Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing

Area of Science:

• Plant biotechnology
• Molecular biology
• Genome editing

Background:

• CRISPR-Cas9 and Cas12a are key genome editing tools.
• Traditional plant CRISPR expression uses separate Pol II and Pol III promoters.
• Coordinated expression from a single Pol II promoter is desirable for compact vectors and efficient gene editing.

Purpose of the Study:

• To develop novel single transcript unit (STU) CRISPR 2.0 systems for plant genome editing.
• To enhance CRISPR-Cas9 and CRISPR-Cas12a expression and editing efficiency in plants.
• To expand the CRISPR toolbox for advanced plant genetic applications.

Main Methods:

• Developed two new STU-Cas9 systems (STU-Cas9-Csy4 and STU-Cas9-tRNA) and one STU-Cas12a system.
• Utilized Csy4 ribonuclease and endogenous tRNA processing for sgRNA maturation in STU-Cas9 systems.
• Applied STU systems for transient expression and stable transgenesis in rice, including base editing comparisons.

Main Results:

• STU CRISPR 2.0 systems demonstrated robust genome editing efficiencies in rice, outperforming the first-generation STU-Cas9-RZ and conventional dual promoter systems.
• The STU-Cas9-tRNA system coupled with PmCDA1 showed high efficiency for C to T base editing in rice.
• The STU-Cas12a system successfully expressed single and multiple guide RNAs from a single transcript unit.

Conclusions:

• The STU CRISPR 2.0 systems offer improved and versatile tools for plant genome editing.
• These systems facilitate compact vector design and coordinated expression of CRISPR components.
• The developed systems significantly expand the capabilities for precise genetic modification in plants.