CRISPR Editing Fusarium with High Efficiency and Target Rate

Fusarium is a large cosmopolitan genus of imperfect fungi and is of interest primarily because numerous species are important plant pathogens (Nelson et al. 1981), produce a wide range of secondary metabolites, and/or cause opportunistic infections in humans. Although Fusarium research over the past 100 years has advanced our understanding of this important group of fungi, many aspects of its biology still need to be addressed. That’s why researchers are interested in conducting gene-editing in Fusarium including knockout, knock-in and point mutation, etc.

 

Most Fusarium species are soil fungi and have a worldwide distribution. Some are plant pathogens, causing root and stem rot, vascular wilt, or fruit rot. Several species have emerged as important opportunistic pathogens in humans causing hyalohyphomycosis (especially in burn victims and bone marrow transplant patients), mycotic keratitis, and onychomycosis (Guarro 2013). Other species cause storage rot and are important mycotoxin producers. Gene knockout in Fusarium services might be different from those in cell lines. Gene-editing such as CRISPR in Fusarium will be more complicated but will also bring many benefits to human life.

 

CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum

Fusarium oxysporum is an economically important pathogen that widely exists in the environment and is capable of causing serious problems in crop production and animal/human health. One important step for the characterization of a fungal protein with an unknown function is to determine its subcellular localization within the cell. To facilitate the study of important functional regulators or key virulence factors, a CRISPR/Cas9-mediated endogenous gene tagging (EGT) system based on two different strategies was developed, homology-independent targeted integration (HITI) and homology-dependent recombination integration (HDRI). The results indicate that this EGT system is efficient and can be another molecular tool to resolve the function(s) of proteins and infection strategies of F. oxysporum.

 

Generation of Fusarium graminearum Knockout Mutants by the Split-marker Recombination Approach

Fusarium graminearum is a destructive phytopathogen and shows an impressive metabolic diversity. Gene deletion is an important and useful approach for gene function study. Here a protocol for generating gene deletion mutant by applying a “split-marker” deletion strategy (Catlett et al., 2003) with PEG-mediated protoplast transformation (Yuan et al., 2008; Martín, 2015) was present. For generating single-gene deletion mutants, a variety of genes conferring resistance to antibiotics are available, e.g., genes resistant to Hygromycin B, Geneticin/G418, Bialaphos/Phosphothricin, Nourseothricin, Blasticidin, and Phleomycin. Among these, the gene resistant to Hygromycin B is the most widely used. In this protocol, HPH worked as a resistant gene. Other markers can also be chosen according to the experiment as well (Turgeon et al., 2010).

 

Biochemical Characterization and Knockout Mutant In Fusarium graminearum

F. graminearum and other species can produce auxin, and auxin levels are increased in Fusarium infected plants (Kidd et al., 2011; Wang et al., 2018). Since it had been reported that ethylene insensitivity in transgenic wheat increased Fusarium resistance and reduced the content of the mycotoxin deoxynivalenol (DON) in infected wheat, researchers generated single and double knockout mutants of both genes in the F. graminearum strain PH-1.

Six PCR confirmed double-knockout strains were chosen for the virulence test with 10 replicates per strain. The progress of infection was observed over 16 days followed by the analysis of DON and D3G after harvesting. No statistically significant effect of the gene disruptions on the fungal spread or mycotoxin content was detected, indicating that the ability of the fungus to manipulate the production of the gaseous plant hormones ethylene and H2S is dispensable for full virulence.

 

Ubigene developed CRISPR-B™  which optimizes the microbial gene-editing vectors and process. The efficiency and accuracy are much higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi. Easily achieve microbial gene knockout (KO), point mutation (PM), and knockin (KI).

 

References:

CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum. Qiang Wang, Jeffrey J. Coleman. Fungal Genetics and Biology, Volume 126, May 2019, Pages 17-24.

Wang, W., and Tang, W. (2018). Generation of Fusarium graminearum Knockout Mutants by the Split-marker Recombination Approach. Bio-101: e2976.

Svoboda T, Parich A, Güldener U, Schöfbeck D, Twaruschek K, Václavíková M, Hellinger R, Wiesenberger G, Schuhmacher R and Adam G (2019) Biochemical Characterization of the Fusarium graminearum Candidate ACC-Deaminases and Virulence Testing of Knockout Mutant Strains. Front. Plant Sci. 10:1072. doi: 10.3389/fpls.2019.01072