Auxin affects gene editing efficiency through regulating chromatin accessibility and plant regeneration process

Abstract

Improving gene editing efficiency has been a prominent research focus with the increasing application of CRISPR/Cas9 in crop genetic enhancement. In this study, we demonstrated that increasing exogenous auxin levels during in vitro tissue culture significantly enhances gene editing efficiency, leading to a higher frequency of functionally edited T₀ plants. While higher auxin levels promoted callus growth, it also delayed shoot initiation and slightly decreased shoot regeneration. Subsequent RNA-Seq analysis revealed significant alterations in the expression of plant developmental regulatory genes and chromatin remodeling genes at two plant regeneration stages. Further analysis using nuclei staining and Transposase-Accessible Chromatin using sequencing showed that excessive auxin resulted in a more relaxed chromatin structure in callus cells, thus enhancing the genomic DNA accessibility to Cas9. Additionally, the prolonged growth period of dedifferentiated callus cells and the delay in shoot initiation likely provided additional time for Cas9 to exert its function, explaining the improved gene editing efficiency due to excessive auxin application. To mitigate the inhibitory effects of excessive auxin on shoot regeneration, a ‘two-phase’ culture strategy was developed and validated using tomatoes, in which the explants were first cultured in media containing excessive auxin to promote calli growth and gene editing, then transferred to the media with lower auxin concentrations to promote the following shoots regeneration. Overall, our research has revealed novel aspects of auxin function in gene editing, offering new insights and a theoretical basis for future studies. Furthermore, the proposed culture method could accelerate the application of gene editing across various plant species.

Acknowledgements

We thank Matthew Creech and Chi Nguyen for their invaluable assistance in purchasing chemicals, maintaining seed stocks, and managing equipment and facilities.

Funding

This project is sponsored by the USDA-NIFA grant 2019-67013-29236 and USDA HATCH program FLA-MFC-006387, awarded to H.H. This work was also supported by the Natural Science Basic Research Program of Shaanxi (Program No. 2024JC-YBQN-0184) awarded to Z.L.

Author contributions

H.H., L.L., Z. L. and G.W. conceived and designed experiments; G.W. performed the tobacco auxin screening and collected gene editing data in tobacco. T.J. performed analysis in tomatoes. L.Y., Z.H., H.W., and P.H contributed to sample collection and histological analysis; L.L. conducted bioinformatic analysis and provided critical suggestion in design and implementation of experiments. Z.L. and H.H. wrote the manuscripts and H.G. read and revised the manuscript.

Data availability

The datasets analyzed in this study are publicly available with the accession numbers PRJNA1097301, GSE264167 and GSE280743.

Conflict of interest statement

The authors declare no conflict of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

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