Evolutionary Stability Optimizer (ESO): A Novel Approach to Identify and Avoid Mutational Hotspots in DNA Sequences While Maintaining High Expression Levels

[Image: see text] Modern synthetic biology procedures rely on the ability to generate stable genetic constructs that keep their functionality over long periods of time. However, maintenance of these constructs requires energy from the cell and thus reduces the host’s fitness. Natural selection results in loss-of-functionality mutations that negate the expression of the construct in the population. Current approaches for the prevention of this phenomenon focus on either small-scale, manual design of evolutionary stable constructs or the detection of mutational sites with unstable tendencies. We designed the Evolutionary Stability Optimizer (ESO), a software tool that enables the large-scale automatic design of evolutionarily stable constructs with respect to both mutational and epigenetic hotspots and allows users to define custom hotspots to avoid. Furthermore, our tool takes the expression of the input constructs into account by considering the guanine-cytosine (GC) content and codon usage of the host organism, balancing the trade-off between stability and gene expression, allowing to increase evolutionary stability while maintaining the high expression. In this study, we present the many features of the ESO and show that it accurately predicts the evolutionary stability of endogenous genes. The ESO was created as an easy-to-use, flexible platform based on the notion that directed genetic stability research will continue to evolve and revolutionize current applications of synthetic biology. The ESO is available at the following link: https://www.cs.tau.ac.il/~tamirtul/ESO/.