A tRNA-gRNA multiplexing system for CRISPR genome editing in Marchantia polymorpha

The liverwort Marchantia polymorpha is a widely used model organism for studying land plant biology, which has also proven to be a promising testbed for bioengineering. CRISPR/Cas9 technology has emerged as 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 efficiency and 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. This approach significantly improves the efficiency 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 versatile, time-saving and straightforward tool for advancing functional genomics in M. polymorpha , enabling more comprehensive genetic modifications and genome engineering.