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High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

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Published on: April 30, 2016

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

Tien Van Vu¹, Ngan Thi Nguyen¹, Jihae Kim¹

¹Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Republic of Korea.

Horticulture Research

|February 5, 2025

Summary

This study enhances CRISPR-Cas gene targeting (GT) in plants by using a dominant-negative ku80 mutant protein (KUDN) to favor homologous recombination (HR) repair. This method significantly boosts GT efficiency for precise plant breeding.

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Published on: July 12, 2013

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.