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.

The study shows that silencing of NOR2 rRNA genes in Arabidopsis thaliana depends primarily on CHH-context cytosine methylation, particularly mediated by CMT2 and the chromatin remodeler DDM1, rather than CG or CHG methylation. Comparative promoter analysis revealed a prevalence of CHH sites in plant rDNA promoters, explaining why CHH methylation mutants disrupt NOR2 silencing more strongly, while NOR2 loci are hyper‑methylated and more condensed than NOR4.