caa a22 4500 528791931 20190713071704.0 cr unu---uuuuu 180924s2016 xx s 000 0 eng 10.3929/ethz-b-000119720 doi 10.1038/ncomms12535 doi (ETHRESEARCH)oai:www.research-collection.ethz.ch:20.500.11850/288673 (ETHRESEARCH)oai:www.research-collection.ethz.ch:20.500.11850/119720 Immunogenomic engineering of a plug-and-(dis)play hybridoma platform [Elektronische Daten] [Mark Pogson, Cristina Parola, William J. Kelton, Paul Heuberger, Sai T. Reddy] Nat Commun Open access ethresearch 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. Creative Commons Attribution 4.0 International http://creativecommons.org/licenses/by/4.0 ethresearch Pogson Mark joint author Parola Cristina joint author Kelton William J. joint author Heuberger Paul joint author Reddy Sai T. joint author Nature Communications London : Nature Publishing Group 7, p. 12535 2041-1723 http://hdl.handle.net/20.500.11850/288673 text/html WWW-Backlink auf das Repository (Open access) 1 Journal Article ethresearch ETHRESEARCH 856 40 http://hdl.handle.net/20.500.11850/288673 text/html WWW-Backlink auf das Repository (Open access) ETHRESEARCH 773 0- Nature Communications London : Nature Publishing Group 7, p. 12535 2041-1723 ETHRESEARCH 700 1- Pogson Mark joint author ETHRESEARCH 700 1- Parola Cristina joint author ETHRESEARCH 700 1- Kelton William J. joint author ETHRESEARCH 700 1- Heuberger Paul joint author ETHRESEARCH 700 1- Reddy Sai T. joint author ETHRESEARCH 856 40 http://hdl.handle.net/20.500.11850/119720 text/html WWW-Backlink auf das Repository (Open access) ETHRESEARCH 773 0- Nature Communications London : Nature Publishing Group 7, p. 12535 BK010053 XK010053 XK010000 ETHRESEARCH ETHRESEARCH ETHRESEARCH Journal Article Open access ETHRESEARCH ETHRESEARCH ETHRESEARCH Journal Article Open access