Senescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6 mediated  gene editing in hematopoietic stem cells [RNA-seq]
Gene editing (GE) using homology-directed repair (HDR) in hematopoietic stem and progenitor cells (HSPCs) offers promise for long-range gene correction of inherited genetic disorders. 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-?B 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: Cord-blood (CB) hematopoietic stem and progenitor cells (HSPCs) were cultured for three days in early active and stem-preserving medium and at day 3 we performed the following treatments: the electroporation with an RNP with no predictive activity in the human genome in presence (RNP NEG + ANAK) or absence  (RNP NEG) of Anakinra and the standard gene editing procedure with HS RNP and transduction with an AAV6 carrying the DNA donor template for HDR in presence (HS/AAV6 + ANAK) or absence (HS/AAV6) of Anakinra. After 24 hours post treatments, HSPCs were collected for RNA-seq analysis.