CRISPR/Cas9 in allergic and immunologic diseases

Figures & data

Figure 1. Basic CRISPR/Cas9 systems for allergic and immunologic diseases. (a) Conventional CRISPR/Cas9 system for gene editing. sgRNA and Cas9 form a ribonucleoprotien complex. sgRNA target sequence is complementary to a specific genomic location and allows binding of the RNP at that loci. Cas9 then creates a double-strand break. Cellular DNA repair mechanisms repair the break. A proportion of these repairs will result in gene knockouts or, if a donor DNA sequence is provided, point mutations or large insertions. Donor DNA sequences contain the desired change flanked by regions homologous to the DNA sequence proximal and distal to the genomic mutation site. Deactivated Cas9 (dCas9) systems can be used to modulate gene expression. Various enhancers or repressors can be fused to Cas9 itself (b), or aptamer technology can be used to allow binding of an enhancer or repressor to the sgRNA (c). After the RNP binds to a specific locus, the enhancer or repressor can modulate the expression of a nearby gene. (d) Using CRISPR/Cas9-aptamer-based gene regulation, it should be possible to achieve multiplex modulation of the expression of transcription factors and cytokine mediators, allowing for repression of the Type 2 T helper (Th2) phenotype associated with atopic disease and promoting development of either a Type 1 T helper (Th1) or a regulatory T cell (Treg) response. Diagrammed are potential targets for such a system. For example, GATA binding protein 3 (GATA-3) is a transcription factor important in the development of Th2 cells and forkhead box p3 (FoxP3) is a transcription factor important in the development of Treg cells. Using CRISPR/Cas9 to repress (blue minus sign) GATA-3 or induce (orange plus sign) FoxP3 expression, it may be possible to skew T cell development away from Th2 development and towards Treg development, respectively. T-box transcription factor TBX21 (T-bet), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 13 (IL-13), interferon γ (IFN-γ), interleukin 2 (IL-2), cytotoxic T lymphocyte–associated protein 4 (CTLA-4), interleukin 10 (IL-10), transforming growth factor β (TGF-β), peripheral blood mononuclear cell (PBMC).

Table 1. Basic CRISPR/Cas9 systems with potential uses in clinical allergy and immunology.

CRISPR/Cas9 approach	Example target	Target cells	Goal
Gene knockout	CCR5 knockout	HSPCs, T cells	Resistance to R5-tropic HIV
Point mutations	ADA-deficiency SCID Asthma-associated SNPs	HSPCs HSPCs, T cells	Correction of ADA mutations leading to SCID Change SNPs that have been associated with asthma
DNA insertion	IL2RG defect (X-SCID)	HSPCs	Insertion of the entire IL2RG cDNA into the mutated IL2RG locus or insertion of an IL2RG transgene into a genomic safe harbor
Gene activation/ repression	Modulate expression of transcription factors and cytokine mediators associated with Th2 phenotype (e.g. GATA-3, FoxP3, and T-bet)	HSPCs, epidermal stem cells, T cells, tissue-specific cells (e.g. macrophages, dendritic cells, etc.)	Activating expression of transcription factors and cytokine mediators associated with Treg or Th1 response and repressing those associated with Th2 response to transition away from an atopic Th2 phenotype (e.g. repressing GATA-3 expression while potentiating expression of FoxP3, IL-10, and/or TGF-β)

Examples of current or potential use are provided.

CCR5: C–C chemokine receptor type 5; R5-tropic HIV: strain of HIV that enters and infects a host CD4 cell by attaching to the CCR5 coreceptor; ADA: adenosine deaminase; SCID: severe combined immunodeficiency; SNPs: single-nucleotide polymorphisms; IL2RG: interleukin 2 receptor subunit gamma; X-SCID: X-linked severe combined immunodeficiency; Treg: regulatory T cell; Th1: Type 1 T helper; Th2: Type 2 T helper; GATA-3: GATA-binding protein 3; FoxP3: forkhead box p3; T-bet: T-box transcription factor TBX21; HSPCs: hematopoietic stem/progenitor cells; IL-10: interleukin 10; TGF-β: transforming growth factor β.