Abstract One of the most predominant hallmarks driving cancer development is genome instability. It creates genome- wide diversity that enables cells to acquire additional capabilities required for cancer development and progression. Most of the ~400 genes known to be mutated and implicated in cancer development are a direct result of increased genome instability. Therefore, understanding the molecular mechanisms of genome instability in cancer cells is imperative for the development of novel treatment strategies. Fanconi Anemia (FA) is a hereditary disorder caused by mutations in at least 22 genes and clinically characterized by bone marrow failure and predisposition to cancer. This proposal focuses on FANCA, a gene that is mutated in ~64% of the entire FA patient population. During the preliminary studies, we found that FANCA promotes error-prone DNA repair that drives genome instability; its expression is upregulated in many cancer types, and the expression level is strongly associated with breast cancer progression and inversely correlates with cancer patient survival. Intriguingly, FANCA recruitment to double strand breaks and DNA damage sites requires active transcription in a KillerRed live cell analysis. More importantly, knockout of FANCA in a triple negative breast cancer cell MDA-MB-231 initiates cell cycle arrest and cellular senescence and abolishes breast cancer formation in mice. Based on these preliminary data, we hypothesize that high expression of FANCA in cancer cells promotes error-prone repair, genome instability, and cell cycle progression. To delineate the role of FANCA in genome instability and cancer development, we will use a biochemically defined in vitro system, a transcription-coupled DSB repair reporter system, a KillerRed live cell imaging system, a xenograft mouse model, and genome-wide instability analysis to accomplish three aims: Aim 1 is to determine the molecular mechanism of how FANCA contributes to R-loop-mediated genome instability; Aim 2 is to study the role of FANCA in DSB-mediated genome instability and how FANCA is regulated; Aim 3 is to determine the relationship between FANCA-mediated genome instability and cell cycle progression. Completion of this proposal will define a novel role for FANCA in genome instability. This work will also elucidate the significance of FANCA as a unique, rationale-driven target for cancer treatment. The outcome of this proposal will expand treatment strategies for cancer patients with elevated FANCA expression and genome instability.