High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis
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Autor(in)
Alle anzeigen
Datum
2018-08-21Typ
- Journal Article
ETH Bibliographie
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. Mehr anzeigen
Persistenter Link
https://doi.org/10.3929/ethz-b-000294306Publikationsstatus
publishedExterne Links
Zeitschrift / Serie
Nucleic Acids ResearchBand
Seiten / Artikelnummer
Verlag
Oxford University PressOrganisationseinheit
03952 - Reddy, Sai / Reddy, Sai
Förderung
679403 - Vaccine profiling and immunodiagnostic discovery by high-throughput antibody repertoire analysis (EC)
ETH Bibliographie
yes
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