Abstract

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.

Highlights

  • Diatoms are found in diverse environments, from polar to tropical, in both marine and freshwater bodies, in aquatic and soil ecosystems

  • We have demonstrated the creation of a large homozygous deletion in the Stramenopiletype lipid droplet protein (StLDP) gene via a multiplexed CRISPR/Cas9 array expressed from the diatom episome

  • This report describes the essential cloning steps and E. coli strains required for successful accomplishment of multiplexed genome editing

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Summary

Introduction

Diatoms are found in diverse environments, from polar to tropical, in both marine and freshwater bodies, in aquatic and soil ecosystems. In ecosystems with high nutrient availability, diatoms rapidly multiply, followed by a collapse in Multiplexed CRISPR of the StLDP Gene in Phaeodactylum tricornutum population, displaying typical “boom and bust” cycles. Diatoms constitute a significant portion of phytoplankton, with estimated primary productivity of 45%, and they contribute nearly 20–40% of the global oxygen production. This success and dominance in several environments may be ascribed to their widespread horizontal gene transfer (Vancaester et al, 2020) and their distinct evolutionary origin from red algal secondary endosymbiosis (Armbrust, 2009)

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