Project Summary The long-term goal of this research program is to understand how genetic interaction among loci (or ‘epistasis’) contributes to phenotypic variation. To achieve this objective, we will pursue two parallel research trajectories. First, we will continue our ongoing work to characterize the genetic and molecular mechanisms that cause spontaneous and induced mutations to show different effects across genetically distinct individuals. These ‘background effects’ are important to human health because they complicate efforts to predict, prevent, and treat disease based on personalized genomic data. We recently used combinatorial DNA barcoding and CRISPR interference (‘CRISPRi’) to screen thousands of protein-coding genes in yeast for background effects in 170 progeny from a cross. With these data, we identified 815 background effects, quantified how background effects shape the phenotypic variance in populations, and mapped a subset of loci causing these background effects. Here, we will extend our work by comprehensively dissecting the genetic bases of hundreds of these already identified background effects, examining the environmental contextuality of these background effects, and determining how certain ‘hotspot’ loci shape the effects of many different genetic perturbations. Second, we have developed an approach for cloning segments of natural chromosomes and assembling them into synthetic chromosomes that can replace the native chromosomes in cells. This method opens new opportunities for characterizing epistasis without a need for mating or meiosis. As proof-of-principle, we showed that a single chromosome could explain nearly the entire difference in the abilities of two yeast species to grow at high temperature. Half of this effect was due to epistasis between different chromosomal segments. Here, we will improve the scalability, versatility, and portability of our technique, and will use it to determine how epistasis shapes trait differences and isolation between yeast species and genera. Our two research directions will produce detailed insights into epistasis that advance efforts to understand and predict the relationship between genotype and phenotype.