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Developing a CRISPR/FrCas9 system for core promoter editing in rice.

Hui Wang1,2, Jian Ding1,2, Jingyan Zhu1,2

  • 1College of Agronomy, Anhui Agricultural University, Hefei, 230036 China.

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|July 8, 2024
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Summary

A new FrCas9 system from Faecalibaculum rodentium enables precise plant genome editing, targeting TATA sequences for efficient core promoter modification and gene expression control.

Keywords:
Base editingCRISPRCore promoterFrCas9Genome editing

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Area of Science:

  • Plant Biotechnology
  • Genome Editing
  • Molecular Biology

Background:

  • Core promoter mutations can significantly alter gene expression.
  • Targeting TA-rich sequences in promoters is challenging for existing Cas9 systems.
  • Need for novel Cas9 variants with different PAM specificities for plant genome editing.

Purpose of the Study:

  • To engineer and characterize a novel FrCas9 system from Faecalibaculum rodentium for plant genome editing.
  • To evaluate the efficiency and specificity of FrCas9 targeting TATA sequences in rice core promoters.
  • To develop FrCas9-based tools for precise gene expression tuning and base editing in plants.

Main Methods:

  • Engineering of the FrCas9 system derived from Faecalibaculum rodentium.
  • Application of FrCas9 for rice genome editing, utilizing TATA sequences as PAMs.
  • Analysis of mutation efficiency, types (homozygous/biallelic), and impact on gene expression and traits (amylose content).

Main Results:

  • FrCas9 efficiently edited rice core promoters using TATA sequences as PAMs, active against all 16 NNTA PAMs.
  • Achieved up to 35.3% editing efficiency in calli and 31.3% homozygous/biallelic mutations in T0 plants.
  • Demonstrated FrCas9's capability for multiplex editing, bidirectional editing, and development of base editors for A·T to G·C conversion.

Conclusions:

  • The FrCas9 system provides a versatile toolset for plant core promoter editing.
  • This technology enables fine-tuning of gene expression and the creation of novel germplasms.
  • FrCas9 expands the toolkit for precise genome engineering in plants, overcoming limitations of previous systems.