Efficient prime editors for heritable multiplex precision genome editing in soybean.

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genetics

Background:

• Prime editing (PE) is a powerful genome editing technology.
• Low efficiency of PE in dicot plants, including soybean, limits its application.
• Development of enhanced PE systems is crucial for crop improvement.

Purpose of the Study:

• To develop an optimized and highly efficient prime editing system for soybean.
• To establish a multiplex prime editing system for simultaneous gene editing in soybean.
• To provide versatile tools for precise and heritable genome editing in soybean breeding.

Main Methods:

• Engineered the prime editor (PE) by modifying Cas9 and reverse transcriptase domains and incorporating a viral protein.
• Co-expressed a dominant-negative GmMLH1 allele to enhance editing.
• Utilized a tRNA processing system and AtU6 promoter for sgRNA expression optimization.
• Developed a Csy4-mediated multiplex PE system (CMMPE) for simultaneous editing.

Main Results:

• The optimized system, GmPEplus, achieved up to 81.3% editing efficiency in stable transgenic soybean lines.
• Optimizing sgRNA expression boosted editing efficiency by 13.1-fold.
• CMMPE enabled simultaneous editing of 2-12 genes in soybean hairy roots and up to 3 genes in stable lines.

Conclusions:

• GmPEplus significantly enhances prime editing efficiency and versatility in soybean.
• CMMPE allows for multiplex and simultaneous genome editing in soybean.
• These advanced editing tools are valuable for soybean breeding and genetic research.