Adenine Phosphoribosyltransferase Is a Universal Counter-Selectable Marker for DNA-Free Genome Editing in Oomycetes

Abstract

CRISPR-Cas genome editing is a powerful tool for understanding the pathogenicity of oomycetes, a group that includes several destructive plant parasites. While few Phytophthora species have benefited from plasmid-based transformation methods for gene overexpression and RNAi silencing, these techniques remain inefficient for other oomycete genera such as Pythium and Aphanomyces. Here, we explored the applicability of DNA-free endogenous counter-selection in filamentous oomycetes, using CRISPR-Cas9 ribonucleoproteins (RNPs). We used biolistics to deliver RNPs targeting the Adenine phosphoribosyltransferase (APT) gene, and generated selectable 2-fluoroadenine-resistant mutants in Aphanomyces, Pythium, and Phytophthora species. Targeted mutagenesis resulted in various deletions at the expected cut-sites, confirming efficient genome editing. Knockout mutants exhibited no alterations in growth or virulence, making APT a suitable selectable marker gene for oomycete research. Whole genome comparative analyses on CRISPR-edited mutants revealed no or very few additional mutations in A. euteiches and P. oligandrum, and substantial off-target effects in P. capsici. This one-step approach circumvents the need for protoplast generation and can be broadly applied to oomycetes producing zoospores or oospores.