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High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis

View ORCID ProfileDerek M Mason, View ORCID ProfileCédric R Weber, View ORCID ProfileCristina Parola, Simon M Meng, View ORCID ProfileVictor Greiff, View ORCID ProfileWilliam J Kelton, View ORCID ProfileSai T Reddy
doi: https://doi.org/10.1101/285015
Derek M Mason
1Department of Biosystems Science and Engineering, ETH Zürich, Basel 4058, Switzerland
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Cédric R Weber
1Department of Biosystems Science and Engineering, ETH Zürich, Basel 4058, Switzerland
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Cristina Parola
1Department of Biosystems Science and Engineering, ETH Zürich, Basel 4058, Switzerland
2Life Science Graduate School, Systems Biology, ETH Zürich, University of Zurich, Zurich 8057, Switzerland
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Simon M Meng
1Department of Biosystems Science and Engineering, ETH Zürich, Basel 4058, Switzerland
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Victor Greiff
1Department of Biosystems Science and Engineering, ETH Zürich, Basel 4058, Switzerland
3Department of Immunology, University of Oslo, Oslo 0372, Norway
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William J Kelton
1Department of Biosystems Science and Engineering, ETH Zürich, Basel 4058, Switzerland
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Sai T Reddy
1Department of Biosystems Science and Engineering, ETH Zürich, Basel 4058, Switzerland
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  • For correspondence: sai.reddy@ethz.ch
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ABSTRACT

Antibody engineering is performed to improve therapeutic properties by directed evolution, usually by high-throughput screening of phage or yeast display libraries. Engineering antibodies in mammalian cells offers advantages associated with expression in their final therapeutic format (full-length glycosylated IgG), however, the inability to express large and diverse libraries severely limits their potential throughput. To address this limitation, we have developed homology-directed mutagenesis (HDM), a novel method which extends the concept of CRISPR/Cas9-mediated homology-directed repair (HDR). HDM leverages oligonucleotides with degenerate codons to generate site-directed mutagenesis libraries in mammalian cells. By improving HDM efficiency (>35-fold) and combining mammalian display screening with next-generation sequencing (NGS), we validated this approach can be used for key applications in antibody engineering at high-throughput: rational library construction, novel variant discovery, affinity maturation, and deep mutational scanning (DMS). We anticipate that HDM will be a valuable tool for engineering and optimizing antibodies in mammalian cells, and eventually enable directed evolution of other complex proteins and cellular therapeutics.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted March 19, 2018.
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High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis
Derek M Mason, Cédric R Weber, Cristina Parola, Simon M Meng, Victor Greiff, William J Kelton, Sai T Reddy
bioRxiv 285015; doi: https://doi.org/10.1101/285015
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High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis
Derek M Mason, Cédric R Weber, Cristina Parola, Simon M Meng, Victor Greiff, William J Kelton, Sai T Reddy
bioRxiv 285015; doi: https://doi.org/10.1101/285015

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