CRISPR CAS 9 use to characterize Kenyan filamentous Fungi
Studies suggest that there has been a rapid increase in number of fully sequenced fungal genomes. However, most genomic tools that are use to sequence and understand the features of fungal genome are poorly developed. Hence, there is a strong necessity for developing versatile methods for genetic manipulation of non-model filamentous fungi. The CRISPR-Cas9 genomic system has been used to sequence to filamentous fungi. The system is used to administer RNA-directed mutagenesis by transforming a target fungus with the help of a single plasmid. The CRISPR-Cas9 genomic system contains four CRISPR-Cas9 vectors. These vectors are equipped with common fungal markers which allow the selection of a broad range of fungi. Previous studies have reported the development of a script which allows identification of protospacers in multiple species containing target gene homologs (Nødvig et al., 2015). Such approaches facilitate the introduction of common mutations across different species of filamentous fungi. In this study, a wild-type Kenyan filamentous fungus was selected for genomic analysis. The CRISPR-Cas9 system was used to generate a strain that contained a specific marker. The resultant strain was successfully used for iterative gene targeting. The CRISPR-Cas9 endonuclease was loaded by a single guiding RNA to the targeted site of the fungal genome. The transfer was achieved through competent plasmids that were tested for transformation with the CRISPR-Cas9 endonuclease genome. The CRISPR-Cas9 system mediated mutagenesis in the Kenyan filamentous fungus was validated by targeting the selected candidate genes for the respective fungus. Such approach provided promising results for screening and scrutinizing the virulent and non-virulent attributes of the defined fungus. Different approaches such genetic polymorphisms and function of gene families was evaluated to identify the virulent and non-virulent attributes of the Kenyan filamentous fungi.
Nødvig CS, Nielsen JB, Kogle ME, Mortensen UH (2015) A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. PLoS ONE 10(7): e0133085.
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