Tuning plant phenotypes by precise, graded downregulation of gene expression

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.

Author information

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

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.

Corresponding author

Correspondence to
Caixia Gao.

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Competing interests

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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Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.

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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 uORF_OsBRI1(−99, 28aa).

(a) Editing efficiencies of pegRNAs with PPE2 used to generate uORF_OsBRI1(−120, 35aa) or uORF_OsBRI1(−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 uORF_OsBRI1(−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 uORF_OsDLT(−589, 56aa).

(a) Editing efficiencies of sgRNAs with ABE8e to generating uORF_OsDLT(−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 uORF_OsDLT(−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 uORF_OsDLT(−402, 27aa), uORF_OsDLT(−540, 73aa), uORF_OsDLT(−141, 42aa) and uORF_OsDLT(−105, 30aa) in the endogenous 5′ UTR of OsDLT.

(a) Editing efficiencies of pegRNAs with plant prime editor (PPE2) used to generate uORF_OsDLT(−402, 27aa), uORF_OsDLT(−141, 42aa) and uORF_OsDLT(−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 uORF_OsDLT(−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.

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

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• Received: 06 November 2022

• Accepted: 07 February 2023

• Published: 09 March 2023

• DOI: https://doi.org/10.1038/s41587-023-01707-w

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