CRISPR-Cas9敲减鹅硬脂酰辅酶A去饱和酶基因的慢病毒质粒构建

doi:10.3785/j.issn.1008-9209.2020.02.151

浙江大学学报（农业与生命科学版）

2020, Vol. 46

Issue (5): 529-538 DOI: 10.3785/j.issn.1008-9209.2020.02.151

生物科学与技术

CRISPR-Cas9敲减鹅硬脂酰辅酶A去饱和酶基因的慢病毒质粒构建

袁鑫(

),李亮,何桦,胡深强,王继文(

)

四川农业大学，畜禽遗传资源发掘与创新利用四川省重点实验室，成都 611130

Construction of a CRISPR-Cas9 knockdown lentiviral plasmid of goose (Anas platyrhynchos) stearoyl-coenzyme A desaturase gene

Xin YUAN(

),Liang LI,Hua HE,Shenqiang HU,Jiwen WANG(

)

Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China

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摘要：

为进一步探究鹅卵泡颗粒细胞内源性脂肪酸合成代谢机制，利用CRISPR-Cas9技术构建靶向鹅硬脂酰辅酶A去饱和酶（stearoyl-coenzyme A desaturase,SCD）基因的慢病毒敲减质粒并进行慢病毒包装。首先设计鹅SCD基因的单链指导RNA（single-guide RNA, sgRNA）序列，其次体外合成并验证裂解效率，最后利用psPAX2和pMD2.G包装质粒制备psgRNA-mCherry-T2A-Puro和pLenti-Cas9-T2A-EGFP慢病毒质粒。结果表明，慢病毒质粒被成功构建，且其感染中国仓鼠卵巢（Chinese hamster ovary, CHO）细胞后筛选出能同时表达红色荧光蛋白和增强绿色荧光蛋白的强双阳性细胞群。这为后续感染鹅原代颗粒细胞并敲减其SCD基因研究奠定了基础。

关键词： 硬脂酰辅酶A去饱和酶基因; CRISPR-Cas9技术; 基因定点突变; 鹅

Abstract:

In order to further explore the mechanism of endogenous fatty acid synthesis and metabolism in goose granulosa cells, we used CRISPR-Cas9 technology to construct knockdown plasmids of a goose targeted stearoyl-coenzyme A desaturase (SCD) gene and package lentivirus. First, we designed the sequence of single-guide RNA (sgRNA) of goose SCD gene; second, synthesized in vitro and tested the lysis efficacy of sgRNA to target DNA site by endonuclease cleavage assays; finally, prepared the lentiviral plasmid of psgRNA-mCherry-T2A-Puro and pLenti-Cas9-T2A-EGFP using psPAX2 and pMD2.G as package plasmids. Results showed that the lentiviral plasmid was successfully constructed, and the strong double positive cell groups co-expressing the red fluorescent protein (mCherry) and enhanced green fluorescent protein (EGFP) were screened out when the lentiviral plasmid infected the Chinese hamster ovary (CHO) cells. The above results lay the foundation for infecting goose primary granulosa cells and targeting knockdown the SCD gene.

Key words: stearoyl-coenzyme A desaturase gene CRISPR-Cas9 technology gene site-directed mutagenesis goose (Anas platyrhynchos)

收稿日期: 2020-02-15 出版日期: 2020-11-19

CLC:

S 835

基金资助: 国家自然科学基金(31672424)

通讯作者: 王继文 E-mail: nihaoyuanxin88@outlook.com;wjw2886166@163.com

作者简介: 袁鑫（https://orcid.org/0000-0002-7130-5990），E-mail：nihaoyuanxin88@outlook.com

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引用本文:

袁鑫,李亮,何桦,胡深强,王继文. CRISPR-Cas9敲减鹅硬脂酰辅酶A去饱和酶基因的慢病毒质粒构建[J]. 浙江大学学报（农业与生命科学版）, 2020, 46(5): 529-538.

Xin YUAN,Liang LI,Hua HE,Shenqiang HU,Jiwen WANG. Construction of a CRISPR-Cas9 knockdown lentiviral plasmid of goose (Anas platyrhynchos) stearoyl-coenzyme A desaturase gene. Journal of Zhejiang University (Agriculture and Life Sciences), 2020, 46(5): 529-538.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2020.02.151 或 http://www.zjujournals.com/agr/CN/Y2020/V46/I5/529

