LaFleur et al. developed and validated four novel CHimeric IMmune Editing (CHIME) CRISPR-Cas9 bone marrow delivery systems for rapid combinatorial (C), inducible (I), lineage-specific (L), and sequential (S) in vivo KO of single genes in hematopoietic cells of adult mice, enabling the study of genes in distinct contexts. C-CHIME KO of the highly homologous PTPN1 and PTPN2 non-receptor tyrosine phosphatases – which impact T cell immunity and for which embryonic KO is lethal – during immune development resulted in lethality associated with bone marrow hypoplasia, and after immune development, (S-CHIME) led to enteritis and lethality.
Contributed by Paula Hochman
ABSTRACT: Annotation of immunologic gene function in vivo typically requires the generation of knockout mice, which is time consuming and low throughput. We previously developed CHimeric IMmune Editing (CHIME), a CRISPR-Cas9 bone marrow delivery system for constitutive, ubiquitous deletion of single genes. Here we describe X-CHIME, four new CHIME-based systems for modular and rapid interrogation of gene function combinatorially (C-CHIME), inducibly (I-CHIME), lineage-specifically (L-CHIME) or sequentially (S-CHIME). We use C-CHIME and S-CHIME to assess the consequences of combined deletion of Ptpn1 and Ptpn2, an embryonic lethal gene pair, in adult mice. We find that constitutive deletion of both PTPN1 and PTPN2 leads to bone marrow hypoplasia and lethality, while inducible deletion after immune development leads to enteritis and lethality. These findings demonstrate that X-CHIME can be used for rapid mechanistic evaluation of genes in distinct in vivo contexts and that PTPN1 and PTPN2 have some functional redundancy important for viability in adult mice.
Author Info: (1) Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA. Gene Lay Institute of Immunology and Inflammation, Brigham and Women's Hospital, Massach
Author Info: (1) Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA. Gene Lay Institute of Immunology and Inflammation, Brigham and Women's Hospital, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. (2) Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA. Gene Lay Institute of Immunology and Inflammation, Brigham and Women's Hospital, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. (3) Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA. Gene Lay Institute of Immunology and Inflammation, Brigham and Women's Hospital, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. (4) Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA. Gene Lay Institute of Immunology and Inflammation, Brigham and Women's Hospital, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. (5) Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA. Gene Lay Institute of Immunology and Inflammation, Brigham and Women's Hospital, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. (6) Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA. Gene Lay Institute of Immunology and Inflammation, Brigham and Women's Hospital, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. (7) Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA. Gene Lay Institute of Immunology and Inflammation, Brigham and Women's Hospital, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. (8) Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA. Gene Lay Institute of Immunology and Inflammation, Brigham and Women's Hospital, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. (9) Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA. Gene Lay Institute of Immunology and Inflammation, Brigham and Women's Hospital, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. (10) Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA. Gene Lay Institute of Immunology and Inflammation, Brigham and Women's Hospital, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, USA. (11) Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA. Gene Lay Institute of Immunology and Inflammation, Brigham and Women's Hospital, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, USA. Dana-Farber Cancer Institute, Boston, MA, USA. (12) MicRoN Core, Harvard Medical School, Boston, MA, USA. (13) MicRoN Core, Harvard Medical School, Boston, MA, USA. (14) Arsenal Biosciences, South San Francisco, CA, USA. (15) Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, USA. (16) Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA. Arlene_Sharpe@hms.harvard.edu. Gene Lay Institute of Immunology and Inflammation, Brigham and Women's Hospital, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. Arlene_Sharpe@hms.harvard.edu. Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, USA. Arlene_Sharpe@hms.harvard.edu.
