(632c) Development of a CRISPR Diversifying Base Editor in Saccharomyces Cerevisiae for Rapid Antibody Enhancement and Selection.
AIChE Annual Meeting
2022
2022 Annual Meeting
Food, Pharmaceutical & Bioengineering Division
General Topics in Synthetic Biology and Metabolic Engineering
Thursday, November 17, 2022 - 1:06pm to 1:24pm
We will highlight three key results from our efforts: (1) Fluorescence-based, mutational assays that have allowed us to estimate the rate and breadth of mutations produced by the DBE, (2) Protein engineering efforts that enhanced the mutation rate of the DBE 5-fold, and (3) Optimization of the guide RNA scaffold sequences to further enhance mutation rate up to 15-fold (Figure 1). Finally, we will describe ongoing experiments employing our optimized DBE and yeast display for the in situ enhancement of antibody affinity and isolation of these sequences in a manner that mimics B cell somatic hypermutation. Ultimately, we foresee our system becoming a rapid, inexpensive, and easily implemented tool for antibody therapeutic development pipelines, as antibodies can be improved by applying multiple rounds of DBE-mediated mutagenesis and FACS selection.
Figure 1. A CRISPR diversifying base editing (DBE) causes localized mutations in a targeted gene. The DBE is composed of a nuclease dead Cas9 (dCas9), a deaminase fusion, and a guide RNA with aptamers. By engineering the guide RNA and deaminase, we have increased the mutation rate of the DBE.