Enhancing CRISPR-Cas-based gene targeting in tomato using a dominant-negative ku80

Area of Science:

• Plant biotechnology
• Molecular biology
• Genetics

Background:

• CRISPR-Cas gene targeting (GT) enables precise genomic DNA modification via homologous recombination (HR).
• Plant somatic cells predominantly use canonical non-homologous end-joining (cNHEJ) to repair double-stranded breaks (DSBs), limiting HR-mediated GT.
• Developing methods to enhance HR-mediated GT is crucial for precise plant breeding.

Purpose of the Study:

• To improve gene targeting efficiency in plant somatic cells by shifting the DNA repair pathway preference towards homologous recombination (HR).
• To evaluate the efficacy of a dominant-negative ku80 mutant protein (KUDN) in disrupting canonical non-homologous end-joining (cNHEJ) and promoting HR-mediated gene targeting.

Main Methods:

• Implementation of a dominant-negative ku80 mutant protein (KUDN) to inhibit cNHEJ initiation.
• Application of the KUDN-based approach for gene targeting in plant somatic cells.
• Assessment of gene targeting efficiency at specific tomato loci (SlHKT1;2, SlEPSPS1, SlCAB13).

Main Results:

• KUDN expression resulted in a 1.71- to 3.55-fold increase in GT efficiency at the callus stage.
• A more significant increase in GT efficiency, ranging from 1.62- to 9.84-fold, was observed at the SlHKT1;2 and SlEPSPS1 loci.
• The KUDN-enhanced GT tool successfully facilitated a 9-bp insertion at the SlCAB13 locus, demonstrating practical efficiency.

Conclusions:

• The KUDN-based approach effectively shifts DNA repair towards HR, significantly enhancing gene targeting efficiency in plant somatic cells.
• This method offers a promising tool for more efficient and precise plant breeding applications.
• The successful modification at multiple loci highlights the versatility and potential of this enhanced gene targeting strategy.