β-hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia are debilitating, painful diseases and a major cause of global mortality and health disparities. Currently, there is no cure for the majority of patients with β-hemoglobinopathies and therapeutic options are limited. We have developed novel approaches to curing β-hemoglobinopathies using CRISPR/Cas9 based ex vivo genome editing of β-globin (HBB) gene in patients’ hematopoietic stem and progenitor cells (HSPCs). Although gene-editing strategies, including correction of the sickle mutation, targeted insertion of the β-globin gene and induction of fetal hemoglobin are very promising in curing β-hemoglobinopathies, significant safety concerns exist, including off-target effects, large deletions and insertions in HBB, and chromosomal rearrangements. In Aim 1, we optimized the design of CRISPR gRNA and short single-strand oligonucleotide donor template to correct the sickle mutation, and demonstrated high rates of gene correction in SCD HSPCs. Erythrocytes derived from gene-edited cells showed high levels of normal hemoglobin expression and a marked reduction of sickle cells. We found that gene-corrected HSPCs retained the ability to engraft in mouse models, and the off-target effects could be significantly reduced by using HiFi-Cas9. In Aim 2, we developed and validated two next-generation sequencing-based assays, LongAmp-seq (long-range PCR amplification based sequencing) and NEW-seq (nuclease-activity identified by gEnome-wide sequencing), to comprehensively investigate the gene-editing outcomes and the potential consequences of unexpected mutations. We performed a thorough analysis of gene-editing outcomes, including large deletions and insertions at the HBB cut site not previously reported. The LongAmp-seq and NEW-seq also have the potential to detect and quantify chromosomal rearrangements including inversions, translocations and large chromosomal deletions. To aid the development of new therapies for β-hemoglobinopathies, in Aim 3, we applied genome editing to establish erythroid cell models for SCD and β-thalassemia that exhibit disease phenotypes. These cell models are reliable, reproducible and low-cost in performing disease studies, including validation of genome editing based therapies and screening of pharmacological drugs. The systematic studies of the efficiency and safety of the gene-editing approaches, and the cell models developed for discovery of therapeutic agents may significantly facilitate the clinical translation of gene editing based therapies for β-hemoglobinopathies.