Multiplex Evolution of Antibody Fragments Utilizing a Yeast Surface Display Platform

Oh, Eun Joong; Liu, Rongming; Liang, Liya; Freed, Emily F.; Eckert, Carrie A.; Gill, Ryan T.

doi:10.1021/acssynbio.0c00159

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Multiplex Evolution of Antibody Fragments Utilizing a Yeast Surface Display Platform

Oh, Eun Joong·Liu, Rongming·Liang, Liya·Freed, Emily F.·Eckert, Carrie A.·Gill, Ryan T.

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Abstract

Advances in high-throughput synthetic biology technologies based on the CRISPR/Cas9 system have enabled a comprehensive assessment of mutations conferring desired phenotypes, as well as a better understanding of genotype-phenotype correlations in protein engineering. Engineering antibodies to enhance properties such as binding affinity and stability plays an essential role in therapeutic applications. Here we report a method, multiplex navigation of antibody structure (MINAS), that combines a CRISPR/Cas9-based trackable editing method and fluorescent-activated cell sorting (FACS) of yeast-displayed libraries. We designed mutations in all of the complementarity-determining and framework regions of a well-characterized scFv antibody and mapped the contribution of these regions to enhanced properties. We identified specific mutants that showed higher binding affinities up to 100-fold compared to the wild-type. This study expands the applicability of CRISPR/Cas9-based trackable protein engineering by combining it with a surface display platform.

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59 BASIC BIOLOGICAL SCIENCES CRISPR/Cas9 Saccharomyces cerevisiae antibody engineering fluorescent-activated cell sorting yeast display

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Oh, Eun Joong, Liu, Rongming, Liang, Liya, Freed, Emily F., Eckert, Carrie A., Gill, Ryan T.. 2020-06-18. Multiplex Evolution of Antibody Fragments Utilizing a Yeast Surface Display Platform. https://doi.org/10.1021/acssynbio.0c00159

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