Fungus Genome-editing Services including Knockout, Knock-in, etc.

Fungus, plural fungi, any of about 144,000 known species of organisms of the kingdom Fungi, which includes the yeasts, rusts, smuts, mildews, molds, and mushrooms. There are also many funguslike organisms, including slime molds and oomycetes (water molds), that do not belong to kingdom Fungi but are often called fungi. Many of these fungus-like organisms are included in the kingdom Chromista. Fungi are among the most widely distributed organisms on Earth and are of great environmental and medical importance. Many fungi are free-living in soil or water; others form parasitic or symbiotic relationships with plants or animals.

Fungal pathogens are the main factors responsible for the most severe diseases affecting plants, leading to a significant reduction in yield and crop quality and causing enormous economic losses worldwide. It is estimated that around 30% of the emerging diseases are caused by fungi (Giraud et al., 2010) thus requiring new strategies to improve their management. The arrival of the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR Associated protein 9) genome-editing technique (including gene knockout, knock-in, point-mutation, etc.) enabled researchers to modify genomic sequences in a more precise way. The use of CRISPR-Cas not only provides a time-saving path to perform genomic functional analyses but also could provide new fungal genotypes, that can be used as potential competitors of plant pathogens and/or in the priming of plant defense responses. It is possible to induce the activation of unknown clusters in beneficial fungi by using CRISPR-Cas9, allowing the discovery of new secondary metabolites that could interact with plants or phytopathogens. This could result in new interesting biocontrol strains to be released in the field avoiding the introduction of transgenes in the environment.

 

Applying CRISPR/Cas9 in Yeasts: a Helpful Genetic Changes Tool

The advancement that is driving the rate of adoption of CRISPR–Cas9 for many organisms is its capability of targeting a specific site in a genome: by and large, trying to manipulate a piece of DNA using a homologous fragment of DNA is highly inefficient. One exception to this occurs in the yeast Saccharomyces cerevisiae, where gene editing can be achieved by transforming into cells fewer than 40 nucleotides of sequence divided on either side of a selectable marker. The high proportion of homologous targeting of exogenous DNA into the S. cerevisiae genome is one reason why yeast is such a powerful experimental tool. Indeed, relatively soon after its genome sequence was completed, every gene in the organism was deleted and those sets of mutants made available to researchers. Likewise, by using homologous integration all proteins were tagged with a green fluorescent protein to understand subcellular protein localizations and epitope-tagged to facilitate the global analysis of protein complexes. Thus, this gene-editing system is already considerably beyond just a tool for making genetic changes.

 

Control the Incidence of FHB by CRISPR/Cas9 Silenced Mutants in Fusarium

One possible scenario for the application of CRISPR-Cas9 silenced mutants could be Fusarium Head Blight (FHB), one of the most destructive diseases of grain cereal crops worldwide caused by different Fusarium spp., with F. graminearum and F. culmorum as the most common and aggressive agents. In FHB, while yield loss derives from the sterility of infected florets, grain quality reduction is mainly due to the accumulation of trichothecenes—coded by the fungal tri genes cluster—highly toxic for humans and animals. Previous studies reported that iRNA (interference RNA) Δtri6 mutants of F. culmorum showed reduced disease indices ranging from 40 to 80% on durum wheat (Scherm et al., 2011). Besides, classic knocked-out Δtri5 and Δtri6 mutants of F. graminearum were unable to spread the disease to the adjacent spikelets and grains on wheat and corn, respectively, and also induced plant defense responses (Ravensdale et al., 2014). Likewise, Δmap1 mutants of F. graminearum showed a two-fold reduction of mycotoxin production and were unable to produce perithecia as well as to penetrate in wheat tissues, while the ability to colonize the straw was not affected (Urban et al., 2003). Considering that competition for space and nutrients between virulent and non-virulent strains could reduce the disease, the field release of non-virulent CRISPR-mutant strains of F. graminearum and F. culmorum might help to control the incidence of FHB.

 

Gene knockout Accelerate Functional Genomic Studies in P. chlamydosporia

Gene knockout techniques are useful molecular tools to study gene functions. However, cultures of P. chlamydosporia are resistant to high levels of a range of fungal inhibitors, which makes the gene knockout technique difficult in this fungus. Fortunately, we found that the wild P. chlamydosporia strain PC-170 could not grow on medium containing 150 μg ml−1 G418 sulfate, representing a new selectable marker for P. chlamydosporia. Researchers knocked out one chitinase gene, VFPPC_01099, and two protease genes (VFPPC_10088, VFPPC_06535). Afterward, they obtained approximately 100 suspected mutants after each transformation. After screening by PCR, the average rate of gene knockout was 13%: 11% (VFPPC_01099), 13% (VFPPC_10088), and 15% (VFPPC_06535). This efficient and convenient technique will accelerate functional genomic studies in P. chlamydosporia.

 

Reference:

Muñoz Isabel Vicente, Sarrocco Sabrina, Malfatti Luca, Baroncelli Riccardo, Vannacci Giovanni. CRISPR-Cas for Fungal Genome Editing: A New Tool for the Management of Plant Diseases. Frontiers in Plant Science. Volume 10, 2019, 135. ISSN: 1664-462X.

Idnurm, A., Meyer, V. The CRISPR revolution in fungal biology and biotechnology, and beyond. Fungal Biol Biotechnol 5, 19 (2018).

Scherm B, Orrù M, Balmas V, Spanu F, Azara E, Delogu G, Hammond TM, Keller NP, Migheli Q. Altered trichothecene biosynthesis in TRI6-silenced transformants of Fusarium culmorum influences the severity of crown and foot rot on durum wheat seedlings. Mol Plant Pathol. 2011 Oct; 12(8):759-71.

Ravensdale M, Rocheleau H, Wang L, Nasmith C, Ouellet T, Subramaniam R. Components of priming-induced resistance to Fusarium head blight in wheat revealed by two distinct mutants of Fusarium graminearum. Mol Plant Pathol. 2014 Dec; 15(9):948-56.

Urban M, Mott E, Farley T, Hammond-Kosack K. The Fusarium graminearum MAP1 gene is essential for pathogenicity and development of perithecia. Mol Plant Pathol. 2003 Sep 1; 4(5):347-59.

Baoming Shen, Jiling Xiao, Liangying Dai, Yonghong Huang, Zhenchuan Mao, Runmao Lin, Yurong Yao, Bingyan Xie. Development of a high-efficiency gene knockout system for Pochonia chlamydosporia. Microbiological Research. Volume 170. January 2015. Page 18-26.