Exploiting the efficient Exo:Cas12i3-5M fusions for robust single and multiplex gene editing in rice

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genome Editing

Background:

• CRISPR/Cas12i3 is a promising gene editing system due to its small size and flexible PAM recognition.
• Previous Cas12i3 systems showed lower editing efficiency, limiting their application in multiplex gene editing in plants.
• Enhancing Cas12i3 efficiency is crucial for functional genomics and pyramiding beneficial traits in crop improvement.

Purpose of the Study:

• To develop a more efficient single and multiplex gene editing system in rice using CRISPR/Cas12i3.
• To systematically evaluate the performance of different exonuclease (Exo) fusions with an optimized Cas12i3 variant (Cas12i3-5M).
• To establish a robust gene editing tool for facilitating crop improvement through pyramiding agronomically important traits.

Main Methods:

• Fused four 5' exonucleases (T5E, UL12, PapE, ME15) to the N-terminus of an optimized Cas12i3 variant (Cas12i3-5M).
• Systematically evaluated the editing efficiencies of these Exo:Cas12i3-5M fusions across six endogenous targets in rice stable lines.
• Assessed both single and multiplex gene editing capabilities of the developed fusion systems.

Main Results:

• Exo:Cas12i3-5M fusions significantly increased gene editing efficiencies, up to 12.46-fold compared to Cas12i3.
• The UL12:Cas12i3-5M fusion achieved high single gene editing efficiencies (90.42%-98.61%) across six targets.
• UL12:Cas12i3-5M demonstrated superior multiplex editing, achieving efficiencies of 82.76% for three genes and 51.06% for six genes.

Conclusions:

• The UL12:Cas12i3-5M fusion system is a robust and highly efficient tool for both single and multiplex gene editing in rice.
• This enhanced system expands the available plant genome editing toolkits.
• It will facilitate the pyramiding of desirable traits for accelerated crop improvement.