Editing the b-globin locus in hematopoietic stem cells is an alternative therapeutic approach for gene therapy of b-thalassemia and sickle cell disease. Using the CRISPR/Cas9 system, we genetically modified human hematopoietic stem and progenitor cells (HSPCs) to mimic the large rearrangements in the b-globin locus associated with hereditary persistence of fetal hemoglobin (HPFH), a condition that mitigates the clinical phenotype of patients with b-hemoglobinopathies. We optimized and compared the efficiency of plasmid-, lentiviral vector (LV)-, RNA-, and ribonucleoprotein complex (RNP)based methods to deliver the CRISPR/Cas9 system into HSPCs. Plasmid delivery of Cas9 and gRNA pairs targeting two HPFHlike regions led to high frequency of genomic rearrangements and HbF reactivation in erythroblasts derived from sorted, Cas9 + HSPCs but was associated with significant cell toxicity. RNA-mediated delivery of CRISPR/Cas9 was similarly toxic but much less efficient in editing the b-globin locus. Transduction of HSPCs by LVs expressing Cas9 and gRNA pairs was robust and minimally toxic but resulted in poor genome-editing efficiency. Ribonucleoprotein (RNP)-based delivery of CRISPR/Cas9 exhibited a good balance between cytotoxicity and efficiency of genomic rearrangements as compared to the other delivery systems and resulted in HbF upregulation in erythroblasts derived from unselected edited HSPCs.