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bioRxiv · 10.1101/2025.04.18.649274

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

Frangedakis, E.·Yelina, N.·Eeda, S. K.·Romani, F.·Fragkidis, A.·Haseloff, J.·Hibberd, J.
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Abstract

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.

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Frangedakis, E., Yelina, N., Eeda, S. K., Romani, F., Fragkidis, A., Haseloff, J., Hibberd, J.. 2025-04-20. A tRNA-gRNA multiplexing system for CRISPR genome editing in Marchantia polymorpha.. https://doi.org/10.1101/2025.04.18.649274

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