Manipulating DNA repair for improved genetic engineering in Aspergillus

Christina Spuur Nødvig

    Research output: Book/ReportPh.D. thesisResearch

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    Abstract

    Aspergillus is a genus of filamentous fungi, which members includes industrial producers of enzymes, organic acids and secondary metabolites, important pathogens and a model organism. As such no matter the specific area of interest there are many reasons to perform genetic engineering, whether it is metabolic engineering to create better performing cell factory, elucidating pathways to study secondary metabolism etc. In this thesis, the main focus is on different ways to manipulate DNA repair for optimizing gene targeting, ultimately improving the methods available for faster and better genetic engineering strategies.
    Chapter 1 gives an introduction to the genus Aspergillus and some of the tools relevant to fungal genetic engineering. It also contains a short introduction to DNA repair and its interplay with gene targeting and finally an overview over the different genome editing technologies, providing a background for the other chapters.
    Aspergillus nidulans is a model organism, with a range of genetic tools developed, and therefore the approach in this thesis has been to use it for proof of concept, and once a method has been established in A. nidulans it can be transferred to other Aspergilli.
    In chapter 2, the focus is on a concept for allowing simultaneous, but transient disruptions of genes of interest, with the main goal being creating a strain with transient disruptions of both pyrG and nkuA. This would yield a strain with a robust selection marker and high rates of gene targeting. However, since the same trait, while beneficial for genetic engineering, is a detriment during a fermentation process where a robust DNA repair system and a prototrophic background are important traits. By inserting a marker gene into an intron, flanked by loxP sites, its presence will interfere with intron splicing, however it can be excised by the cre recombinase, leaving only a single loxP scar in the intron, which if placed correctly will not interfere with intron-splicing and restore function. This was demonstrated to work in two different introns in the pigment gene yA in A. nidulans as a proof of concept, and the concept was expanded to include the tetON promoter controlling the cre recombinase gene as part of the insert and also testing whether it is tolerated to insert or replace an intron from one gene to another, which were partial successful. Finally, a transient pyrG mutation was introduced in a marker free strain of A. nidulans, and successfully tested and similarly a disruption of nkuA was made.
    Genome editing is not a new concept, but never has it been as accessible as it is now due to the CRISPR-Cas9 technology. In chapter 3, a versatile CRISPR-Cas9 system for use in various Aspergillus species was made, consisting of four vectors each with a different selection marker. To successfully express the two components of CRISPR-Cas9, Cas9 was codon-optimized to A. niger, and a ribozyme based strategy was used for gRNA expression. With a functional system in place, I demonstrated how it could be used to disable genes by mutagenesis, but also how it could be used to greatly enhance gene targeting frequencies in wild type strains, similar to what is observed when using non-homologous end-joining (NHEJ) deficient strains. Furthermore, a perl script for identifying protospacers in common for gene homologs across multiple species was developed. While the initial experiments were made in A. nidulans and A. aculeatus, this was used to quickly demonstrate that the system could be used in more species, and mutagenesis was done in four additional species.
    In chapter 4 the focus stays on CRISPR-Cas9, focusing on three aspects. Alternative methods for gRNA expression, strategies for limiting off-targeting effects, and how to combine CRISPR-Cas9 with the traditional strategy of disabling NHEJ for even greater results. Four other promoters were tested for their ability to express functional gRNA, chosen based on which has been shown to work in other species. However, none of them worked in A. nidulans and the ribozyme based strategy remained the most effective. Next two strategies for limiting potential off-targeting effects using CRISPR-Cas9 were explored. One was based on using a shorter protospacer than the 20 bp that is normally used. 17 bases of length was tested in A. nidulans, but lead to aberrant sporeless colonies. The other strategy was based on inactivating one of the cleavage domains in the Cas9 protein, turning it into a nickase and then use two gRNAs to create two nicks in close proximity on opposite strands rather than a single double-strand break. Results indicated that it could be a viable strategy to stimulate gene targeting while lowering potential off-targeting. Finally I demonstrated by combining CRISPR-Cas9 with an NHEJ deficient background, it was possible to do gene targeting without selection for DNA to be integrated and furthermore that, for small changes, such as for introducing point mutations, it was possible to use short single-stranded oligos.
    Overall several very useful tools for genetic engineering of various Aspergillus species were developed as a part of this thesis, and especially the CRISPR-Cas9 based tools have the potential to transform genetic engineering strategies.
    Original languageEnglish
    PublisherDepartment of Systems Biology, Technical University of Denmark
    Number of pages113
    Publication statusPublished - 2015

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