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

Simona Florea,Christopher L Schardl,Jolanta Jaromczyk

doi:10.3390/toxins13020153

Abstract

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.

Highlights

A few species of filamentous fungi have been genetic models of choice since the 1950s due to their haploid growth stage, facile sexual cycles, abundant sporulation, rapid growth and, with time, large repertoires of mutants and molecular transformation systems
The genome of E. coenophiala e19 wild-type strain was previously sequenced by a combination of pyrosequencing (Roche) and Sanger sequencing of fosmid-cloned ends [24]
The 196.2 kb complete sequence of the EAS1 cluster was identified on a 676 kb scaffold that ended with a telomere repeat array, and the 75.2 kb EAS2 cluster was identified on a 2.6 Mb scaffold sequence where it was flanked by housekeeping genes

Summary

Introduction

A few species of filamentous fungi have been genetic models of choice since the 1950s due to their haploid growth stage, facile sexual cycles, abundant sporulation, rapid growth and, with time, large repertoires of mutants and molecular transformation systems. Given the importance of fungi in medicine, agriculture, and ecosystems, considerable efforts have been invested over several decades to establish molecular transformation and targeted mutation systems for a much broader range of species. These include the Epichloë species (family Clavicipitaceae, order Hypocreales), which are systemic, constitutive, and often seed-transmitted symbionts (endophytes) of cool-season grasses Cas9-sgRNA (single guide and tracrRNA) ribonucleoprotein complexes (RNPs) have been employed in a wide range of fungi [5,6]

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Discussion

Conclusion