Bioengineering of fungal endophytes through the CRISPR/Cas9 system

Abstract

The CRISPR/Cas9 system is a genome-editing tool that allows for precise and efficient modifications to the DNA of a cell. This technology can be used in endophytic fungi, which live within plants and can have beneficial effects on their host, making them important for agriculture. Using CRISPR/Cas9, researchers can introduce specific genetic changes into endophytic fungal genomes, allowing them to study the function of genes, improve their plant-growth-promoting properties, and create new, more beneficial endophytes. This system works by using the Cas9 protein, which acts as a pair of molecular scissors, to cut DNA at specific locations determined by a guide RNA. Once the DNA is cut, the cell's natural repair mechanisms can be used to insert or delete specific genes, allowing for precise editing of the fungal genome. This article discusses the mechanism and applications of CRISPR/Cas9 to fungal endophytes.

Keywords: CRISPR/Cas9 technology; bioactive compounds; bioengineering; fungal endophytes; metabolite.

Figure 1

Illustration of Cas9/sgRNA-based genome editing. The diagram showing the sgRNA-mediated Cas9 protein binding to the target sequence and knocking out the double strand of DNA (A) and the self-repair mechanism of the cell after DNA double-strand breakage (B). A non-homologous end-joining repair pathway results in random loss, introduction, and replacement of bases at damaged points, thus resulting in gene mutation. By using the homology repair pathway (HR), the gene of interest will be precisely edited based on fragments of donor DNA (Song et al., 2019).

Figure 2

(A) Showing RNP-based transformation and disruption of EAS1 and EAS2 gene clusters in Epichloë coenophiala using the CRISPR/Cas9 technique; (Ba,b) communicating CRISPR/Cas9-based single locus gene manipulation in Pestalotiopsis fici; (c) showing dual loci gene editing in Pestalotiopsis fici using CRISPR/Cas9 technology; (C) showing knocking out of PmkkA gene in Phomopsis liquidambaris using CRISPR/Cas9 tool (D) conveys the role of CRISPR/Cas9 to knockout PmkkA gene using double gRNA in Phomopsis liquidambaris resulting in the production of flavonoids.