A versatile tool for gene editing in the diatom Thalassiosira pseudonana

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ID: 319275
2026
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Abstract
Diatoms are major contributors to marine primary production and global CO2 fixation, with the centric diatom Thalassiosira pseudonana a powerful model for understanding biogeochemical processes including carbon fixation and silicification. Whilst there are molecular tools available for fluorescent protein (FP) tagging and CRISPR/Cas9 genome editing in T. pseudonana, these require the delivery of multiple vectors or have limited versatility. Additionally, scarless endogenous tagging, that results in a fluorescent protein fusion expressed from its native genomic location, has yet to be developed. Here we describe a versatile modular Golden Gate-based toolkit for T. pseudonana that through the delivery of a single-episome via bacterial conjugation enables: [1] FP tagging, [2] dual FP tagging, [3] CRISPR/Cas9 genome editing, [4] simultaneous FP tagging with gene editing, and [5] scarless endogenous FP tagging. We further expand the available parts for T. pseudonana by validating three additional FPs and two untested promoter/terminator pairs. We demonstrate the versatility of our system by knocking out Diatom Pyrenoid Component 1 (DPC1), whilst simultaneously GFP tagging the Rubisco small subunit (rbcS); and by endogenously GFP tagging the bestrophin-like protein BST2. Whilst DPC1 knock-out does not result in a major pyrenoid structural defect due to unperturbed rbcS-GFP localization to the pyrenoid, we confirm that BST2 localizes to the pyrenoid and exhibits increased fluorescence under low CO2 - supporting a role in diatom carbon fixation. Our developed genetic tools provide a robust framework for exploring cellular processes in diatoms, accelerating routine studies and enabling systematic, quantitative and large-scale studies.
Reference Key
openalex_W7166799452 Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Onyou Nam, Irina Grouneva, Luke C. M. Mackinder
Journal Plant physiology and biochemistry : PPB
Year 2026
DOI
10.1093/plphys/kiag385
URL
https://doi.org/10.1093/plphys/kiag385
Keywords Keywords not found

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