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Tuning plant phenotypes by precise, graded downregulation of gene expression

Nature Biotechnology volume 41, pages 1758–1764 (2023)Cite this article

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Abstract

The ability to control gene expression and generate quantitative phenotypic changes is essential for breeding new and desired traits into crops. Here we report an efficient, facile method for downregulating gene expression to predictable, desired levels by engineering upstream open reading frames (uORFs). We used base editing or prime editing to generate de novo uORFs or to extend existing uORFs by mutating their stop codons. By combining these approaches, we generated a suite of uORFs that incrementally downregulate the translation of primary open reading frames (pORFs) to 2.5–84.9% of the wild-type level. By editing the 5′ untranslated region of OsDLT, which encodes a member of the GRAS family and is involved in the brassinosteroid transduction pathway, we obtained, as predicted, a series of rice plants with varied plant heights and tiller numbers. These methods offer an efficient way to obtain genome-edited plants with graded expression of traits.

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Fig. 1: Introduced uORFs repress protein expression in protoplasts and plants.
Fig. 2: Extending original uORFs by base editing reduces protein expression in protoplasts and plants.
Fig. 3: Producing uORFs with diverse inhibitory activities to downregulate protein expression in a graded fashion.
Fig. 4: Obtaining mutants with the expected quantitative traits.

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Data availability

All data supporting the findings of this study are available in the article, extended data figures and supplementary information or are available from the corresponding author upon reasonable request. Sequence data are present in The Arabidopsis Information Resource (https://seqviewer.arabidopsis.org/) or Phytozome databases (https://phytozome-next.jgi.doe.gov/) under the following accession numbers: AtABI1 (AT4G26080), AtPYR1 (AT4G17870), AtBRI1 (AT4G39400), OsBRI1 (LOC_Os01g52050), OsGW7 (LOC_Os07g41200), OsDLT (LOC_Os06g03710), OsCKX2 (LOC_Os01g10110), OsTCP19 (LOC_Os06g12230) and OsTB1 (LOC_Os03g49880). The deep sequencing data have been deposited in a National Center for Biotechnology Information BioProject database (accession code PRJNA931443)44. Plasmids for pH-ABE8e and pH-ABE8e-spG will be made available through Addgene. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Key Research and Development Program (2022YFF1002802 to C.G.), the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24020102, to C.G.), the Ministry of Agriculture and Rural Affairs of China to C.G., the National Natural Science Foundation of China (31788103 to C.G. and 31971370 to K.C.), the R&D Program in Key Areas of Guangdong Province (2018B020202005 to C.G.) and the Schmidt Science Fellows to K.T.Z.

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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, Chinese Academy of Sciences, Beijing, China

    Chenxiao Xue, Fengti Qiu, Yuxiang Wang, Boshu Li, Kunling Chen & Caixia Gao

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

    Chenxiao Xue, Yuxiang Wang, Boshu Li & Caixia Gao

  3. Qi Biodesign, Beijing, China

    Kevin Tianmeng Zhao

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Contributions

C.X., K.C. and C.G. designed the project. C.X., F.Q. and Y.W. performed the experiments. B.L. performed rice transformation. C.X., K.T.Z. and C.G. wrote the manuscript. C.G. supervised the project. All authors reviewed the manuscript.

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Correspondence to Caixia Gao.

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The authors have submitted a patent application based on the results reported in this paper. K.T.Z. is a founder and employee at Qi Biodesign.

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Extended data

Extended Data Fig. 1 Creating uORFs in 5′ UTRs.

(a) 5′ UTR and part of CDS of AtABI1 and OsBRI1. Lowercase is the non-uORF sequence in 5′ UTR; black uppercase is the sequence of uORF; gold uppercase is the sequence of pORF; red bold base is the upstream ATG (uATG) sites to be created. (b) Schematic diagram of the dual-luciferase reporter system with or without de novo ATG in 5′ UTR upstream the CDS of LUC. (c) RNA expression of LUC relative to REN in protoplasts. The data were normalized to control (n = 3). All data are presented as mean ± s.e.m. *P < 0.05 by two-tailed Student’s t-test.

Extended Data Fig. 2 Genotypes of prime-edited rice mutants carrying uORFOsBRI1(−99, 28aa).