表1 靶向位点及sgRNA寡核苷酸序列

图1 鹅SCD基因sgRNA靶点设计示意图sgRNA1～6靶点分别位于鹅SCD基因上的不同位置。

图2 sgRNA体外线性化裂解1～6：实验组（样品sgRNA1～6）；7：阴性对照组（sgRNANC）；M：DL1000 DNA标志物。

图3 插入sgRNA1和sgRNA3序列的测序图红色框内的核苷酸序列代表插入的sgRNA寡核苷酸链。

图4 共同感染psgRNA-mCherry-T2A-Puro和pLenti-Cas9-T2A-EGFP的CHO细胞的荧光显微镜观察标尺为50 μm。

图5 CHO细胞感染效率的流式分析Q1：表达红色荧光蛋白（mCherry）细胞的感染效率；Q2：共同表达mCherry和增强绿色荧光蛋白（EGFP）细胞的感染效率；Q3：未表达荧光蛋白细胞的感染效率；Q4：表达EGFP细胞的感染效率。

图6 流式分选出的强双阳性细胞群的荧光显微镜观察标尺为50 μm。

1	BARRANGOU R, FREMAUX C, DEVEAU H, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science, 2007,315(5819):1709-1712. DOI:10.1126/science.1138140 doi: 10.1126/science.1138140
2	JINEK M, EAST A, CHENG A, et al. RNA-programmed genome editing in human cells. Elife, 2013,2:e00471. DOI:10.7554/eLife.00471 doi: 10.7554/eLife.00471
3	XUE W, CHEN S D, YIN H, et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature, 2014,514(7522):380-384. DOI:10.1038/nature13589 doi: 10.1038/nature13589
4	CHANG N N, SUN C H, GAO L, et al. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Research, 2013,23(4):465-472. DOI:10.1038/cr.2013.45 doi: 10.1038/cr.2013.45
5	BAI Y C, HE L J, LI P C, et al. Efficient genome editing in chicken DF-1 cells using the CRISPR/Cas9 system. G3:Genes, Genomes, Genetics, 2016,6(4):917-923. DOI:10.1534/g3.116.027706 doi: 10.1534/g3.116.027706
6	JINEK M, CHYLINSKI K, FONFARA I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012,337(6096):816-821. DOI:10.1126/science.1225829 doi: 10.1126/science.1225829
7	GASIUNAS G, BARRANGOU R, HORVATH P, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. PNAS, 2012,109(39):E2579-E2586. DOI:10.1073/pnas.1208507109 doi: 10.1073/pnas.1208507109
8	MAKAROVA K S, ARAVIND L, WOLF Y I, et al. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biology Direct, 2011,6(1):38. DOI:10.1186/1745-6150-6-38 doi: 10.1186/1745-6150-6-38
9	MAKAROVA K S, HAFT D H, BARRANGOU R, et al. Evolution and classification of the CRISPR-Cas systems. Nature Reviews Microbiology, 2011,9(6):467-477. DOI:10.1038/nrmicro2577 doi: 10.1038/nrmicro2577
10	HAFT D H, SELENGUT J, MONGODIN E F, et al. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Computational Biology, 2005,1(6):e60. DOI:10.1371/journal.pcbi.0010060 doi: 10.1371/journal.pcbi.0010060
11	DOUDNA J A, CHARPENTIER E. The new frontier of genome engineering with CRISPR-Cas9. Science, 2014,346(6213):1258096. DOI:10.1126/science.1258096 doi: 10.1126/science.1258096
12	PATON C M, NTAMBI J M. Biochemical and physiological function of stearoyl-CoA desaturase. American Journal of Physiology: Endocrinology and Metabolism, 2009,297:E28-E37. DOI:10.1152/ajpendo.90897.2008 doi: 10.1152/ajpendo.90897.2008
13	HARDY S, LANGELIER Y, PRENTKI M. Oleate activates phosphatidylinositol 3-kinase and promotes proliferation and reduces apoptosis of MDA-MB-231 breast cancer cells, whereas palmitate has opposite effects. Cancer Research, 2000,60(22):6353-6358.
14	CASTRO L F, WILSON J M, GONCALVES O, et al. The evolutionary history of the stearoyl-CoA desaturase gene family in vertebrates. BMC Evolutionary Biology, 2011,11(1):132. DOI:10.1186/1471-2148-11-132 doi: 10.1186/1471-2148-11-132
15	DOBRZYN A, NTAMBI J M. The role of stearoyl-CoA desaturase in the control of metabolism. Prostaglandins Leukot Essent Fatty Acids, 2005,73(1):35-41. DOI:10.1016/j.plefa.2005.04.011 doi: 10.1016/j.plefa
16	张立敏,张猛,周正奎,等.基于高密度SNP芯片技术对中国西门塔尔牛SCD1基因与肉质性状的相关性分析. 畜牧兽医学报,2011,43(6):878-886. ZHANG L M, ZHANG M, ZHOU Z K, et al. Association of SCD1 gene with meat quality traits in Chinese simmental based on high-density SNP chip. Acta Veterinaria Et Zootechnica Sinica, 2011,43(6):878-886. (in Chinese with English abstract)
