Efficient CRISPR/Cas9 Gene Editing in Uncultured Naive Mouse T Cells for In Vivo Studies [NOVEL IMMUNOLOGICAL METHODS]

Key Points

  • Cas9 RNP electroporation deletes genes in naive CD8+ T cells transferred in vivo.

  • High efficiency gene deletion occurs even in the absence of T cell activation.

  • Multiple gene KO and HDR edited cells can be generated using this method.

Visual Abstract

Figure1

Abstract

CRISPR/Cas9 technologies have revolutionized our understanding of gene function in complex biological settings, including T cell immunology. Current CRISPR-mediated gene editing strategies in T cells require in vitro stimulation or culture that can both preclude the study of unmanipulated naive T cells and alter subsequent differentiation. In this study, we demonstrate highly efficient gene editing within uncultured primary naive murine CD8+ T cells by electroporation of recombinant Cas9/sgRNA ribonucleoprotein immediately prior to in vivo adoptive transfer. Using this approach, we generated single and double gene knockout cells within multiple mouse infection models. Strikingly, gene deletion occurred even when the transferred cells were left in a naive state, suggesting that gene deletion occurs independent of T cell activation. Finally, we demonstrate that targeted mutations can be introduced into naive CD8+ T cells using CRISPR-based homology-directed repair. This protocol thus expands CRISPR-based gene editing approaches beyond models of robust T cell activation to encompass both naive T cell homeostasis and models of weak activation, such as tolerance and tumor models.

Footnotes

  • This work was supported by Human Frontier Science Program Young Investigators Grant RGY0065/2018 and by program, fellowship, and project grant support from the National Health and Medical Research Council of Australia.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    Cas9
    CRISPR-associated protein 9
    CRISPR
    clustered, regularly interspaced short palindromic repeats
    F
    forward
    HDR
    homology-directed repair
    KO
    knockout
    LCMV-Cl13
    lymphocytic choriomeningitis virus Clone 13 strain
    LM-OVA
    OVA-transgenic Listeria monocytogenes
    NHEJ
    nonhomologous end joining
    p.i.
    postinfection
    R
    reverse
    sgRNA
    single guide RNA
    SNP
    single nucleotide polymorphism.
  • Received November 22, 2019.
  • Accepted February 10, 2020.

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