利用单碱基编辑器定点突变猪肌肉生长抑制素基因的研究

doi:10.16431/j.cnki.1671-7236.2022.08.004

中国畜牧兽医 ›› 2022, Vol. 49 ›› Issue (8): 2880-2887.doi: 10.16431/j.cnki.1671-7236.2022.08.004

利用单碱基编辑器定点突变猪肌肉生长抑制素基因的研究

王晶^1,2, 朱喆², 张鹏¹, 毕延震²

1. 中南民族大学生命科学学院, 武汉 430074;
2. 湖北省农业科学院畜牧兽医研究所, 动物胚胎工程与分子育种湖北重点实验室, 武汉 430064

收稿日期:2022-02-22 出版日期:2022-08-05 发布日期:2022-07-21
通讯作者: 张鹏, 毕延震 E-mail:zhangpenghust@126.com;sukerbyz@126.com
作者简介:王晶,E-mail:631474815@qq.com。
基金资助:
国家自然科学基金(31772577);湖北省农业科技创新中心项目(2019-620-000-001-20);湖南创新型省份建设专项经费(2019RS1068);重点领域研发计划(2020WK2030);外国青年人才计划(QN20200127002);中国科学院战略性先导科技专项(XDA24030204);碱基编辑技术介导的MSTN"基因仿生"羊新品种培育;湖北省科技创新人才及服务专项任务书(国际科技合作类)(2021EHB023)

Site-directed Mutagenesis of Porcine Myostatin Gene Using Single Base Editor

WANG Jing^1,2, ZHU Zhe², ZHANG Peng¹, BI Yanzhen²

1. College of Life Sciences, South-Central University for Nationalities, Wuhan 430074, China;
2. Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Institute of Animal Science and Veterinary Medicine, Hubei Academy of Agricultural Sciences, Wuhan 430064, China

Received:2022-02-22 Online:2022-08-05 Published:2022-07-21

摘要/Abstract

摘要： 【目的】利用单碱基编辑器在宁乡花猪肌肉生长抑制素(myostatin,MSTN)基因第2外显子处引入终止密码子,以获得MSTN基因表达沉默的肾成纤维细胞系,为后期培育MSTN碱基编辑猪奠定基础。【方法】首先在MSTN基因的第2外显子处设计1条单向导RNA (single guide RNA,sgRNA),将其连接至pMLM3636-puro质粒,形成重组表达载体pMLM3636-puro-MSTN,与含有红色荧光碱基编辑器YE1-BE3-FNLS共转入宁乡花猪肾成纤维细胞中,在红色荧光和嘌呤霉素双筛选条件下,挑取单克隆细胞,测序验证后,分析阳性单克隆细胞的蛋白表达情况。【结果】在MSTN基因第2外显子处发生了碱基定点突变,目标位点的氨基酸序列由色氨酸(TGG)转变成终止密码子(TAA),且G→A突变率为5.5%。Western blotting检测结果表明,试验组10号单克隆细胞的MSTN蛋白表达量与野生型相比降低了60%。【结论】本研究运用单碱基编辑技术在宁乡花猪MSTN基因编码区引入终止密码子,使翻译提前终止,导致蛋白表达量显著降低,为后期MSTN基因碱基编辑猪的生产奠定基础。

关键词: 单碱基编辑; 宁乡花猪; MSTN基因; 终止密码子

Abstract: 【Objective】 Using a single base editor to introduce a stop codon at the exon 2 of myostatin(MSTN) gene in Ningxiang pig,and obtaining kidney fibroblast cells with MSTN gene silencing,which was the foundation for the later breeding of MSTN base-editing pigs.【Method】 In this study,a single guide RNA (single guide RNA,sgRNA) was designed at the exon 2 of MSTN gene,and connected to the pMLM3636-puro plasmid to form a recombinant expression vector pMLM3636-puro-MSTN,co-transfected into kidney fibroblast cells with YE1-BE3-FNLS containing the red fluorescent base editor.Under the conditions of red fluorescence and puromycin,single cell colonies were picked.After confirmed by sequencing,the protein expression of the target mutant cell lines was analyzed.【Result】 Base site directed mutation occurred in the exon 2 of MSTN gene,the amino acid sequence of the target site was changed from tryptophan (TGG) to a stop codon (TAA),and the G→A mutation rate was 5.5%.The results of Western blotting showed that the MSTN protein expression in the experimental group No.10 single clone cells decreased by 60% compared with the wild type.【Conclusion】 In this study,single base editing technology was used to introduce stop codon in the coding region of MSTN gene of Ningxiang pig,so that translation was terminated early,resulting in a significant decrease in protein expression,which laid a foundation for the production of MSTN base mutant pigs.

