利用CRISPR-Cas9基因编辑技术制备牛MSTN基因编辑胚胎
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摘要
为了给双肌肉牛品种的培育提供新的遗传素材,拟利用CRISPR-Cas9基因编辑技术制备牛MSTN基因编辑胚胎。从采集到的86对牛卵巢中收集卵母细胞进行成熟培养以及体外受精,并使用在线软件设计能够在体外高效编辑牛MSTN基因的gRNA序列,将验证后所得的体外切割活性最高的gRNA与Cas9 mRNA的混合物显微注射牛的受精卵,培养至囊胚后,利用T7EI核酸内切酶法检测囊胚中MSTN基因的编辑情况,然后将基因编辑阳性的囊胚样品进行测序验证。结果表明,牛胚胎体外培养平均囊胚率为21.28%;在设计的多条gRNA序列中,gRNA5的体外切割活性最高,为96.00%;将Cas9 mRNA和gRNA5的混合物显微注射受精卵并培养至囊胚后,T7EI核酸内切酶检测表明囊胚MSTN基因切割阳性率为15.40%;对MSTN基因编辑囊胚阳性样品测序表明,在gRNA5靶位点存在部分片段缺失。综上,说明利用CRISPR-Cas9基因编辑技术成功获得了牛MSTN基因编辑胚胎。
In order to provide new genetic materials for the double-muscle cattle breeding in the future,the CRISPR-Cas9 gene editing technology was used to prepare bovine MSTN gene-editing embryos in this study.Oocytes were collected from 86 pairs of bovine ovaries collected for maturation and fertilization in vitro,and the gRNAs capable of efficiently editing the bovine MSTN gene within CRISPR-Cas9 system were designed using online software.Then the mixture of gRNAs with validated high cleavage activity and Cas9 mRNA were microinjected into the fertilized eggs.After the embryos were cultured to blastocysts,the MSTN gene editing in the blastocysts samples was detected by T7 EI endonuclease digestion,and then the positive samples with gene editing were verified by sequencing.The results showed that the average blastocysts rate of cattle embryo cultured in vitro was 21.28%.In designed gRNA sequences,the gRNA5 had the highest cleavage activity in vitro,which was 96.00%.After microinjection of the mixture of Cas9 mRNA and gRNA5,the T7 EI endonuclease digestion showed that the positive rate of blastocysts with MSTN gene editing was 15.40%,and the sequencing of MSTN gene-edited blastocysts positive samples showed that fragment deletion was observed at the gRNA5 target site.In conclusion,the bovine MSTN gene edited embryos were successfully obtained by CRISPR-Cas9 gene editing technology.
In order to provide new genetic materials for the double-muscle cattle breeding in the future,the CRISPR-Cas9 gene editing technology was used to prepare bovine MSTN gene-editing embryos in this study.Oocytes were collected from 86 pairs of bovine ovaries collected for maturation and fertilization in vitro,and the gRNAs capable of efficiently editing the bovine MSTN gene within CRISPR-Cas9 system were designed using online software.Then the mixture of gRNAs with validated high cleavage activity and Cas9 mRNA were microinjected into the fertilized eggs.After the embryos were cultured to blastocysts,the MSTN gene editing in the blastocysts samples was detected by T7 EI endonuclease digestion,and then the positive samples with gene editing were verified by sequencing.The results showed that the average blastocysts rate of cattle embryo cultured in vitro was 21.28%.In designed gRNA sequences,the gRNA5 had the highest cleavage activity in vitro,which was 96.00%.After microinjection of the mixture of Cas9 mRNA and gRNA5,the T7 EI endonuclease digestion showed that the positive rate of blastocysts with MSTN gene editing was 15.40%,and the sequencing of MSTN gene-edited blastocysts positive samples showed that fragment deletion was observed at the gRNA5 target site.In conclusion,the bovine MSTN gene edited embryos were successfully obtained by CRISPR-Cas9 gene editing technology.
引文
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[2] 王建起,曹文广.Myostatin基因及其与动物双肌性状间关系的研究进展[J].中国农业科学,2014,47(8):1577-1587.
[3] 刘博,韩志国,高腾云.牛双肌基因遗传机制及研究进展[J].中国农学通报,2011,27(1):333-336.
[4] 汪新建,樊爽爽,翁少亭,等.AAV-SaCas9敲除MSTN基因病毒的构建及鉴定[J].河南农业科学,2018,47(1):118-121.
[5] 马毅,何志全,张金龙,等.肌肉抑制素与“双肌”表型关系的研究进展[J].中国畜牧杂志,2008,44(3):60-64.
[6] 邵华,郭旭东,旭日干.精子载体法转基因动物研究进展[J].内蒙古大学学报(自然科学版),2000,31(3):338-342.
[7] 李君,张毅,陈坤玲,等.CRISPR/Cas系统:RNA靶向的基因组定向编辑新技术[J].遗传,2013,35(11):1265-1273.
[8] 孙丹,龙华洋,庄英华,等.利用CRISPR-Cas9构建syncytinA条件敲除小鼠[J].实验动物科学,2015,32(5):1-6.
[9] CHEN F,WANG Y,YUAN Y,et al.Generation of B cell-deficient pigs by highly efficient CRISPR/Cas9-mediated gene targeting[J].Journal of Genetics & Genomics,2015,42(8):437-444.
[10] 汤贝贝.CRISPR/Cas9介导MSTN基因敲除绵羊制备[D].扬州:扬州大学,2016.
[11] 肖小帅.利用CRISPR/Cas9技术制备MSTN基因敲除绵羊[D].郑州:河南农业大学,2017.
[12] 吴姣.绵羊卵母细胞体外成熟、孤雌激活及核移植胚胎发育的研究[D].郑州:河南农业大学,2014.
[13] 施巧婷.北山羊体细胞异种克隆技术的研究[D].乌鲁木齐:新疆农业大学,2004.
[14] 王艳萍,曾维斌,薛建华,等.不同处理方法对牛卵母细胞体外受精效果的影响[J].畜牧与兽医,2015,47(1):40-42.
[15] 佟桂芝,宋斌,王洪宝,等.反刍动物体外受精技术的研究进展[J].现代畜牧科技,2017(1):8-9.
[16] 于学颖,郭芹芹,郝海生,等.谷胱甘肽促进牛体外受精胚胎发育的转录组初探[J].畜牧兽医学报,2016,47(7):1363-1372.
[17] 徐礼杰,李钟淑,方南洙.吡格列酮对MZT期小鼠胚胎发育阻滞的影响[J].畜牧兽医学报,2016,47(11):2240-2247.
[18] 李晓霞,刘燕,曹平华,等.褪黑素在牛卵母细胞体外受精中的抗氧化作用[J].家畜生态学报,2015,36(7):57-62.
[19] 刘志国.CRISPR/Cas9系统介导基因组编辑的研究进展[J].畜牧兽医学报,2014,45(10):1567-1583.
[20] 张冬杰,刘娣,张旭,等.利用CRISPR-Cas9系统定点突变猪MSTN基因的研究[J].畜牧兽医学报,2016,47(1):207-212.