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

ABSTRACT 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 bioproduction of recombinant proteins, supported by a human-like N -linked glycosylation pathway. However, its α (1,3)-linked core fucose is immunogenic in humans and thus limits 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 ( Ph aeodactylum tricornutum Cs y 4- C as9 m u ltiplex t ool), a versatile plasmid-based CRISPR/Cas9 system that uses the Csy4 endoribonuclease to process multi-guide 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 markedly 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.