Genetic Editing Monkeys And CRISPR/Cas9 System Application Optimization Were Constructed Using The CRISPR/Cas9 System

The rapid development of gene editing techniques in recent years,especially the emergence of CRISPR/Cas9 gene editing,has ushered in the new vane of gene editing history.There are three major endonucleases ZFN,TALEN and CRISPR/Cas9 among the current fixed-point gene editing tools.Therein,the CRISPR/Cas9 system has been widely used in genetic research,for its advantages as convenient construction,high knock-out efficiency and various usages in cells and organisms.In current medical research,animal model plays an indispensable part in life science research.Every breakthrough of each model animal research has brought along tremendous developments in genetics,molecular biology and developmental biology.With the deepening of the scientific research,exploring on complicated life phenomenon,as well as human being facing serious diseases such as cancer,HIV/AIDS,cardiovascular diseases,neurological diseases,and so on,the lower animal models haven't been able to meet the needs of simulating human major diseases.Nonhuman primates,as human relatives,having the similar immune system,nervous system and metabolic system as human beings,are supposed to be the ideal,sometimes the exclusive animal model,whose research achievements even can be directly applied to human disease treatment and health security.Therefore,to apply the CRISPR/Cas9 technology to constructing nonhuman primates gene-editing model is an urgent need for life science research.This topic applied CRISPR/Cas9 gene editing system to design five sgRNAs on three genes Ppar-y,NrObl and Rag1,of which Ppar-y is associated with fat metabolism,NrObl is associated with early gonad development,and Ragl is associated with immune system.We successfully constructed the world's first two cases of targeted gene knockout monkeys by microinjecting Cas9 mRNA and sgRNA into cynomolgus monkeys' eggs,then transplanting them into the surrogate monkeys.Afterwards,we designed several sgRNAs in 3'UTR region of Oct4(Pou5f1)gene,constructed U6 sgRNA and verified their efficiency in COS7 cell line,finally confirmed sgRNA efficiency and performed in vitro transcription.Cas9 mRNA,sgRNA,and donor plasmid 5' Arm-IRES-hrGFP-3' Arm were microinjected into the zygote of cynomolgus monkey,using homologous recombination repair mechanisms to insert exogenous hrGFP into target gene Oct43'UTR region,and constructed site specific knock-in monkeys.In this study,the 293T cell line with single copy BFP gene will be used as a tool to optimize the design of ssODN.Different ssODNs and sgRNAs were designed for differential site of BFP and GFP,and the efficiency recombination was evaluated by the ratio of cell fluorescence chenge after recombination.By comparing the ssODN of different designs and combinations of ssODN and sgRNA,we found that the recombination rate was higher when the complementary strand ssODN was used and the mutation site located at 3' position of DSB position,then using the CRISPR/Cas9 system,combined with a single stranded oligonucleotide donor(ssODN)for embryo injections,and successfully constructed three directional point mutant mice on genes Usp42,Cep41,Fbxo47,which laid the foundation for gene editing of nonhuman primates gene-editing model based on ssODN.In this study,the CRISPR/Cas9 gene editing system was applied to nonhuman primates,cynomolgus monkeys,combined with the microinjection technology,iand produced two cases of nonhuman primates simulating human disease or abnormal function.In terms of construction of point-mutation mice,we summarize the design efficienies of point mutations by comparing positive recombinant mutation rate with different design approaches,and assessed clinical prospects of CRISPR/Cas9 technology.