Andrew Cazier, Ph.D. Candidate
2024 Suddath Award Winner
John Blazeck, Ph.D., Advisor 

ABSTRACT
The yeast Saccharomyces cerevisiae is an excellent host for directed evolution experiments, but classical methods to introduce mutations can be time-consuming or inefficient. To address these limitations, we have built a CRISPR-based, yeast diversifying base editor (yDBE) that can generate DNA mutant libraries in situ. This system can cause substantial and diverse mutations in a 100-bp window near each targeted locus and can target multiple loci simultaneously. To demonstrate the capabilities of this platform, we have successfully applied yDBE to rapidly enhance and isolate antibody sequences with up to 100-fold higher affinity and greatly increase the strength of a weak, exogenous promoter. We will highlight our optimization strategies which significantly improved the mutation rate or breadth, including 1) applying an improved deaminase variant, 2) modifying the CRISPR guide RNA (gRNA) scaffold, and 3) building multi-gRNA cassettes. We will also outline ongoing experiments to employ yDBE such as to isolate high affinity antibodies from a naïve library. Ultimately, we will show that our system is a rapid, inexpensive, and easily-incorporated tool for directed evolution in yeast.

The F. L. "Bud" Suddath and Frances "Lee" Gafford Suddath Fellowship Award was established by family and friends in recognition of Bud Suddath's contributions as a Georgia Tech faculty member and to support Bud and Lee's shared interest in graduate education in the fields of biology, biochemistry and biomedical engineering. The fellowship award is given annually to Georgia Tech Ph.D. students, typically within one year of graduation, who have demonstrated significant achievement in research of fundamental importance in the fields of biology, biochemistry, or biomedical engineering and are working in laboratories affiliated with Georgia Tech's Institute of Bioengineering and Biosciences (IBB).