PROJECT SUMMARY Sickle cell disease (SCD) is a single-gene disorder, affecting ~100,000 Americans and millions worldwide. Existing pharmacological treatments may alleviate SCD severity but do provide a cure for SCD patients, and the chronic transfusion therapy poses challenges due to alloimmunization and iron overload risks. Currently, the only curative therapy for SCD is an allogeneic hematopoietic stem cell transplant (HSCT), typically from a matched related donor, which is available to only ~15% of SCD patients. Recently, gene therapy and gene editing-based autologous HSCT have emerged as potential curative avenues, offering an alternative approach to address donor availability and graft-versus-host disease. Several CRISPR/Cas9 gene editing based therapies for SCD are currently in clinical trials, by inducing fetal hemoglobin through BCL11A erythroid-specific enhancer targeting or correcting the sickle mutation on the beta-globin gene (HBB). Although these SCD clinical trials show promise, our recent studies found that three of the CRISPR gRNAs designed for treating SCD all induced unintended large gene modifications with high frequencies at the Cas9 on-target cut-sites in SCD HSPCs, including large deletions (LDs), insertions and complex local rearrangements. Given that two of these gRNAs (R-02 and BCL11A) are being used in the ongoing clinical trials, it is imperative to comprehensively assess the biological and clinical implications of these unintended gene modifications, and establish new strategies to mitigate potential adverse effects, which are the primary goals of the Parent R01 grant. This Diversity Supplement will expand upon the research proposed in the Parent R01 by supporting an underrepresented minority student to conduct gene editing research for treating SCD. Aim 1 of this Diversity Supplement aims to expand the first aim of the Parent R01, to determine the consequences of small indels, especially the frequencies and genotype- phenotype relations of small in-frame indels at the Cas9 cut-site in HBB. Aim 2 of the Diversity Supplement will expand Aim 3 of the Parent R01 to investigate the possibility of enhancing homology directed repair (HDR) through the modulation of DNA repair pathways using small molecules, while simultaneously reducing LDs and small in-frame mutations. It is expected that the studies supported by the Diversity Supplement will help establish a better understanding of the consequences of HBB gene editing by CRISPR/Cas9, thus facilitating the translation of genome editing based treatment of SCD.