Efficient methods for target gene manipulation in haematopoietic stem cell derived human neutrophils.

Neutrophils are the most abundant leukocyte in humans and the principal effectors of the innate immue response. Genetic modification of human neutrophils is challenging due to their short lifespan and tendency to activate in response to even minor perturbation. However, genetic manipulation of haematopoietic progenitor cells and subsequent directed differentation into neutrophils represents a potential avenue to study the contributions of individual genes and pathways to human neutrophil function. Here we present a method of directed granulocytic CD34+ progenitor differentiation into neutrophils capable of key functions such as priming and neutrophil extracellular trap (NET) formation. We further show that differentiating progenitors can be efficiently and stably modified by lentiviral gene delivery and Cas9-gRNP nucleofection to produce potent and activation-free gene knockdown in mature neutrophils, thereby providing new tools for understanding the contribution of neutrophils to health and disease. Using this model we have shown that, contrary to previous reports, CD11b is not required for phagocytosis of serum-opsonised bacterial particles.