Multiplex Evolution of Antibody Fragments Utilizing a Yeast Surface Display Platform

Eun Oh, Rongming Liu, Liya Liang, Emily Freed, Carrie Eckert, Ryan Gill

Research output: Contribution to journalArticlepeer-review

7 Scopus Citations

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 languageAmerican English
Pages (from-to)2197-2202
Number of pages6
JournalACS Synthetic Biology
Volume9
Issue number8
DOIs
StatePublished - 2020

NREL Publication Number

  • NREL/JA-5100-77053

Keywords

  • antibody engineering
  • CRISPR/Cas9
  • fluorescent-activated cell sorting
  • Saccharomyces cerevisiae
  • yeast display

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