Multiplex Evolution of Antibody Fragments Utilizing a Yeast Surface Display Platform

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

doi:10.1021/acssynbio.0c00159

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 journal › Article › peer-review

8 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 language	American English
Pages (from-to)	2197-2202
Number of pages	6
Journal	ACS Synthetic Biology
Volume	9
Issue number	8
DOIs	https://doi.org/10.1021/acssynbio.0c00159
State	Published - 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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10.1021/acssynbio.0c00159

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title = "Multiplex Evolution of Antibody Fragments Utilizing a Yeast Surface Display Platform",

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.",

keywords = "antibody engineering, CRISPR/Cas9, fluorescent-activated cell sorting, Saccharomyces cerevisiae, yeast display",

author = "Eun Oh and Rongming Liu and Liya Liang and Emily Freed and Carrie Eckert and Ryan Gill",

year = "2020",

doi = "10.1021/acssynbio.0c00159",

language = "American English",

volume = "9",

pages = "2197--2202",

journal = "ACS Synthetic Biology",

issn = "2161-5063",

publisher = "American Chemical Society",

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T1 - Multiplex Evolution of Antibody Fragments Utilizing a Yeast Surface Display Platform

AU - Oh, Eun

AU - Liu, Rongming

AU - Liang, Liya

AU - Freed, Emily

AU - Eckert, Carrie

AU - Gill, Ryan

PY - 2020

Y1 - 2020

N2 - 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.

AB - 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.

KW - antibody engineering

KW - CRISPR/Cas9

KW - fluorescent-activated cell sorting

KW - Saccharomyces cerevisiae

KW - yeast display

U2 - 10.1021/acssynbio.0c00159

DO - 10.1021/acssynbio.0c00159

M3 - Article

SN - 2161-5063

VL - 9

SP - 2197

EP - 2202

JO - ACS Synthetic Biology

JF - ACS Synthetic Biology

IS - 8

ER -

Multiplex Evolution of Antibody Fragments Utilizing a Yeast Surface Display Platform

Abstract

NREL Publication Number

Keywords

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