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

Diatoms are globally significant microalgae that contribute ∼20% of oxygen production and exhibit remarkable metabolic diversity. The marine diatom <i>Phaeodactylum tricornutum</i> has emerged as a promising synthetic biology platform for the bioproduction of recombinant proteins, supported by a human-like <i>N</i>-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 <i>P. tricornutum</i> is the lack of a robust system for simultaneous CRISPR/Cas9 editing at multiple sites. To overcome this limitation, we develop PHYCUT (<i><b>Ph</b>aeodactylum tricornutum</i> Cs<b>y</b>4-<b>C</b>as9 m<b>u</b>ltiplex <b>t</b>ool), 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 <i>FucT</i> genes responsible for α(1,3) fucosylation in <i>P. tricornutum</i>, yielding strains with reduced fucosylation of secreted proteins. PHYCUT enables facile, multiplexed genome engineering in diatoms and provides a foundation for humanizing the <i>P. tricornutum</i> glycosylation pathway to support next-generation algal biotechnology.