Abstract: My laboratory is interested in studying signaling mechanisms for the regulation of neutrophil biology. Neutrophils are a type of polymorphonuclear leukocytes and are well recognized as one of the major players during inflammation. Although neutrophils are required for normal immune responses, they are also responsible for many inflammation-related diseases as the effector molecules released by neutrophils are toxic to host tissues. Mature neutrophils are known for their short lifespan (half-life is generally believed less than a day), and their lifespan may be expanded in inflammatory and disease conditions. Thus, the control of neutrophil development and lifespan can be a key step in regulating neutrophil-related functions in normal physiology as well as disease conditions. However, not much is known about the mechanisms underlying neutrophil development and lifespan regulation. While transcription factors like Cebpe are known to play a role in neutrophil development, investigations into these mechanisms have been limited to individual studies. Therefore, a comprehensive genetic screen would provide comprehensive knowledge of neutrophil development. In addition, although various death mechanisms, particularly apoptosis, play roles in neutrophil death, their blockades only had moderate effects, suggesting unknown mechanisms may exist. Thus, we hypothesize that there might be novel mechanisms for the regulation of neutrophil lifespan. Primary neutrophils have a very short lifespan and are unable to expand perpetually in vitro. Together with their being refractory to transfection or viral transduction, neutrophils have never been used in any large-scale genetic screen in mice. The HoxB8-ER fusion protein has been used to immortalize mouse hematopoietic progenitor cells (HoxB8-HPCs), which can differentiate into neutrophils both in vitro and in vivo. The HoxB8-HPC-derived neutrophils were functionally similar to freshly isolated mouse neutrophils. Because HoxB8-HPCs can be unlimited expanded and transduced with lentivirus, they are amenable to genetic screens. In our preliminary studies, we have established Hoxb8-HPCs from a Cas9 knock-in mouse line. These cells can be transduced with lentivirus with sufficient efficiency to meet our screening goals. We also did a pilot in vivo screen using one of the sub-libraries, which demonstrated the feasibility of our screen strategy and method. Therefore, in this exploratory R21 proposal, we propose to perform the first CRISPR-Cas9-based screens for key regulators that control neutrophil differentiation from HoxB8-HPCs and lifespan of differentiated neutrophils in mice. Our proposed work will not only establish a valuable research tool but also yield insights into neutrophil biology that cannot be predicted from existing knowledge. Importantly, the tool we are developing and validating here can be readily adapted for screens with many other possible readouts.