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CRISPR-Combo–mediated orthogonal genome editing and transcriptional activation for plant breeding

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Abstract

CRISPR–Cas nuclease systems, base editors, and CRISPR activation have greatly advanced plant genome engineering. However, the combinatorial approaches for multiplexed orthogonal genome editing and transcriptional regulation were previously unexploited in plants. We have recently established a single Cas9 protein-based CRISPR-Combo platform, enabling efficient multiplexed orthogonal genome editing (double-strand break–mediated genome editing or base editing) and transcriptional activation in plants via engineering the single guide RNA (sgRNA) structure. Here, we provide step-by-step instructions for constructing CRISPR-Combo systems for speed breeding of transgene-free, genome-edited Arabidopsis plants and enhancing rice regeneration with more heritable targeted mutations in a hormone-free manner. We also provide guidance on designing efficient sgRNA, Agrobacterium-mediated transformation of Arabidopsis and rice, rice regeneration without exogenous plant hormones, gene editing evaluation and visual identification of transgene-free Arabidopsis plants with high editing activity. With the use of this protocol, it takes ~2 weeks to establish the CRISPR-Combo systems, 4 months to obtain transgene-free genome-edited Arabidopsis plants and 4 months to obtain rice plants with enrichment of heritable targeted mutations by hormone-free tissue culture.

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Fig. 1: Schematic of Cas9-Act3.0 and CBE-Cas9n-Act3.0 CRISPR-Combo systems.
Fig. 2: Workflow and timeline of the CRISPR-Combo experiments.
Fig. 3: Schematic illustrations of assembling multiplexed sgRNA expression vectors.
Fig. 4: Anticipated results of CBE-Cas9n-Act3.0-mediated speed breeding of transgene-free, genome-edited Arabidopsis plants via activation of AtFT.
Fig. 5: Anticipated results of Cas9-Act3.0-mediated rice regeneration and heritable mutations in a hormone-free manner via Combo-mediated activation of OsBBM1.

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Data availability

No new data were generated for this protocol, and all presented data in this protocol are available in the primary supporting paper23.

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Acknowledgements

This work was supported by NSF Plant Genome Research Program grants (award nos. IOS-1758745 and IOS-2029889), a USDA-NIFA Biotechnology Risk Assessment grant (award no. 2020-33522-32274), the USDA-AFRI Agricultural Innovations Through Gene Editing Program (award no. 2021-67013-34554), National Natural Science Foundation of China (31972417) and the Starry Night Science Fund of Zhejiang University Shanghai Institute for Advanced Study (SN-ZJU-SIAS-0011).

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Authors and Affiliations

  1. Department of Horticulture, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China

    Changtian Pan

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

    Changtian Pan & Yiping Qi

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

    Yiping Qi

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C.P. wrote the manuscript. Y.Q. and C.P. revised the manuscript.

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Correspondence to Changtian Pan or Yiping Qi.

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Competing interests

Y.Q. and C.P. are inventors on a US patent application that has been filed on the CRISPR-Combo system in this protocol. Y.Q. is a consultant for Inari Agriculture.

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Nature Protocols thanks Caixia Gao, Diego Orzaez and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Pan, C. et al. Nat. Plants 7, 942–953 (2021): https://doi.org/10.1038/s41477-021-00953-7

Pan, C. et al. Nat. Plants 8, 513–525 (2022): https://doi.org/10.1038/s41477-022-01151-9

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Pan, C., Qi, Y. CRISPR-Combo–mediated orthogonal genome editing and transcriptional activation for plant breeding. Nat Protoc 18, 1760–1794 (2023). https://doi.org/10.1038/s41596-023-00823-w

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