PHYCUT: Scalable Multiplex CRISPR/Cas9 Editing for Genome Engineering in the Diatom Phaeodactylum tricornutum

Diatoms are globally significant microalgae that contribute ∼20% of oxygen production and exhibit remarkable metabolic diversity. The marine diatom Phaeodactylum tricornutum has emerged as a promising synthetic biology platform for the bioproduction of recombinant proteins, supported by a human-like N-linked glycosylation pathway. However, its α(1,3)-linked core fucose is potentially immunogenic in humans and thus limits its biopharmaceutical applications. One hurdle to efficient genome engineering in P. tricornutum is the lack of a robust system for simultaneous CRISPR/Cas9 editing at multiple sites. To overcome this limitation, we develop PHYCUT (Phaeodactylum tricornutum Csy4-Cas9 multiplex tool), a versatile plasmid-based CRISPR/Cas9 system that uses the Csy4 endoribonuclease to process multiguide RNA arrays. To highlight PHYCUT applications, we demonstrate multiplex editing of all three FucT genes responsible for α(1,3) fucosylation in P. tricornutum, yielding strains with reduced fucosylation of secreted proteins. PHYCUT enables facile, multiplexed genome engineering in diatoms and provides a foundation for humanizing the P. tricornutum glycosylation pathway to support next-generation algal biotechnology.