Cas9-PE: a robust multiplex gene editing tool for simultaneous precise editing and site-specific random mutation in...

Area of Science:

• Plant Science
• Molecular Biology
• Genetics

Background:

• Advanced gene editing tools are crucial for molecular design breeding, enabling simultaneous gene introduction and elimination.
• Current prime editing (PE) efficiency limits simultaneous editing of multiple mutation types.

Purpose of the Study:

• To develop a versatile and efficient multiplex gene editing system for rice molecular breeding.
• To overcome the limitations of simultaneous multi-type gene editing in plants.

Main Methods:

• Replaced Cas9 nickase (nCas9) with Cas9 to create a Cas9-mediated PE (Cas9-PE) system in rice.
• Established a transgene-free multiplex gene editing system using a co-editing strategy.
• Utilized Agrobacterium-mediated transient expression of edited ALS gene for herbicide resistance selection.

Main Results:

• The Cas9-PE system enables precise editing and site-specific random mutation in rice.
• A transgene-free multiplex gene editing system was successfully established.
• Herbicide resistance was conferred using a precisely edited ALS gene as a selection marker.

Conclusions:

• The developed Cas9-PE system is a versatile and efficient tool for multiplex gene editing in rice.
• This technology advances molecular design breeding by enabling simultaneous editing of multiple genes.
• The co-editing strategy offers a practical approach for transgene-free multiplex gene editing.