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CRISPR/Cas9-mediated knockout of Rag-2 causes systemic lymphopenia with hypoplastic lymphoid organs in FVB mice

Abstract

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. Rag-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of Rag-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of Rag-2, we recently established a new Rag-2 knockout FVB mouse line (Rag-2-/-) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. Rag-2-/- mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in Rag-2-/- mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in Rag-2-/- mice. These findings indicate that our Rag-2-/- mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.

References

  1. 1.

    Nalefski EA, Kasibhatla S, Rao A. Functional analysis of the antigen binding site on the T cell receptor alpha chain. J Exp Med 1992; 175(6): 1553–1563.

    CAS  Article  Google Scholar 

  2. 2.

    Tonegawa S. Somatic generation of antibody diversity. Nature 1983; 302(5909): 575–581.

    CAS  Article  Google Scholar 

  3. 3.

    Gellert M. V(D)J recombination: RAG proteins, repair factors, and regulation. Annu Rev Biochem 2002; 71(1): 101–132.

    CAS  Article  Google Scholar 

  4. 4.

    McBlane JF, van Gent DC, Ramsden DA, Romeo C, Cuomo CA, Gellert M, Oettinger MA. Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell 1995; 83(3): 387–395.

    CAS  Article  Google Scholar 

  5. 5.

    Oettinger MA, Schatz DG, Gorka C, Baltimore D. RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science 1990; 248(4962): 1517–1523.

    CAS  Article  Google Scholar 

  6. 6.

    Schatz DG, Oettinger MA, Baltimore D. The V(D)J recombination activating gene, RAG-1. Cell 1989; 59(6): 1035–1048.

    CAS  Article  Google Scholar 

  7. 7.

    Xu K, Liu H, Shi Z, Song G, Zhu X, Jiang Y, Zhou Z, Liu X. Disruption of the RAG2 zinc finger motif impairs protein stability and causes immunodeficiency. Eur J Immunol 2016; 46(4): 1011–1019.

    CAS  Article  Google Scholar 

  8. 8.

    Villa A, Santagata S, Bozzi F, Imberti L, Notarangelo LD. Omenn syndrome: a disorder of Rag1 and Rag2 genes. J Clin Immunol 1999; 19(2): 87–97.

    CAS  Article  Google Scholar 

  9. 9.

    Shinkai Y, Rathbun G, Lam KP, Oltz EM, Stewart V, Mendelsohn M, Charron J, Datta M, Young F, Stall AM, et al. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 1992; 68(5): 855–867.

    CAS  Article  Google Scholar 

  10. 10.

    Ito M, Hiramatsu H, Kobayashi K, Suzue K, Kawahata M, Hioki K, Ueyama Y, Koyanagi Y, Sugamura K, Tsuji K, Heike T, Nakahata T. NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells. Blood 2002; 100(9): 3175–3182.

    CAS  Article  Google Scholar 

  11. 11.

    Skarnes WC. Is mouse embryonic stem cell technology obsolete? Genome Biol 2015; 16(1): 109.

    Article  Google Scholar 

  12. 12.

    Valera A, Perales JC, Hatzoglou M, Bosch F. Expression of the neomycin-resistance (neo) gene induces alterations in gene expression and metabolism. Hum Gene Ther 1994; 5(4): 449–456.

    CAS  Article  Google Scholar 

  13. 13.

    Scacheri PC, Crabtree JS, Novotny EA, Garrett-Beal L, Chen A, Edgemon KA, Marx SJ, Spiegel AM, Chandrasekharappa SC, Collins FS. Bidirectional transcriptional activity of PGK-neomycin and unexpected embryonic lethality in heterozygote chimeric knockout mice. Genesis 2001; 30(4): 259–263.

    CAS  Article  Google Scholar 

  14. 14.

    Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 2013; 153(4): 910–918.

    CAS  Article  Google Scholar 

  15. 15.

    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science 2013: 339(6121): 819–823.

    CAS  Article  Google Scholar 

  16. 16.

    Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 2013; 31(3): 233–239.

    CAS  Article  Google Scholar 

  17. 17.

    Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012; 337(6096): 816–821.

    CAS  Article  Google Scholar 

  18. 18.

    Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 2013; 154(6): 1380–1389.

    CAS  Article  Google Scholar 

  19. 19.

    Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 2014; 346(6213): 1258096.

    Article  Google Scholar 

  20. 20.

    Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 2014; 157(6): 1262–1278.

    CAS  Article  Google Scholar 

  21. 21.

    Lee JH, Park JH, Nam TW, Seo SM, Kim JY, Lee HK, Han JH, Park SY, Choi YK, Lee HW. Differences between immunodeficient mice generated by classical gene targeting and CRISPR/Cas9-mediated gene knockout. Transgenic Res 2018: 241–251.

    Google Scholar 

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Correspondence to Byeong-Cheol Kang.

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Kim, J., Park, J., Kim, H. et al. CRISPR/Cas9-mediated knockout of Rag-2 causes systemic lymphopenia with hypoplastic lymphoid organs in FVB mice. Lab Anim Res 34, 166–175 (2018). https://doi.org/10.5625/lar.2018.34.4.166

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Keywords

  • CRISPR/Cas9
  • Rag-2
  • histopathology
  • immunodeficiency
  • splenic atrophy
  • thymic atrophy