GE-EAS1/EAS2 Epichloë coenophiala was generated by CRISPR-Cas editing of the ergot alkaloid synthesis (eas) gene cluster.
Epichloë coenophiala is a seed-transmissible symbiotic fungus (endophyte) of the forage grass tall fescue (Festuca arundinacea). The endophyte is important for stand longevity and maximal forage productivity of tall fescue. However, the most common endophyte strains produce ergot alkaloids, which are responsible for episodic toxicosis to livestock.
All genes required for production of ergot alkaloids are located in the gene cluster named eas. Because E. coenophiala is polyploid, it has two alleles of the gene cluster, designated eas1 and eas2.
CRISPR-Cas9 technique was used to generate an E. coenophiala deletion mutant, which eliminates the two alleles eas1 and eas2. A ribonucleoprotein preparation of two guide RNA molecules and a modified Cas9 nuclease were used for simultaneous deletion of both alleles. A selectable plasmid vector was introduced to allow for selection of transient transformants. Protoplast transformation by the polyethylene glycol method was used to introduce the gene editing components and the plasmid vector into E. coenophiala.
PCR screens indicated that the alleles eas1 and eas2 were deleted and that the selectable marker was absent from the genome edited E. coenophiala.
The EAS1/EAS2 deletion mutant is unable to produce any ergot alkaloids.
The deletion mutant will be introduced into an elite breeding line of tall fescue by meristem inoculation of seedlings. Field performance between tall fescue symbiotic with the mutant, tall fescue symbiotic with the wild-type parent, and tall fescue without symbiotic endophyte will be compared. If field performance is acceptable, the tall fescue lines will be used to establish paddocks to compare animal performance when grazed on the three tall fescue lines. If animal performance is good, then tall fescue seeds with the mutant will be made available for distribution to growers.
Source:
USDA APHIS
