Multiplex evolution of antibody fragments utilizing a yeast surface display platform

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

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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.
Original languageEnglish
JournalACS Synthetic Biology
Volume9
Issue number8
Pages (from-to)2197-2202
Number of pages6
ISSN2161-5063
DOIs
Publication statusPublished - 2020

Keywords

  • CRISPR/Cas9
  • Saccharomyces cerevisiae
  • Yeast display
  • Fluorescent-activated cell sorting
  • Antibody engineering

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