Abstract Neutrophils comprise a crucial component of the innate immune system and rely on chemotaxis to track down, engulf, and destroy pathogens. However, neutrophils also migrate to sites of developing tumors where they may promote angiogenesis and set the stage for metastasis to occur. The WAVE regulatory complex (WRC) serves a central role in mediating chemotaxis, as it links the actin dynamics powering cell movement to the signaling cascade which responds to environmental stimuli. Actively migrating neutrophils must respond quickly (within seconds) to changes in their environment; unfortunately, traditional methods one may use to alter WRC activity (e.g, RNAi and gene deletion) require hours/days to take effect. Consequently, while we know that WRC is generally required for efficient chemotaxis, its specific functions during each temporal phase, such as establishment and maintenance of a chemotactic response, remain unknown. Furthermore, the roles of WRC in this process are convoluted by additional actin assembly and assembly-promoting factors that act alongside WRC to drive neutrophil movement: Cells may compensate for prolonged loss of WRC through altering the activities of one or more of these factors. Here, I employed CRISPR to knockout the HEM1 subunit of WRC in neutrophil-like HL60 cells. Lacking functional WRC, these cells exhibit impaired chemotaxis, similar to cells where HEM1 expression is knocked-down. Unexpectedly however, the knockout neutrophils display additional defects in morphology and actin organization that are never observed in knockdown cells, despite knockdown cells containing less than 10% of normal Hem1 levels. I have also, in parallel, generated and validated optogenetic tools that can be used to rapidly and reversibly recruit WRC to different subcellular locations. When expressed in neutrophils lacking HEM1, these tools will open up the possibility of acutely ‘activating’ or ‘inactivating’ WRC during different temporal stages of chemotaxis (e.g., polarity establishment or maintenance). Ultimately, a more nuanced understanding of neutrophil chemotaxis should lead to the identification of new drug targets that may be used to block the accumulation of neutrophils at sites of tumor growth. Citation Format: Brian R. Graziano, Orion D. Weiner. Dissecting the spatiotemporal functions of WAVE complex during chemotaxis using CRISPR and optogenetics. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A181.