An Integrative Approach to HighThroughput CRISPR Genetic Engineering in Filamentous Fungi

The ability to perform precise genetic engineering through CRISPR technology has changed the landscape of biology, but despite this advancement, our knowledge of the genome is still quite limited. Gene knockout libraries are the logical next step to survey a greater biological space, but to efficiently produce these libraries, end-to-end strain design and build high throughput systems are required. Such high throughput genetic engineering systems have been demonstrated in single celled and single nucleated organisms such as Escherichia coli and Saccharomyces cerevisiae allowing breakthroughs in basic and applied research. Filamentous fungi are vital in human society producing common products such as penicillin, citric acid, and soy sauce as well as causing detrimental issues such as aspergillosis, house rot, and food spoilage. The application of such technologies to filamentous fungi are slower in their development due to issues inherent in the biology of the organisms such as multinucleation and morphology. In this thesis, I address these issues in filamentous fungi in order to develop an end-to-end high throughput CRISPR based genetic engineering platform for filamentous fungi using Aspergillus niger as a demonstration.

In this thesis, I present a three-step process that I developed in order to obtain a high-throughput CRISPR Cas-9 genetic engineering pipeline for A. niger. 1) Comparative proteomics of both the mycelia and spore which produced a list of interesting gene knockout targets, 2) proof-of-concept for CRISPR engineering compatible with a high-throughput setup using cell wall genes as targets, and 3) development and implementation of high-throughput gene deletion system. The developed pipeline will allow for rapid and systematic creation of large mutant deletion collections in such filamentous fungi as A. niger, a resource type that has proven to accelerate research for organisms like Saccharomyces cerevisiae. The technology and creation of a singlegene mutant library sets the stage for accelerated gene discovery within pathogen research and biotechnology, as the knowledge from these libraries provides new insight and allow for rapid innovation cycles.

The presented work is valuable to both fundamental and applied research because it provides a technological guide for the systematic genome-wide investigation into the functions of the individual genes and corresponding proteins in A. niger. From the perspective of fundamental biology, it provides information for generating new insights into the cellular biology of A. niger, and areas ripe for focus include the cell wall proteins (that make good drug targets), functionally unannotated proteins, transcription factors, and intracellular trafficking proteins. From an applied perspective, the system provides the methods for the efficient engineering of A. niger to arrive at a better production host (e.g., increased protein secretion levels or improved fermentation properties).

While A. niger is an ideal model organism for its balance between industrial relevance and academic use, the work in this thesis could be used in a broader context in other filamentous fungi. From faster drug discovery in A. fumigatus and Penicillium spp. to industrial enzyme production in A. oryzae and Trichoderma reesei, high throughput CRISPR platforms open up a new era of academic and industrially-timely biological engineering.