Preparation And Evaluation Of Multiplex Gene Knocked Out Tan Sheep Via The CRISPR/Cas9 Gene Editing System

Based on the histological analysis techniques,such as high-throughput sequencing technology and GWAS,the functional genes related to the excellent economic traits and stress resistance of livestock were excavated.And then based on the conventional breeding techniques and the molecular breeding techniques,it is becoming more and more possible to speed up the breeding process and raise the level of livestock breeding.Especially in recent years,with the development and application of CRISPR/Cas9,a popular gene editing technology,it has already became a reality that breeders could make the site-specific,accurate and efficient modification of given genes in the livestock genome,and promote the animal's phenotypes toward the desired goal of human.At the same time,it is possible to achieve multiple gene editing and polymerization effect of phenotypes,to shorten the breeding cycle and to improve breeding effect.In this study,the knockout efficiency of CRISPR/Cas9 gene editing system was verified at the cellular level and embryo level respectively.After that,the Cas9 mRNA and 6 sgRNAs targeting the CDS regions of the MSTS,ASIP and BCO2 genes were directed to the cytoplasm of 1-cell fertilized egg.After the in vitro culture and embryo transfer,the multiple gene knockout Tan sheep were successfully obtained.And following systematic analyses of knockout situation,off-target effects,hereditary capacity and phenotypes were conducted with the gene knockout Tan sheep.The main findings are as follows:1.Verification of editing efficiency on fibroblasts.Two sgRNAs were designed for CDS region of each gene,and the cell expression vector was constructed and transfected into fibroblasts with the expression vector of Cas9.Compared with single sgRNA knockout strategy,the double sgRNA knockout strategy could produce more mutant genotypes and larger fragment deletion,and could have a stable editing efficiency to MSTN,ASIP and BCO2,between 50% and 80%.At the same time,all sgRNAs designed were not detected with off-target effects.2.Verification of editing efficiency on embryos.The in vitro expression vector of each sgRNA was constructed individually and Cas9 mRNA and sgRNAs were transcribed in vitro.After that,Cas9 mRNA and 6 sgRNA were injected into the cytoplasm of 1-cell fertilized egg through co-injection.Two sgRNAs were targeting to the CDS region of same gene.The knockout efficiency of MSTN,ASIP,BCO2 were 35%,50% and 50% individually,while the co-knockout efficiency was 20%.3.Construction of gene knockout Tan sheep.After an estrus sychronization,613 fertilized eggs were collected from 45 donor Tan sheep,of which 588 fertilized eggs were conducted with cytoplasmic injection.578 divisive fertilized eggs were transplanted to 82 recipient ewes,54 lambs were obtained,36 survived,26 live lambs were successfully edited,and three lambs were knocked out MSTN,ASIP,BCO2 three genes at the same time.4.Detection of tissue editing status in gene knockout Tan sheep.8 kinds of tissue(heart,liver,spleen,lung,kidney,skin,muscle,testis)differentiated from three embryonic germ layers were collected to screen the editing status.It was found that all the tissues were successfully edited,indicating that the gene knockout method can be used to construct systemic knockout Tan sheep.In addition,the muscle tissue of the MSTN knockout Tan sheep,the skin tissue of the ASIP knockout Tan sheep and the adipose tissue of the BCO2 knockout beach sheep were detected,and the mutations were found in functional tissues of this three gene respectively.5.Detection of heredity in gene knockout Tan sheep.The testicular tissue of gene knockout Tan sheep was detected by H&E staining method,showing that there were active germ cells in the testicular tissue of the gene knockout Tan sheep.After that,the germ cells were separated by stereoscope and detected for editing.The results showed that the germ cells of the gene knockout Tan sheep contained mutations.The above results suggested that the constructed gene knockout Tan sheep could produce active mutant germ cells,and that the gene mutant Tan sheep have genetic potential.6.Phenotypic analysis of BCO2 knockout Tan sheep.In this study,according to whether the wild genotype was included,the gene knockout beach sheep was divided into two groups,double allele knockout group and single allele knockout group.The color of Tan sheep fat was identified by RGB color standard and the expression of protein was compared by western blotting,showing that only the double allele knockout Tan sheep could not express BCO2 protein.And only the double allele mutated of BCO2 gene could lead to the change of fat color.7.Phenotypic analysis of MSTN knockout Tan sheep.A continuous observation was conducted with MSTN knockout Tan sheep to collect the data of body weight.The result was showing that birth weight and average daily gain of MSTN gene knockout Tan was significantly higher than that of wild type control group(P<0.05),while the body weight of MSTN knockout Tan sheep was significantly higher than that of wild type control group in the two periods of D0-D30 and D150-D240(P<0.05).The morphological differences of muscle tissue of MSTN gene knockout Tan sheep were observed by H&E staining and transmission electron microscopy.It was found that the fineness of muscle fibers of MSTN knockout Tan sheep was significantly greater than that of wild type Tan sheep.The expression of MSTN protein was analyzed by western blotting.The results showed that gene knockout down-regulated the expression of MSTN protein.In conclusion,this study has successfully prepared single gene or multi-gene knockout Tan sheep of MSTN,ASIP and BCO2,using the gene precision editing technique.The method of multiple genes editing in Tan sheep via CRISPR/Cas9 has been built,laying a technical foundation for breeding the polygenes polymerized new varieties(lines)and providing breeding materials.