A Primary Study On The Rabbit GDF8 Using CRISPR-Cas9 System

Growth differentiation factor 8(GDF-8),also known as myostatin(MSTN),is a negative feedback factor for muscle growth which mainly expresses in the skeletal muscle.Animals naturally or artificially mutated in the GDF-8 gene will exhibit the skeletal muscle hypertrophy,and the muscle content will significantly increase,which means great economic value to the meat livestock industry.Gene editing technology is an important method for studying the gene function.In recent years,the CRISPR-Cas system has been widely used in editing of various animal and plant genes due to its flexibile,convenient and low cost.The CRISPR-Cas system is a complex of nuclease and non-coding small RNA derived from ancient bacteria.It mainly relies on a single strand guide RNA(SgRNA)to bind to the specific sequence on the gene strands and to achieve targeting DNA specific sequence.The cleavage will be performed at the target site,and causing DNA double-strand breaks(DSBs),and then the gene editing is performed under the action of the cell's own DNA repair mechanism.In this study,we use the CRISPR-Cas system to konck out the California rabbit GDF8.First,we verify the editing efficiency of the selected site in HEK293T cells and rabbit fibroblasts cells,then we inject the Cas9 mRNA and sgRNA into the rabbit embryo,and culture to the blastocyst stage to verify the editing efficiency.Finally,.the microinjected embryo will be transplanted back into the female fallopian in order to give birth to gene-edited fetal rabbits.The min results are as follow:1.The gene targeting system consisting of hSpCas9 eukaryotic expression vector and gRNA eukaryotic expression vector was successfully constructed,and it was verified at the HEK293T cells and embryonic level that the system can accurately perform gene editing on the target site.2.We successfully screened two target sites E1 and E2 that can efficiently bind gRNA eukaryotic expression vectors in rabbit GDF8 gene.Through the detection of RGS-CR reporter vector in HEK293T cells transfection experiments and analysis by flow cytometry,both gRNA loci were efficiently mediated to form DSBs,and the efficiency of gRNA E1 reached 64.0%.The efficiency of gRNA E2 is slightly higher than E1,reaching 64.4%.3.Rabbit primary fibroblasts were successfully isolated and the GDF8 gene was edited in rabbit fibroblasts using the established targeting system.The results of T7E1 digestion showed that the editing efficiency of gRNA E1 inn fibroblasts was 27.1%,and the editing efficiency of gRNA E2 in fibroblasts was 7.4%.Sequencing results showed that the editing efficiency of gRNA E1 reached 45.3%,and the gene editing efficiency of gRNA E2 in fibroblasts was 25.0%.4.The GDF8 gene was successfully edited in rabbit one-cell embryos using the constructed targeting system.Using a two-site gene targeting strategy,hspCas9mRNA and sgRNA were obtained by in vitro transcription and then microinjected into rabbit one-cell stage embryos at a concentration ratio of 100:50 ng/?L and cultured to the blastocyst stage and amplified by PCR.Sequencing verification and sequencing results showed that the editing efficiency of gRNA E1 and gRNA E2 targeting rabbit endogenous embryos reached 20.0%.5.Using the constructed target system to produce transgenic rabbits,a total of 28 embryos were transplanted to two female rabbits.After 10 days,both of them were diagnosed were pregnant by palpation.One of them was miscarried on the 21st day of pregnancy and left 6 stillbirths.Another pregnant rabbit abortion on the 23 rd day of pregnancy and left 3 stillbirths.Then the thigh muscle tissue of these nine stillbirths was cut off,the tissue genome was extracted,and PCR amplification was performed and sequencing verification.The sequencing results showed that no gene mutation was detected at the target site.