(a) Editing efficiencies of pegRNAs with PPE2 used to generate uORFOsBRI1(−120, 35aa) or uORFOsBRI1(−99, 28aa) at the endogenous 5′ UTR of OsBRI1 in protoplasts (n = 2). (b) Schematic representation of the pH-ePPE-epegRNA vector. The black arrows indicate three pairs of primers used to detect transgene-free mutants. (c) Sanger sequencing chromatograms of representative prime-edited mutants carrying uORFOsBRI1(−99, 28aa). Red arrows represent the desired edits.

Extended Data Fig. 3 Extending uORFs in 5′ UTRs.

(a) 5′ UTR and part of the CDS of AtABI1, AtPYR1, AtBRI1, OsDLT, OsCKX2 and OsGW7. Lowercase is the non-uORF sequence in 5′ UTR; underlined uppercase is the CDS of uORF; gold uppercase is the CDS of pORF; red bold base is the stop codons to be mutated. (b) Schematic diagram of the dual-luciferase reporter system with original or extended uORF in 5′ UTR upstream the CDS of LUC.

Extended Data Fig. 4 Effects of extended uORFs on LUC/REN mRNA levels in dual-luciferase assay.

RNA expression of LUC relative to REN in protoplasts. The data were normalized to control (n = 3). All data are presented as mean ± s.e.m. *P < 0.05 by two-tailed Student’s t-test.

Extended Data Fig. 5 Genotypes of base-edited rice mutants containing uORFOsDLT(−589, 56aa).

(a) Editing efficiencies of sgRNAs with ABE8e to generating uORFOsDLT(−589, 56aa) at the endogenous 5′ UTR of OsDLT in protoplasts (n = 3). All data are presented as mean ± s.e.m. (b) Schematic representation of the pH-ABE8e-spG vector. (c) Sanger sequencing chromatograms of representative base-edited mutants containing uORFOsDLT(−589, 56aa). Red arrows indicate the desired edits.

Extended Data Fig. 6 5′ UTR and part of the CDS of OsDLT, OsTCP19 and OsTB1.

Lowercase is non-uORF sequence in 5’ UTR; underlined uppercase is the CDS of uORF; gold uppercase is the CDS of pORF; red bold base is the uATG site to be created or stop codon to be mutated.

Extended Data Fig. 7 Editing efficiencies of pegRNAs and sgRNAs used to generate uORFOsDLT(−402, 27aa), uORFOsDLT(−540, 73aa), uORFOsDLT(−141, 42aa) and uORFOsDLT(−105, 30aa) in the endogenous 5′ UTR of OsDLT.

(a) Editing efficiencies of pegRNAs with plant prime editor (PPE2) used to generate uORFOsDLT(−402, 27aa), uORFOsDLT(−141, 42aa) and uORFOsDLT(−105, 30aa) in the endogenous 5′ UTR of OsDLT in protoplasts (n = 2). (b) Editing efficiencies of sgRNAs with adenine base editor (ABE8e) used to generate uORFOsDLT(−540, 73aa) in the endogenous 5′ UTR of OsDLT in protoplasts (n = 3). The data are presented as mean ± s.e.m. (c) Schematic representation of the pH-ABE8e vector.

Extended Data Fig. 8 Sanger sequencing chromatograms of representative mutants containing uORFOsDLT(−402, 27aa), uORFOsDLT(−540, 73aa), uORFOsDLT(−141, 42aa) and uORFOsDLT(−105, 30aa), respectively.

Red arrows indicate the desired edits.

Extended Data Fig. 9 Detection of transgene-free mutants with three pairs of primers based on the pH-ePPE-epegRNA, pH-ABE8e-spG and pH-ABE8e binary vector.

Lanes with no bands generated by the three pairs of primers indicate transgene-free T1 mutants. M represents a DNA molecular weight ladder.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–8 and sequences.

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Source data

Source Data Fig. 1

Unprocessed western blots for Fig. 1.

Source Data Fig. 2

Unprocessed western blots for Fig. 2c.

Source Data Fig. 3

Unprocessed western blots for Fig. 3d.

Source Data Extended Data Fig. 9

Unprocessed agarose gels for Extended Data Fig. 9.

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Xue, C., Qiu, F., Wang, Y. et al. Tuning plant phenotypes by precise, graded downregulation of gene expression. Nat Biotechnol 41, 1758–1764 (2023). https://doi.org/10.1038/s41587-023-01707-w

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