A versatile tool for gene editing in the diatom Thalassiosira pseudonana.

Diatoms are major contributors to marine primary production, with the centric diatom Thalassiosira pseudonana a powerful model for understanding biogeochemical processes including carbon fixation and silicification. While 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 FP 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: (i) FP tagging, (ii) dual FP tagging, (iii) CRISPR/Cas9 genome editing, (iv) simultaneous FP tagging with gene editing, and (v) scarless endogenous FP tagging. We further expand the available parts for T. pseudonana by validating 3 additional FPs and 2 untested promoter/terminator pairs. We demonstrate the versatility of our system by knocking out diatom pyrenoid component 1 (DPC1) while simultaneously GFP tagging the rbcS (Rubisco small subunit), and by endogenously GFP tagging the bestrophin-like protein BST2. While DPC1 knockout 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.