Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac

Abstract

β-thalassemia, one of the most common genetic diseases worldwide, is caused by mutations in the human hemoglobin beta (HBB) gene. Creation of human induced pluripotent stem cells (iPSCs) from β-thalassemia patients could offer an approach to cure this disease. Correction of the disease-causing mutations in iPSCs could restore normal function and provide a rich source of cells for transplantation. In this study, we used the latest gene-editing tool, CRISPR/Cas9 technology, combined with the piggyBac transposon to efficiently correct the HBB mutations in patient-derived iPSCs without leaving any residual footprint. No off-target effects were detected in the corrected iPSCs, and the cells retain full pluripotency and exhibit normal karyotypes. When differentiated into erythroblasts using a monolayer culture, gene-corrected iPSCs restored expression of HBB compared to the parental iPSCs line. Our study provides an effective approach to correct HBB mutations without leaving any genetic footprint in patient-derived iPSCs, thereby demonstrating a critical step toward the future application of stem cell-based gene therapy to monogenic diseases.

Figure 1.

Strategy for seamless correction of the β-thalassemia mutations using piggyBac and Cas9. (A) Locations of the two mutations at −28 with A/G substitution and the codon 41/42 with 4-bp deletion. (B) The DSB at intron 1 following Cas9 cleavage. (C) The targeting construct of the piggyBac transposon carrying the selectable markers, puro∆tk and Neo, flanked by 500 bp of wild-type genomic sequences. (D) Insertion of the piggyBac following homologous recombination. (E) After selection with puromycin, clones with mutation-corrected lines were identified and transiently transfected with transposase expression plasmids, followed by treatment with FIAU to eliminate piggyBac-containing clones, and the seamless mutation-corrected clones were isolated.

Figure 2.

Site-specific homologous recombination (HR) mediated by CRISPR/Cas9 in β-thalassemia iPSCs. (A) PCR analyses using two pairs of primers, P4/P5 and P1/P2, each pair with one in the piggyBac and the other in the HBB outside the targeting construct to detect homologous recombination. (B) Bacterial artificial chromosome (BAC) containing the piggyBac cassette inserted in the TTAA site located in intron 1 of the wild-type HBB used as a positive control; (P) parental line, (M) marker. (B) Homologous recombination confirmed by Southern blot analysis. Southern blot analysis after HindIII digestion of genomic DNA from puromycin-resistant clones using a 5′ genomic probe outside the targeting construct (left) and a Neo probe inside the piggyBac (right). Clone 75 shows the correct 13-kb band marked by an asterisk (*), detected by both the 5′ globin gene and Neo probes, indicating site-specific homologous recombination at the globin locus. The 8-kb bands are from the endogenous HBB. Other bands seen with the Neo probe are due to random integrations.

Figure 3.

Identification of gene correction in β-thalassemia iPSCs. (A) Nucleotides changed (in red) in PCR primers to generate restriction enzyme sites to distinguish the normal from the mutant alleles. For the –28 location, replacing nucleotide “a” with “t” at –31 generates a DdeI site for the mutant allele. At 41/42, replacing “cc” with “ga” generates an XmnI site for the wild-type allele. (B) PCR amplification followed by restriction enzyme digestion reveals a normal sequence for clones 28 and 33 at the –28 site and clones 75, 29, 36, 63, and 5 at the 41/42 location. (P) Parental iPSCs, (B) BAC, (M) marker. (C) Sequences of the two mutation sites showing correction of the heterozygous states (upper) to the normal sequences (lower).

Figure 4.

Precise excision of piggyBac in mutation-corrected iPSC clones. (A) Primers P4/P8 and P6/P7 amplify the CAG promoter and puro∆TK, respectively. No amplification by both pairs indicates no piggyBac reintegration either at the targeting site or other chromosomal locations in clones 75-5 and 75-7. P1/P3 amplifies the junction between the piggyBac insert and the globin gene sequence to the 3′ genomic sequences. No amplification by these primers in these two clones indicates the removal of the piggyBac insert from HBB, while positive amplification (B) with P2/P9 further confirms PB removal. (C) Sequence analysis showing the junction between the ITR of the piggyBac and genomic sequences before transposon removal and the restoration of the normal intron 1 sequence after removal. Note the TTAA sequence of the piggyBac that is used for insertion and excision from the genome.

Figure 5.

Hematopoietic differentiation of gene-corrected iPSCs. (A) Schematic representation of a stepwise hematopoietic differentiation strategy for iPSCs. (B) Representative morphology changes at day 7 and day 13 in hematopoietic differentiation of corrected iPSC lines. (C) Flow cytometric analysis of piPSCs (parental lines), 75RiPSCs, and 59RiPSCs lines harvested at day 14 showing the specific hematopoietic stem and progenitor markers: CD34⁺, CD43⁺, and CD45⁺. (D) Colony-forming assay for differentiated cells at day 10 revealed various types of hematopoietic colonies as well as nucleated cells, including erythroblasts. Blue arrows indicate erythroid cells. (E) HBB and HBG gene expression (normalized to GAPDH) were measured by quantitative RT-PCR in hematopoietic differentiation of parental lines (piPSCs-E), −28 mutation-corrected (59RiPSCs) lines, and 41/42 mutation-corrected (75RiPSCs) lines (data represent mean ± SEM, n = 3).