CRISPR-Cas9 gene insertion in Epichloë species

Abstract

The dairy, meat, and fibre industries in several regions within New Zealand are heavily reliant on selected strains of endophytic fungi, within the genus Epichloë, which confer resistance to a range of insect pests and environmental pressures when in symbiosis with pasture cultivars. Unfortunately, some fungal strains are historically intractable to genetic manipulation, therefore preventing investigation into novel traits. Only recently with the development of CRISPR-Cas systems, a revolutionary gene editing tool, was CRISPR-Cas9 successfully used on one of these intractable strains, Epichloë sp. LpTG-3 strain AR37, to create targeted gene disruptions. This study focused on CRISPR-Cas9 targeted gene insertion capabilities in Epichloë spp. CRISPR-Cas9 was successfully deployed to precisely insert 236 bp of coding sequence from a critical condensation domain of the perA gene, missing in the genetically intractable Epichloë festucae var. lolii strain AR48. CRISPR-Cas9 was also successfully deployed to insert the reporter gene gfp into a precise location within the indole diterpene pathway, a known secondary metabolite pathway in AR37. This research illustrated the ability of CRISPR-Cas9 to repair or insert genes in genetically intractable Epichloë species, with the potential for reconstruction of secondary metabolite pathways for novel compound production and delivery into New Zealand’s pasture-based agricultural system.