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 (EAS1) and 75 kb (EAS2), 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 EAS1 and EAS2 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.
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This research was funded by the U.S. Department of Agriculture National Institute of Food and Agriculture Hatch project KY012044, and by the Mycological Society of America.
Patent Pending: Schardl, C.L.; Florea, S.; Farman, M.L. Fungal chromosome-end knockoff strategy. US 2017 /0349899 Al, 2017.
The following are available online at https://www.mdpi.com/2072-6651/13/2/153/s1, Figure S1: PCR tests of putative CRISPR/Cas9-mediated deletion mutants, Table S1: Genome assembly statistics for wild-type Epichloë species, Table S2: Genome assembly statistics for CRISP/Cas9-mediated mutants. These materials are also available for download as the additional file listed at the end of this record.
Florea, Simona; Jaromczyk, Jolanta; and Schardl, Christopher L., "Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi" (2021). Computer Science Faculty Publications. 38.