Targeted Knockout Study Of Goat MSTN Gene By CRISPR/Cas9 Technology

As a negative regulator of muscle development,Myotatin gene(MSTN)is a important candidate gene for inhibiting the proliferation of myoblast.It can effectively increase muslce and inhibit adipocyte differentiation.Though the research on it has important application value on improving meat production and meat performance.Gene Knock-Out Animal Model has been an important tool for gene function research.To get transgenic goats,we usually need to construct target vectora,screening cell and transfer the nuclear.The process is complicated.Since CRISPR/Cas9 is simple,low cost and high effectively,it has been developed as a genetic modification technique in recent years.Up to now,there were few reports about the research of using CRISPR/Cas9 technology to edit goat MSTN gene.We intend to use the CRISPR/Cas9 technology,in vitro transcription and micro injection method to obtain MSTN gene knock out goat in a short time.In this study,according to the genome database in NCBI and Ensemble,two sgRNAs targeted on coding regions of MSTN genloci were designed.It is successful to microinject the Cas9 and sgRNA mRNAs into the goat zygotes.Then the MSTN KO goats were constructed by reimplantation of zygotes.In this study,495 oocytes were collected from 41 goats.248 of them were injected with Cas9 mRNA and sgRNA1.Finally we transplant 166 embryos into 20 receipts,the embryo survival rate was 66.94%and the pregnancy rate was 70%.Three male fetues(115d)were got from one recipient by Cesarean section.while 230 embros were injected with Cas9 mRNA and sgRNA2.180 embryos were transplanted into 17 receipts,the embryo survival rate was 78.26%and the pregnancy rate was 70.59%.Sequencing results indicated that two of the goat fetus were MSTN KO fetus(number:M1,M2),another was not(number:M3).The rusult showed that M1 was biallelically modified,with one allele containing 12 basic deletions and 1 base replacement;another allele containing 3 bases deletions.M2 contained one mutant allele,resulting in 26 bases loss of MSTN.M3 didn't contained mutant gene,the results showed that the MSTN KO has been fully achived.the first 309,340,372,374 cysteines of MSTN gene were missing at different degree in Ml,M2.The destroy of the cysteine knot finally caused the inactivation of MSTN gene.Off-target analysis showed that no off-target effect was detected.Compared with M3,Ml and M2 were heavier and bigger.Double-muscled phenotype was found in M1 and M2.The histological analysis showed that the increased muscle mass in MSTN KO goats resulted from fiber hyperplasia and hypertrophy.In all,this study successfully obtained double muscled-goat fetus by CRISPR/Cas9 and zygote injection.The results showed that the sgRNA 1 has activity.CRISPR/Cas9 technology can be used for the building of MSTN knock out goat model,by which we make a found for using CRISPR/Cas9 to edit other gene.