CRISPR-Cas9-mediated genome editing of the extracellular matrix gene TNC in human fibroblastsTools Brunetti, Giulio (2017) CRISPR-Cas9-mediated genome editing of the extracellular matrix gene TNC in human fibroblasts. MRes thesis, University of Nottingham.
AbstractMammalian cells exist in complex three-dimensional (3D) microenvironments, in which diverse signalling molecules and support structures dynamically interact forming a complex network or extracellular matrix (ECM). Beyond providing structural support to cells, the matrix conveys environmental signals that direct many aspects of normal cell behaviour, including shape, differentiation, survival and migration. Tenascin-C (TN-C), an ECM glycoprotein absent in most healthy adult tissues, but persistently expressed during chronic inflammatory diseases and in cancer, has been recently shown to be rapidly induced during the inflammatory response to infection. TN-C is required for an effective pro-inflammatory response to bacterial lipopolysaccharide (LPS) in vivo. It sustains the synthesis of the pro-inflammatory cytokine TNF-α by regulating the expression of the microRNA miR-155 in primary murine macrophages upon LPS stimulation. These data highlight a novel, regulatory function of the ECM molecule TN-C that controls inflammatory gene expression by regulating microRNA levels in the mouse. However, we still do not know whether this occurs in human cells and how. Here, I have applied the CRISPR-Cas9-mediated genome editing technology to knock-out TNC gene expression in human fibroblast cells, which are a natural and abundant source of ECM. Once validated, these will be used to generate a cell-free fibroblast-derived matrix on which primary human macrophages will be cultured in the presence or absence of infection. This system will allow to investigate whether human TN-C modulates macrophage gene expression by regulating microRNA levels. The HEK 293 cell line was used to design and optimise a CRISPR-Cas9 system that can efficiently knock-out the TNC gene. Three different pairs of oligonucleotides were designed to guide the Cas9 endonuclease to the target gene sequence. The oligonucleotides were cloned into a mammalian expression vector and delivered to HEK 293 cells together with Cas9. PCR amplification and sequencing, in combination with fluorescence in situ hybridization (FISH), were used to validate the genome edit. One oligonucleotide pair edited the TNC gene in HEK 293 cells by introducing a premature stop codon at the translation start site. Subsequently, three different approaches were tested to deliver the oligonucleotides and Cas9 to the difficult-to-transfect human fibroblasts. I was able to transfect them with a transfection efficiency of 40-50%. Single cell sorting of transfected cells was performed to generate single-cell clones and a number of single-fibroblast clones are currently growing. Their expansion will allow validation of the genome edit. Together this work has generated a CRISPR-Cas9 system for the specific knock-out of the TNC gene in mammalian cells and single-fibroblast clones whose TNC expression may be ablated.
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