High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis.
Nucleic Acids Res
; 46(14): 7436-7449, 2018 08 21.
Article
en En
| MEDLINE
| ID: mdl-29931269
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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Ingeniería de Proteínas
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Mutagénesis Sitio-Dirigida
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Sistemas CRISPR-Cas
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Anticuerpos
Límite:
Animals
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Humans
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2018
Tipo del documento:
Article
País de afiliación:
Suiza