RT Journal Article SR Electronic T1 Cas9-generated auxotrophs of Phaeodactylum tricornutum are characterized by small and large deletions that can be complemented by plasmid-based genes JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.04.12.038471 DO 10.1101/2020.04.12.038471 A1 Samuel S. Slattery A1 Helen Wang A1 Daniel J. Giguere A1 Csanad Kocsis A1 Bradley L. Urquhart A1 Bogumil J. Karas A1 David R. Edgell YR 2020 UL http://biorxiv.org/content/early/2020/04/13/2020.04.12.038471.1.abstract AB The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Editing events are characterized by loss of heterozygosity and by the occurrence of large deletions of up to ~2.7-kb centred on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-FOA and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyl transferase activity in knockout strains. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains.Competing Interest StatementThe authors have declared no competing interest.