SSA-mediated selection marker gene activation enhances relative gene targeting efficiency in plants

Abstract

The precise manipulation of genome sequences through gene targeting (GT) is beneficial; however, the low efficiency of homology-directed repair (HDR) in seed plants has made GT difficult to achieve. Generation of double-strand breaks (DSBs) at the target DNA site of interest represents a promising approach to facilitate HDR-mediated GT in organisms. Despite recent advances, GT remains a significant challenge in seed plants. To address these challenges, we propose that the efficiency of CRISPR/Cas9-mediated GT could be enhanced by the exclusive selection of plants that exhibit high levels of HDR activity. To test this hypothesis, a surrogate screening system was developed, which consists of a nonfunctional split-selection marker gene. In this system, DSBs generated by CRISPR/Cas9 at the linker sequence of the tandem repeat will be repaired via single-strand annealing (SSA), a subtype of HDR, resulting in the achievement of antibiotic resistance in plants. This approach allows for a 2- to 23-fold increase in precise and heritable GT efficiency in Arabidopsis and rice. The results indicate that screening with SSA-mediated surrogate system can enrich cells and plants with high HDR activity as well as DSB activity, thus facilitating the establishment of highly efficient GTs at target loci in these plants.

Acknowledgements

Expressions of gratitude are extended to all members of the laboratories belonging to the following groups: Epigenetics and Genome Engineering, the Shanghai Center for Plant Stress Biology, CAS Centre for Excellence in Molecular Plant Sciences, and the SUAT Institute of Emerging Agricultural Technology, Shenzhen University of Advanced Technology. The assistance provided by these groups is greatly appreciated. This work was supported by the Shanghai Science and Technology Innovation Plan (20ZR1467000 and 23WZ2500800), the Foreign Expert Project (G202201355L), and the Chinese Academy of Sciences to D.M.

Author contributions

D.L.K. and D.M. designed the research; D.L.K., Y.Q.C., Y.P.K., and D.M. performed the experiments with assistance from X.F.D., X.L., C.N.W.W., C.F.H., and R.Q.Y.; D.L.K. and D.M. wrote the paper. All authors read and approved of the manuscript.

Data availability

The plasmids harboring the Ba-ar and NP-PTII have been deposited with Addgene and are available from their website (Addgene IDs 226 815 and 226 816, respectively). All data supporting the results of this study are presented in the manuscript, including Supplementary information. The datasets generated or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of interest statement

The authors declare that they have no competing interests.

Supplementary data

Supplementary data is available at Horticulture Research online.

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