The authors adapted OpenPlant kit CRISPR/Cas9 tools to enable multiplex gRNA expression from a single transcript using tRNA sequences in the liverwort Marchantia polymorpha, markedly enhancing editing efficiency and scalability. They coupled this vector system with a simplified, optimized thallus transformation protocol, providing a rapid and versatile platform for generating CRISPR/Cas9 mutants and advancing functional genomics in this model species.

Phylogenetic analysis reveals that non‑seed plants, exemplified by the liverwort Marchantia polymorpha, possess a streamlined repertoire of cyclin and CDK genes, with only three cyclins active in a phase‑specific manner during vegetative development. Single‑cell RNA‑seq and fluorescent reporter assays, combined with functional overexpression studies, demonstrate the distinct, non‑redundant roles of MpCYCD;1, MpCYCA, and MpCYCB;1 in G1 entry, S‑phase progression, and G2/M transition, respectively.