Efficient Genomic Correction Of Mutants In Compound Alpha-and Beta-thalassaemia Major IPSCs Using CRISPR/Cas9 System

OBJECTIVE: To correct mutants in compound alpha-and beta-thalassaemia major iPSCs by CRISPR/Cas9 system.To test the pluripotency and the hematopoietic function of the corrected iPSCs.METHODS: In this study,the β41-42/β41-42 & ααws/αα＿iPS was selected as experimental subject.The β-sg RNA and α-sg RNA were respectively designed in600～bp downstream of the third exon of HBB and 200～bp downstream of the third exon of HBA2,which were inserted into vectors PX330 and p TP53-GFP-reporter,thus plasmids HBB-sg RNA,HBA2-sg RNA,HBB-GFP-reporter-sg RNA,HBA2-GFP-reporter-sg RNA were constructed.HBB-sg RNA/ HBB-GFP-reporter-sg RNA and HBA2-sg RNA/ HBA2-GFP-reporter-sg RNA were respectively transfected into293 T cells by calcium transfection,and then the activity of β-and α2-sg RNA detected by fluorescent microscope observation and flow cytometry analysis after 48 hours.Secondly,the left(2kb)and right(1.5Kb)arms were amplified from normal genomic DNA and flanked into anti-puromycin gene of Psimple-18 T vector,construced HBB-Donor.Meanwhile,the left(800bp)and right(790bp)arms were amplified from normal genomic DNA and flanked into anti-neomycin gene of PUC-57 vector,then the plasmid HBA2-Donor construced.In the end,the linearized HBB-Donor and HBB-sg RNA were electroporated into compound alpha-and betaThalassaemia Major iPSCs.The sg RNA identified the target site and cutted,then the homologous recombination occured.Finally,the β41-42 M corrected iPS cells(βN&ααws/αα＿corrected iPS)derived by drug screening,PCR detection and sequencing.And then the linearized HBA2-Donor and HBA2-sg RNA were transfected intoβ41-42 M corrected iPS.Ultimately,the compound alpha-and beta-Thalassaemia Major iPSCs were corrected entirely.In addition,the stem cell markers expression,karyotyping,teratoblastoma formation and hematopoietic induced differentiation were studied.RESULTS: The mutants of β41-42 and westmead were successfully corrected.The corrected efficiency of β41-42 homozygous mutations to heterozygous mutation was90%,but the entirely corrected efficiency of β41-42 homozygous mutations was only 1%,and the corrected efficiency of westmead was 14%.Compared with uncorrected iPS cells,the relative expression of OCT4,SOX2,NANOG genes inβN/βN & αα/αα＿corrected iPS has no significant difference.Teratoblastoma formation assay clearly revealed endoderm,mesoderm and ectoderm.Karyotype analysis showed a nomal female karyotype,46,XX.The corrected iPS cells were co-cultured with OP9 cells,and the genes relative expression of OCT4,SOX2,NANOG,GATA4,T,PAX6 were tested at D0,D2,D4,D6,D8,D10,D12.The relative expression quantity of pluripotency genes OCT4,SOX2,NANOG gradually reduced as time gone on.The mesoderm gene T was expressed in advance.The outcome of flow cytometry analysis of cells at D8,D10,D12 showed that CD34+CD43+ positive cells significantly increased compared with uncorrected iPS cells.And the CFU-E also significantly increased,and the flow cytometry analysis of CFU-E proved that the quantity of red blood cells and β-globin protein had an obvious increase.CONCLUSIONS: iPSCs and CRISPR/Cas9 provide an effective treatment for thalassemia.If the corrected iPS cells of patients was transplanted,the immune rejection of hematopoietic stem cell transplantation could be avoided,and the disease could be cured successfully.