Targeted JAK2V617F Point Mutation In Human Induced Pluripotent Stem Cells Based On CRISPR/Cas9 Technology

Background The transfusion treatment of red blood cell is becoming more and more extensive.However,the disparity between the aging population pushing up demand and a relatively smaller donor base has made none remunerated blood donations far from meeting the clinical treatment.It has been reported that the JAK2V617 F mutation could be detected in the majority of patients with polycythemia vera.Thus,this study intends to interfere with JAK2 to realize JAK2V617 F mutation in induced pluripotent stem cells(i PS)by CRISPR/Cas9-mediated genome editing so that we can establish i PS cell lines containing JAK2V617 F mutation,which may bring a new horizon for improving the yield of red blood cells derived from i PS cells..Methods Under the guidande of CRISPR/Cas9 technology,we designed 3single-guide RNAs(sgRNA)targeting the V617 F mutation of JAK2,and synthesized a single-stranded oligo deoxynucleotide(ss ODN)centered on the mutation site as the homologous directed repair(HDR)donor template.After detecting the editing efficiency of 3 sgRNAs,we used the optimal sgRNA to construct a CRISPR plasmid expressing Cas9/sgRNA.To edit JAK2 to accurately cause JAK2V617 F mutation,the CRISPR plasmid and ss ODN donor were delivered into i PS cells in vitro by lentivirus packaging system.Sanger sequencing was further used to detect whether the mutation of JAK2V617 F in i PS cells was successful..Results we successfully realized the JAK2V617 F mutation in i PS cells through HDR-mediated CRISPR/Cas9 gene editing and no off-target effects were detected.Both forward sequencing and reverse sequencing showed that the guanine at position1849 of the JAK2 gene was replaced by thymine,which caused the mistranslation of valine at position 617 to phenylalanine(V617F).Conclusions It is feasible to achieve JAK2V617 F mutation in i PS cells by CRISPR/Cas9 gene editing technology mediated by HDR.We have successfully constructed a i PS cell line containing JAK2V617 F mutation via this method,which laid the foundation for subsequent red blood cells directed differentiation experiments in vitro.