Clinical application of gene therapy for beta-thalassemia is currently based on gene disruption for reactivation of fetal hemoglobin (HbF) or on HBB gene addition, both of which have potential drawbacks for severe mutations. As alternative, specific correction of sufficiently common causative mutations is economically of interest, but is as yet not efficient enough for clinical application when based on homology-mediated precision repair. Employing both TALEN and CRISPR/Cas designer nucleases, we have established proof of concept for homology-independent mutation-specific repair of the exceedingly common beta-thalassemia mutation HBB{IVSI-110(G>A)}, which creates an aberrant HBB splice acceptor site. We have shown that in transgenic cell models and patient-derived primary cells, non-homologous-end-joining (NHEJ)-mediated disruption of the mutation or of its context sequences efficiently restores normal splicing, beta-globin levels and cell morphology. This project will perform preclinical assessment of our TALEN-, CRISPR/Cas- and BE-based mutation-specific therapeutics in primary cells in vitro and in chimeric mouse models, to validate efficacy, safety and long-term repopulation potential of modified cells, and suitability for clinical trials. Critically, the project will directly compare all three platforms with clinically applied disruption-based HbF reactivation (NCT03745287), in order to establish the relative merits and potential for commercialization of our HBB{IVSI-110(G>A)} mutation-specific therapeutics.