title: Immunogenomic engineering of a plug-and-(dis)play hybridoma platform.
abstract: 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.
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, Basel 4058, Switzerland.
authors.forename: Mark
authors.initials: M
authors.lastname: Pogson
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, Basel 4058, Switzerland.|Life Science Zurich Graduate School, Systems Biology, ETH Zurich, University of Zurich, Zurich 8057, Switzerland.
authors.forename: Cristina
authors.initials: C
authors.lastname: Parola
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, Basel 4058, Switzerland.
authors.forename: William J
authors.initials: WJ
authors.lastname: Kelton
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, Basel 4058, Switzerland.
authors.forename: Paul
authors.initials: P
authors.lastname: Heuberger
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, Basel 4058, Switzerland.
authors.forename: Sai T
authors.initials: ST
authors.lastname: Reddy
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism; D000918:Antibody Specificity; D000941:Antigens / Q000378:metabolism; D002460:Cell Line; D000072669:Gene Editing; D005818:Genetic Engineering / Q000379:methods*; D006825:Hybridomas / Q000276:immunology*; D051379:Mice; D015203:Reproducibility of Results
chemical_list: D000906:Antibodies; D000941:Antigens