CRISPR Cas9 mediated gene knock-in in Candida glabrata

Scharte, Michelle (2020) CRISPR Cas9 mediated gene knock-in in Candida glabrata. Master's Thesis / Essay, Biomolecular Sciences.

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Abstract

Although modifications of the genome have been a standard procedure for decades, homologous recombination dependent gene knock-ins still prove to be problematic. Development of the clustered regularly interspaced short palindromic repeats CRISPR associated (CRISPR Cas9) toolbox has led to higher preciseness and efficiency by introducing a double stranded break at a specific location and recruiting the cell’s homology directed repair (HDR) mechanism to the target site. However, many organisms prefer the non-homologous end joining (NHEJ) repair pathway resulting in low knock-in efficiency. One of these species is Candida glabrata (C. glabrata), the most common cause of candidiasis after Candida albicans. Increasing occurrences of resistance to antifungals stress the need for effective gene editing tools to expand the knowledge on this pathogen in order to develop new treatments. Since most CRISPR Cas9 techniques are not developed in fungi specifically, methods from mammalian cells are the major source of inspiration. This essay first gives an overview over the CRISPR Cas9 method, deoxynucleic acid repair mechanisms and explains how the application varies in different organisms. Further, several methods of HDR enhancement are evaluated in order to propose an efficient knock-in strategy in C. glabrata. Finally, the essay arrives at the conclusion that knockout of Ligase IV, a crucial part of the NHEJ machinery, would be able to impede NHEJ and thereby enhance HDR in C. glabrata.

Item Type:	Thesis (Master's Thesis / Essay)
Supervisor name:	Billerbeck, S.K.
Degree programme:	Biomolecular Sciences
Thesis type:	Master's Thesis / Essay
Language:	English
Date Deposited:	11 Feb 2020 13:55
Last Modified:	11 Feb 2020 13:55
URI:	https://fse.studenttheses.ub.rug.nl/id/eprint/21529

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