Key words: single base editing; Ningxiang pig; MSTN gene; stop codon

中图分类号:

S826.2

王晶, 朱喆, 张鹏, 毕延震. 利用单碱基编辑器定点突变猪肌肉生长抑制素基因的研究[J]. 中国畜牧兽医, 2022, 49(8): 2880-2887.

WANG Jing, ZHU Zhe, ZHANG Peng, BI Yanzhen. Site-directed Mutagenesis of Porcine Myostatin Gene Using Single Base Editor[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 2880-2887.

参考文献

[1] PETERSEN B.Basics of genome editing technology and its application in livestock species[J].Reproduction in Domestic Animals,2017,52(Suppl 3):4-13.
[2] RUAN J,XU J,CHEN-TSAI R Y,et al.Genome editing in livestock:Are we ready for a revolution in animalbreeding industry?[J].Transgenic Research,2017,26(6):715-726.
[3] ZHAO J,LAI L,JI W,et al.Genome editing in large animals:current status and future prospects[J].National Science Review,2019,6(3):402-420.
[4] KOSICKI M,TOMBERG K,BRADLEY A.Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements[J].National Biotechnology,2018,36(8):765-771.
[5] KOMOR A C,KIM Y B,PACKER M S,et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage[J].Nature,2016,533(7603):420-424.
[6] BILLON P,BRYANT E E,JOSEPH S A,et al.CRISPR-mediated base editing enables efficient disruption of eukaryotic genes through induction of STOP codons[J].Molecular Cell,2017,67(6):1068-1079.
[7] KOBLAN L W,DOMAN J L,WILSON C,et al.Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction[J].National Biotechnology,2018,36(9):843-846.
[8] HUANG T P,ZHAO K T,MILLER S M,et al. Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors[J].National Biotechnology,2019,37(6):626-631.
[9] YUAN H,YU T,WANG L,et al.Efficient base editing by RNA-guided cytidine base editors (CBEs) in pigs[J].Cell Molecular Life Science,2020,77(4):719-733.
[10] QIN W,LU X,LIN S.Programmable base editing in zebrafish using a modified CRISPR-Cas9 system[J]. Methods,2018,150:19-23.
[11] LIU Z,SHAN H,CHEN S,et al.Improved base editor for efficient editing in GC contexts in rabbits with an optimized AID-Cas9 fusion[J].FASEB Journal,2019,33(8):9210-9219.
[12] GROBET L,MARTIN L J,PONCELET D,et al.A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle[J].Nature Genetics,1997,17(1):71-74.
[13] BI Y,HUA Z,LIU X,et al.Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP[J].Science Reports,2016,6(1):31729.
[14] XU S,KIM J,TANG Q,et al.CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway[J].Protein Cell,2020,11(5):352-365.
[15] PAN J S,LIN Z S,WEN J C,et al.Application of the modified cytosine base-editing in the cultured cells of bama minipig[J].Biotechnology Letters,2021,43(9):1699-1714.
[16] ZUO E,SUN Y,YUAN T,et al.A rationally engineered cytosine base editor retains high on-target activity while reducing both DNA and RNA off-target effects[J].Nature Methods,2020,17:600-604.
[17] LAPINAITE A,KNOTT G J,PALUMBO C M,et al.DNA capture by a CRISPR-Cas9-guided adenine base editor[J].Science,2020,369(6503):566-571.
[18] JEONG Y K,LEE S,HWANG G H,et al.Adenine base editor engineering reduces editing of bystander cytosines[J].National Biotechnology,2021,39(11):1426-1433.

利用单碱基编辑器定点突变猪肌肉生长抑制素基因的研究

Site-directed Mutagenesis of Porcine Myostatin Gene Using Single Base Editor

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