Repair Of ?-thalassemia Caused By The IVS2-654 (C>T) Mutation In HiPS Cells Using TALEN And CRISPR Editing

Beta(?)-Thalassemia is caused by either point mutations or deletions in the ?-globin(HBB)gene.The reduction or absence of synthesizing the beta chains of hemoglobin that results in outcomes ranging from severe anemia to clinically asymptomatic individuals.?-Thalassemia is a kind of recessive hereditary disease.The morbidity of ?-thalassemia is much higher in the south Pacific region,Africa,Asia.There are some treatments clinically for ?-Thalassemia,including blood transfusion,bone marrow transplants and splenectomy,which need treat patients for lifelong time,and make them painful,all of these methods can not cure the?-Thalassemia thoroughly.The induced pluripotent stem cells(iPSC)technology was pioneered by Shinya Yamanaka's lab in 2006,who generated mouse iPSC from mouse embryonic fibroblast.Following this,many species including human iPSC have been successfully prepared.There are two advantages in iPSC compaired to embryonic stem cells:one is that it has no ethical problems,the other is that patient can obtain iPSC from themselves,thus no immunological rejection problem.The major concern with the potential clinical application of iPSCs is their propensity to form tumors.In recent years,new genome editing methods,like transcription activator like effector nucleases(TALEN)and clustered regularly interspaced short palindromic repeats(CRISPR),have been developed rapidly.Compared to ZFNs(zinc-finger nucleases),TALENs and CRISPR/Cas9 are easier to be design and flexible in genome editing.TALEN and CRISPR/Cas9 can work as genome editing technologies.In the presence of exogenous DNA repair templates,nuclease-induced DNA double-strand breaks(DSBs)can be repaired by homology-directed repair.By this method,we can achieve the goal of accurate gene therapy.The IVS2-654(C>T)mutation is one common disease mutation of ?-thalassemia in Southeast Asia.In this study,we selected TALENs and CRISPR/Cas9 for the HBB intron2 IVS2-654 C>T mutation and successfully corrected point mutation in hiPSC.We analyzed the cleavage efficiency and off-target efficiency between TALEN and CRISPR/Cas9.TALENs mediated a higher homologous gene targeting efficiency compared to CRISPR/Cas9.In addition,more obvious off-target events were observed with CRISPR/Cas9 compared to TALENs.Finally,TALENs-corrected iPSC clones were selected for follow-up studies.TALENs-corrected iPSC have normal pluripotency like wide-type iPSC.TALENs-corrected iPSC clones were selected for erythroblast differentiation using the OP9 co-culture system and detected relatively higher transcription of HBB than the uncorrected cells.TALENs or CRISPR/Cas9 to correct specific HBB mutations in patient-derived iPSCs will guide future applications of TALENs-or CRISPR/Cas9-based gene therapies in monogenic diseases.