title: Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange.
abstract: The development of programmable nucleases has enabled the application of new genome engineering strategies for cellular immunotherapy. While targeted nucleases have mostly been used to knock-out or knock-in genes in immune cells, the scarless exchange of entire immunogenomic alleles would be of great interest. In particular, reprogramming the polymorphic MHC locus could enable the creation of matched donors for allogeneic cellular transplantation. Here we show a proof-of-concept for reprogramming MHC-specificity by performing CRISPR-Cas9-assisted cassette exchange. Using murine antigen presenting cell lines (RAW264.7 macrophages), we demonstrate that the generation of Cas9-induced double-stranded breaks flanking the native MHC-I H2-Kd locus led to exchange of an orthogonal H2-Kb allele. MHC surface expression allowed for easy selection of reprogrammed cells by flow cytometry, thus obviating the need for additional selection markers. MHC-reprogrammed cells were fully functional as they could present H2-Kd-restricted peptide and activate cognate T cells. Finally, we investigated the role of various donor template formats on exchange efficiency, discovering that templates that underwent in situ linearization resulted in the highest MHC-reprogramming efficiency. These findings highlight a potential new approach for the correcting of MHC mismatches in cellular transplantation.
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: William
authors.initials: W
authors.lastname: Kelton
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: Ann Cathrin
authors.initials: AC
authors.lastname: Waindok
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: Theresa
authors.initials: T
authors.lastname: Pesch
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: Mark
authors.initials: M
authors.lastname: Pogson
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: Kyle
authors.initials: K
authors.lastname: Ford
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: Cristina
authors.initials: C
authors.lastname: Parola
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.
authors.forename: Sai T
authors.initials: ST
authors.lastname: Reddy
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D002460:Cell Line; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D005347:Fibroblasts; D005434:Flow Cytometry; D006183:H-2 Antigens / Q000235:genetics / Q000276:immunology; D008264:Macrophages / Q000276:immunology; D008285:Major Histocompatibility Complex / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C
chemical_list: D006183:H-2 Antigens