Delivery of the Cas9 or TevCas9 System into Phaeodactylum tricornutum via Conjugation of Plasmids from a Bacterial Donor

Abstract

Diatoms are an ecologically important group of eukaryotic microalgae with properties that make them attractive for biotechnological applications such as biofuels, foods, cosmetics and pharmaceuticals. Phaeodactylum tricornutum is a model diatom with defined culture conditions, but routine genetic manipulations are hindered by a lack of simple and robust genetic tools. One obstacle to efficient engineering of P. tricornutum is that the current selection methods for P. tricornutum transformants depend on the use of a limited number of antibiotic resistance genes. An alternative and more cost-effective selection method would be to generate auxotrophic strains of P. tricornutum by knocking out key genes involved in amino acid biosynthesis, and using plasmid-based copies of the biosynthetic genes as selective markers. Previous work on gene knockouts in P. tricornutum used biolistic transformation to deliver CRISPR-Cas9 system into P. tricornutum. Biolistic transformation of non-replicating plasmids can cause undesired damage to P. tricornutum due to random integration of the transformed DNA into the genome. Subsequent curing of edited cells to prevent long-term overexpression of Cas9 is very difficult as there is currently no method to excise integrated plasmids. This protocol adapts a new method to deliver the Cas9 or TevCas9 system into P. tricornutum via conjugation of plasmids from a bacterial donor cell. The process involves: 1) design and insertion of a guideRNA targeting the P. tricornutum urease gene into a TevCas9 expression plasmid that also encodes a conjugative origin of transfer, 2) installation of this plasmid in Escherichia coli containing a plasmid (pTA-Mob) containing the conjugative machinery, 3) transfer of the TevCas9 expression plasmid into P. tricornutum by conjugation, 4) screening of ex-conjugants for urease knockouts using T7 Endonuclease I and phenotypic screening, and 5) curing of the plasmid from edited cells.