• CRISPRa downstream effects – now it gets interesting!

    Detecting signaling effects of IL1R2 overexpression by CRISPRa with synthetic crRNAs

    CRISPRa downstream effects – now it gets interesting!

    The CRISPR-Cas9 system has been adapted to up-regulate any gene in its endogenous context, enabling gain-of-function or gene activation experiments while avoiding the use of exogenous over-expression plasmids. CRISPR activation (CRISPRa) provides new tools to identify gene functions that might otherwise go undetected using loss-of-function studies through down-regulation of gene expression or gene knockout.

    CRISPRa uses deactivated or “dead” Cas9 (dCas9), a mutant variant of the Cas9 protein that is nuclease-deficient due to mutations in the RuvCI and HNH domains1. Edit-R CRISPRa utilizes dCas9-VPR where the dCas9 is fused to three transcriptional activators VP64, p65, and Rta (VPR) on the C-terminus 2. Guide RNAs that target upstream of the transcriptional start site (TSS) of a desired gene can bind the dCas9-VPR and guide the complex to the DNA target site and enable up-regulation of the target gene. Dharmacon Edit-R CRISPRa guide RNA designs are based on a published CRISPRa v2 algorithm 3 and are different than the Edit-R guide RNAs which are optimized for functional gene knockout. The CRISPRa guide RNA can be either single guide RNA (sgRNA) or synthetic CRISPR RNA (crRNA) complexed with a trans-activating CRISPR RNA (tracrRNA) (Figure 1) enabling development of wide range of phenotypic assays for functional gene analysis.

    Analysis of downstream gene expression following IL1R2 activation

    IL1R2 gene encodes for a cytokine receptor that belongs to the interleukin 1 receptor family. IL1R2 is a negative regulator of interleukin 1 (IL1) signaling in several ways (reviewed in 4). It is competing with interleukin 1 receptor, type I (IL1R1) for IL1 ligand and it complexes with interleukin 1 receptor accessory protein (IL1RAP), thereby sequestering both the ligand and the accessory protein required for signal transduction that leads to production of cytokines IL6 and IL8 5, 6, 7. Other studies have shown that IL1R2 plays role in regulation of cell morphology and migration and some of its functions are exuded through regulation of ZEB2 and GEMIN2 expression levels 8, 9 (Figure 2A).

    In our study, we looked at downstream effects of IL1R2 transcriptional activation by CRISPRa in U2OS cells that stably express dCas9-VPR. IL1R2 is not expressed in U2OS cells, and upon transfection with an Edit-R CRISPRa crRNA pool (four individual synthetic crRNAs) targeting the promoter region we observe strong activation of more than 10,000 fold (Figure 2B). This IL1R2 upregulation leads to down-regulation of IL6 and IL8 (Figure 2C) and up-regulation of ZEB2 and GEMIN2 (Figure 2D), confirming literature predicted downstream gene effects and demonstrating that signaling of IL1R2 can be studied using CRISPRa.

    Highlights

    • CRISPRa enables researchers to up-regulate any gene in its endogenous context, even from non-detectable levels
    • Dharmacon predesigned synthetic crRNAs for CRISPRa cause strong target gene activation and can be used for functional gene analysis suitable for short-term assay development in an arrayed format
    • CRISPRa mediated gene activation leads to up- or down-regulation of other downstream genes in the signaling pathway

    Authors: Eldon Chou, Maren Mayer Gross and Žaklina Strezoska

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    References

    1. M. Jinek et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 377, 816-21 (2012).
    2. A. Chavez et al., Highly efficient Cas9-mediated transcriptional programming. Nat Methods. 12, 326-8 (2015).
    3. M. A. Horlbeck et al., Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife. 5, e19760 (2016).
    4. V. A. Peters et al., IL-1 receptor 2 (IL-1R2) and its role in immune regulation. Brain Behav Immun. 32, 1–8 (2013).
    5. D. Malinowski et al., Interleukin-1 receptor accessory protein interacts with the type II interleukin-1 receptor. FEBS Lett. 3, 299-302 (1998).
    6. D. Lang et al., The type II IL-1 receptor interacts with the IL-1 receptor accessory protein: a novel mechanism of regulation of IL-1 responsiveness. J Immunol. 12, 6871-7 (1998).
    7. J. E. Sims et al., Interleukin 1 signaling occurs exclusively via the type I receptor. P Natl Acad Sci USA. 90, 6155-9 (1993).
    8. O. Leshem et al., TMPRSS2/ERG Promotes Epithelial to Mesenchymal Transition through the ZEB1/ZEB2 Axis in a Prostate Cancer Model. PLoS ONE. 6, e21650 (2011).
    9. S. Y. Chang et al., Ectopic expression of interleukin-1 receptor type II enhances cell migration through activation of the pre-interleukin 1alpha pathway. Cytokine. 45, 32-8 (2009).

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