The green algal genus Micromonas is globally distributed in marine ecosystems and phylogenetically diverse. Some species within genus such as Micromonas commoda have been proposed as potential sentinels for monitoring ocean changes due to their ecological prevalence and environmental sensitivity. Originating from primary endosymbiosis, Micromonas position within the green lineage (Chlorophyta) enables valuable insights into the early evolution of green algae and the origins of land plants. Moreover, this prasinophyte has a small genome, low gene family expansion, and contains key regulators such as phytochrome that are lacking in chlorophyte model organisms. However, functional genomics studies have been constrained by the lack of efficient and stable genome editing tools. Here, we present a robust and highly efficient transformation method for delivering DNA into M. commoda. Using Golden Gate cloning, we engineered a plasmid containing the selective marker ShBle and the reporter gene enhanced Yellow Fluorescent Protein (eYFP), which was subsequently introduced into the cells. Transformed cell lines grew in seawater agarose plates containing zeocin and expressed eYFP as observed by fluorescence microscopy, while maintaining a maximum growth rate comparable to wild-type cells. Furthermore, we assembled a CRISPR/Cas construct via Golden Gate cloning, achieving a 76-nt deletion in the coding region of targeted gene, resulting in a frameshift mutation in M. commoda. This represents the first report of a stable CRISPR/Cas-mediated gene knockout in any prasinophyte, demonstrating the feasibility of genome editing in this important lineage. Thus, our highly operable transformation and genome editing system paves the way for functional genomic studies in prasinophytes.

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