High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis
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Date
2018-08-21
Publication Type
Journal Article
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yes
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Abstract
Antibody engineering is often performed to improve therapeutic properties by directed evolution, usually by high-throughput screening of phage or yeast display libraries. Engineering antibodies in mammalian cells offer 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 HDR to a robust efficiency of 15–35% and combining mammalian display screening with next-generation sequencing, 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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published
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Journal / series
Volume
46 (14)
Pages / Article No.
7436 - 7449
Publisher
Oxford University Press
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Organisational unit
03952 - Reddy, Sai / Reddy, Sai
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Funding
679403 - Vaccine profiling and immunodiagnostic discovery by high-throughput antibody repertoire analysis (EC)