Developing a highly efficient CGBE base editor in watermelon

Abstract

Cytosine and adenosine base editors (CBEs and ABEs) are novel genome-editing tools that have been widely utilized in molecular breeding to precisely modify single-nucleotide polymorphisms (SNPs) critical for plant agronomic traits and species evolution. However, conventional BE editors are limited to achieve C-to-T and A-to-G substitutions, respectively. To enhance the applicability of base editing technology in watermelon, we developed an efficient CGBE editor (SCGBE2.0) by removing the uracil glycosylase inhibitor (UGI) unit from the commonly used hA3A-CBE and incorporating the uracil-DNA glycosylase (UNG) component. Seven specific guide RNAs (sgRNAs) targeting five watermelon genes were designed to assess the editing efficiency of SCGBE. The results obtained from stably transformed watermelon plants demonstrated that SCGBE2.0 could efficiently induce C-to-G mutations at positions C5–C9 in 43.2% transgenic plants (with a maximum base conversion efficiency of 46.1%) and C-to-A mutation at position C4 in 23.5% transgenic plants (with a maximum base conversion efficiency of 45.9%). These findings highlight the capability of our integrated SCGBE2.0 editor to achieve C-to-G/A mutations in a site-preferred manner, thus providing an efficient base editing tool for precise base modification and site-directed saturated mutagenesis in watermelon.

Acknowledgements

We thank Dr Huanbin Zhou for his technical advisement and support. This work was supported by the National Youth Talent Program (A279021801), Earmarked Fund for China Agriculture Research System (CARS-25), Key-Area R&D Program of Guangdong Province (2022B0202060001), Key R&D Program of Shaanxi province (2023-YBNY-008), the Natural Science Foundation of Shaanxi Province (2022JM-112), the Fundamental Research Funds for the Central Universities(2452022111) and the Science and Technology Innovation Team of Shaanxi (2021TD-32).

Author contributions

L.Y. conceived the study. D.W., Y.C., T.Z., J.W., M.L., S.T. and J.W. executed the experiments. L.Y. and D.W. wrote the manuscript.

Data availability statement

All data generated in this study are presented in the paper or the supplementary material. Additional data related to this paper may be requested from the authors.

Conflict of interests

We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

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