Engineering an optimized hypercompact CRISPR/Cas12j-8 system for efficient genome editing in plants.

Area of Science:

• Molecular Biology
• Biotechnology
• Plant Science

Background:

• The Cas12j-8 nuclease, a compact Type V CRISPR system component, offers potential for crop genome editing due to its small size and TTN PAM recognition.
• However, its low editing efficiency in plants has limited its practical application.

Purpose of the Study:

• To enhance the genome editing efficiency of the Cas12j-8 system in plants.
• To develop a more robust and versatile CRISPR tool for plant biotechnology.

Main Methods:

• Rational engineering of both the Cas12j-8 nuclease and its associated crRNA.
• Testing the engineered system's editing activity in soybean and rice.
• Development of cytosine base editors utilizing the engineered Cas12j-8 system.

Main Results:

• Engineered Cas12j-8 and crRNA significantly improved genome editing efficiency in plants.
• The combined system demonstrated robust activity in soybean and rice, editing previously inaccessible sites.
• Editing efficiency in some cases matched or exceeded SpCas9 and outperformed the Cas12j-2 variant.
• Developed base editors showed a 5.36- to 6.85-fold increase in base-editing efficiency (C to T) without indels.

Conclusions:

• The engineered hypercompact CRISPR/Cas12j-8 system is a highly efficient tool for plant genome editing.
• This enhanced system facilitates both nuclease-mediated cleavage and precise base editing in plants.
• The findings pave the way for advanced crop improvement strategies using CRISPR technology.