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

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.