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Consolidating Ulva functional genomics: gene editing and new selection systems.
Jonas Blomme1,2,3, Júlia Arraiza Ribera2,3, Olivier De Clerck1
1Department of Biology, Phycology Research Group, Ghent University, 9052, Ghent, Belgium.
The New Phytologist
|March 15, 2025
Summary
Researchers developed new molecular tools for the green seaweed Ulva compressa, enabling efficient gene function studies. Blasticidin deaminases serve as selectable markers, and CRISPR-Cas systems facilitate precise gene editing for functional genomics in Ulva.
Area of Science:
- Marine biology
- Functional genomics
- Molecular biology
Background:
- Ulva compressa is a valuable model organism for functional biology research.
- Existing -omics data and transformation tools are available, but more efficient gene function study methods are needed.
- Development of advanced molecular toolkits is crucial for advancing Ulva research.
Purpose of the Study:
- To expand the molecular toolkit for Ulva compressa to enable efficient gene function studies.
- To establish new selectable markers and genome editing tools for Ulva.
- To generate gain- and loss-of-function mutants in Ulva.
Main Methods:
- Screened 14 selective agents to identify Blasticidin deaminases (BSD) as effective selectable markers.
- Utilized Cas9 and Cas12a ribonucleoproteins (RNPs) for targeted mutagenesis and large genomic deletions (up to 20 kb) using ADENINE PHOSPHORIBOSYLTRANSFERASE (APT).
- Employed homology-directed repair and co-editing strategies for targeted insertion of selectable markers into non-marker genes.
- Evaluated 31 vector configurations to optimize mutant generation, identifying bicistronic Cas9-resistance fusions and intron-containing Cas9 as most effective.
- Generated mutants in three non-marker genes using a co-editing strategy.
Main Results:
- Established Blasticidin deaminases (BSD) as a reliable selectable marker for generating stable transgenic Ulva lines.
- Demonstrated the efficacy of Cas9 and Cas12a RNPs for targeted mutagenesis, achieving genomic deletions up to 20 kb.
- Successfully performed targeted gene insertion using homology-directed repair and co-editing strategies.
- Identified optimal vector configurations for efficient mutant generation in Ulva.
- Generated targeted mutants in three distinct non-marker genes.
Conclusions:
- The expanded molecular toolkit significantly enhances the ability to study gene function in Ulva compressa.
- The developed methods enable reliable generation of gain- and loss-of-function mutants.
- Further optimization is needed for vector-based multiplex genome editing in Ulva.
