Research Of CRISPR/Cas9–mediated MSTN Gene Editing In Sheep

Objective:Myostatin(MSTN)also known as growth differentiation factor 8(GDF-8)is a member of TGF-?superfamily.Myostatin make a down regulation to muscle cells growth and MSTN gene mutation will result in animal double muscle effect which improve the domestic animal meat production.CRISPR/Cas9 system is a very good technology to gene-editing.It's simple,more efficiency,low cost,and could make kinds of complex gene editings in a short time.In my study,we used CRISPR/Cas9 system,designed sgRNA and ssDNA to edit sheep's MSTN gene on its third exon,hope to lay the foundation of make target gene editing sheep.Methods:1.Make pX330-EGFP-sgRNA plasmid.Using Gibson Assembly method to incorporate the 2A+enhanced green fluorescent protein(EGFP)DNA fragment into pX330plasmid get pX330-EGFP,then using golden gate method incorporate 12 sgDNA oligo separately into pX330-EGFP plasmid and named pX330-EGFP-sgRNA.The sgDNA will transcript into sgRNA which was designed to target the MSTN third exon.2.Choose the most efficient sgRNA.Separately electroporated the pX330-EGFP-sgRNA vectors into sheep fibroblasts.Using micro manipulating system collected 100 expressing green fluorescent protein cells.The other cells were collected to extract genome for PCR.Targeting result was detected with SURVEYOR analysis method.The genome of 100 GFP cells that belong to the SURVEYOR analysis positive group was extacted for PCR.Purified PCR production was cloned for sequencing,and 10 samples were sequenced in each group.According to the results of sequencing find the most efficient sgRNA.3.Find the best concentions of Cas9 protein and sgRNA.Mix the most efficient pX330-EGFP-sgRNA and Cas9 protein in three different concentration groups,then separately microinjected into the 200 sheep parthenogenetic zygotes.After 72 hours,collected morula and extracted genome DNA for PCR.The purified PCR products were cloned for sequencing and 10 samples in each group.According to the sequencing results,find the optimal concentrations of sgRNA and Cas9 protein for Microinjection.4.Sheep MSTN targeting.Designed a 120 base ssDNA which is homologous with sheep MSTN gene target site,but it have a 11 base deletion and contains a restriction enzyme site(Xba?).There are experimental and control group in this experiment.The ssDNA and best concentration of sgRNA,Cas9 protein were microinjected into the in vitro fertilized oocytes.And the only ssDNA was for the control group.After 72 hours the embryos'genome was extracted for PCR,and the PCR product was cloned for sequencing.According to the result of sequence analysis to find if make the targeting.Results:1.The sequencing results and agarose electrophoresis detecting show that pX330-EGFP plasimd and pX330-EGFP-sgRNA plasmids were correct.The pX330-EGFP plasimd is 9261 bp and the pX330-EGFP-sgRNA plasmid is 9281 bp.2.pX330-EGFP-sgRNA plasmids were electroporated into sheep fibroblasts,and three sgRNA(T2,T9,Q2)with high target efficiency were successfully screened by SURVEYOR analysis and sequencing,and the target efficiency was 40%,40%and 60%respectively.In this experiment we also optimized electrporation conditions:in 20?L electroporation suspension,2×10~5 cells and 4 ug plasmid is the best choose.3.Sequencing results show that when injection concentrations of sgRNA and Cas9 protein were 200 ng/?L and 1000 ng/?L could get the hightest targeting efficiency.4.sgRNA and Cas9 protein mixed with ssDNA(67 ng/?L)was microinjected into fertilized oocytes,and sheep genome was successfully precise editing with 11 bp deletion and a restriction enzyme site inserted.And the editing efficiency is 20%.Conclusion:We successfully designed sgRNA and ssDNA to target sheep MSTN third exon and after electroporated into sheep fibroblasts three pX330-EGFP-sgRNA plasmids successfully make the gene editing,the best editing efficiency is 60%.Find the best concentions of sgRNA and Cas9 protein for micoroinjection is 200 ng/?L and 1000 ng/?L.Use the ssDNA(67 ng/uL)and optimized concentration of sgRNA and Cas9 protein we successfully made the sheep MSTN targeting.