基于CRISPR/Cas9基因编辑系统建立WIP1基因g.37536832 C＞A位点突变的ST细胞系

doi:10.16431/j.cnki.1671-7236.2025.05.003

摘要/Abstract

摘要： 【目的】利用CRISPR/Cas9基因编辑技术获得野生型p53诱导磷酸酶1(wild-type p53-induced phosphatase 1，WIP1)基因与精子活力显著相关的位点g.37536832 C＞A突变的猪睾丸(swine testis，ST)细胞系，为在细胞层面上进一步探讨WIP1基因与公猪精液品质性状之间的关系奠定基础。【方法】利用CRISPOR在线网站在猪WIP1基因g.37536832 C＞A位点附近区域设计3条向导RNA(single guide RNA，sgRNA)，并将其连接至pX458-GFP载体；通过T7E1酶切试验检测3条sgRNA活性，选择效率较高的sgRNA。构建Donor载体，并将其与sgRNA载体共同转染至ST细胞，采用流式细胞术将表达绿色荧光蛋白(green fluorescent protein，GFP)的阳性细胞进行富集和单克隆细胞筛选，并对筛选出的单克隆细胞进行基因型鉴定。【结果】质粒测序结果表明，成功将3条sgRNA连接至pX458-GFP载体上；T7E1酶切结果显示，转染pX458-GFP-sgRNA1和pX458-GFP-sgRNA2质粒组产生了切割，效率分别为24%和27%，而转染pX458-GFP-sgRNA3质粒组未检测到切割，因此选择pX458-GFP-sgRNA2质粒进行试验。成功构建Donor载体，并将pX458-GFP-sgRNA2和Donor载体共转染至ST细胞。基因型鉴定结果显示，获得的269株单克隆中有205株细胞发生了基因编辑，基因编辑效率为76%，其中9株为WIP1基因g.37536832 C>A位点纯合突变的ST细胞，精确突变效率为3%。【结论】本研究利用CRISPR/Cas9基因编辑技术成功获得了WIP1基因g.37536832 C＞A位点突变的ST细胞系，为深入研究WIP1基因对公猪精液品质的影响提供了良好的细胞模型。

关键词: CRISPR/Cas9; WIP1基因; ST细胞系

Abstract: 【Objective】 The purpose of this experiment was to establish the wild-type p53-induced phosphatase 1 (WIP1) gene g.37536832 C＞A mutation swine testis (ST) cell line which was significantly associated with sperm motility by using CRISPR/Cas9 gene editing technology,and laid a solid foundation for further research on the relationship between the WIP1 gene and the semen quality traits of boar semen at the cellular level.【Method】 Three single guide RNA (sgRNA) were designed near the g.37536832 C＞A locus of porcine WIP1 gene using CRISPOR online website and connected to pX458-GFP vector.The activities of three sgRNA vectors were detected by T7E1 enzymatic cleavage assay,and the sgRNA with higher efficiency was selected.Donor vector was constructed and transfected into ST cells together with sgRNA vector.Positive cells expressing green fluorescent protein (GFP) were enriched and monoclonal cells were screened using flow cytometry,and the genotypes of the selected monoclonal cells were identified. 【Result】 Plasmid sequencing results showed that three sgRNAs were successfully ligated into the pX458-GFP vector.The results of T7E1 enzyme digestion showed that pX458-GFP-sgRNA1 and pX458-GFP-sgRNA2 transfected plasmids produced cleavage,and the efficiency was 24% and 27%,respectively,while no cleavage was detected in pX458-GFP-sgRNA3.Therefore,pX458-GFP-sgRNA2 plasmid was selected for the experiment.Donor vector was successfully constructed,and pX458-GFP-sgRNA2 and Donor vector were transfected into ST cells.Genotype identification showed that 205 of the 269 monoclones obtained had gene editing,and the gene editing efficiency was 76%.Among them,9 clones were ST cells with homozygous mutation at g.37536832 C>A of WIP1 gene,and the precise mutation efficiency was 3%.【Conclusion】 In this study,the ST cell line with the mutation of g.37536832 C＞A locus of WIP1 gene was successfully obtained using CRISPR/Cas9 gene editing technology,which provided a favourable cell model for further study of the effect of WIP1 gene on the semen quality of boars.

