Genome editing is fast becoming an accessible molecular tool for researchers working on non-model species. Dr Andi Wilson has established a CRISPR-Cas9-based genome editing protocol for use in Huntiella omanensis, a tree-inhabiting fungus belonging to the Ceratocystidaceae family. 

Model species are organisms, such as mice, Drosophila fruit flies, Saccharomyces cerevisiae and the bacterium Escherichia coli, that have been used extensively to study important biological processes. They are typically easy to work with, have short generation times and are amenable to experimental manipulation in the lab. For this reason, the study of these species is generally well supported, both financially and scientifically. There is much data available for these organisms, for example, model species often have high quality, chromosome-level genome assemblies available, as well as transcriptomic data from a wide variety of life stages and experimental conditions. Additionally, the molecular toolkits available for these species are impressive, with well-developed and highly optimized protocols for everything from DNA extraction and gene annotation to genome editing and phenotypic screening. 

In contrast, working on non-model species, such as Huntiella omanensis, can pose quite a challenge. While next generation sequencing data is becoming far cheaper and easier to produce, the molecular toolkits of these species remain limited. A fairly new genome editor, the CRISPR-Cas9 system, is slowly changing this by providing a much easier method of genome editing. The system, based on a bacterial defense mechanism, boasts high efficiency and accuracy. It is also flexible, which enables the system to be adapted and manipulated for the establishment in a wide variety of different organisms.  

Dr Wilson, a postdoctoral researcher at the University of Pretoria’s Forestry and Agricultural Biotechnology Institute, developed a CRISPR-Cas9-based genome editing protocol for use in H. omanensis, a saprobic species belonging to the Ceratocystidaceae family, during her PhD study. The development of this protocol came after various unsuccessful attempts at genome editing using more established technologies. Use of the CRISPR-Cas9 protocol enabled highly specific and targeted editing of the genome of H. omanensis. The protocol was used to produce mutant strains by knocking out a gene thought to be important for mating in H. omanensis. Once the mutant strains were allowed to interact with suitable mating partners, it became apparent that the gene was indeed essential for mating, as these fungi were unable to produce ascomata (mature sexual structures). This work was published in Fungal Genetics and Biology. Genome and transcriptomic sequencing of these mutant strains to further our understanding of sexual reproduction will follow from this study. 

In the future, we hope to expand the use of this protocol into other species in the Ceratocystidaceae family, many of which cause serious diseases in plants. Using the CRISPR-Cas9 system, we will be able to identify and begin to characterize genes that are involved in determining important biological characteristics such as virulence, growth rate and host specificity. 

This work was completed under the supervision of Professor Brenda Wingfield, Prof. Mike Wingfield, Dr Magriet van der Nestand Dr Markus Wilken. 

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