Engineering innate immunity: CRISPR-Cas9 MPO knockout in hematopoietic stem cells abolishes chlorination and PMA-induced NET formation while preserving non-oxidative effector functions

Introduction Myeloperoxidase (MPO) is a key neutrophil heme peroxidase that contributes to antimicrobial oxidative killing. Emerging evidence implicates MPO in diverse inflammatory disorders, including atherosclerosis, autoimmune diseases, and neurodegenerative diseases. Since inherited MPO - deficiency is typically compatible with preserved host defense, inhibition or genetic deletion of MPO represents a promising strategy to mitigate MPO-driven tissue injury. Methods Human HSPCs were edited using CRISPR-Cas9 to generate MPO -knockout ( MPO -KO) neutrophils, followed by in vitro differentiation. We systematically evaluated canonical neutrophil effector functions to determine which of them require MPO. Additionally, RAG2 -KO was generated and used as a control for knockout procedure. Results MPO-KO did not significantly impair major neutrophil effector functions, including neutrophil elastase activity, fMLP-induced NE and MMP9 degranulation, phagocytosis of E . coli and S. aureus bioparticles, and superoxide production. In contrast, MPO-dependent chlorination was abolished and PMA-induced neutrophil extracellular trap (NET) release was profoundly reduced in MPO-KO neutrophils, while RAG2-KO control cells retained all of these functions. Conclusion This strategy may offer a durable means to attenuate MPO-driven inflammatory damage, warranting further preclinical evaluation in relevant disease models.