Dissection of the epoxyjanthitrem pathway in Epichloë sp. LpTG-3 strain AR37 by CRISPR gene editing

Epichloë festucae var. lolii and Epichloë sp. LpTG-3 are filamentous fungal endophytes of perennial ryegrass (Lolium perenne) that have a substantial impact on New Zealand’s agricultural economy by conferring biotic advantages to the host grass. Epichloë endophyte strain AR37 provides ryegrass with improved agronomic performance, insect protection and plant persistence, with this endophyte alone estimated to contribute NZ$3.6 billion to the New Zealand economy over a 20-year period. This strain produces secondary metabolites, including epoxyjanthitrems, which are a class of indole diterpenes, associated with the observed effects of AR37 on livestock and insect pests. Until very recently, AR37 was intractable to genetic modification but this has changed with the application of CRISPR-Cas9 based gene editing techniques. We used gene inactivation by CRISPR-Cas9 to deconvolute the genetic basis for epoxyjanthitrem biosynthesis and manipulate this secondary metabolite pathway to reduce or remove endophyte-induced mammalian toxicity whilst retaining insect activity against some important agricultural pests. This provides a step change in the future use of animal safe Epichloë strains in New Zealand pastures which will significantly reduce chemical inputs and increase animal welfare.

We also show that gene editing of Epichloë can be achieved without off-target events or introduction of foreign DNA (footprint-less) through an AMA1-based plasmid that simultaneously expresses the CRISPR-Cas9 system and selectable marker. Furthermore, since a nucleic acid template was not used to guide Cas9 and the repair mechanism was through NHEJ, gene edits generated with this delivery system are classified as site-directed nuclease 1 (SDN-1), which are not regulated as genetically modified organisms (GMO) in selected jurisdictions, including Australia. These SDN-1 technology edits have already been grown out of containment in the field in Australia for seed multiplication in preparation for agronomy trials which is a significant step forward for creating impact in Australia and New Zealand.