title: Rapid generation of novel models of RAG1 deficiency by CRISPR/Cas9-induced mutagenesis in murine zygotes.
abstract: Mutations in the Recombination Activating Gene 1 (RAG1) can cause a wide variety of clinical and immunological phenotypes in humans, ranging from absence of T and B lymphocytes to occurrence of autoimmune manifestations associated with expansion of oligoclonal T cells and production of autoantibodies. Although the mechanisms underlying this phenotypic heterogeneity remain poorly understood, some genotype-phenotype correlations can be made. Currently, mouse models of Rag deficiency are restricted to RAG1-/- mice and to knock-in models carrying severe missense mutations. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system is a novel and powerful gene-editing strategy that permits targeted introduction of DNA double strand breaks with high efficiency through simultaneous delivery of the Cas9 endonuclease and a guide RNA (gRNA). Here, we report on CRISPR-based, single-step generation and characterization of mutant mouse models in which gene editing was attempted around residue 838 of RAG1, a region whose functional role had not been studied previously.
authors.affiliation: Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.|Pediatric Immunology, Wilhelmina Children's Hospital, Utrecht University Medical Center, Utrecht, The Netherlands.
authors.forename: Lisa
authors.initials: L
authors.lastname: Ott de Bruin
authors.affiliation: Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, MD, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.affiliation: Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: Kelly
authors.initials: K
authors.lastname: Capuder
authors.affiliation: Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: Yu Nee
authors.initials: YN
authors.lastname: Lee
authors.affiliation: Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: Maddalena
authors.initials: M
authors.lastname: Antolini
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.
authors.forename: Robin
authors.initials: R
authors.lastname: Meyers
authors.affiliation: Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, MD, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Gellert
authors.affiliation: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.|Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA.
authors.forename: Kiran
authors.initials: K
authors.lastname: Musunuru
authors.affiliation: Division of Transfusion Medicine, Department of Laboratory Medicine, Boston Children's hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: John
authors.initials: J
authors.lastname: Manis
authors.affiliation: Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.|Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA.
authors.forename: Luigi
authors.initials: L
authors.lastname: Notarangelo
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D000072669:Gene Editing / Q000379:methods*; D018398:Homeodomain Proteins / Q000235:genetics*; D051379:Mice; D008817:Mice, Mutant Strains / Q000235:genetics*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D015053:Zygote
chemical_list: D018398:Homeodomain Proteins; C064658:RAG-1 protein
keywords: CRISPR/Cas9; RAG1; genome editing; immunodeficiency; recombination activation gene 1