Science & Nature
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.
References
Gao, C. Genome engineering for crop improvement and future agriculture. Cell 184, 1621–1635 (2021).
Song, X. et al. Targeting a gene regulatory element enhances rice grain yield by decoupling panicle number and size. Nat. Biotechnol. 40, 1403–1411 (2022).
Wang, Y. et al. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat. Biotechnol. 32, 947–951 (2014).
Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).
Hannon, G. J. RNA interference. Nature 418, 244–251 (2002).
Bowman, E. K. et al. Bidirectional titration of yeast gene expression using a pooled CRISPR guide RNA approach. Proc. Natl Acad. Sci. USA 117, 18424–18430 (2020).
Rodríguez-Leal, D., Lemmon, Z. H., Man, J., Bartlett, M. E. & Lippman, Z. B. Engineering quantitative trait variation for crop improvement by genome editing. Cell 171, 470–480 (2017).
Hendelman, A. et al. Conserved pleiotropy of an ancient plant homeobox gene uncovered by cis-regulatory dissection. Cell 184, 1724–1739 (2021).
Xue, C., Zhang, H., Lin, Q., Fan, R. & Gao, C. Manipulating mRNA splicing by base editing in plants. Sci. China Life Sci. 61, 1293–1300 (2018).
Yuan, J. et al. Genetic modulation of RNA splicing with a CRISPR-guided cytidine deaminase. Mol. Cell 72, 380–394 (2018).
Barbosa, C., Peixeiro, I. & Romao, L. Gene expression regulation by upstream open reading frames and human disease. PLoS Genet. 9, e1003529 (2013).
Srivastava, A. K., Lu, Y., Zinta, G., Lang, Z. & Zhu, J. K. UTR-dependent control of gene expression in plants. Trends Plant Sci. 23, 248–259 (2018).
Zhang, H. et al. Genome-wide maps of ribosomal occupancy provide insights into adaptive evolution and regulatory roles of uORFs during Drosophila development. PLoS Biol. 16, e2003903 (2018).
Zhang, T., Wu, A., Yue, Y. & Zhao, Y. uORFs: important cis-regulatory elements in plants. Int. J. Mol. Sci. 21, 6238 (2020).
Tran, M. K., Schultz, C. J. & Baumann, U. Conserved upstream open reading frames in higher plants. BMC Genomics 9, 361 (2008).
Niu, R. et al. uORFlight: a vehicle toward uORF-mediated translational regulation mechanisms in eukaryotes. Database (Oxford) 2020, baaa007 (2020).
Chen, Y. et al. PsORF: a database of small ORFs in plants. Plant Biotechnol. J. 11, 2158–2160 (2020).
Ferreira, J. P., Overton, K. W. & Wang, C. L. Tuning gene expression with synthetic upstream open reading frames. Proc. Natl Acad. Sci. USA 110, 11284–11289 (2013).
Lin, Y. et al. Impacts of uORF codon identity and position on translation regulation. Nucleic Acids Res. 47, 9358–9367 (2019).
Kozak, M. Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol. Cell. Biol. 7, 3438–3445 (1987).
Kozak, M. Constraints on reinitiation of translation in mammals. Nucleic Acids Res. 29, 5226–5232 (2001).
Wang, J., Zhang, X., Greene, G. H., Xu, G. & Dong, X. PABP/purine-rich motif as an initiation module for cap-independent translation in pattern-triggered immunity. Cell 185, 3186–3200 (2022).
Zhang, H. et al. Genome editing of upstream open reading frames enables translational control in plants. Nat. Biotechnol. 36, 894–898 (2018).
Xing, S. et al. Fine-tuning sugar content in strawberry. Genome Biol. 21, 230 (2020).
Si, X., Zhang, H., Wang, Y., Chen, K. & Gao, C. Manipulating gene translation in plants by CRISPR–Cas9-mediated genome editing of upstream open reading frames. Nat. Protoc. 15, 338–363 (2020).
Yamamuro, C. et al. Loss of function of a rice brassinosteroid insensitive1 homolog prevents internode elongation and bending of the lamina joint. Plant Cell 12, 1591–1605 (2000).
Lin, Q. et al. Prime genome editing in rice and wheat. Nat. Biotechnol. 38, 582–585 (2020).
Zong, Y. et al. An engineered prime editor with enhanced editing efficiency in plants. Nat. Biotechnol. 40, 1394–1402 (2022).
Nelson, J. W. et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. 40, 402–410 (2022).
Morinaka, Y. et al. Morphological alteration caused by brassinosteroid insensitivity increases the biomass and grain production of rice. Plant Physiol. 141, 924–931 (2006).
Richter, M. F. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat. Biotechnol. 38, 883–891 (2020).
Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR–Cas9 variants. Science 368, 290–296 (2020).
Tong, H. et al. DWARF AND LOW-TILLERING, a new member of the GRAS family, plays positive roles in brassinosteroid signaling in rice. Plant J. 58, 803–816 (2009).
Tong, H. et al. DWARF AND LOW-TILLERING acts as a direct downstream target of a GSK3/SHAGGY-like kinase to mediate brassinosteroid responses in rice. Plant Cell 24, 2562–2577 (2012).
Tong, H. & Chu, C. Functional specificities of brassinosteroid and potential utilization for crop improvement. Trends Plant Sci. 23, 1016–1028 (2018).
Hellens, R. P. et al. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 1, 13 (2005).
Li, C. et al. Expanded base editing in rice and wheat using a Cas9–adenosine deaminase fusion. Genome Biol. 19, 59 (2018).
Lin, Q. et al. High-efficiency prime editing with optimized, paired pegRNAs in plants. Nat. Biotechnol. 39, 923–927 (2021).
Jin, S., Lin, Q., Gao, Q. & Gao, C. Optimized prime editing in monocot plants using PlantPegDesigner and engineered plant prime editors (ePPEs). Nat. Protoc. https://doi.org/10.1038/s41596-022-00773-9 (2022).
Zong, Y. et al. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat. Biotechnol. 36, 950–953 (2018).
Shan, Q. et al. Rapid and efficient gene modification in rice and Brachypodium using TALENs. Mol. Plant 6, 1365–1368 (2013).
Zhai, Z., Jung, H. I. & Vatamaniuk, O. K. Isolation of protoplasts from tissues of 14-day-old seedlings of Arabidopsis thaliana. J. Vis. Exp. 30, e1149 (2009).
Hiei, Y., Ohta, S., Komari, T. & Kumashiro, T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6, 271–282 (1994).
Xue, C. et al. Tuning plant phenotypes by precise, graded downregulation of gene expression. National Center for Biotechnology Information (NCBI) https://dataview.ncbi.nlm.nih.gov/object/PRJNA931443 (2023).
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.
Ethics declarations
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.
Peer review
Peer review information
Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
Supplementary information
Source data
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
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
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41587-023-01707-w
Read More
Chenxiao Xue