(337h) Developing a CRISPR Base Editor for Multiplex Directed Evolution in Saccharomyces Cerevisiae.

Directed evolution is a critical design paradigm used to modify or enhance a desired protein characteristic by creating then screening a library of mutants. The yeast Saccharomyces cerevisiae is routinely used for these experiments due to its high rate of growth and ease of cloning. Nonetheless, classical methods to introduce mutations can be laborious, time-consuming, or inefficient, especially when targeting multiple loci, targeting genomic loci, or applying sequential rounds of mutagenesis. To address these limitations, we have built a yeast-based platform that is capable of generating DNA mutant libraries in situ by using the first CRISPR-based, diversifying base editor (yDBE) to be applied in yeast. This system creates intense, localized mutations on a targeted gene or genes while minimizing off-target effects. To build our DBE, we integrated a nuclease-dead Cas9 protein along with human activation-induced cytidine deaminase (AID) fused to MS2 coat protein (MCP). We can add a custom CRISPR guide RNA (gRNA) with embedded MS2 aptamers, which recruit the MCP-AID fusion to the targeted locus. Using yDBE, any DNA-encoded element can be improved by inducing rounds of mutagenesis, given a suitable high-throughput screen exists.

We will present high-throughput sequencing results that have allowed us to estimate the rate and breadth of mutations produced by yDBE. Furthermore, we will describe our efforts to improve the activity of this platform, emphasizing two domains. First, we have developed a human AID mutant with 5-fold improved base editing rate relative to previously described deaminases. Second, we have optimized the mutation rate and mapped the positional mutation preference of gRNA variants with altered MS2 aptamers. Altogether, yDBE is highly tractable while exhibiting mutation rates that rival or exceed previously described techniques. To demonstrate the capability of this platform, we will show how we successfully applied yDBE to rapidly enhance and isolate antibody sequences with up to 100-fold higher affinity. We will also detail additional experiments to employ the DBE, such as to isolate high affinity antibodies from a naïve library as well as in situ promoter engineering. We anticipate this platform will be able to eliminate many cloning steps typically needed for targeted mutagenesis and library selection of antibodies and other proteins. Ultimately, we will demonstrate that our system is a rapid, inexpensive, and easily incorporated tool for directed evolution in yeast.