title: Generation of a Double KO Mouse by Simultaneous Targeting of the Neighboring Genes Tmem176a and Tmem176b Using CRISPR/Cas9: Key Steps from Design to Genotyping.
abstract: The CRISPR/Cas9 system has been tailored to a revolutionary genetic tool because of its remarkable simplicity and efficacy. While complex genome editing in the mouse since the 1990s has been dominated by the use of embryonic stem (ES) cells, CRISPR/Cas9 now offers a versatile and fast approach to precisely modify virtually any DNA regions directly in mouse zygotes. Yet, this relative simplicity does not preclude a conscientious preparatory work that is often neglected when initiating a project. Here, we describe the key steps leading to successful generation of a double knockout (KO) mouse by simultaneously targeting two homolog genes, Tmem176a and Tmem176b, which are located in the same genomic locus. Additionally, we show that similar efficiency can be obtained in a mixed genetic background or directly in the C57BL/6 inbred strain. Thus, presented as a detailed case study that should be helpful to the non-specialists, we focus on the genotyping strategy to anticipate the various possibilities.
authors.affiliation: INSERM UMR 1064, Center for Research in Transplantation and Immunology, Université de Nantes, CHU Nantes, Institut de Transplantation Urologie Néphrologie (ITUN), Nantes 44093, France.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Lemoine
authors.affiliation: Institut Pasteur, Plate-Forme Technologique Centre d'Ingénierie Génétique Murine, 25-28 rue du Dr. Roux, Paris 75724, France.
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Chauveau-Le Friec
authors.affiliation: Institut Pasteur, Plate-Forme Technologique Centre d'Ingénierie Génétique Murine, 25-28 rue du Dr. Roux, Paris 75724, France.
authors.forename: Francina
authors.initials: F
authors.lastname: Langa
authors.affiliation: INSERM UMR 1064, Center for Research in Transplantation and Immunology, Université de Nantes, CHU Nantes, Institut de Transplantation Urologie Néphrologie (ITUN), Nantes 44093, France. Electronic address: cedric.louvet@univ-nantes.fr.
authors.forename: Cédric
authors.initials: C
authors.lastname: Louvet
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D059766:DNA End-Joining Repair / Q000235:genetics; D018703:Founder Effect; D055786:Gene Knockout Techniques / Q000379:methods*; D060005:Genotyping Techniques; D008565:Membrane Proteins / Q000172:deficiency* / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout
chemical_list: D008565:Membrane Proteins; C552365:Tmem176A protein, mouse; C552366:Tmem176B protein, mouse
keywords: C57BL/6; CRISPR/Cas9; Deletion; Double KO; Genotyping; Inversion; Mosaicism; NHEJ; Off-targets