Breaking the cell wall for efficient DNA delivery to diatoms.

Diatoms produce 20% of the world's fixed organic carbon yet remain underutilized as cell factories due to limited genetic engineering tools. Here, we present optimized electroporation and polyethylene glycol (PEG) transformation methods for the model diatom Phaeodactylum tricornutum, enabling delivery of DNA and Cas9 ribonucleoprotein complexes with high efficiency. Transformants are recovered with as little as 1 ng of DNA, and linear or circular episomes as large as 55.6 kb are successfully introduced. The optimized electroporation protocol also reveals an unexpected capability: episomes can be assembled directly in the algal cell through non-homologous or homology-driven repair mechanisms, a process we term diatom in vivo assembly (DIVA). In addition, the PEG approach is adapted to successfully transform Thalassiosira pseudonana, demonstrating the applicability of our methods for engineering other diatom species. These tools could be used to accelerate diatom synthetic biology projects and, therefore, the development of sustainable technologies.