Abstract
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice are an immunodeficient strain that enables human cell xenografts. However, NSG mice possess a complex genetic background that would complicate cross-breeding with other inbred transgenic or knockout mouse strains to establish a congenic strain with a desired genetic modification in the NSG background. Newly developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology enables modification of the mouse genome at the zygote stage without the need for extensive cross-breeding or the use of embryonic stem cells. In this chapter, we use the knockout of the X-linked Cybb gene as an example to describe our procedures for genetically modifying NSG mice using the CRISPR/Cas9 method. Briefly, two sgRNAs were designed and made to target exon 1 and exon 3 of the Cybb gene, and either sgRNA was then microinjected together with Cas9 mRNA into fertilized eggs collected from NSG mice. The injected embryos are subsequently transferred into the oviducts of pseudopregnant surrogate mothers. Offspring born to the foster mothers were genotyped by PCR and DNA sequencing. In this chapter, we describe our experiment procedures in detail and report our genotyping results for demonstrating that NSG mice can be genetically modified using the CRISPR/Cas9 technology in a highly efficient manner.
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References
Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, Chaleff S, Kotb M, Gillies SD, King M, Mangada J, Greiner DL, Handgretinger R (2005) Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2Rγnull mice engrafted with mobilized human hemopoietic stem cells. J Immunol 174(10):6477–6489
Billerbeck E, Barry WT, Mu K, Dorner M, Rice CM, Ploss A (2011) Development of human CD4+FoxP3+ regulatory T cells in human stem cell factor-, granulocyte-macrophage colony-stimulating factor-, and interleukin-3-expressing NOD-SCID IL2Rγnull humanized mice. Blood 117(11):3076–3086
Takagi S, Saito Y, Hijikata A, Tanaka S, Watanabe T, Hasegawa T, Mochizuki S, Kunisawa J, Kiyono H, Koseki H, Ohara O, Saito T, Taniguchi S, Shultz LD, Ishikawa F (2012) Membrane-bound human SCF/KL promotes in vivo human hematopoietic engraftment and myeloid differentiation. Blood 119(12):2768–2777
Brehm MA, Racki WJ, Leif J, Burzenski L, Hosur V, Wetmore A, Gott B, Herlihy M, Ignotz R, Dunn R, Shultz LD, Greiner DL (2012) Engraftment of human HSCs in nonirradiated newborn NOD-scid IL2rγnull mice is enhanced by transgenic expression of membrane-bound human SCF. Blood 119(12):2778–2788
Capecchi MR (2005) Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6(6):507–512
Landel CP, Dunlap J, Patton JB, Manser T (2013) A germline-competent embryonic stem cell line from NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Transgenic Res 22(1):179–185
Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153(4):910–918
Bae S, Kweon J, Kim HS, Kim J-S (2014) Microhomology-based choice of Cas9 nuclease target sites. Nat Methods 11(7):705–706
Yang H, Wang H, Shivalila Chikdu S, Cheng Albert W, Shi L, Jaenisch R (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154(6):1370–1379
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826
Sung YH, Kim JM, Kim H-T, Lee J, Jeon J, Jin Y, Choi J-H, Ban YH, Ha S-J, Kim C-H, Lee H-W, Kim J-S (2014) Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res 24(1):125–131
Sweeney CL, Choi U, Liu C, Koontz S, Ha S-K, Malech HL (2017) CRISPR-mediated knockout of Cybb in NSG mice establishes a model of chronic granulomatous disease for human stem-cell gene therapy transplants. Hum Gene Ther 28(7):565-575. https://doi.org/10.1089/hum.2017.005
Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JRJ, Joung JK (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31(3):227–229
Auer TO, Duroure K, Concordet J-P, Del Bene F (2014) CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish. Nat Protoc 9:2823-2840. https://doi.org/10.1038/nprot.2014.187
Kaneko T, Sakuma T, Yamamoto T, Mashimo T (2014) Simple knockout by electroporation of engineered endonucleases into intact rat embryos. Sci Rep 4:6382. https://doi.org/10.1038/srep06382
Hashimoto M, Takemoto T (2015) Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Sci Rep 5:11315. https://doi.org/10.1038/srep11315
Takahashi G, Gurumurthy CB, Wada K, Miura H, Sato M, Ohtsuka M (2015) GONAD: genome-editing via oviductal nucleic acids delivery system: a novel microinjection independent genome engineering method in mice. Sci Rep 5:11406. https://doi.org/10.1038/srep11406
Qin W, Wang H (2018) Delivery of CRISPR-Cas9 into mouse zygotes by electroporation. In: Liu C, Du Y (eds) Microinjection: methods and protocols. Humana, New York, NY
Acknowledgements
This work was supported by the Intramural Research Program of the NIH, NIAID and NHLBI.
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Du, Y., Xie, W., Zhang, F., Choi, U., Liu, C., Sweeney, C.L. (2019). Using CRISPR/Cas9 for Gene Knockout in Immunodeficient NSG Mice. In: Liu, C., Du, Y. (eds) Microinjection. Methods in Molecular Biology, vol 1874. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8831-0_8
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DOI: https://doi.org/10.1007/978-1-4939-8831-0_8
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