A tRNA-gRNA multiplexing system for CRISPR genome editing in <i>Marchantia polymorpha</i>

The liverwort Marchantia polymorpha is a widely used model organism for studying land-plant biology, and it has also proven to be a promising testbed for bioengineering. CRISPR/Cas9 technology has become a transformative tool for precise genome modifications in M. polymorpha; however, a robust method for the simultaneous expression of multiple gRNAs, which is crucial for enhancing the versatility of CRISPR/Cas9-based genome editing, has yet to be fully developed. In this study, we introduce an adaptation from the OpenPlant kit CRISPR/Cas9 tools that facilitates expression of multiple gRNAs from a single transcript through incorporation of tRNA sequences. The ability to deliver multiple gRNAs simultaneously significantly improves the capacity and scalability of genome editing in M. polymorpha. Additionally, by combining this vector system with a simplified and optimized protocol for thallus transformation, we further streamline the generation of CRISPR/Cas9 mutants in M. polymorpha. The resulting gene-editing system offers a multipurpose, time-saving, and straightforward tool for advancing functional genomics in M. polymorpha, enabling more comprehensive genetic modifications and genome engineering.