| Background Since 1953,two American scientists Watson and Crick proposed the double helix model of DNA molecules,the molecular biology of gene technology has been developing rapidly,the epoch-making discovery laid the foundation for modern molecular biology,and so that people study the structure and function of the gene into the nucleotide level.Since then studies have found that the occurrence of certain human diseases related to nucleotide sequence and composition,from then on,people look forward to treat human diseases by the changes in nucleotide change.Thus sprouted out of the basic concepts of gene therapy.Subsequently,a series of important discoveries of molecular biologists has prompted the idea of gene therapy is gradually becoming a reality.The rapid development of gene cloning,gene recombination,gene transfer and gene expression technology,promoting gene therapy entered the testing stage in the early 1980s.The pioneer of this phase is the University of California,Los Angeles Martin Clineprofessor.He led the laboratory import the globin gene into mouse bone marrow cells and get the expressed,began the pilot phase of gene therapy.Then,They implant the transduced bone marrow cells into mice that geted full irradiation.This is a milestone in the history of gene therapy research.Gene therapy can be used for genetic diseases,especially single-gene genetic diseases,after decades of development,formally approved and ongoing clinical trials of gene therapy protocols have been widely covered by genetic diseases,cancer,infectious diseases and fields of cardiovascular disease,respiratory system,digestive system,nervous system.Hemophilia is a X-chains recessive genetic disease.Cause of the disease is due to the defective clotting factor gene,lead the expression of plasma coagulation factor protein reduced or absent,then the coagulation system become hampered.CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeats,CRISPR)gene technology is a kind of new biological technology emerging in 2013.We get the sgRNA by designed RNA,guiding the Cas9 protein at the target site-directed cleavage DNA.Cas9 protein gene causing double-strand breakage,cause the DNA repair.DNA double helix structure is very suitable for restoration,If a chain get damaged,complementary strand still retain a complete copy of the same information,and this copy is often used to restore the correct nucleotide sequence of the damage chain.DSB repair has two repair mode,Homologous recombination and Non homologous end joining.Objective We use the CRISPR-Cas9 gene editing technology and homologous recombination technology,verify the gene therapy could be succeed use for repair the mutation of FⅧ gene,by using the adenovirus vecteur.Methods We build the Plasmid vector and adenovirus vector that targeting the cos-7 cell,we knockout the FⅧ subject sequence by using the CRISPR/Cas9 gene editing technology,causing the DNA double-strand breaks,then we import the homologous recombination template,promoting the homologous recombination in the form of DNA repair,In order to achieve the purpose of replacement of the base.In terms of animal models,we build the Plasmid vector and adenovirus vector for the Macaca fascicularis.Results We successfully build the plasmid vector and adenovirus vector that targeting the FⅧ subject sequence of cos-7 cell,successfully build the homologous recombination template.we have detected the knockout the FⅧsubject sequence of cos-7 cell.But the rate of the homologous recombination is too low,we failed to detecte the homologous recombination.We successfully build the Plasmid vector and adenovirus vector for the Macaca fascicularis.Conclusion CRISPR/Cas9 gene editing system can effectively targeting the COS-7 cells FⅧ gene,cause genetic damage and repair,and operation is simple and efficient.Homologous recombination plays an important role in DSB repair,but the rate is too low.We can further improve the efficiency of homologous recombination to provide technical support to repair gene mutations,but also for labor detect treatment-related diseases provides a potential means. |
