We have studied the role of cold storage and post-transfusion hemolysis as one of the pathways that define the red blood cell (RBC) storage lesion, and how inherited genetic polymorphisms have a great effect on RBC function and transfusion outcomes. These studies have catalyzed new fields of research, encompassing the biophysics that govern hemolysis-related impairments in NO/ROS equilibrium, studies of the transfusion- recipient physiological and pathological responses, and interactions with donor genetics on RBC transfusion outcomes. In this renewal we have focused on a simple observation, that the variability in donor RBC stability and function during cold storage and post-transfusion recovery is more dependent on the donor, than on the actual time the unit is in storage. For example, common mutations like sickle cell trait (HbAS), G6PD deficiency, and SNPs in SEC14L4 and MYO9B exhibit enhanced storage hemolysis and lower post-transfusion recovery. These observations inform our overarching hypothesis that evolved variability in genes encoding RBC proteins significantly modulates RBC function and recovery after transfusion. Few studies have explored how these common or rare, single or polygenic, variants modulate donor RBC function during storage and post-transfusion. To address this question, in collaboration with the REDS-III program, we performed the largest genome-wide association study (GWAS) to date of >12,000 healthy human blood donors (Page et al. JCI 2021). We identified 27 candidate SNPs at genome-wide significance (P<10-8) that were associated with enhanced or reduced responses to hemolytic perturbations. The identified variants were in candidate genes known to modulate erythrocyte structure, metabolism, and ion channels; in genes encoding antioxidant enzymes; and in proteins with unknown function (SEC14L4 and MYO9B). Many SNPs were observed in polygenic combinations associated with higher risk of hemolysis. We also confirmed that many of the identified SNPs were associated with higher rates of steady state hemolysis in patients with sickle cell disease and were associated with lower post-transfusion RBC recovery in large patient populations (Roubinian et al, JCI Insight 2022). Finally, we have developed and established colonies of novel CRISPR-Cas9 genetically engineered “humanized” mouse models carrying the human SNPs for the common A- African American G6PD deficiency variant (Zuchelkowski et al, PLoS One, 2020) and a SNP with unknown function in SEC14L4. In this renewal we propose to now explore the function of these candidate SNPs in vitro and in vivo, using our novel “humanized” SNP mouse models and our validated and FDA-approved high sensitivity autologous red cell biotin labeled transfusion-recovery protocols in humans. We anticipate that these studies will identify new signaling pathways that modulate RBC function, and inform a Precision Transfusion Medicine field, in which RBC donor genotype determines the time limits of blo...