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

Area of Science:

• Plant Biology
• Molecular Biology
• Bioengineering

Background:

• Marchia polymorpha is a key model organism for plant biology and bioengineering.
• CRISPR/Cas9 enables precise genome editing, but simultaneous expression of multiple guide RNAs (gRNAs) is limited.
• Enhanced CRISPR/Cas9 versatility is crucial for advanced genetic modifications.

Purpose of the Study:

• To develop a robust method for simultaneous expression of multiple gRNAs in M. polymorpha.
• To improve the capacity and scalability of CRISPR/Cas9-based genome editing.
• To streamline the generation of CRISPR/Cas9 mutants for functional genomics.

Main Methods:

• Adapted OpenPlant kit CRISPR/Cas9 tools for multiple gRNA expression.
• Incorporated transfer RNA (tRNA) sequences for single-transcript expression of gRNAs.
• Developed a simplified and optimized protocol for M. polymorpha thallus transformation.

Main Results:

• Successfully facilitated simultaneous delivery of multiple gRNAs from a single transcript.
• Significantly improved the capacity and scalability of genome editing in M. polymorpha.
• Streamlined the generation of CRISPR/Cas9 mutants through optimized transformation.

Conclusions:

• The developed gene-editing system enhances CRISPR/Cas9 versatility in M. polymorpha.
• This tool offers a time-saving and straightforward approach for functional genomics.
• Enables more comprehensive genetic modifications and genome engineering in this model organism.