• CRISPR CAS9
• Fusarium
• Fusarium Graminearu
• Fusarium Head Blight

Fusarium graminearum Schwabe (teleomorph: Gibberella zeae (Schwein.) Petch) is a plant pathogen that causes several diseases on small-grain cereals, such as seedling blight and foot rot, Fusarium Head Blight (FHB) and ear rot. FHB is one of the most economically worldwide devastating diseases of wheat (Triticum aestivum L.) and other cereal crops. In recent years the disease has caused extensive agricultural damage through direct losses in yield and quality due to the presence of Fusarium damaged kernels and their associated mycotoxins such as the trichothecene deoxynivalenol (DON). Despite of the availability of different strategies, such as crop rotation, tillage, the application of fungicides and the use of resistant cultivars or biopesticides, no one of these alone are able to control the disease.
The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) technique has emerged in recent years as a powerful and fast-growing genome-editing technique and it is applied to different organisms thanks to its high efficiency, easy operations, and the possibility of multi-gene editing. The gene knock-out (KO) induced by the CRISPR-Cas9 is produced by small insertions or deletions (InDels) of nucleotides at the Double Strand Break (DSB) site which derives from the Cas9 endonuclease activity. In many published papers this technique was applied to plants, animals and viruses, as well as in human medicine.
Genome editing of filamentous fungi through the CRISPR-Cas9 technology has increased in recent years but, actually, very few reports about CRISPR-engineered filamentous fungi and only a minor number refers to plant pathogenic fungi.
The main purpose of this master thesis was to set up a protocol for the genome editing of a Fusarium graminearum mycotoxigenic isolate in order to demonstrate the applicability of the CRISPR-Cas9 technique on this organism. During the master thesis the genome of this isolate (ITEM124) was sequenced, annotated and released thus making easier all the bioinformatics work needed. A gene encoding a polyketide-synthase (PKS12) - which disruption is detected easily at a phenotypic level as slow growth and reduced sporulation - was chosen as target gene and used to design the RNA-guides to be included in the RGR-cassette. The cassette was then assembled in a Cas9 expressing plasmid containing also a marker gene (Hygromycin resistance) and a shortened AMA1 sequence, which allows to quickly removing the plasmid from the edited strain simply by reducing the selection pressure. The resulting vectors, one for each of the designed RNA-guide, were used for fungal transformation by protoplasts, these latter obtained according to a protocol optimized in the present work for our isolate. Putative mutants, resulting from different cloning and transformation experiments, were analyzed phenotypically and molecularly in order to verify the knockout of the selected gene.
The ability to manipulate, at a genetic level, beneficial and plant pathogenic isolates with these technique represents a valid and promising tool to study how plant pathogens interact with their hosts as well as to improve the performance of beneficial isolates for the management of plant diseases.
Edited strains need to be checked for the presence of foreign DNA, to contribute to the debate about the exclusion of this type of genetically manipulated microorganisms from GMO.