An Intein-Mediated Split-nCas9 System for Base Editing in Plants.

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genome Editing

Background:

• Virus-assisted delivery of CRISPR/Cas systems offers a promising avenue for plant genome editing.
• The large size of CRISPR/Cas9 components poses challenges for packaging into viral vectors with limited capacity.

Purpose of the Study:

• To develop a strategy for packaging the CRISPR/Cas9 system into viral vectors by splitting its components.
• To demonstrate the efficacy of a split CRISPR-Cas9 system for targeted genome editing in plant cells.

Main Methods:

• A split intein-based strategy was employed to divide CRISPR/Cas9 components across a dual-vector system.
• The system was optimized in plant cells, with successful demonstration of split-eYGFPuv expression.
• A plant-based biosensor was utilized to assess the efficiency of split nCas9 in base editing.

Main Results:

• The split intein system enabled the reassembly of active CRISPR/Cas9 components upon co-infection for plant genome editing.
• Efficient base editing in plant cells was achieved using the split nCas9 system.
• Several split sites were identified, offering potential for future biodesign strategies.

Conclusions:

• The developed split CRISPR-Cas9 strategy effectively overcomes viral vector packaging limitations for plant genome editing.
• This approach facilitates the integration of various CRISPR/Cas9 tools, including base editors and prime editors, with virus-mediated gene editing technologies.
• The study opens new avenues for advancing plant gene editing applications through viral delivery systems.