment knockout method, and the recombinant plasmid PX459-ACE2-sgRNA was successfully constructed. The monoclonal cell line was successfully screened by puromycin, sequencing confirmed the occurrence of fragment knockout, and ACE2 protein was not expressed in the ACE2 knockout cell line. CRISPR-Cas9 technology was used to successfully construct ACE2 knockout Huh7 cell line, which lays a foundation for future research on the mechanism of ACE2 in HCC.

Key words: angiotensin converting enzyme 2, hepatocellular carcinoma（HCC）, CRISPR-Cas9

张祖霖, 刘方芳, 周青鸟, 赵瑞强, 贺菽嘉, 林文珍. 基于CRISPR/Cas9技术构建与鉴定敲除ACE2基因的Huh7肝癌细胞株[J]. 生物技术通报, 2023, 39(6): 181-188.

ZHANG Zu-lin, LIU Fang-fang, ZHOU Qing-niao, ZHAO Rui-qiang, HE Shu-jia, LIN Wen-zhen. Construction and Identification of Huh7 Hepatoma Cell Line with ACE2 Gene Knockout Based on CRISPR/Cas9 Technology[J]. Biotechnology Bulletin, 2023, 39(6): 181-188.

图/表 9

表1 测序引物和PCR序列

Table 1 Sequencing primers and PCR sequences

名称Primer name	引物序列Primer sequence（5'-3'）
U6-F	ATGGACTATCATATGCTTACCGTA
CMV-R	TCGTTGGGCGGTCAGC
验证-F Verification-F	GACGACTTCCTGACAGCTCATCAT
验证-R Verification-R	AGTGTCACCTGACCCAAGGAAAT

图1 鉴定出的ACE2结构域 A: NCBI CD-search鉴定的ACE2结构域，红框标出主要结构域；B：Esembl数据库查找ACE2最大转录本编码的蛋白质结构域，红框标出第9、10外显子及其所在的结构域

Fig. 1 Identified ACE2 domain A: ACE2 domain identified by NCBI CD-search, with major domains marked in red boxes. B: The Esembl database searches for the protein domain encoded by the largest transcript of ACE2. Exons 9 and 10 and their domain are marked in red boxes

表2 靶向ACE2基因的两条sgRNA序列

Table 2 Sequences of two sgRNAs targeting ACE2 gene

sgRNA	序列Sequence（5'-3'）
sgRNA1F	CACCGATCGGGTGACAGAAGACCAA
sgRNA1R	AAACTTGGTCTTCTGTCACCCGATC
sgRNA2F	CACCGTCGTGAGTGCTTGTTTGAGC
sgRNA2R	AAACGCTCAAACAAGCACTCACGAC

图2 两条sgRNA寡核苷酸片段特异性评分和脱靶分析 A：sgRNA1特异性评分及脱靶分析；B：sgRNA2特异性评分及脱靶分析

Fig. 2 Specificity score and off-target analysis of two sgRNA oligonucleotide fragments A: sgRNA1 specific score and off-target analysis; B: sgRNA2 specific score and off-target analysis

图3 PX459-sgRNA重组载体菌落验证 M: DL500 DNA marker；1-8泳道为PX459-ACE2-sgRNA1; 9-16泳道为PX459-ACE2-sgRNA2

Fig. 3 Colony validation of PX459-sgRNA recombinant vector M: DL500 DNA marker. Lane 1 to 8 are PX459-ACE2-sgRNA1. Lane 9 to 16 are PX459-ACE2-sgRNA2

图4 重组质粒菌液双向测序结果 A:PX459-ACE2-sgRNA1测序峰图；B：PX459-ACE2-sgRNA2测序峰图

Fig. 4 Results of bidirectional sequencing of recombinant plasmid liquid A: PX459-ACE2-sgRNA1 sequencing peaks. B: PX459-ACE2-sgRNA2 sequencing peaks

图5 琼脂糖凝胶电泳验证基因编辑效果 M:DL10000 marker；1-5泳道分别为：Huh7野生型；PXNC（对照组）；PX1#、PX2#、PX3#（敲除组）

Fig. 5 Agarose gel electrophoresis to verify the effect of gene editing M: DL10000 marker. Lane 1-5 are Huh7 wild-type; PXNC（control group）; PX1 #， PX2 #， PX3#（knockout group）

图6 基因编辑结果 A: PXNC（对照组）基因片段；B: PX2#（敲除组）在sgRNA作用于特定外显子后，造成基因片段缺失，以非同源重组的方式连接形成修复的DNA片段；C: Blast比对片段敲除的PX2#单克隆细胞株和PXNC测序结果，部分外显子和内含子的碱基序列缺失，红色为编辑后的PX2#基因序列；与PXNC相比，片段敲除的PX2#基因断裂

Fig. 6 Results of gene editing A: PXNC（control group）gene fragment. B: PX2#（knockout group）, gene fragment deletion after sgRNA acts on specific exons, which is connected to form repaired DNA fragment by non-homologous recombination. C: Blast comparison of PX2# monoclonal cell line and PXNC sequencing results showed that some exons and introns were missing, and the edited PX2# gene sequence was shown in red. Compared with PXNC, the PX2# gene with fragment knockout was fragmented

图7 Western Blot验证蛋白表达水平

Fig. 7 Protein expression level verified by Western Blot

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