17	陈忠琦,李转见,屈玉娇,等.SCD基因在两个奶山羊品种中的酶切多态性分析. 中国草食动物,2010():169-171. DOI:10.3969/j.issn.2095-3887.2010.z1.061 CHEN Z Q, LI Z J, QU Y J, et al. Analysis of SCD gene polymorphism in two dairy goat breeds. China Herbivores, 2010():169-171. (in Chinese) doi: 10.3969/j.issn.2095-3887.2010.z1.061
18	ALVARENGA R R, ZANGERONIMO M G, PEREIRA L J, et al. Lipoprotein metabolism in poultry. World’s Poultry Science Journal, 2011,67(3):431-440. DOI:10.1017/s0043933911000481 doi: 10.1017/s004393
19	RILEY J K, BOLZENIUS J K, LEWIS W G, et al. Fatty acid synthase is expressed and enzymatically active in the mouse ovary and human granulosa cells. Fertility and Sterility, 2013,100(3):S345. DOI:10.1016/j.fertnstert.2013.07.867 doi: 10.1016/j.fertnstert.2013.07.867
20	LIU Z L, RUDD M D, HERNANDEZ-GONZALEZ I, et al. FSH and FOXO1 regulate genes in the sterol/steroid and lipid biosynthetic pathways in granulosa cells. Molecular Endocrinology, 2009,23(5):649-661. DOI:10.1210/me.2008-0412 doi: 10.1210/me.2008-
21	MAGALH?ES-PADILHA D M, GEISLER-LEE J, WISCHRAL A, et al. Gene expression during early folliculogenesis in goats using microarray analysis. Biology of Reproduction, 2013,89(1):19. DOI:10.1095/biolreprod.112.106096 doi: 10.1095/biolreprod.112
22	AUCLAIR S, UZBEKOV R, ELIS S, et al. Absence of cumulus cells during in vitro maturation affects lipid metabolism in bovine oocytes. American Journal of Physiology: Endocrinology and Metabolism, 2013,67:E599-E613. DOI:10.1152/ajpendo.00469.2012 doi: 10.1152/ajpendo.00469.2012
23	XIAO A, CHENG Z C, KONG L, et al. CasOT: a genome-wide Cas9/gRNA off-target searching tool.Bioinformatics, 2014,30(8):1180-1182. DOI:10.1093/bioinformatics/btt764 doi: 10.1093/bioinformatics/btt764
24	WEN R, HU S Q, XIAO Q H, et al. Leptin exerts proliferative and anti-apoptotic effects on goose granulosa cells through the PI3K/Akt/mTOR signaling pathway. Journal of Steroid Biochemistry and Molecular Biology, 2015,149:70-79. DOI:10.1016/j.jsbmb.2015.01.001 doi: 10.1016/j.jsbmb.2015.01.001
25	LI Q, HU S Q, WANG Y S, et al. mRNA and miRNA transcriptome profiling of granulosa and theca layers from geese ovarian follicles reveals the crucial pathways and interaction networks for regulation of follicle selection. Frontiers in Genetics, 2019,10:988. DOI:10.3389/fgene.2019.00988 doi: 10.3389/fgene.2019
26	张利敏,武晓红,姚军虎.产蛋鸡血浆卵黄前体物及其受体研究进展. 中国家禽,2009,31(20):40-44. ZHANG L M, WU X H, YAO J H. Research advances on yolk precursors and oocyte vitellogenesis receptor of layering hens. China Poultry, 2009,31(20):40-44. (in Chinese)
27	LI H, WANG T A, XU C L, et al. Transcriptome profile of liver at different physiological stages reveals potential mode for lipid metabolism in laying hens. BMC Genomics of the United States of America, 2015,16(1):763. DOI:10.1186/s12864-015-1943-0 doi: 10.1186/s12864-015-1943-0
28	NTAMBI J M, MIYAZAKI M, STOEHR J P, et al. Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity. PNAS, 2002,99(17):11482-11486. DOI:10.1073/pnas.132384699 doi: 10.1073/pnas.132384699
29	IGAL R A. Stearoyl-CoA desaturase-1: a novel key player in the mechanisms of cell proliferation, programmed cell death and transformation to cancer. Carcinogenesis, 2010,31(9):1509-1515. DOI:10.1093/carcin/bgq131 doi: 10.1093/carcin/bgq131
30	COHEN P, FRIEDMAN J M. Leptin and the control of metabolism: role for stearoyl-CoA desaturase-1 (SCD-1). The Journal of Nutrition, 2004,134(9):2455S-2463S. DOI:10.1093/jn/134.9.2455s doi: 10.1093/jn/134.9.2455s
31	RAHMAN S M, DOBRZYN A, DOBRZYN P, et al. Stearoyl-CoA desaturase 1 deficiency elevates insulin-signaling components and down-regulates protein-tyrosine phosphatase 1B in muscle. PNAS, 2003,100(19):11110-11115. DOI:10.1073/pnas.1934571100 doi: 10.1073/pnas.1934571100

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