Key words: CRISPR/Cas9; WIP1 gene; ST cell line

中图分类号:

S828.9

王楠, 杜伟伟, 王婉洁, 王悦, 袁茂莎, 聂雨欣, 孙亚茹, 刘志国, 吴添文, 牟玉莲. 基于CRISPR/Cas9基因编辑系统建立WIP1基因g.37536832 C＞A位点突变的ST细胞系[J]. 中国畜牧兽医, 2025, 52(5): 1966-1976.

WANG Nan, DU Weiwei, WANG Wanjie, WANG Yue, YUAN Maosha, NIE Yuxin, SUN Yaru, LIU Zhiguo, WU Tianwen, MU Yulian. Establishment of ST Cell Lines with WIP1 Gene g.37536832 C＞A Mutation Using the CRISPR/Cas9 Gene Editing System[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(5): 1966-1976.

参考文献

[1] 雷润雨,张宇,冯越,等.EFNA3和ITGA9基因多态性与公猪精液品质性状的关联分析[J].中国畜牧杂志,2024,60(8):233-239.LEI R Y,ZHANG Y,FENG Y,et al.Association analysis of EFNA3 and ITGA9 gene polymorphisms with semen quality traits in boars[J].Chinese Journal of Animal Science,2024,60(8):233-239.(in Chinese)
[2] MCPHERSON F J,NIELSEN S G,CHENOWETH P J.Semen effects on insemination outcomes in sows[J].Animal Reproduction Science,2014,151(1-2):28-33.
[3] MYROMSLIEN F D,TREMOEN N H,ANDERSEN-RANBERG I,et al.Sperm DNA integrity in Landrace and Duroc boar semen and its relationship to litter size[J].Reproduction in Domestic Animals, 2019,54(2):160-166.
[4] XU K,YANG L,ZHANG L,et al.Lack of AKAP3 disrupts integrity of the subcellular structure and proteome of mouse sperm and causes male sterility[J].Development,2020,147(2):dev181057.
[5] WIDLAK W,VYDRA N.The role of heat shock factors in mammalian spermatogenesis[J].Advances in Anatomy Embryology and Cell Biology,2017,222:45-65.
[6] HAN F,DONG M Z,LEI W L,et al.Oligoasthenoteratospermia and sperm tail bending in PPP4C-deficient mice[J].Molecular Human Reproduction,2021,27(1):gaaa083.
[7] LI Y,LI C,LIN S,et al.A nonsense mutation in Ccdc62 gene is responsible for spermiogenesis defects and male infertility in repro29/repro29 mice[J].Biology of Reproduction,2017,96(3):587-597.
[8] LEEM J,BAI G Y,OH J S.The capacity to repair sperm DNA damage in zygotes is enhanced by inhibiting WIP1 activity[J].Frontiers in Cell and Developmental Biology,2022,10:841327.
[9] 吴雨,周迪,陈琨,等.种公猪精液品质候选基因的研究进展[J].中国畜禽种业,2023,19(6):105-113.WU Y,ZHOU D,CHEN K,et al.Research progress on candidate genes for semen quality in boars[J].The Chinese Livestock and Poultry Breeding,2023,19(6):105-113.(in Chinese)
[10] YU Z,SONG Y,CAI M,et al.PPM1D is a potential prognostic biomarker and correlates with immune cell infiltration in hepatocellular carcinoma[J].Aging,2021,13(17):21294-21308.
[11] WEI Y,GAO Q,NIU P,et al.Integrative proteomic and phosphoproteomic profiling of testis from Wip1 phosphatase-knockout mice:Insights into mechanisms of reduced fertility[J].Molecular & Cellular Proteomics,2019,18(2):216-230.
[12] 王楠,冯保亮,郑云曦,等.WIP1基因对3T3-L1前脂肪细胞增殖分化的影响及其在小鼠不同生长阶段的表达[J].中国畜牧兽医,2022,49(8):2869-2879.WANG N,FENG B L,ZHENG Y X,et al.Effects of WIP1 gene on proliferation and differentiation of 3T3-L1 preadipocytes and its expression in different growth stages of mice[J].China Animal Husbandry & Veterinary Medicine,2022,49(8):2869-2879.(in Chinese)
[13] CHOI J,NANNENGA B,DEMIDOV O N,et al.Mice deficient for the wild-type p53-induced phosphatase gene (WIP1) exhibit defects in reproductive organs,immune function,and cell cycle control[J].Molecular and Cellular Biology,2002,22(4):1094-1105.
[14] XU K,ZHANG X,LIU Z,et al.A transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection[J].Science China-Life Sciences,2022,65(8):1535-1546.
