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Manipulating gene translation in plants by CRISPR–Cas9-mediated genome editing of upstream open reading frames

Nature Protocols volume 15, pages 338–363 (2020)Cite this article

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Abstract

Gene expression is regulated by multiple processes, and the translation of mRNAs into proteins is an especially critical step. Upstream open reading frames (uORFs) are widespread cis-elements in eukaryotic genes that usually suppress the translation of downstream primary ORFs (pORFs). Here, we describe a protocol for fine-tuning gene translation in plants by editing endogenous uORFs with the CRISPR–Cas9 system. The method we present readily yields transgene-free uorf mutant offspring. We provide detailed protocols for predicting uORFs and testing their effects on downstream pORFs using a dual-luciferase reporter system, designing and constructing single guide RNA (sgRNA)–Cas9 vectors, identifying transgene-free uorf mutants, and finally comparing the mRNA, protein and phenotypic levels of target genes in uorf mutants and controls. Predicting uORFs and confirming their effects in protoplasts takes only 2–3 weeks, and transgene-free mutants with edited target uORFs controlling different levels of pORF translation can be obtained within 4 months. Unlike previous methods, our strategy achieves fine-tuning of gene translation in transgene-free derivatives, which accelerates the analysis of gene function and the improvement of crop traits.

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Fig. 1: Schematic illustration of how gene translation is manipulated by editing upstream open reading frames.
Fig. 2: Overview of manipulation of gene translation by editing uORFs with the CRISPR–Cas9 system.
Fig. 3: Categories of uORFs according to the positions of their start codons and stop codons.
Fig. 4: The anticipated effects of predicted uORFs on pORFs in transient assays.
Fig. 5: Anticipated effects of CRISPR editing on gene translation in uorf mutants.

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Data availability

The authors declare that all data supporting the findings of this study are available in the original paper. Other supporting data are available upon reasonable request to the corresponding author.

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Acknowledgements

This work was supported by grants from the National Transgenic Science and Technology Program (2018ZX0801002B, 2019ZX08010-001, 2018ZX0800102B and 2019ZX08010-003) and the National Natural Science Foundation of China (31788103 and 31971370).

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Authors and Affiliations

  1. State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, the Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China

    Xiaomin Si, Huawei Zhang, Yanpeng Wang, Kunling Chen & Caixia Gao

  2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China

    Xiaomin Si & Caixia Gao

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  1. Xiaomin Si
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Contributions

X.S. performed the experiments; Y.W., X.S. and K.C. designed figures; C.G. supervised the project; X.S., H.Z. and C.G. wrote the manuscript.

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Correspondence to Caixia Gao.

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Peer review information Nature Protocols thanks Jian Lu, Yiping Qi, Changfu Zhu, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Zhang, H. et al. Nat. Biotechnol. 36, 894–898 (2018): https://doi.org/10.1038/nbt.4202

Li, T. et al. Nat. Biotechnol. 36, 1160–1163 (2018): https://doi.org/10.1038/nbt.4273

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Si, X., Zhang, H., Wang, Y. et al. Manipulating gene translation in plants by CRISPR–Cas9-mediated genome editing of upstream open reading frames. Nat Protoc 15, 338–363 (2020). https://doi.org/10.1038/s41596-019-0238-3

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