Abstract Over their ~1.5-billion-year evolution from a bacterial endosymbiont, mitochondria have retained genes essential for energy metabolism on their own distinct genome (mtDNA) in almost all eukaryotes. Human mtDNA encodes 13 core proteins in the respiratory chain and varies up to 10,000-fold in copy number from ~100 in blood to ~half a million in the unfertilized egg, with each cell maintaining an intrinsic, optimal setpoint via yet unknown mechanisms. A decline in this setpoint causes rare but severe disorders with no proven therapies and underlies the common age-related mitochondrial dysfunction with relevance to Parkinson’s and Alzheimer’s Disease. In my K99 phase, I propose to address two major unmet needs in clinical and basic mitochondrial biology – to overcome the detrimental consequences of mtDNA loss and to understand homeostatic regulation of mtDNA copy number. Genetic studies in yeast, trypanosomes, and my published work in human cells, reveal a conserved link between membrane potential and tolerance to mtDNA loss. In aim 1, I will determine the mechanism by which boosting membrane potential rescues the growth of mtDNA-depleted cells. My completed multi-omic profiling of mtDNA depletion and repletion states has identified a small number of candidate effectors of membrane potential which I will test using a drug that targets stress signaling, a metabolite that can be supplemented exogenously, and a protein prosthetic for redox manipulation pioneered by my mentor’s lab. In aim 2, I will take a more unbiased approach and systematically identify nuclear regulators of human mtDNA copy number for the first time using a FACS-based genome-wide CRISPR knockout screen, which I conceptualized and developed in my early postdoctoral work. While my K99 focuses on mtDNA sensing and regulation within the mitochondrial matrix, my R00 (aim 3) examines the pathways and immunogenic consequences of mtDNA leakage to the cytosol which is highly relevant to autoimmune disorders. Thus, my independent research program aims at a synergistic culmination of postdoctoral training in mitochondrial systems biology and PhD expertise in innate immunity. Through my K99 goals I will master the theory and experimentation of bioenergetics and get hands-on training in high-throughput genetics, from screen design to computational analysis. My mentor, Dr. Vamsi Mootha, is a world leader with >20 years of experience in precisely the field of my proposed training – mitochondrial systems biology – and has propelled over a dozen trainees into thriving academic research careers. I have a diverse advising committee with local and national experts relevant to my proposal, including one junior faculty member in my department and one immunologist for specific guidance at the R00 transition. Thus, with the NIH Pathway to Independence award, I will train towards my long-term goal of launching my own laboratory and discovering basic molecular mechanisms underlying mitochondrial a...