Cas9-generated auxotrophs ofPhaeodactylum tricornutumare characterized by small and large deletions that can be complemented by plasmid-based genes

Abstract

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. pathways of P. tricornutum and show for the first time that plasmid-based copies of the intact PtUMPS and PtPRA-PH/CH genes can complement the uracil-and histidine-requiring phenotypes, respectively. Individual auxotroph strains are characterized by loss of heterozygosity at the edited alleles, and Nanopore sequencing of the edited population reveals large, heterogeneous deletions up to ∼ 2.7 kb. The uracil and histidine auxotrophs and their respective complementation markers are a potential alternative to antibiotic-based selection of plasmids in P. tricornutum. Our results also suggest a simple methodology to cure plasmids from uracil auxotrophs to enable strain and genome engineering.