Novel techniques for targeted DNA diversification and directed evolution in Saccharomyces cerevisiae
Author(s)
Cazier, Andrew Paul
Advisor(s)
Editor(s)
Collections
Supplementary to:
Permanent Link
Abstract
Directed evolution is a powerful strategy for enhancing genes and proteins. This design paradigm involves creating diversity in genetic sequences in vitro and then selecting for improved function in a host. A desirable extension of these techniques is in vivo or continuous diversification, which would eliminate many laborious cloning steps typically involved in directed evolution. Developing B cells naturally carry out a process of continuous directed evolution of antibodies by applying two main processes: V(D)J recombination and somatic hypermutation. However, B cells are challenging to culture and manipulate. Therefore, we engineered a more tractable, eukaryotic organism, Saccharomyces cerevisiae, to carry out V(D)J recombination or somatic hypermutation and apply these customized yeast for in vivo mutagenesis and evolution.
First, to implement V(D)J recombination in yeast, we integrated the key murine recombination activating genes, RAG1 and RAG2. Using fluorescence microscopy, we identified that RAG1 has poor nuclear localization but that this could be overcome by truncating the protein. By using a novel split antibiotic resistance assay with homology-directed repair, we demonstrated that yeast can make coding joints after RAG cutting. We increased the rate of our yeast’s assisted recombination by over 500-fold by employing codon optimization, co-expressing the HMGB1 DNA-binding protein, improving protein nuclear localization and expression, and evaluating RAG1 truncations. We further showed that our platform could assay the severity of several disease-causing RAG1 mutations. Finally, we used our engineered yeast to simultaneously generate up to three unique fluorescent proteins or two distinct antibody fragments starting from an array of nonfunctional gene segments.
Second, to recapitulate somatic hypermutation, we developed and optimized a CRISPR diversifying base editor for yeast (yDBE). This system functions in vivo and utilizes a dCas9-directed cytidine deaminase 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 aptamer-embedded guide RNAs. By performing high-throughput sequencing, we showed that our base editor enables a mutation rate of up to 8.4×10-4 substitutions/bp/generation over a window of 100 bp. We then demonstrated the ability of the yDBE platform to improve the affinity of a displayed antibody fragment. Next, we built gRNA-tRNA arrays that could express up to six gRNAs simultaneously and used them to target up to three loci at once. Lastly, we applied the base editor to engineer noncoding DNA, creating promoters with both increased and decreased transcriptional activity.
By following B-cell antibody diversification, we have developed two complementary tools for in vivo directed evolution in yeast. Each can facilitate a variety of directed evolution experiments for both antibodies and almost any user-specified gene.
Sponsor
Date
2024-12-02
Extent
Resource Type
Text
Resource Subtype
Dissertation (PhD)