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

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 <i>Phytophthora</i> 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 <i>Pythium</i> and <i>Aphanomyces</i>. 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 (<i>APT</i>) gene and generated selectable 2-fluoroadenine-resistant mutants in <i>Aphanomyces</i>, <i>Pythium</i>, and <i>Phytophthora</i> 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 <i>APT</i> a suitable selectable marker gene for oomycete research. Whole-genome comparative analyses on CRISPR-edited mutants revealed no or very few additional mutations in <i>A</i>. <i>euteiches</i> and <i>Pythium oligandrum</i> and substantial off-target effects in <i>Phytophthora capsici.</i> This one-step approach circumvents the need for protoplast generation and can be broadly applied to oomycetes producing zoospores or oospores. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.