利用CRISPR-Cas9基因编辑技术制备牛MSTN基因编辑胚胎

Preparation of Bovine MSTN Gene Edited Embryos Using CRISPR-Cas9 Gene Editing Technology

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 T7EI 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 T7EI 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.