Pairwise and higher-order genetic interactions during the evolution of a tRNA

A central question in genetics and evolution is the extent to which the outcomes of mutations change depending on the genetic context in which they occur^1-3. Pairwise interactions between mutations have been systematically mapped within^4-18 and between ¹⁹ genes, and have been shown to contribute substantially to phenotypic variation among individuals ²⁰ . However, the extent to which genetic interactions themselves are stable or dynamic across genotypes is unclear^{21, 22}. Here we quantify more than 45,000 genetic interactions between the same 87 pairs of mutations across more than 500 closely related genotypes of a yeast tRNA. Notably, all pairs of mutations interacted in at least 9% of genetic backgrounds and all pairs switched from interacting positively to interacting negatively in different genotypes (false discovery rate < 0.1). Higher-order interactions are also abundant and dynamic across genotypes. The epistasis in this tRNA means that all individual mutations switch from detrimental to beneficial, even in closely related genotypes. As a consequence, accurate genetic prediction requires mutation effects to be measured across different genetic backgrounds and the use of  higher-order epistatic terms.