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An efficient CRISPR–Cas12a promoter editing system for crop improvement

Nature Plants volume 9, pages 588–604 (2023)Cite this article

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Abstract

Promoter editing represents an innovative approach to introduce quantitative trait variation (QTV) in crops. However, an efficient promoter editing system for QTV needs to be established. Here we develop a CRISPR–Cas12a promoter editing (CAPE) system that combines a promoter key-region estimating model and an efficient CRISPR–Cas12a-based multiplexed or singular editing system. CAPE is benchmarked in rice to produce QTV continuums for grain starch content and size by targeting OsGBSS1 and OsGS3, respectively. We then apply CAPE for promoter editing of OsD18, a gene encoding GA3ox in the gibberellin biosynthesis pathway. The resulting lines carry a QTV continuum of semidwarfism without significantly compromising grain measures. Field trials demonstrated that the OsD18 promoter editing lines have the same yield performance and antilodging phenotype as the Green Revolution OsSD1 mutants in different genetic backgrounds. Hence, promoter editing of OsD18 generates a quantitative Green Revolution trait. Together, we demonstrate a CAPE-based promoter editing and tuning pipeline for efficient production of useful QTV continuum in crops.

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Fig. 1: Rational design of the CAPE system.
Fig. 2: Generation of a QTV continuum of the starch content of rice by promoter editing of OsGBSS1.
Fig. 3: Generation of a QTV continuum of rice grain size by promoter editing of OsGS3.
Fig. 4: Generation of a QTV continuum of rice plant height by promoter editing of OsD18.
Fig. 5: Comparison of the levels of active gibberellins and differentially expressed genes between three OsD18-PE lines and controls.
Fig. 6: Field trials of OsD18 promoter editing lines in different genetic backgrounds.
Fig. 7: Summary of genome editing outcomes in this study towards engineering semidwarfism in rice.

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Data availability

Sequence conservation data were downloaded from PlantRegMap. The TF binding motif datasets were downloaded from JASPAR (2020 Core Plants collections). Genetic variations and phenotype data of rice were downloaded from MBKbase. The rice reference genome (MSU/Tigr7) was downloaded from the Rice Genome Annotation Project. The tomato reference genome (SL4.0) was downloaded from the Solanaceae Genomics Network. The maize reference genome (AGPv4) was downloaded from MaizeGDB. Public open chromatin and histone modification datasets were retrieved from the NCBI under the accession numbers GSE26610 and GSM2084219 for rice, GSE164297 and PRJNA381300 for tomato, and PRJNA599454 and PRJNA417726 for maize.

Code availability

The instructions of feature data processing and the complete pipeline of CAPE for calculating aggregate scores and detecting KRs have been deposited to GitHub (https://github.com/zhangtaolab/CAPE).

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Acknowledgements

We thank Q. Qian (China National Rice Research Institute, Chinese Academy of Agricultural Sciences) for providing xiaowei, a OsD18-null mutant in a slightly different Nipponbare background. This research was supported by the National Natural Science Foundation of China (award nos. 32270433, 32101205, 32072045 and 31960423) to Y. Zhang, X.Z. and X.T.; and the National Agricultural Major Science and Technology Program of China (award no. NK2022010204) to Y. Zhang and J.Z.; the Technology Innovation and Application Development Program of Chongqing (award no. CSTC2021JSCX-CYLHX0001) to X.T., J.Z. and Y. Zhang; the Sichuan Science and Technology Program (award nos. 2021JDRC0032, 2021YFH0084 and 2021YFYZ0016) to J.Z. and Y. Zhang; and the Open Foundation of Jiangsu Key Laboratory of Crop Genetics and Physiology (award no. YCSL202009) to J.Z., Y. Zhang and T.Z. It was also supported by the NSF Plant Genome Research Program (grant nos. IOS-2029889 and IOS-2132693) and the MAES Competitive Grants Program (CGP) to Y.Q.

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  1. These authors contributed equally: Jianping Zhou, Guanqing Liu.

Authors and Affiliations

  1. Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China

    Jianping Zhou, Yuxin Zhao, Rui Zhang, Xu Tang, Ling Li, Xinyu Jia, Yachong Guo, Yao He, Qinqin Han, Han Yang, Xuelian Zheng & Yong Zhang

  2. Chongqing Key Laboratory of Plant Resource Conservation and Germplasm Innovation, Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, School of Life Sciences, Southwest University, Chongqing, China

    Jianping Zhou & Yong Zhang

  3. Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding/Jiangsu Key Laboratory of Crop Genetics and Physiology, Agricultural College, Yangzhou University, Yangzhou, China

    Guanqing Liu, Yuechao Wu, Yangshuo Han, Yu Bao, Tao Zhang & Yong Zhang

  4. Key Laboratory of Plant Functional Genomics of the Ministry of Education/Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, China

    Guanqing Liu, Yuechao Wu, Yangshuo Han, Yu Bao & Tao Zhang

  5. Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, China

    Guanqing Liu, Yuechao Wu, Yangshuo Han, Yu Bao & Tao Zhang

  6. Department of Plant Science and Landscape Architecture, University of Maryland, College Park, Rockville, MD, USA

    Yiping Qi

  7. Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, USA

    Yiping Qi

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Contributions

Y. Zhang proposed the project. Y. Zhang, T.Z., Y.Q. and J.Z. conceived and designed the experiments. G.L. and T.Z. developed the CAPE estimating system. J.Z., X.T. and Y. Zhao designed guide RNA and generated all the constructs for CAPE. G.L., Y. Han and T.Z. built the genome browser for CAPE. Y. Zhao, R.Z., L.L., X.J., Y.G., Y. He, H.Y. and X.Z. performed the rice stable transformation and plant material assays. J.Z., R.Z., Y. Zhao, Q.H. and X.J. performed the mRNA-seq and qPCR experiments. Y.W., Y. Han and Y.B. assisted with data analysis. Y. Zhang, Y.Q., T.Z., J.Z. and G.L. analysed the data and wrote the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Yiping Qi, Tao Zhang or Yong Zhang.

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Nature Plants thanks Pengcheng Wei and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Table 1

Supplementary Table 1. The differential expression of rice genes via mRNA-Seq. Table 2. Gibberellin-response genes in rice. Table 3. Gibberellin biosynthesis genes in rice. Table 4. Comparison of promoter editing efficiency in rice. Table 5. Oligonucleotides used in this study.

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Zhou, J., Liu, G., Zhao, Y. et al. An efficient CRISPR–Cas12a promoter editing system for crop improvement. Nat. Plants 9, 588–604 (2023). https://doi.org/10.1038/s41477-023-01384-2

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