Integrating a Diversifying CRISPR Base-Editor with a Synthetic DNA Library to Develop Novel Antibodies
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Yellayi, Sreenivas
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Abstract
The yeast strain Saccharomyces cerevisiae, or baker’s yeast, is used for a variety of directed evolution studies primarily for the purpose of screening protein variants for enhanced features and desirable qualities. In previous work with the Blazeck Lab, we created a diversifying base editor in yeast (yDBE) capable of rapidly enhancing the affinity of an antibody candidate. Here, we present a further application of yDBE for the rapid diversification of an scFv library. The main goal was to introduce greater diversity to a library using yDBE following traditionally utilized methods of manual mutagenesis. The results were obtained from flow-activated cell sorting (FACS), in which each variant’s expression and affinity for prostate stem-cell antigen (PSCA) was tested and plotted.
In earlier unpublished work, we were unable to isolate a binding variant from a library generated through yDBE diversification of a single, starting scFv sequence. Therefore, we generated an initial starting library with VHCDR3 sequences diversified using traditional methods of mutagenesis. After conducting two experimental trials, we isolated a high-affinity variant from the yDBE-mutated library within five sorts. However, this required more sorts than a control which only used traditional mutagenesis. This occurred despite conducting an additional magnetic-activated cell sorting (MACS) protocol prior to the main FACS protocol and varying both the starting library size and gRNA arrangement between experiments. A wide variety of factors could account for this shortcoming, ranging from protocol errors to limitations in base editor functionality. While the isolation of a high-affinity variant was not possible in this work, future efforts could focus on introducing more targeted genetic diversity or ensuring more optimized base editor mutagenicity to develop a more robust antibody discovery platform in yeast.
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Undergraduate Research Option Thesis