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Open AccessArticle

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

1
Department of Plant Pathology, University of Kentucky, Lexington, KY 40546, USA
2
Computer Science Department, University of Kentucky, Lexington, KY 40546, USA
*
Author to whom correspondence should be addressed.
Received: 30 October 2020 / Revised: 18 November 2020 / Accepted: 21 November 2020 / Published: 16 February 2021
(This article belongs to the Special Issue Global Impact of Ergot Alkaloids)
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. View Full-Text
Keywords: CRISPR/Cas9; non-transgenic engineered fungi; genome editing; genome sequencing; MinION; nanopore; secondary metabolites CRISPR/Cas9; non-transgenic engineered fungi; genome editing; genome sequencing; MinION; nanopore; secondary metabolites
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MDPI and ACS Style

Florea, S.; Jaromczyk, J.; Schardl, C.L. Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi. Toxins 2021, 13, 153. https://0-doi-org.brum.beds.ac.uk/10.3390/toxins13020153

AMA Style

Florea S, Jaromczyk J, Schardl CL. Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi. Toxins. 2021; 13(2):153. https://0-doi-org.brum.beds.ac.uk/10.3390/toxins13020153

Chicago/Turabian Style

Florea, Simona; Jaromczyk, Jolanta; Schardl, Christopher L. 2021. "Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi" Toxins 13, no. 2: 153. https://0-doi-org.brum.beds.ac.uk/10.3390/toxins13020153

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