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The Epichloë species of fungi include seed-borne symbionts (endophytes) of cool-season grasses that enhance plant fitness, although some also produce alkaloids that are toxic to livestock. Selected or mutated toxin-free endophytes can be introduced into forage cultivars for improved livestock performance. Long-read genome sequencing revealed clusters of ergot alkaloid biosynthesis ( EAS ) genes in Epichloë coenophiala strain e19 from tall fescue ( Lolium arundinaceum ) and Epichloë hybrida Lp1 from perennial ryegrass ( Lolium perenne ). The two homeologous clusters in E. coenophiala -a triploid hybrid species-were 196 kb ( EAS 1) and 75 kb ( EAS 2), and the E. hybrida EAS cluster was 83 kb. As a CRISPR-based approach to target these clusters, the fungi were transformed with ribonucleoprotein (RNP) complexes of modified Cas9 nuclease (Cas9-2NLS) and pairs of single guide RNAs (sgRNAs), plus a transiently selected plasmid. In E. coenophiala , the procedure generated deletions of EAS 1 and EAS 2 separately, as well as both clusters simultaneously. The technique also gave deletions of the EAS cluster in E. hybrida and of individual alkaloid biosynthesis genes ( dmaW and lolC ) that had previously proved difficult to delete in E. coenophiala . Thus, this facile CRISPR RNP approach readily generates non-transgenic endophytes without toxin genes for use in research and forage cultivar improvement.

Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi