Advancing PAM-less genome editing in soybean using CRISPR-SpRY

Abstract

Although CRISPR-Cas9 technology has been rapidly applied in soybean genetic improvement, it is difficult to achieve the targeted editing of the specific loci in the soybean complex genome due to the limitations of the classical protospacer adjacent motif (PAM). Here, we developed a PAM-less genome editing system mediated by SpRY in soybean. By performing targeted editing of representative agronomic trait targets in soybean and evaluating the results, we demonstrate that the SpRY protein can achieve efficient targeted mutagenesis at relaxed PAM sites in soybean. Furthermore, the SpRY-based cytosine base editor SpRY-hA3A and the adenine base editor SpRY-ABE8e both can accurately induce C-to-T and A-to-G conversion in soybean, respectively. Thus, our data illustrate that the SpRY toolbox can edit the soybean genomic sequence in a PAM-free manner, breaking restrictive PAM barriers in the soybean genome editing technology system. More importantly, our research enriches soybean genome editing tools, which has important practical application value for precise editing and molecular design in soybean breeding.

Acknowledgements

This research was supported by National Natural Science Foundation of China awards 32301801 and U21A20215 to X.C. and X.F., and National Natural Science Foundation of China awards 32270433, 32072045, and 31960423 to Yong Zhang and X.Z.

Author contributions

X.F. and Yong Zhang proposed the project and designed the experiments. Z.Z. and X.T. designed and constructed all the plasmids. X.C. and S.Y. performed the soybean transformation. X.C., S.W., Y.L. and Ye Zhang tested the soybean mutants. X.C. and Z.Z. analyzed the experimental data. X.F., Yong Zhang, X.C., Z.Z., X.T., and Y.H.Z. wrote the manuscript with input from other authors. All authors read and approved the final manuscript.

Conflict of interests

The authors declare no competing interests.

Data availability statement

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

Supplementary data

Supplementary data are available at Horticulture Research online.

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