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However, adverse cellular responses induced by CRISPR-Cas9/AAV6 engineering impair the long-term repopulating potential of HDR-edited HSPCs, limiting clinical translation. Our study uncovers a senescence-like response in genetically-engineered HSPCs triggered by p53 and IL-1/NF-κB activation, which restricts graft size and clonal diversity in long-term transplantation assays. We show that transient p53 inhibition or blocking inflammatory pathways can mitigate senescence-associated responses, enhancing the repopulating capacity of edited HSPCs. Importantly, we identify Anakinra, an IL-1 signaling antagonist, as a safer and effective strategy to enhance polyclonal output in HDR-edited cells while minimizing genotoxicity risks associated with the gene-editing procedure. These findings present strategies to overcome key hurdles in HDR-based HSPC therapies, providing a framework for enhancing the efficacy and safety of these approaches in future clinical applications. Overall design: Targeted deep sequencing on Bone Marrow HSPC donors, treated with different editing conditions (i.e., standard editing, + ANAK, +GSE56, adn +SC514).ENAfalseSenescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6 mediated gene editing in hematopoietic stem cells [NHEJ]Senescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6 mediated gene editing in hematopoietic stem cells [NHEJ]2025-09-242025-03-22PRJNA1214732GSE287805960640466639