[15] 陈宗见,陈远昆,甘麦邻,等.品种和季节效应对公猪精液品质的影响[J].猪业科学,2022,39(2):114-116.CHEN Z J,CHEN Y K,GAN M L,et al.Influence of breed and seasonal effects on semen quality in boars[J].Swine Industry Science,2022,39(2):114-116.(in Chinese)
[16] MANKOWSKA A,BRYM P,PAUKSZTO L,et al.Gene polymorphisms in boar spermatozoa and their associations with post-thaw semen quality[J].International Journal of Molecular Sciences,2020,21(5):1902.
[17] FAN Z,MU Y,LI K,et al.Safety evaluation of transgenic and genome-edited food animals[J].Trends in Biotechnolog,2022,40(4):371-373.
[18] 许美娜,朱奕舟,林思远,等.CRISPR/Cas9基因编辑技术在猪育种中的研究进展[J].广东农业科学,2022,49(8):87-96.XU M N,ZHU Y Z,LIN S Y,et al.Progress of the application of CRISPR/Cas9 gene editing technology in pig breeding[J].Guangdong Agricultural Sciences,2022,49(8):87-96.(in Chinese)
[19] XIANG G,REN J,HAI T,et al.Editing porcine IGF2 regulatory element improved meat production in Chinese Bama pigs[J].Cellular and Molecular Life Sciences,2018,75(24):4619-4628.
[20] 车晶晶,徐奎,张秀玲,等.基于CRISPR/Cas9技术的WIP1基因敲除ST细胞的建立[J].畜牧兽医学报,2021,52(10):2814-2821.CHE J J,XU K,ZHANG X L,et al.Establishment of Wip1-knockout ST cells mediated by CR1SPR/Cas9 system[J].Acta Veterinaria et Zootechnica Sinica,2021,52(10):2814-2821.(in Chinese)
[21] 王悦.猪WIP1基因与精液品质的关联研究及对ST细胞增殖的影响[D].广州：佛山科学技术学院,2022.WANG Y.Association between porcine WIP1 gene and semen quality and its effect on the proliferation of ST cells[D].Guangzhou：Foshan University,2022.(in Chinese)
[22] 吴建忠.猪遗传育种技术研究进展[J].畜禽业,2004，3:58-60.WU J Z.Progress in pig genetic breeding technology[J].Livestock and Poultry Industry,2004，3:58-60.(in Chinese)
[23] ZUIDEMA D,KERNS K,SUTOVSKY P.An exploration of current and perspective semen analysis and sperm selection for livestock artificial insemination[J].Animals (Basel),2021,11(12):3563.
[24] WANG X,SIPILA P,SI Z,et al.CDK5RAP2 loss-of-function causes premature cell senescence via the GSK3beta/beta-catenin-WIP1 pathway[J].Cell Death & Disease,2021,13(1):9.
[25] EREN M K,KARTAL N B,PILEVNELI H.Oncogenic WIP1 phosphatase attenuates the DNA damage response and sensitizes p53 mutant Jurkat cells to apoptosis[J].Oncology Letters,2021,21(6):479.
[26] LEEM J,KIM J S,OH J S.WIP1 phosphatase suppresses the DNA damage response during G2/prophase arrest in mouse oocytes[J].Biology of Reproduction,2018,99(4):798-805.
[27] PARK D S,YOON G H,KIM E Y,et al.WIP1 regulates Smad4 phosphorylation and inhibits TGF-beta signaling[J].EMBO Reports,2020,21(5):e48693.
[28] FILIPPONI D,MULLER J,EMELYANOV A,et al.WIP1 controls global heterochromatin silencing via ATM/BRCA1-dependent DNA methylation[J]. Cancer Cell,2013,24(4):528-541.
[29] CHO S J,CHA B S,KWON O S,et al.WIP1 directly dephosphorylates NLK and increases Wnt activity during germ cell development[J].Biochimica et Biophysica Acta-Molecular Basis of Disease,2017,1863(4):1013-1022.
[30] BLICHARSKA D,SZUCKO-KOCIUBA I,FILIP E,et al.CRISPR/Cas as the intelligent immune system of bacteria and archea[J].Postepy Biochemii,2022,68(3):235-245.
[31] LEE H,YOON D E,KIM K.Genome editing methods in animal models[J].Animal Cells and Systems,2020,24(1):8-16.
[32] WELLS K D,PRATHER R S.Genome-editing technologies to improve research,reproduction,and production in pigs[J].Molecular Reproduction and Development,2017,84(9):1012-1017.
