Cordyceps militaris serves as a critical microbial cell factory for high-value bioactive compounds; however, the scarcity of versatile and sophisticated genome-editing toolsets significantly restricts its systematic metabolic engineering. This study aimed to develop OmniEdit, a unified and highly efficient CRISPR/Cas9-based platform, to streamline diverse and complex genetic modifications for strain engineering. We integrated multiple editing modalities into a standardized workflow utilizing an AMA1-based CRISPR/Cas9 system combined with customized homologous donor templates. The platform's robustness was systematically validated through five distinct engineering tasks delivered via PEG-mediated transformation, including precise point mutation, in situ fluorescent protein tagging, large biosynthetic gene cluster (BGC) deletion, regulatory upstream open reading frame (uORF) disruption, and one-step multiplexed gene knockout. OmniEdit achieved the first CRISPR-based precise point mutation (∼4–5% efficiency) in this fungus. It enabled the surgical excision of an entire ∼26 kb BGC (∼10% efficiency) and efficient one-step dual-gene knockout (∼30% efficiency). Furthermore, targeted uORF editing was implemented to modulate translational efficiency, alongside flexible protein tagging for subcellular analysis. By overcoming technical bottlenecks, OmniEdit provides a standardized, powerful toolkit for functional genomics and the systematic enhancement of C. militaris as a high-performance bioproduction platform. • OmniEdit, a unified CRISPR/Cas9 platform, was developed for Cordyceps militaris . • First reported point mutation and uORF editing in mushroom-forming fungi. • Precise excision of a 26 kb gene cluster and one-step multiplexed knockout achieved. • A standardized toolkit for building high-performance microbial cell factories.
Liu et al. (Sun,) studied this question.