PROJECT SUMMARY/ABSTRACT The regulation of human cholesterol plays a critical role in determining a person’s lifetime risk of cardiovascular disease. Identification of the genes and proteins that underlie this process has improved our fundamental understanding of how cardiovascular disease develops, and led to the development of cardiovascular drugs that have greatly benefited public health. While many key regulators of cholesterol homeostasis have been identified, the majority of the heritable risk of hypercholesterolemia cannot be explained by currently recognized variants, suggesting the presence of additional unknown mediators. Recently, breakthroughs in CRISPR-mediated genome editing and massively parallel sequencing have allowed high-throughput, unbiased functional testing of the entire human genome. During my postdoctoral research, I developed a genome-scale CRISPR screen for novel regulators of the extracellular secretion of PCSK9, a process that plays a critical role in modulating plasma cholesterol levels. This led to my discovery of SURF4 as a cargo receptor that promotes efficient PCSK9 secretion in vitro. However, the physiologic relevance and mechanistic basis of the interaction between SURF4 and PCSK9 remains unclear. This proposal builds upon my prior work through the generation of mice with liver-specific deletion of Surf4 and characterization of the consequences to cholesterol homeostasis (Aim 1), and by altering the coding sequence of human SURF4 to determine which domains mediate its interaction with PCSK9 (Aim 2). In addition to elucidating the basic biology of PCSK9 secretion, these studies will inform the potential of SURF4 as a therapeutic target and serve as proof-of-principle for the power of CRISPR screening to identify novel regulators of protein secretion. This research will be conducted under the guidance of a primary mentor who has extensive expertise in protein trafficking, cardiovascular physiology, and genetics as well as a long track record of mentorship for early career physician-scientists who have successfully transitioned to scientific independence. This work, along with the mentorship and training activities described in my proposal, will facilitate my development toward my ultimate goal of becoming an academic physician-scientist and independent investigator.