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

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.

Efficient Gene Editing and Overexpression of Gametophyte Transformation in a Model Fern

“…Previous studies showed that a single Pol II promoter (either constitutive or inducible) can also achieve effective gene editing (Hassan et al, 2021; Cardi et al, 2023) in O. sativa (Tang et al, 2016; Ren et al, 2019), T. aestivum (Luo et al, 2021), Hordeum vulgare, S. lycopersicum, Medicago truncatula (Čermák et al, 2017), and the diatom Phaeodactylum tricornutum (Taparia et al, 2022). The Actin promoter isolated from P. vittata was able to function efficiently in both P. vittate and Ceratopteris thalictroides (Muthukumar et al, 2013).…”

Chemoproteomic profiling of serine hydrolases reveals the dynamic role of lipases inPhaeodactylum tricornutum

“…Fatty acid methyl ester analysis of lipids was performed as described by (Taparia et al, 2022). In brief, 50 x 10 6 cells were collected every 12 h during recovery from N stress and freeze dried.…”

Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-Associated Protein and Its Utility All at Sea: Status, Challenges, and Prospects

“…The lipid accumulation could form lipid droplets (LDs) in microalgae, and the size and structure of LD were related to the LD protein ( LDP ) [ 202 ]. The role of one of the most abundant LDP s, stramenopile-type LDP ( StLDP ), was investigated in diatom using CRISPR/Cas9-mediated genome editing [ 203 , 204 ]. The StLDP mutants showed an expansion in LD size and a decrease in LD number per cell under N-depleted conditions, indicating the role of StLDP as an LD scaffold to regulate LD size and lipid homeostasis in P. tricornutum [ 203 , 204 ].…”