Preparation Of FS?-?-? Site-specific Integrated Gene Modified Pigs Using CRISPR/Cas9 Mediated HMEJ Strategy

As a robust antagonist of myostatin(MSTN),follistatin(FST)is a critical regulator of skeletal muscle development.Crystallographic analyses reveals that the mature FST is organized into N-terminal,C-terminal and three functional(FS?,FS?,FS?)domains,which are highly conserved among different species.Each domain has different ligand binding activity,and the N-terminal and FS? show the highest affinity for MSTN.Up regulation expression of the FS? domain or deletion of the FS? domain could increase the binding ablity of MSTN.In addition,transgenic mice in which FST was driven by a skeletal muscle specific promoter exhibited increased skeletal muscle mass.Importantly,when FS?-?-overexpressing mice were crossed with Duchenne muscular dystrophy mice,the resultant DMD/FS?-? mice showed enlarged skeletal muscles.Thus,the FST overexpression in skeletal muscle is a potential therapeutic strategy for muscular dystrophy.CRISPR/cas9 gene editing technology has been widely used in various fields due to its high knockout efficiency.However,CRISPR/cas9 mediated homologous recombination is usually inefficient which limits the precise genome modification.In HEK293 T cells,primary astrocytes and neurons cells,homology-mediated end joining(HMEJ)-based strategy directed by CRISPR/Cas9 is more efficient than traditional HDR,which can achieve precise single base mutation,gene knock-out and gene insertion in the genome.HMEJ-based strategy requires a repair template containing a guide RNA target site on the outer portion of the HA.In our study,we aimed to site-specifically integrate FS?-?-?,including the signal peptide,complete N-terminus and three FS? domains,into the last codon of the porcine MSTN gene using a homology-mediated end joining(HMEJ)-based strategy mediated by CRISPR/Cas9.The expression of FS?-?-? was driven by porcine endogenous MSTN promoter.The two genes were linked by P2A.Under the self shearing effect of P2A,FS?-?-? and MSTN were expressed at similar levels in developing muscle,without affecting their activities.Our study mainly includes three aspects:Firstly,the transfection efficiency in porcine fetal fibroblasts and C2C12 was improved by optimizing the electroporation system.Furthermore,the screened sgRNAs at porcine ?-Actin gene,mouse MSTN gene site and mouse ?-Actin gene were effective.We constructed HMEJ and HDR homologous targeting vectors for the four gene loci,and then compared the knock in efficiency of CRISPR/cas9 mediated HMEJ strategy and HDR strategy.Results show that,the knock-in efficiency resutling from HMEJ at the pig Rosa26 locus(p Rosa26)and the pig ?-Actin locus(pACTB)(20.83%±0.7881% and 15.40%±0.3606%)was significantly higher than that resulting from HDR(7.7%±0.3331% and 5.0%±0.2961%)in PFFs.The knock-in efficiency resutling from HMEJ at the mouse ?-Actin locus(m ACTB)was significantly higher than HDR(13.30%±0.1856% versus 5.83%±0.1417%).That indicates the knock-in efficiency resutling from HMEJ was significantly higher than that resulting from HDR in pig fetal fibroblasts and C2C12.Finally,we successfully obtained FS?-?-? knock in pigs using CRISPR/cas9 mediated HMEJ strategy and SCNT technology.In knock in pigs,there was no selective marker gene,no exogenous promoter,no off target effect,no random insertion,and the transgene could be stably transmitted to offspring.Importantly,FS?-?-? knock-in pigs exhibited hypertrophic muscle tissue,principally in the hindquarters,with intramuscular boundaries and visible grooves under the skin.The H&E staining of cross-sections of the longissimus dorsi showed larger myofiber size in FS?-?-? knock-in pigs than in the controls.In addition,compared with wild-type pigs,the smad signaling pathway and Erk pathway are inhibited,while PI3k/AKT pathway is activated in FS?-?-? knock-in pigs.Myogenic regulatory factors MyoD,Myf5 and MyoG were up regulated and MRF4 was down regulated in FS?-?-? knock-in pigs.These results indicate that FS?-?-? gene mediates skeletal muscle hypertrophy through MSTN related signal pathway and the expression of myogenic regulatory factors.Altogether,these findings suggested that the HMEJ-based strategy which greatly improved the knock-in efficiency in PFFs is a powerful tool in the generation of genetically engineered pigs.Pigs are considered as an important material for human disease models because of its similar genetic,physiological and anatomical characteristics with human.The FS?-?-? overexpression in pigs enhances skeletal muscle mass and hold great promise to serve as a therapy model of human muscular dystrophies and diabetes.