Multiplexed Genome Editing via an RNA Polymerase II Promoter-Driven sgRNA Array in the Diatom Phaeodactylum tricornutum: Insights Into the Role of StLDP.

CRISPR/Cas9-mediated genome editing has been demonstrated in the model diatom P. tricornutum, yet the currently available genetic tools do not combine the various advantageous features into a single, easy-to-assemble, modular construct that would allow the multiplexed targeting and creation of marker-free genome-edited lines. In this report, we describe the construction of the first modular two-component transcriptional unit system expressing SpCas9 from a diatom episome, assembled using the Universal Loop plasmid kit for Golden Gate assembly. We compared the editing efficiency of two constructs with orthogonal promoter-terminator combinations targeting the StLDP gene, encoding the major lipid droplet protein of P. tricornutum. Multiplexed targeting of the StLDP gene was confirmed via PCR screening, and lines with homozygous deletions were isolated from primary exconjugants. An editing efficiency ranging from 6.7 to 13.8% was observed in the better performing construct. Selected gene-edited lines displayed growth impairment, altered morphology, and the formation of lipid droplets during nutrient-replete growth. Under nitrogen deprivation, oversized lipid droplets were observed; the recovery of cell proliferation and degradation of lipid droplets were impaired after nitrogen replenishment. The results are consistent with the key role played by StLDP in the regulation of lipid droplet size and lipid homeostasis.

A novel endogenous selection marker for the diatom Phaeodactylum tricornutum based on a unique mutation in phytoene desaturase 1.

Phaeodactylum tricornutum is a well-developed model diatom for both marine ecology and microalgal biotechnology, which has been enabled by the sequenced genome and the availability of gene delivery tools, such as biolistic transformation and E. coli-mediated conjugation. Till now, these tools have mainly relied on two selectable markers of bacterial origin which confer resistance to antibiotics Zeocin and nourseothricin. An alternative cost-effective and preferably endogenous selectable marker would facilitate gene stacking efforts through successive transformation or conjugation. We performed UV-mutagenesis of P. tricornutum to obtain mutations in the phytoene desaturase (PDS) gene, conferring resistance to the bleaching herbicide norflurazon. Two mutants displaying high tolerance to norflurazon and carrying unique mutations in PtPDS1 (PHATRDRAFT_45735) were selected. These mutants revealed novel point mutations at a conserved residue Gly290 to Ser/Arg. Homology-based structural modeling of mutated PDS1, over a resolved crystallographic model of rice PDS1 complexed with norflurazon, suggests steric hindrance by bulkier residue substitution may confer herbicide resistance. We report the characterization of PtPDS1 mutants and the development of the first endogenous selectable marker in diatoms suitable for industrial strain development, with the added benefit of biocontainment. The plasmid carrying the mutated PDS1 as a selection marker and eGFP as a reporter was created. An optimized biolistic transformation system is reported which allowed the isolation of positive transgenic events at the rate of 96.7%. Additionally, the ease of in vivo UV-mutagenesis may be employed as a strategy to create PDS-norflurazon-based selectable markers for other diatoms.