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 sole electroporation (MOCK EL in the paper), electroporation with an RNP complex with a guide RNA with high specificity (HS in the paper) and the standard gene editing procedure with HS RNP and transduction with an AAV6 carrying the DNA donor template for HDR (HS/AAV6 in the paper). After 24 hours post treatments, HSPCs were collected for RNA-seq analysis.