Abstract
The yeast Saccharomyces cerevisiae is commonly used to interrogate and screen protein variants and to perform directed evolution studies to develop proteins with enhanced features. While several techniques have been described that help enable the use of yeast for directed evolution, there remains a need to increase their speed and ease of use. Here we present yDBE, a yeast diversifying base editor that functions in vivo and employs a CRISPR-dCas9-directed cytidine deaminase base editor to diversify DNA in a targeted, rapid, and high-breadth manner. To develop yDBE, we enhanced the mutation rate of an initial base editor by employing improved deaminase variants and characterizing several scaffolded guide constructs. We then demonstrate the ability of the yDBE platform to improve the affinity of a displayed antibody scFv, rapidly generating diversified libraries and isolating improved binders via cell sorting. By performing high-throughput sequencing analysis of the high-activity yDBE, we show that it enables a mutation rate of 2.13 × 10-4 substitutions/bp/generation over a window of 100 bp. As yDBE functions entirely in vivo and can be easily programmed to diversify nearly any such window of DNA, we posit that it can be a powerful tool for facilitating a variety of directed evolution experiments.