Toward a CRISPR–Cas9 gene editing system for the Dutch Elm Disease pathogen Ophiostoma novo-ulmi
Strategies employed to establish a CRISPR–Cas9 system to efficiently disrupt target genes in the tree pathogen Ophiostoma novo-ulmi are being described. Different vectors are being constructed. The efficiency of vectors containing a type II RNA ploymerase bidirectional promoter driving the expression of O. novo-ulmi codon-optimized Cas9 gene and sgRNA flanked by ribozymes will be compared to vectors containing O. novo-ulmi codon-optimized Cas9 gene and a suitable sgRNA under the control of the O. novo-ulmi U6 snRNA. Intact conidia and protoplasts of O. novo-ulmi will be transformed by Agrobacterium and PEG-mediated insertion respectively. Furthermore, we are testing, in O. novo-ulmi, the ability of a linear plasmid with telomeric ends to behave as an artificial acentric minichromosome, rapidly lost under non-selective conditions. This would allow recycling of the selection marker, and prevent the potential negative effects of constitutive Cas9 expression. A CRISPR–Cas9 gene editing system would be an important advance to investigate gene function in O. novo-ulmi, a species with extremely low rates of homologous recombination.