Generation Of Heterozygous Mutation FOP FF-iPSCs By CRISPR/Cas9 And NgAgo Mediated Genome Editing

Background: Fibrodysplasia ossificans progressiva(FOP)syndrome is a rare and devastating genetic disease of heterotopic endochondral ossification(HEO),and no effective therapies are available for this disease.FOP is caused by a mutation of the gene ACVR1.Mutation of ACVR1 is capable of continuously activating the bone morphogenetic protein type I receptor,thereby inducing the patient to produce heterotopic ossification.In many cases,injuries can give raise to joints to become permanently frozen in place.Surgical removal of the extra bone growths has been shown causing the body to "repair" the affected area with even more bone.Therefore,the acquisition of cells from the patient to carry out research will make the patient's condition worse.To overcome the limitation,we endeavor to generate the heterozygous point mutaion ACVR1-FF-iPSCs by tranfected CRISPR/Cas9 and NgAgo technologies,including single-stranded oligodeoxynucleotides(ssODNs),into human FF-FF-iPSCs.Thus it was provided a new approach to the study of FOP disease,but also for the better studying of the pathogenesis of FOP and the development of new therapeutic drugs.NgAgo is a new gene editing technology,however,in addition to Chunyu Han's lab which can prove that NgAgo can make gene edit,many labs fail to prove it can make gene edit.Therefore,it will has very important significance to verify NgAgo can make gene edit or not.Objective: To explore NgAgo a new gene editing technology can make gene edit on human genome or not;to establish heterozygous point mutation FF-iPSCs cell line with ACVR1 gene mutation,and the mutation site is on G328 E of ACVR1 gene.We will provide a new approach for the study of FOP disease,but also for the better study of the pathogenesis of FOP and the development of new therapeutic drugs.Methods: Designing gDNA that target the exon region of ACVR1 gene.The gDNA and NgAgo were transfected into human umbilical vein endothelial cells(HUVEC),and selected with neomycin(G418).After screening,PCR amplify gDNA target site from the DNA genomic.In order to verify whether NgAgo do gene edit or not,we will use T7 EI and sequencing methods to detect it.Designing gRNA target sequence and ssODNs around ACVR1 gene G328 E region,and cloning gRNA into pSpCas9(BB)-2A-Puro vector.The gRNA pSpCas9(BB)-2A-Puro vectors were cotransfected into FF-iPSCs and selected by puromycin.We were picked up some colonies from the selected FF-iPSCs and established of monoclonal cell lines.PCR amplify the gRNA targeted site and using the PCR products to build library for Next Generation Sequence.Through the Next Generation Sequence we can detect whether have heterozygous point mutation on the monoclonal cell lines.Result: It was proved that NgAgo failed to carry out gene editing on the human genome;we were successful made gRNA pSpCas9(BB)-2A-Puro vector,gRNA pSpCas9(BB)-2A-Puro vector was transfected to FF-iPSCs and established 16 monoclonal cell lines.Through the Next Generation Sequence we were detected heterozygous point mutation on the monoclonal cell lines.However,the mutation rates were very low,which may be due to the failure to successfully select monoclonal cell lines when the monoclonal cell lines were picked,resulting in each cell line has WT FF-iPSCs mixed in monoclonal cell lines.Therefore,it is necessary to further screening and sequencing the obtained heterozygous point mutant cell lines.Conclusions: This study was proved that NgAgo gene editing technology failed to carry out gene editing on the human genome;the heterozygous mutation of ACVR1 gene was successfully achieved in FF-iPSCs cells by CRISPR/Cas9 technology,and a monoclonal cell line with heterozygous mutation was obtained.The results have laid the foundation for the in-depth study of the pathogenesis of FOP and the development of new therapeutic drugs.