Expanding the scope of precision editing in seaweeds through the application of a novel CRISPR-Associated Nuclease 12a (Cas12a) -aligned CRISPR system in <i>Ulva prolifera</i>

Abstract Seaweeds, such as the fast-growing green alga Ulva prolifera, can be harnessed as valuable marine crops. The lack of scalable genome-editing tools hampers functional genomics to explore and elucidate algal molecular pathways with industrial importance. Here, we expanded precision genome modification in seaweeds by successfully demonstrating gene editing with a transgene-free AT-rich-targeting Cas system in U. prolifera. By evaluating various delivery buffers, comparing different Cas systems, and optimizing incubation temperatures, we determined suitable conditions for higher applicability of a novel Cas12a-aligned ST8 editor. We obtained more than 50 ST8-mediated knock-out mutants of a toxin-based endogenous marker gene, UpAPT, at 28℃ post-delivery incubations. Our work diversified the applicable genome editing tools in seaweeds, advancing algal functional genomics, and providing more strategies to precisely target unexplored seaweed resources.