Both TALENs and CRISPR/Cas9 directly target the HBB IVS2–654 (C > T) mutation in β-thalassemia-derived iPSCs

Abstract

β-Thalassemia is one of the most common genetic blood diseases and is caused by either point mutations or deletions in the β-globin (HBB) gene. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations may be a potential therapeutic strategy for this disease. Due to the low efficiency of typical homologous recombination, endonucleases, including TALENs and CRISPR/Cas9, have been widely used to enhance the gene correction efficiency in patient-derived iPSCs. Here, we designed TALENs and CRISPR/Cas9 to directly target the intron2 mutation site IVS2-654 in the globin gene. We observed different frequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENs and CRISPR/Cas9, and TALENs mediated a higher homologous gene targeting efficiency compared to CRISPR/Cas9 when combined with the piggyBac transposon donor. In addition, more obvious off-target events were observed for CRISPR/Cas9 compared to TALENs. Finally, 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. This comparison of using 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.

Combining Single Strand Oligodeoxynucleotides and CRISPR/Cas9 to Correct Gene Mutations in β-Thalassemia-induced Pluripotent Stem Cells

“…Traditional methods of gene correction rely on antibiotic selection and then removal of the marker, which is a lengthy process that increases the risk of generating additional genome instability in human iPSCs (20). CRISPR/Cas9-induced genome editing was recently performed in ␤-Thal iPSCs using a piggyBac or donor vector as the repair template (15,16).…”

Cure for thalassemia major – from allogeneic hematopoietic stem cell transplantation to gene therapy

“…[77][78][79][80][81] One advantage of using iPSCs for gene correction is that it could then be possible to obtain a completely corrected clone of pluripotent stem cells, from which a large number of stem cells or other cells of interest could be derived for transplantation. However, so far it has not been possible to use iPSCderived HSCs to engraft and provide sustained hematopoiesis.…”

CRISPR/Cas9-mediated traceless gene correction and activation of the HBB locus in iPSCs with the beta thalassaemia mutation

“…Moreover, upon screening 624 and 717 potential off-target sites for 12 sgRNA in 293FT cells and iPSCs, respectively, a total of three sites were identified as being associated with two sgRNAs only [87]. On the other hand, Xu et al [138] showed clear off-target activity at seven out of 10 sites after targeting Intron 2 of the HBB gene, and reported a comparable level of off-target activity in 293FT cells and iPSCs. The discrepancy between these studies may be related to the sequence of the sgRNA used and the local environment of the targeted locus.…”

“…A number of studies have successfully used programmable engineered nucleases such as ZFNs, TALENs and RGNs for targeted repair of the disease-causing mutation in iPSCs derived from patients with monogenic disorders affecting the -globin gene, including sickle cell anaemia [140][141][142][143] and thalassaemia [26,35,36,38,138,144]. In contrast to ZFNs and TALENs, the Cas9 system offers the advantage of a higher targeting efficiency using a cost-effective, flexible and versatile system [8].…”

“…The genotoxicity analyses in studies that used Cas9-based systems for gene correction of the beta thalassaemia mutation are mostly limited to a handful of computationallypredicted potential off-target sites that bear high sequence homology to the on-target site. A high rate of off-target activity (7 sites with mutagenesis out of 10 screened sites) was reported in one of those studies [138]. Recent evidence suggests that bioinformatic methods may not accurately predict off-target sites, given the complex milieu of the factors determining functional off-target sequences [65].…”