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1.
Nucleic Acids Res ; 46(14): 7436-7449, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-29931269

RESUMO

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.


Assuntos
Anticorpos/imunologia , Sistemas CRISPR-Cas , Mutagênese Sítio-Dirigida , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Animais , Anticorpos/genética , Anticorpos/metabolismo , Afinidade de Anticorpos/genética , Afinidade de Anticorpos/imunologia , Sequência de Bases , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Hibridomas , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Reparo de DNA por Recombinação
2.
Nat Commun ; 7: 12535, 2016 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-27531490

RESUMO

Hybridomas, fusions of primary mouse B cells and myelomas, are stable, rapidly-proliferating cell lines widely utilized for antibody screening and production. Antibody specificity of a hybridoma clone is determined by the immunoglobulin sequence of the primary B cell. Here we report a platform for rapid reprogramming of hybridoma antibody specificity by immunogenomic engineering. Here we use CRISPR-Cas9 to generate double-stranded breaks in immunoglobulin loci, enabling deletion of the native variable light chain and replacement of the endogenous variable heavy chain with a fluorescent reporter protein (mRuby). New antibody genes are introduced by Cas9-targeting of mRuby for replacement with a donor construct encoding a light chain and a variable heavy chain, resulting in full-length antibody expression. Since hybridomas surface express and secrete antibodies, reprogrammed cells are isolated using flow cytometry and cell culture supernatant is used for antibody production. Plug-and-(dis)play hybridomas can be reprogrammed with only a single transfection and screening step.


Assuntos
Engenharia Genética/métodos , Hibridomas/imunologia , Animais , Anticorpos/metabolismo , Especificidade de Anticorpos , Antígenos/metabolismo , Linhagem Celular , Edição de Genes , Camundongos , Reprodutibilidade dos Testes
3.
Chimia (Aarau) ; 70(6): 439-42, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27363374

RESUMO

The simplicity of the CRISPR/Cas9 technology has been transformative in making targeted genome editing accessible for laboratories around the world. However, due to the sheer volume of literature generated in the past five years, determining the best format and delivery method of CRISPR/Cas9 components can be challenging. Here, we provide a brief overview of the progress that has been made in the ex vivo genome editing of mammalian cells and summarize the key advances made for improving efficiency and delivery of CRISPR/Cas9 in DNA, RNA, and protein form. In particular, we highlight the delivery of Cas9 components to human cells for advanced genome editing applications such as large gene insertion.


Assuntos
Engenharia Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Animais , DNA/genética , Técnicas In Vitro , Mamíferos , RNA Mensageiro/genética , Ribonucleoproteínas/administração & dosagem
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