RNA interference (RNAi)-assisted genome evolution (RAGE) applies directed evolution principles to engineer Saccharomyces cerevisiae genomes. Here, we use acetic acid tolerance as a target trait to describe the key steps of RAGE. Briefly, iterative cycles of RNAi screening are performed to accumulate multiplex knockdown modifications, enabling directed evolution of the yeast genome and continuous improvement of a target phenotype. Detailed protocols are provided on the reconstitution of RNAi machinery, creation of genome-wide RNAi libraries, identification and integration of beneficial knockdown cassettes, and repeated RAGE cycles.
CITATION STYLE
Si, T., & Zhao, H. (2016). RNAi-assisted genome evolution (RAGE) in Saccharomyces cerevisiae. Methods in Molecular Biology, 1470, 183–198. https://doi.org/10.1007/978-1-4939-6337-9_15
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