X-CHIME enables combinatorial, inducible, lineage-specific and sequential knockout of genes in the immune system

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. He...

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Veröffentlicht in:Nature immunology Jg. 25; H. 1; S. 178 - 188
Hauptverfasser: LaFleur, Martin W., Lemmen, Ashlyn M., Streeter, Ivy S. L., Nguyen, Thao H., Milling, Lauren E., Derosia, Nicole M., Hoffman, Zachary M., Gillis, Jacob E., Tjokrosurjo, Qin, Markson, Samuel C., Huang, Amy Y., Anekal, Praju V., Montero Llopis, Paula, Haining, W. Nicholas, Doench, John G., Sharpe, Arlene H.
Format: Journal Article
Sprache:Englisch
Veröffentlicht: New York Nature Publishing Group US 01.01.2024
Nature Publishing Group
Schlagworte:
631/250/232/1997
631/250/2502/248
Animals
Biomedical and Life Sciences
Biomedicine
Bone marrow
CRISPR
CRISPR-Cas Systems - genetics
Enteritis
Gene deletion
Gene Editing
Genes
Hematopoietic stem cells
Hypoplasia
Immune System
Immunology
Infectious Diseases
Lethality
Mice
Mice, Knockout
Protein Tyrosine Phosphatase, Non-Receptor Type 2 - genetics
PTPN2 protein
technical-report
ISSN:1529-2908, 1529-2916, 1529-2916
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Zusammenfassung: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. Sharpe and colleagues devise a conditional gene deletion model in mice for rapid sequential, combinatorial and lineage-specific interrogation of gene function in hematopoietic cells.
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Author Contributions Statement
M.W.L., W.N.H., and A.H.S. conceived the project. M.W.L., A.M.L., I.S.L.S., T.H.N., N.M.D., and J.E.G. designed experiments. M.W.L., A.M.L., I.S.L.S., T.H.N., L.E.M., N.M.D., Z.M.H., J.E.G., and Q.T. acquired and analyzed data. M.W.L., T.H.N., and A.H.S. wrote the manuscript with contributions from A.M.L., I.S.L.S., N.M.D., and L.E.M. All authors edited the manuscript. P.V.A. and P.M.L. assisted with imaging and analysis. S.C.M. and A.Y.H. performed 10x scRNAseq analyses. J.G.D. assisted with methodology. A.H.S. secured funding and supervised the project.
ISSN:1529-2908
1529-2916
1529-2916
DOI:10.1038/s41590-023-01689-6