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Consolidating Ulva functional genomics: gene editing and new selection systems.

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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.

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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.