Adenine Phosphoribosyltransferase Is a Universal Counterselectable 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. Although a few Phytoph-thora species have benefited from plasmid-based transformation methods for gene overexpression and RNA interference silencing, these techniques remain inefficient for other oomycete genera such as Pythium and Aphanomyces. Here, we explored the applicability of DNA-free endogenous counterselection 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 Pythium oligandrum and substantial off-target effects in Phytophthora capsici. This one-step approach circumvents the need for protoplast generation and can be broadly applied to oomycetes producing zoospores or oospores.