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

doi:10.3785/j.issn.1008-9209.2020.02.151

Journal of Zhejiang University (Agriculture and Life Sciences)

2020, Vol. 46

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

Biological sciences & biotechnology

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

Download:

HTML

HTML ( )

PDF(8983KB)
Export: BibTeX | EndNote (RIS)

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)

Received: 15 February 2020 Published: 19 November 2020

CLC:

S 835

Corresponding Authors: Jiwen WANG E-mail: nihaoyuanxin88@outlook.com;wjw2886166@163.com

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Xin YUAN
	Liang LI
	Hua HE
	Shenqiang HU
	Jiwen WANG

Cite this article:

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.

URL:

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

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

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

Table 1 Targeting sites and sequences of sgRNA oligonucleotides

Fig. 1 Schematic diagram of the sgRNA-targeting sites in goose SCD genesgRNA1-6 targets are located in different positions of goose SCD gene.

Fig. 2 Linearization cleavage in vitro of sgRNA1-6: Experimental group (samples sgRNA1-6); 7: Negative control group (sgRNANC); M: DL1000 DNA marker.

Fig. 3 Sequencing diagrams of the inserted sgRNA1 and sgRNA3 sequencesThe nucleotide sequences in the red box indicate the insertion of sgRNA oligonucleotide chain.

Fig. 4 Fluorescence microscope observation on CHO cells by co-transfection of psgRNA-mCherry-T2A-Puro and pLenti-Cas9-T2A-EGFPScale bar is 50 μm.

Fig. 5 Analysis of infection efficiency of CHO cells by fluorescence activated cell sorting (FACs)Q1: Infection efficiency of cells expressing mCherry; Q2: Infection efficiency of cells expressing both mCherry and EGFP; Q3: Infection efficiency of cells no expressing fluorescent protein; Q4: Infection efficiency of cells expressing EGFP.

Fig. 6 Fluorescence microscope observation on strong double positive cell groups sorted by the flow sorterScale bar is 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

[1]	Zhixin YI,Yilong JIANG,Qiuhong WANG,Qilin XU,Xinxing WANG,Guilin MO,Dongmei JIANG,Bo KANG. Effects of spermine on immune organ indexes and expression levels of genes related to immune factors in geese[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(5): 596-602.

[2]	Yuanliang MO,Yushi WANG,Jiwen WANG. Identification for internal reference genes in different periods of granulosa cells of Tianfu meat geese.[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 376-384.

[3]	ZHAO Qianming, ZHU Longji, ZHAN Kang, HUO Yongjiu, ZHAO Guoqi. Effects of alfalfa meal with different particle sizes on production performance and blood biochemical index of Yangzhou goose[J]. Journal of Zhejiang University (Agriculture and Life Sciences), 2017, 43(2): 253-261.

Viewed

Full text

Abstract

Cited

Shared

Discussed