基于CRISPR/Cas9系统构建FOXA2表达示踪体系

doi:10.13560/j.cnki.biotech.bull.1985.2018-0111

摘要/Abstract

摘要： 利用CRISPR/Cas9系统在基因FOXA2蛋白质编码区后插入绿色荧光蛋白核酸序列,实现对FOXA2基因开启和关闭的示踪。通过靶向序列选择、Cas9靶向质粒及Donor片段的克隆、细胞转染并进行流式细胞荧光分选、阳性细胞有限稀释、富集培养及单克隆细胞的鉴定和转录翻译水平验证等5个方面,进行实验研究。在编辑的乳腺肿瘤细胞MCF-7中,绿色荧光蛋白有效表达;乳腺癌细胞内绿色荧光蛋白的表达水平同FOXA2蛋白的表达水平正相关,且经肿瘤细胞上皮间质转化进程诱导因子EGF的诱导,绿色荧光蛋白和FOXA2表达水平同步降低。方法学上CRISPR/Cas9靶向FOXA2并利用细胞内同源性定向修复插入绿色荧光蛋白序列成功;利用报告基因蛋白表达水平示踪目的基因的开启、关闭及表达量高低结果准确、方法简单,效果明显。

关键词: CRISPR/Cas9, 基因编辑, FOXA2, 示踪, 乳腺癌, MCF-7, 上皮间质转化

Abstract: This study enabled the tracing of FOXA2 gene on and off by using CRISPR/Cas9 system to insert a green fluorescent protein（GFP）nucleic acid sequence following the FOXA2 protein coding region. The study was carried out from the following five aspects：targeting sequence selection,cloning of Cas9-targeting plasmids and donor fragment,cells transfection and flow cytometry fluorescence sorting,limited dilution of positive cells,as well as enrichment culture and identification,transcription and translation level verification of monoclonal cells. The results showed that the expression of GFP was positive in the edited breast cancer cell line MCF-7. The expression level of GFP in breast cancer cells was positively correlated with the expression level of FOXA2 protein,and the expression of GFP and FOXA2 decreased synchronously after the induction by EGF,an inducing factor in the process of tumor cells epithelial-mesenchymal transition（EMT）. The results indicate that CRISPR/Cas9 targeting FOXA2 and using intracellular homology directed repair（HDR）to insert GFP sequence are success. Using the expression level of reporter gene protein for tracing the target gene on and off as well as its expression is significantly accurate and convenient.

Key words: CRISPR/Cas9, gene editing, FOXA2, tracing, breast cancer, MCF-7, epithelial-mesenchymal transition

程浩杰,张振旺,谭桂湘,刘昱翔,卜会铜,谭拥军. 基于CRISPR/Cas9系统构建FOXA2表达示踪体系[J]. 生物技术通报, 2018, 34(5): 80-86.

CHENG Hao-jie, ZHANG Zhen-wang, TAN Gui-xiang, LIU Yu-xiang, PIAO Hui-tong, TAN Yong-jun. Construction of FOXA2 Expression Tracer System Based on CRISPR/Cas9 System[J]. Biotechnology Bulletin, 2018, 34(5): 80-86.

参考文献

[1] Jansen R, Embden JD, Gaastra W, et al.Identification of genes that are associated with DNA repeats in prokaryotes[J]. Mol Microbiol, 2002, 43(6):1565-1575.
[2] Andersson AF, Banfield JF.Virus population dynamics and acquired virus resistance in natural microbial communities[J]. Science, 2008, 320(5879):1047-1050.
[3] Bhaya D, Davison M, Barrangou R.CRISPR-Cas systems in bacteria and archaea:versatile small RNAs for adaptive defense and regulation[J]. Annu Rev Genet, 2011, 45:273-297.
[4] Fineran PC, Dy RL.Gene regulation by engineered CRISPRCas systems[J]. Curr Opin Microbiol, 2014, 18:83-89.
[5] Makarova KS, Haft DH, Barrangou R, et al.Evolution and classification of the CRISPR-Cas systems.[J]. Nature Reviews Microbiology, 2011, 9(6):467-477.
[6] Chylinski K, Makarova KS, Charpentier E, et al.Classification and evolution of type II CRISPR-Cas systems[J]. Nucleic Acids Res, 2014, 42(10):6091-6105.
[7] Wang H, Yang H, Shivalila CS, et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas mediated genome engineering[J]. Cell, 2013, 153(4):910-918.
[8] Wan H, Feng C, Teng F, et al.One-step generation of p53 gene biallelic mutant Cynomolgus monkey via the CRISPR/Cas system[J]. Cell Res, 2015, 25(2):258-261.
[9] Johnson RA, Gurevich V, Filler S, et al.Comparative assessments of CRISPR-Cas nucleases’cleavage efficiency in planta[J]. Plant Mol Biol, 2015, 87(1-2):143-156.
[10] Endo M, Mikami M, Toki S.Multigene knockout utilizing offtarget mutations of the CRISPR/Cas9 system in rice[J]. Plant Cell Physiol, 2015, 56(1):41-47.
[11] Jiang W, Zhou H, Bi H, et al.Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice[J]. Nucleic Acids Res, 2013, 41(20):e188.
[12] Jiang Y, Chen B, Duan C, Sun B, et al.Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol, 2015, 81(7):2506-14.
[13] van Pijkeren JP, Britton RA. Precision genome engineering in lactic acid bacteria[J]. Microb Cell Fact, 2014, 13(Suppl1):S10.
[14] Yuen KS, Chan CP, Wong NH, et al.CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells[J]. J Gen Virol, 2015, 96(Pt 3):626-636.
[15] Seeger C, Sohn J A.Targeting Hepatitis B Virus With CRISPR/Cas9[J]. Mol Ther Nucleic Acids, 2014, 3:e216.
[16] Tan Y, Adami G, Costa RH.Maintaining HNF6 Expression Prevents AdHNF3beta-Mediated Decrease in Hepatic Levels of Glut-2and Glycogen[J]. Hepatology, 2002, 35(4):790-8.
[17] Tang Y, Shu G, Yuan X, et al.FOXA2 functions as a suppressor of tumor metastasis by inhibition of epithelial-to-mesenchymal transition in human lung cancers[J]. Cell Res, 2011, 21(2):316-26.
[18] Zhang Z, Yang C, Gao W, et al.FOXA2 attenuates the epithelial to mesenchymal transition by regulating the transcription of E-cadherin and ZEB2 in human breast cancer[J]. Cancer Lett, 2015, 361(2):240-50.
[19] Goldman O, Han S, Hamou W, et al.Endoderm generates endothelial cells during liver development[J]. Stem Cell Reports, 2014, 3(4):556-65.
[20] Alder O, Cullum R, Lee S, et al.Hippo signaling influences HNF4A and FOXA2 enhancer switching during hepatocyte differentiation[J]. Cell Rep, 2014, 9(1):261-71.
[21] von Meyenn F, Porstmann T, Gasser E, et al. Glucagon-Induced Acetylation of Foxa2 Regulates Hepatic Lipid Metabolism[J]. Cell Metab, 2013, 17(3):436-47.
[22] Cyranoski D.CRISPR gene-editing tested in a person for the first time[J]. Nature, 2016, 539(7630):479.
[23] Korkmaz G, Lopes R, Ugalde AP, et al.Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9[J]. Nat Biotechnol, 2016, 34(2):192-8.