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

Abstract

Beta thalassaemia is caused by mutations in the adult haemoglobin gene (HBB) and is one of the most prevalent monogenic blood disorders worldwide. The transplantation of haematopoietic stem cells derived from gene-corrected patient induced pluripotent stem cells (iPSCs) could provide a promising therapeutic strategy. Here, the generation and characterisation of footprint-free iPSCs from dermal fibroblasts of an individual with a homozygous beta thalassaemia-causing mutation affecting splicing is described (Chapter III). Successful gene correction of the disease-causing mutation was achieved by employing a CRISPR/Cas9 dual nickase approach and two donor design strategies aimed at reducing undesired on-target mutagenesis (Chapter IV), followed by the This study combined cutting-edge cellular reprogramming methods to generate beta thalassaemia iPSCs, a double nickase-mediated gene editing approach and piggyBac transposase-aided excision to achieve seamless gene repair, and a CRISPRa approach to model the impact of the diseasecausing mutation and demonstrate restoration of normal transcription following genetic repair.Having established a platform for precise gene correction and for validation of the restoration of the functional allele in this monogenic disease, this strategy may provide a viable avenue for iPSCiii based cell therapy of beta thalassaemic patients provided mature engraftable blood cells can be generated from hiPSCs in the future.iv