POLG deficiency is associated with tremendous allelic heterogeneity and pleiotropy, hence making it very challenging to know which observed variants are benign versus pathogenic. Our goal is to create a "look up table" for POLG that connects all possible coding variants to function. We will leverage next generation DNA synthesis to create a saturation mutagenesis library for POLG and then perform pooled functional genetic screens that quantitatively score POLG variants based on their impact on mitochondrial oxidative phosphorylation. The proposed project takes advantage of our lab's longstanding commitment to mitochondrial disease, expertise in pooled genetic screening for mitochondrial function, as well as the Broad Institute's proven expertise in mammalian genetic screening. Our goal is to create a durable resource that will be useful to the research and clinical communities.