Summary/Abstract Disulfide reductase-driven antioxidant defenses prevent molecular damage that can contribute to inflammatory diseases, neurodegeneration, stem cell depletion, aging, and cancers. NADPH, generated from NADP+ using energetic nutrients, is the electron-donor for most biosynthetic, homeostatic, and cytoprotective reductions, but only two enzymes can use NADPH to reduce cytosolic disulfides: thioredoxin reductase-1 (TrxR1) and glutathione reductase (Gsr). Electrophilic toxins can coincidentally inhibit both the TrxR1 and Gsr pathways in liver. These include environmental metal/metalloids (e.g., arsenic), drugs (e.g., cisplatin) or drug metabolites (e.g., NAPQI from acetaminophen), and many organic toxins from plants or microbes. Mice with liver-specific disruptions of both TrxR1+Gsr (TrxR1/Gsr-null), which provide a useful genetic model for such situations, have revealed surprising robustness in the disulfide reductase systems, including a previously unrecognized methionine (Met)-fueled NADPH-independent system that sustains redox homeostasis in TrxR1/Gsr-null livers. These models reveal that mammals, unlike microbes, have unexpected sources and distribution- mechanisms to supply disulfide reducing power when the canonical pathways are compromised. We hypothesize that realigned metabolic activities and expanded functionality of Trx- and glutaredoxin (Grx)-family members provides support for essential reductase activities, when needed. A better understanding of these systems promises to provide improved therapeutic avenues for rescuing liver- and patient-health following severe oxidative stress or toxic exposures. Testing this hypothesis, however, will require development of innovative approaches to detect the putative complementary activities. Here we propose two specific aims that will use a novel CRISPR/Cas9 gene disruption approach in genetically modified mouse livers to (i) define the respective roles of Grx family members in distributing reducing power when Trx1 is disrupted and (ii) perform an innovative screen to identify genes supporting redox homeostasis upon co-disruption of TrxR1 and Gsr. Synopsis: This project will use innovative approaches for genome-editing-enhanced somatic cell genetic complementation in mouse liver to better define the pathways that support disulfide reductase systems when the canonical pathways become compromised. Consistent with PA-16-141: “Development of animal models and related biological materials for research (R21)”, this project develops a new approach for performing genetic complementation studies in mouse hepatocytes in situ.