[33] LIU H,LI Z,HUO S,et al.Induction of G0/G1 phase arrest and apoptosis by CRISPR/Cas9-mediated knockout of CDK2 in A375 melanocytes[J].Molecular and Clinical Oncology,2020,12(1):9-14.
[34] LIN Z,YAN J,SU J,et al.Novel OsGRAS19 mutant,D26,positively regulates grain shape in rice (Oryza sativa)[J].Functional Plant Biology,2019,46(9):857-868.
[35] ZHOU A,ZHANG W,DONG X,et al.Porcine genome-wide CRISPR screen identifies the golgi apparatus complex protein COG8 as a pivotal regulator of Influenza virus infection[J].CRISPR Journal,2021,4(6):872-883.
[36] SUN X,WANG J,MOU C,et al.Knockout of IRF3 and IRF7 genes by CRISPR/Cas9 technology enhances porcine virus replication in the swine testicular (ST) cell line[J].Biotechnology Journal,2024,19(1):e2300389.
[37] YOUNIS S,NABOULSI R,WANG X,et al.The importance of the ZBED6-IGF2 axis for metabolic regulation in mouse myoblast cells[J].FASEB Journal,2020,34(8):10250-10266.
[38] 夏振涛,王楠,王婉洁,等.pAPN基因敲除的IPEC-J2介导的TGEV感染特征分析[J].畜牧兽医学报,2024,55(8):3395-3407.XIA Z T,WANG N,WNAG W J,et al.Characteristics analysis of TGEV infection mediated by IPEC-J2 with knockout of pAPN gene[J].Acta Veterinaria et Zootechnica Sinica,2024,55(8):3395-3407.(in Chinese)
[39] XU K,ZHOU Y,MU Y,et al.CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance[J].eLife,2020,9:e57132.
[40] GAO X,TAO Y,LAMAS V,et al.Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents[J].Nature,2018,553(7687):217-221.
[41] 李小平,王可品,刘琪帅,等.基因修饰工具猪模型的建立及应用[J].中国实验动物学报,2017,25(3):329-335.LI X P,WANG K P,LIU Q S,et al.Establishment and application of genetically modified pig tool models[J].Acta Laboratorium Animalis Scientia Sinica,2017,25(3):329-335.(in Chinese)
[42] XUE C,GREENE E C.DNA repair pathway choices in CRISPR-Cas9-mediated genome editing[J].Trends in Genetics,2021,37(7):639-656.
[43] SCHUBERT M S,THOMMANDRU B,WOODLEY J,et al.Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair[J].Scientific Reports,2021,11(1):19482.
[44] SALSMAN J,DELLAIRE G.Precision genome editing in the CRISPR era[J].Biochemistry and Cell Biology,2017,95(2):187-201.
[45] DOENCH J G,FUSI N,SULLENDER M,et al.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9[J].Nature Biotechnology,2016,34(2):184-191.
[46] ZHANG J P,LI X L,LI G H,et al.Efficient precise knock in with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage[J].Genome Biology,2017,18(1):35.
[47] HOU M,SUN S,FENG Q,et al.Genetic editing of the virulence gene of Escherichia coli using the CRISPR system[J]. PeerJ,2020,8:e8881.
[48] 赖伟宁.绵羊全基因组编码区功能性SNP的挖掘及验证[D].长春：吉林大学,2023.LAI W N.Detection and validation of functional SNP in coding regions of the whole genome of domesticated sheep[D].Changchun：Jilin University,2023.(in Chinese)
[49] CLAUSSNITZER M,DANKEL S N,KIM K H,et al.FTO obesity variant circuitry and adipocyte browning in humans[J]. New England Journal of Medicine,2015,373(10):895-907.
[50] HUA J T,AHMED M,GUO H,et al.Risk SNP-mediated promoter-enhancer switching drives prostate cancer through lncRNA PCAT19[J].Cell,2018,174(3):564-575.
[51] CHAO T,LIU Z,ZHANG Y,et al.Precise and rapid validation of candidate gene by allele specific knockout with CRISPR/Cas9 in wild mice[J].Frontiers in Genetics,2019,10:124.
[52] CAPON S J,BAILLIE G J,BOWER N I,et al.Utilising polymorphisms to achieve allele-specific genome editing in zebrafish[J].Biology Open,2017,6(1):125-131.