title: Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line.
abstract: Applying clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis to primary mouse immune cells, we used high-fidelity single guide RNAs (sgRNAs) designed with an sgRNA design tool (CrispRGold) to target genes in primary B cells, T cells, and macrophages isolated from a Cas9 transgenic mouse line. Using this system, we achieved an average knockout efficiency of 80% in B cells. On this basis, we established a robust small-scale CRISPR-mediated screen in these cells and identified genes essential for B-cell activation and plasma cell differentiation. This screening system does not require deep sequencing and may serve as a precedent for the application of CRISPR/Cas9 to primary mouse cells.
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany; klaus.rajewsky@mdc-berlin.de vantrung.chu@mdc-berlin.de robin.graf@mdc-berlin.de.
authors.forename: Van Trung
authors.initials: VT
authors.lastname: Chu
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany; klaus.rajewsky@mdc-berlin.de vantrung.chu@mdc-berlin.de robin.graf@mdc-berlin.de.
authors.forename: Robin
authors.initials: R
authors.lastname: Graf
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
authors.forename: Tristan
authors.initials: T
authors.lastname: Wirtz
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
authors.forename: Timm
authors.initials: T
authors.lastname: Weber
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.|Centre d'Immunologie de Marseille-Luminy, Université Aix-Marseille, UM2, 13288 Marseille Cedex 09, France.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Favret
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
authors.forename: Xun
authors.initials: X
authors.lastname: Li
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Petsch
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
authors.forename: Ngoc Tung
authors.initials: NT
authors.lastname: Tran
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.|Centre d'Immunologie de Marseille-Luminy, Université Aix-Marseille, UM2, 13288 Marseille Cedex 09, France.|Institut National de la Santé et de la Recherche Médicale, U1104, Marseille, France.|Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7280, Marseille, France.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Sieweke
authors.affiliation: Deutsches Rheuma-Forschungszentrum, 10117 Berlin, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Berek
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.|Berlin Institute of Health, 10117 Berlin, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Kühn
authors.affiliation: Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany; klaus.rajewsky@mdc-berlin.de vantrung.chu@mdc-berlin.de robin.graf@mdc-berlin.de.
authors.forename: Klaus
authors.initials: K
authors.lastname: Rajewsky
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D000072669:Gene Editing / Q000379:methods*; D008213:Lymphocyte Activation / Q000235:genetics; D008264:Macrophages / Q000378:metabolism*; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016296:Mutagenesis; D010950:Plasma Cells / Q000378:metabolism; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000378:metabolism*
keywords: B cells; CRISPR/Cas9; knockout efficiency; primary cells; sgRNA design