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

Abstract

The CRISPR-Cas-based gene targeting (GT) method has enabled precise modifications of genomic DNA ranging from single base to several kilobase scales through homologous recombination (HR). In plant somatic cells, canonical non-homologous end-joining (cNHEJ) is the predominant mechanism for repairing double-stranded breaks (DSBs), thus limiting the HR-mediated GT. In this study, we implemented an approach to shift the repair pathway preference toward HR by using a dominant-negative ku80 mutant protein (KUDN) to disrupt the initiation of cNHEJ. The employment of KUDN conferred a 1.71- to 3.55-fold improvement in GT efficiency at the callus stage. When we screened transformants, there was a more remarkable increase in GT efficiency, ranging from 1.62- to 9.84-fold, at two specific tomato loci, SlHKT1;2 and SlEPSPS1. With practical levels of efficiency, this enhanced KUDN-based GT tool successfully facilitated a 9-bp addition at an additional locus, SlCAB13. These findings provide another promising method for more efficient and precise plant breeding.

Acknowledgements

This work was supported by the National Research Foundation of Korea (Program 2020M3A9I4038352, 2020R1A6A1A03044344, 2021R1A5A8029490, 2022R1A2C3010331) and the Program for New Plant Breeding Techniques (NBT, Grant PJ01686702), Rural Development Administration (RDA), Korea.

Author contributions

T.V.V. and J.Y.K. conceived and designed the research. T.V.V., N.T.N., J.K., M.H.V., Y.J.S., M.T.T., and Y.W.S. conducted experiments. T.V.V., N.T.N., and J.Y.K. analyzed data. T.V.V., N.T.N., and J.Y.K. wrote the manuscript. T.V.V. and J.Y.K. finalized the manuscript. All authors read and approved the manuscript.

Data availability

The data underlying this article are available in the article and in its online supplementary material.

Conflict of interest statement

J.Y.K is a founder and CEO of Nulla Bio Inc. The remaining authors declare that the work was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. The authors have submitted a patent application based on the results reported in this article.

Supplementary Data

Supplementary data is available at Horticulture Research online.

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