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Plasmid-based complementation of large deletions in Phaeodactylum tricornutum biosynthetic genes generated by Cas9 editing.
- Source :
-
Scientific reports [Sci Rep] 2020 Aug 17; Vol. 10 (1), pp. 13879. Date of Electronic Publication: 2020 Aug 17. - Publication Year :
- 2020
-
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. Nanopore long-read sequencing indicates that editing events are characterized by the occurrence of large deletions of up to ~ 2.7 kb centered 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-fluoroorotic acid 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 phosphoribosyltransferase activity. 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.
- Subjects :
- Drug Resistance, Bacterial genetics
Genetic Engineering
Histidine biosynthesis
Orotate Phosphoribosyltransferase metabolism
Tryptophan biosynthesis
Uracil biosynthesis
Ascomycota genetics
Ascomycota metabolism
CRISPR-Cas Systems
Complement System Proteins genetics
Gene Deletion
Gene Editing methods
Plasmids genetics
Subjects
Details
- Language :
- English
- ISSN :
- 2045-2322
- Volume :
- 10
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Scientific reports
- Publication Type :
- Academic Journal
- Accession number :
- 32807825
- Full Text :
- https://doi.org/10.1038/s41598-020-70769-6