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Functional Interrogation of Primary Human T Cells via CRISPR Genetic Editing (2018)

Discussion in 'Health News and Research unrelated to ME/CFS' started by Milo, Jul 19, 2018.

  1. Milo

    Milo Senior Member (Voting Rights)

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    Functional Interrogation of Primary Human T Cells via CRISPR Genetic Editing

    Dr Unutmaz is one of the principal investigator of this paper.
    http://www.jimmunol.org/content/early/2018/07/18/jimmunol.1701616?papetoc

    Abstract:
    Abstract
    Developing precise and efficient gene editing approaches using CRISPR in primary human T cell subsets would provide an effective tool in decoding their functions.

    Toward this goal, we used lentiviral CRISPR/Cas9 systems to transduce primary human T cells to stably express the Cas9 gene and guide RNAs that targeted either coding or noncoding regions of genes of interest.

    We showed that multiple genes (CD4, CD45, CD95) could be simultaneously and stably deleted in naive, memory, effector, or regulatory T cell (Treg) subsets at very high efficiency. Additionally, nuclease-deficient Cas9, associated with a transcriptional activator or repressor, can downregulate or increase expression of genes in T cells.

    For example, expression of glycoprotein A repetitions predominant (GARP), a gene that is normally and exclusively expressed on activated Tregs, could be induced on non-Treg effector T cells by nuclease-deficient Cas9 fused to transcriptional activators.

    Further analysis determined that this approach could be used in mapping promoter sequences involved in gene transcription.

    Through this CRISPR/Cas9–mediated genetic editing we also demonstrated the feasibility of human T cell functional analysis in several examples: 1) CD95 deletion inhibited T cell apoptosis upon reactivation; 2) deletion of ORAI1, a Ca2+ release–activated channel, abolished Ca2+ influx and cytokine secretion, mimicking natural genetic mutations in immune-deficient patients; and 3) transcriptional activation of CD25 or CD127 expression enhanced cytokine signaling by IL-2 or IL-7, respectively.

    Taken together, application of the CRISPR toolbox to human T cell subsets has important implications for decoding the mechanisms of their functional outputs.
     
    adambeyoncelowe likes this.

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