Nødvig, Christina Spuur

Aspergillus er en slægt filamentøse svampe, som inkluderer flere industrielt relevante arter, som er i stand til at producere enzymer, organiske syrer og sekundære metabolitter, men også arter som er vigtige patogener eller model organismer. Derfor, uanset det specifikke interesseområde, er der mange årsager til, hvorfor det kan være relevant at genmanipulere disse organismer, hvilket inkluderer at lave cellefabriker, undersøge specifikke synteseveje, studere sekundær metabolisme el.lign. Denne afhandling er centreret omkring forskellige måder, DNA reparations mekanismerne kan blive udnyttet til at optimere introduktion af specifikke mutationer og generelt forbedre strategier for at genmanipulere Aspergillus arter.Kapitel 1 er en introduktion til Aspergillus slægten og mange af de relevante redskaber der er til genmanipulation af disse skimmelsvampe. Derudover er der også en kortfattet introduktion til DNA reparation og dets relevans for genmanipulation. Endelig er der en kort oversigt over nogle af de forskellige gen editerings teknologier som eksisterer, hvilket er relevant for de senere kapitler.Aspergillus nidulans er model organisme, for hvilken adskillige genetiske redskaber er blevet udviklet. Derfor har udgangspunktet i denne afhandling været at A. nidulans har været anvendt til at teste koncepter, hvorefter de bliver overført til andre Aspergillus arter.I kapitel 2, var målet en metode for at lave reversible gen afbrydelser, hvoraf hovedformålet var at lave en stamme midlertidigt afbrudt i både pyrG og nkuA i A. nidulans. Dette vil resultere i en stamme som har en mod-selekterbar genetisk selektions markør, men som også inkorporerer heterolog DNA ved hjælp homolog rekombination, hvilke tillader at lave præcise ændringer i svampens genom. De samme to træk er dog en ulempe i fermenterings processer, hvor en god produktionsstamme gerne skal være prototrof og med et robust DNA reparations system. Ideen er at indsætte en dominant markør flankeret af loxP sites ind i intronen af det gen som skal afbryde. Så længe det er indsat i intronen vil dets tilstedeværelse forhindre intron splejsning, i praksis forhindre translation. Til gengæld med cre rekombinasen er det muligt at skære det indsatte DNA ud igen og kun efterlade et enkelt loxP site, hvilket, hvis det er placeret korrekt, ikke interfererer med intron splejsning. Først blev konceptet testet ved succesfuldt at afbryde og restaurere pigment genet yA. Derefter blev konceptet udvidet til at inkludere cre rekombination genet under kontrol af tetON promoter, og derudover blev det testet om det muligt at indsætte eller udskifte en intron fra et gen til et andet, hvilket var delvist succesfuldt. Til sidst blev pyrG succesfuldt midlertidigt afbrudt i en markørfri stamme, og en tilsvarende mutation blev indført i nkuA genet.Genom editerings teknologi er ikke et nyt koncept, men det har aldrig været så tilgængeligt som det er nu. I kapitel 3 blev et fleksibelt CRISPR-Cas9 system udviklet, bestående af fire vektorer med forskellige selektions markører, hvilket kan blive anvendt i forskellige arter. CRISPR-Cas9 kræver to komponenter for at virke. Cas9 var kodon optimeret til at matche A. niger, mens en strategi baseret på ribozymes blev anvendt til at udtrykke gRNA’et. Med et funktionelt system, var det muligt at afbryde gener ved mutagenese, men også booste frekvensen hvorved heterolog DNA bliver integreret ved homolog rekombination, tilsvarende den effekt som ses når man fjerner svampens evne til at reparere DNA dobbelt strengs brud ved hjælp af illegitim rekombination. Derudover blev et Perl script udviklet til at kunne identificere protospacers som fungerer i flere homologer af det samme gen i forskellige arter. Som udgangspunkt blev CRISPR-Cas9 testet i to arter, A. nidulans and A. aculeatus, men scriptet blev udnyttet til at demonstrere at systemet var funktionelt i yderligere fire arter.Kapitel 4 fokuserer på tre forskellige aspekter af anvendelsen af CRISPR-Cas9. Først blev alternative promotere testet for deres evne til succesfuldt at udtrykke gRNA’er. Disse var valgt, baseret på hvad der har virket i andre organismer. Desværre virkede ingen af dem ud over den ribozyme baserede strategi præsenteret i kapitel 3. Derefter blev to forskellige strategier som kan begrænse potentielle problemer med ”off-targeting” testet. Den første var baseret på at anvende protospacere som var 17 baser lange i stedet for 20, men det gav sære sporeløse kolonier og var ikke en succes. Derimod virkede strategien med at omdanne Cas9 til en nickase og anvende to gRNA’er til at lave to nicks i stedet for et enkelt dobbelt strengs brud, til at stimulere integration ved hjælp af homolog rekombination. Endelig viste jeg at man ved at kombinere CRISPR-Cas9 med at afbryde illegitim rekombination, er det muligt at lave gen modificationer uden at efterlade en selektion markør i genomet. Derudover demonstrerede jeg at det er muligt at bruge enkelt strengede oligoer i stedet for konventionelle vektorer når der skal introduceres små modifikationer.Alt i alt har denne afhandling resulteret i adskillige brugbare værktøjer til at lave genetiske modifikationer og især de CRISPR-Cas9 baserede redskaber har potentiale til at ændre konventionelle strategier for genmanipulation i Aspergillus arter.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

Online Research Database In Technology

Manipulating DNA repair for improved genetic engineering in <i>Aspergillus</i>

http://orbit.dtu.dk/ws/files/127587042/Manipulating_DNA.pdf

Manipulating DNA repair for improved genetic engineering in <i>Aspergillus</i>

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