Study On CRISPR/Cas9-Mediated Genome Editing For Rb1 Suppressor Gene In Cynomolgus Monkey Embryos

Background:Tumors have become the most important type of diseases that threaten human life currently,The incidence of which is increasing year by year.Among them,retinoblastoma is the most common intraocular malignant tumor in infants and young children.The incidence of pediatric retinoblastoma in our country is 1100-1500 each year and the prognosis is poor.It is a malignant tumor derived from photoreceptor precursor cells.It is common in children under 3 years of age and has a family genetic predisposition to monocular,bilateral,or both.Currently,in the treatment of retinoblastoma,radiotherapy alone can cure early RB,so quick detection and diagnosis of Rb are essential.About the pathogenesis,located in 13q14 tumor suppressor gene Rbl,double allele mutation at the same time,inactivation,may lead to retinoblastoma.Single-gene function inactivation of this feature has become a genetic engineering method to build the model of retinoblastoma;In recent years,the Rb model of rodents,amphibians and human embryonic stem cells has been constructed using transgenic technology and gene editing technology.Among them,The CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated nuclease 9)gene editing system is a novel gene editing technology developed based on the immune mechanism of archaea against exogenous nucleic acid invasion.CRISPR system gene editing experiments widely used for the study on single gene function provides an effective tool.The CRISPR system plays a key role in the establishment of tumor models.Through the editing of proto-oncogenes and tumor suppressor genes,the development of tumors has provided conditions for studying the mechanism of tumorigenesis and development,as well as providing theoretical guidance and support for the treatment of cancer.the CRISPR system has the characteristics of being more efficient,simple to operate and less cytotoxic and has become the current The best way to build this model of gene editing.Objective:In recent years,the Rb1 tumor suppressor gene research has been gradually developed from rodents to amphibians(such as Xenopus)and then to human embryonic stem cells.Rb1 knockout animal model was constructed to analyze the different species of Rb1 gene function and the gene inactivation of the body to form retinoblastoma.However,the Rb1 knockout animal models that constructed by the researchers successfully before didn't mimic the pathogenesis and phenotype of human retinoblastoma well.Therefore,in view of the similarities between human and non-human primates,we used non-human primates,cynomolgus monkeys,as the research objects to perform Rbl gene editing at the cell and embryo level.Then we can obtain the knockout efficiency and protein expression of the retinoblastoma model.Moreover,it is of importance to further study for the occurrence and development of retinoblastoma mechanism and treatment.Methods:The experiment is divided into two parts:the first part is to test the sgRNA editing efficiency and off-target site prediction in cynomolgus monkey Rb1 gene at the cellular level;the second part is to edit the cynomolgus embryo genome and transfer it to the surrogate mother to obtain offspring animals.To further investigate the relationship between Rb1 gene inactivation and the development of retinoblastoma.The first part:Selected CRISPR editing system as a gene editing tool to non-human primate cynomolgus fibroblasts as experimental subjects.First,cell establishment and cell expansion culture:When the cell volume is sufficient,then What we should do is as follows:Designed sgRNA and SpCas9 co-transfected cells of interest,extracted the target cell genome,designed to contain Rb1 gene 8 exon region of DNA primers,amplify the edited cell genome,selected proper single-plasmid vector,and sequenced the genome,and calculated the gene knockout efficiency.Then,We extracted the edited cell protein and analyzed the transcriptional knockout effect by WB assay.Finally,the knockout efficiency and off-target efficiency of Rbl gene were obtained.The second part:The CRISPR editing technology is imperative.First of all,we deployed a certain ratio of the mixture of sgRNA and SpCas9 configuration.We utilize estrogen to stimulate superovulation of female cynomolgus monkey,in order for the acquisition of mature egg cells.SgRNA and SpCas9 were injected into the egg cells before fertilization of eggs.Projected the next Intracytoplasmic sperm injection.The single-cell embryos were cultured to the four-cell stage.Then the culture was terminated and the embryonic genomes were extracted.The knockout efficiency of each of the different embryos was analyzed,and the type of single-cell knockout of each embryo was analyzed.Next,we transferred the Rbl knockout embryos to surrogate mothers.Results:The first part:Using cynomolgus monkey fibroblasts as experimental subjects,6 sgRNAs were designed for Rbl gene editing at the cellular level,and CRISPR/Cas9 editing technology was used for Rbl knockout efficiency.The efficiency of Rb1-sgRNA-8-1 is 75%and Rbl-sgRNA-8-2 is 85%.The rest of the gRNAs had no editing effect.The result of Rb protein expression via the WB assay was significantly decreased or even no detected.Off-target analysis did not reveal detectable off-target sites.In the second part:RNP was injected into the cytoplasm of oocytes at Oh and-2h before fertilization.For Rbl-sgRNA-8-1,eleven edited embryos(Oh for 6;-2h for 5)were knocked out.The proportion of embryos was 33.3%and 40%,respectively,and the proportion of embryos that were edited by 4-cell was 33.3%and 20%.For Rbl-sgRNA-8-2,26 edited embryos were obtained(of which 0 h 13;-2 h 13),and the proportion of genes in embryos that had been knocked out was 84.62%and 61.54%,respectively.The proportion of embryos which were edited at 4-cell was 53.85%and 23.08%,respectively.Fifteen embryos were injected with RNP and were cultured into blastocysts,and then 5 of them were transplanted into the surrogate mother.Conclusion:For gene editing studies of cynomolgus monkey cells and embryos,the CRISPR/Cas9 gene editing system can be used as an effective tool,and the editing efficiency is high.RNP with a single gRNA transfection can obtain a more efficient gene knockout embryo and decrease the probability of chimerism.Gene-editing embryos with a high blastocyst rate were obtained and transplanted successfully.