Large-scale genome editing in plants: approaches, applications, and future perspectives

Area of Science:

• Plant science
• Genetics
• Biotechnology

Background:

• CRISPR/Cas technology is a revolutionary tool for fundamental research and crop breeding.
• CRISPR screening has advanced to become efficient, simple, and programmable for large-scale genome editing.
• The number of genome-edited crops is rapidly increasing due to CRISPR screening technologies.

Purpose of the Study:

• To describe the general workflow of a CRISPR screen in plants.
• To discuss applications of CRISPR screening in plants, including gene screening, spatiotemporal expression optimization, protein activity evolution, and knockout mutant library establishment.
• To propose a virus-mediated strategy to improve CRISPR screens in plants.

Main Methods:

• Selection of appropriate CRISPR/Cas editors.
• Genome-wide guide RNA design.
• Pooled library construction, massive plant transformation, and high-throughput genotyping.
• Exploration of CRISPR screening applications for gene discovery and trait optimization.
• Consideration of current challenges and limitations in plant CRISPR screening.

Main Results:

• A comprehensive workflow for conducting CRISPR screens in plants is detailed.
• Various applications of CRISPR screening for plant research and breeding are discussed.
• Potential improvements to CRISPR screening methodologies, including a virus-mediated strategy, are proposed.

Conclusions:

• CRISPR screening is a powerful approach for advancing plant science and crop breeding.
• The described workflow and proposed enhancements can accelerate the development of improved plant varieties.
• Further development of CRISPR screening technologies is crucial for future agricultural innovation.