Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice

Area of Science:

• Plant biotechnology
• Genome engineering
• Molecular biology

Background:

• CRISPR/Cas9 and base editors enable targeted genome modification for crop trait improvement.
• Efficient gene editing tools are crucial for advancing plant research.

Purpose of the Study:

• To engineer highly efficient and precise base editors for rice.
• To enhance the efficiency of CRISPR/Cas9-based genome editing in plants.

Main Methods:

• Fused PmCDA1 with CRISPR/Cas9 nickase for cytosine (C)-to-thymine (T) conversion.
• Engineered three high-fidelity SpCas9 variants (eSpCas9(1.1), SpCas9-HF2, HypaCas9) as C-to-T base editors (pBEs).
• Applied a tandemly arrayed tRNA-modified single guide RNA (sgRNA) architecture to improve base-editing efficiency.

Main Results:

• PmCDA1 demonstrated higher efficiency than rAPOBEC1 for C-to-T conversion in rice.
• High-fidelity editors showed distinct multiplex-genome editing efficiencies.
• The tRNA-modified sgRNA architecture enhanced eSpCas9(1.1)-pBE efficiency up to 25.5-fold with acceptable off-target effects.
• Knock-out mutation frequency improved two- to five-fold using high-fidelity Cas9s with the tRNA-modified sgRNA.

Conclusions:

• Engineered a versatile toolkit for efficient and precise rice genome engineering.
• The developed tools offer increased flexibility for plant research and crop improvement applications.