Preparation And Evalution Of MSTN Gene Knockout Tan Sheep

In recent years,CRISPR/Cas9 system,an effective tool for targeting genomic modification,have developed rapidly,which introduces specific DNA double-strand breaks(DSBs)that can then be repaired through endogenous DNA repair mechanisms.The CRISPR/Cas9 system is widely used in animal genetic engineering to generate genomic knockouts,insertions,and point mutations.Compared to traditional ZFN and TALEN gene editing technologies,CRISPR/Cas9 is more efficient and has fewer restrictions on site recognition(only GG is required in the targeted genome).Its construction procedure is simple and convenient for large-scale animal gene editing.Sheep is an important livestock species and an ideal large animal model which is indispensable in the biomedical studies of genetic diseases.Gene editing technologies can be utilized to improve the productive and reproductive performance of livestock species,including sheep,allowing the rapid generation of desirable phenotypes.Muscle development is one of the important economic characteristics in farm animals,and the myostatin(MSTN)gene is considered to be the major gene associated with muscle development and the percentage of lean meat.In most cases,the disruption of gene function can be induced by designing sgRNA,which can mediate gene knockout.However,the induction of gene knockout mutation in only few nucleotides may have a little effect on the gene function.Therefore,it is necessary to investigate the effects of the large fragment deletions in the gene of interest.This experiment aims to use the CRISPR/Cas9 system to modify the ovine MSTN gene to explore the effects of gene disruption.In this study,the MSTN gene was selected as the target gene in Tan sheep and precision,high-efficiency and large fragment deletion were used as research objective.Three sgRNAs targeting MSTN gene were designed then the CRISPR/Cas9 system was used to validate the targeting effects on sheep fetal fibroblasts and in the generation of gene knockout lambs,respectively.Potential off-target sites of fetal fibroblasts and gene editing lambs were detected.Body weight of MSTN knockout founders and wild-type counterparts(WT)were compared,and MSTN expression in sheep muscle tissue was detected.The main results are as follows:(1)Efficiency verification on fetal fibroblasts cell.SgRNAs targeting exon 1,2,and 3 of MSTN gene were designed,and the fibroblasts were simultaneously transfected to obtain cell targeted by CRISPR/Cas9 system.The editing efficiencies of the used sgRNAs were from 40% to 60%,indicating the high efficiency of the designed sgRNAs to target MSTN gene in sheep fibroblasts cell.Moreover,off-target mutations of the tested sgRNAs have been detected.(2)Preparation of knockout sheep.The designed sgRNA and Cas9 vector were constructed and the Cas9 mRNA and sgRNA were obtained by in vitro transcription.Cas9 mRNA and sgRNAs were injected simultaneously in about 640 embryos at the single-cell stage.After embryo maturation,585 embryos with good development were transplanted into 120 surrogate mothers,and 35 live lambs were obtained.Among them,10 lambs had targeted knockout,and the estimated knockout efficiency was 28.6%(10/35).Of the 10 edited founders,3(3/10,30%)was produced with large gene fragment deletions(~ 5 kb).We further tested off-targets,and no off-target effects were detected in all potential sites.(3)Phenotypic analysis of knockout lambs mediated by CRISPR/Cas9 system.Samples of the hip muscles were obtained from MSTN knockout and WT lambs of the F1 generation and were transported to the laboratory in liquid nitrogen then the expression of MSTN protein was detected by Western blotting.The MSTN protein level of the edited lambs was lower than that of the wild-type,indicating that MSTN gene expressed less in skeletal muscle.(4)Body weights(BW)from 0 to 180 days were observed and recorded every 30 days.The birth weight of the knockout lambs was significantly higher than that of the control group(p<0.01).In addition,body weight of the lambs was significantly increased except for the 60 th to 90 th day.The average daily gain of gene edited lambs was 180 g/d and was 142 g/d for the wild-type lambs,indicating the improvement of the muscle growth in the edited lambs.In summary,the MSTN knockout lambs were successfully generated,and lambs with large fragment deletion were obtained.The successful large fragment deletion in sheep through the CRISPR/Cas9 system provides a practical example for the utilization of this strategy in other large animal models,and provides technical support and breeding materials for further research.