Studies On The CRISPR/Cas9 System Applied For Gene Editing In The Mammals

Myostatin(MSTN)is a negative regulatory factor of muscle development,which is coded by growth differentiation factor 8(GDF8)gene.The spontaneous mutations or artificial mutations of GDF8 gene would induce the phenotype of muscle hypertrophy in the mouse,pig,cattle,sheep and goat,even in the human.GDF8 gene plays an important role on the regulation of amyotrophy induced by some diseases.Another,GDF8 gene is important for the development of embryo,adipose and skeleton.The prokaryotic clustered regularly interspaced short palindromic repeat(CRISPR)-associated system(Cas)is from prokaryote as an immune defend system in the bacteria against the exogenous nucleotides.CRISPR/Cas9 system can target the specific site by a guide RNA and cleavage the DNA double strands using the Cas9 nuclease activity to create a DSB,then the DNA repair mechanism in cell would be induced by the DNA damage,some nucleotides will be deleted and inserted at the target sites.As a result,the target gene may be loss of the biological function because of the frameshift mutations of the CDS.In this study,we edited the GDF8 gene of the mouse,rabbit,pig and buffalo by CRISPR/Cas9.And the GDF8-modifiled mice and rabbits were generated using Cas9-gRNA.In addition,we have successfully edited the GDF8 gene in the fibroblasts and embryos of pig and buffalo.Furthermore,we attempted to resolve some problems that the efficiency of precise correction and DNA insertion mediated by CRISPR/Cas9 system is inefficient.Part I Generation of GDF8-modified mouse and rabbit by CRISPR/Cas9 systemWe constructed some DNA plasmids associated CRISPR/Cas9 system,such as hSpCas9 expression plasmid and gRNA backbone expression plasmid.Six gRNA target sites were designed at the CDS of GDF8 gene from rabbit.And the target sequences were cloned into the gRNA backbone expression vector phU6-gRNA,and the target sequences with the PAM(5'-NGG-3')was cloned into the RGS-CR reporter.Efficient gRNAs were screened using the RGS-CR reporter.The gene-editing ability of gRNA E31 and E32 were more efficient than others,up to 78.8%and 75.6%,respectively.And the efficient gRNAs were applied to generate the GDF8-modifiled mouse and rabbit.We obtained the GDF8-modifiled mice by micro-injecting RNA(hSpCas9 mRNA&sgRNA E32)into the cytoplasm of zygotes and embryos transplantation,the positive rate is 23.3%(7/30).Using the CRISPR/Cas9 system,we have successfully edited the GDF8 gene in the fibroblasts and embryos of rabbits.The gene-editing rate is up to 52.5%in fibroblasts and 88.7%in embryos.And there are various editing efficiency in different quality embryos and different types of embryos.Using the CRISPR/Cas9 system,we generated the GDF8-modifiled rabbits by micro-injecting RNA mixture of Cas9 mRNA and sgRNA into the cytoplasm and autotransplantation.Part ? GDF8 gene-editing of buffalo and pig by CRISPR/Cas9 systemWe successfully screened the efficient gRNA using RGS-CR reporter and those gRNAs were applied to editing the GDF8 gene of pig and buffalo.By nucleofection,the hSpCas9 DNA plasmids with efficient gRNAs were delivered into the fibroblasts of pig and buffalo.After nucleofection,the positive cells were enriched by G418,the cells were collected for mutant detection when the most of cells have died.The results showed that the efficiency is up to 87.5%and 78.9%in fibroblasts of pig and buffalo.At the same time,we obtained the single cell clones of pig and buffalo fibroblasts by screening with G418,the positive rate is 53.5%and 72.7%,respectively.We tested the gene-targeting efficiency mediated CRISPR/Cas9 system in the different types of embryos of pig and buffalo,such as IVF embryos of buffalo,and PA embryos,ICSI embryos and IVF embryos of pig.The DNA plasmids or RNA mixture of Cas9 mRNA and sgRNA were applied to edit the GDF8 gene of pig and buffalo.The results showed that the CRISPR/Cas9 system able to edit the GDF8 gene of PA(3%),ICIS(36%)and IVF(25%)embryos.And the RNA could edit the GDF8 gene in the buffalo IVF embryos(18.8%),but the indels at the specific sites in the embryos couldn't be detected by micro-injecting the DNA plasmid into the cytoplasm of IVF embryos.Part ? Optimize the CRISPR/Cas9 systemWe compared the gene-editing efficiency of targeting the sites with same sequences in exogenous DNA plasmids and endogenous gene.The results showed that the efficiency targeting the DNA plasmids(80%)was more efficient than the endogenous(10%).That indicated that the structure and epigenetics of chromosome might influence the ability of CRISPR/Cas9 to bind to the target DNA by space hampered.For improve the efficiency of CRISPR/Cas9-mediated exogenous DNA insert into the specific sites of cell genomic,we attempted to treat the cell with VPA,a histone deacetylase inhibitor.The results demonstrated that VPA could enhance the HR by improving the expression of HR associated factors,such as Mre11,Rad50,Rad51 and BRCA2.At the same time,we found that the concentration of VPA in a certain range could enhance the efficiency of NHEJ,and the VPA could facilitate the expression of NHEJ associated factors,such as LIG4 and XRCC4.