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Development of a CRISPR-based epigenetic screening method

Köferle, A; (2017) Development of a CRISPR-based epigenetic screening method. Doctoral thesis , UCL (University College London).

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Abstract

Millions of chromatin marks have been profiled across the human genome in dif- ferent tissues and cell types. Yet, which and how many of these marks contribute to the establishment and control of gene activities remains incompletely under- stood. The focus of this PhD project is to develop a CRISPR-based epigenetic screening method for the discovery of functional epigenetic marks. The aim of this method is to identify sites in the genome where addition or removal of a particular chromatin mark has an impact on cellular phenotype. To this end, I fused the catalytic domain of a chromatin-modifying enzyme to the nuclease- dead Cas9 protein and introduced it into cells concomitantly with an appropriate library of guide RNAs (gRNAs). Cells that show a change in the phenotype of interest are then separated from the pool of cells by Fluorescence-activated cell sorting (FACS). I designed libraries of gRNAs that are both targeted towards a particular phenotype of interest, targeting regulatory regions around a limited set of genes, as well as more complex, genome-wide libraries, which were generated from fragmented genomic DNA. I used the CRISPR-based screening strategy to identify candidate gRNAs, which, together with the appropriate dCas9-chromatin modifier fusion protein, bring about changes in expression of various cell surface markers.

Type: Thesis (Doctoral)
Title: Development of a CRISPR-based epigenetic screening method
Event: UCL (University College London)
Language: English
UCL classification: UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute
URI: http://discovery.ucl.ac.uk/id/eprint/1536075
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