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Title: Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants

Abstract

Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits. In meeting these challenges we will facilitate transgene expression regulation and support the fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5'UTR of mRNA, we developed a two-component expression–repression system to tightly control synthesis of transgene products. We demonstrated that this regulatory device was functional in monocotyledonous and dicotyledonous plant species, and showed that it can be used to repress transgene expression by >400-fold and to synchronize transgene repression. In addition to tissue-specific transgene repression, this system offers stimuli-dependent expression control. Here, we identified 54 orthologous systems from various bacteria, using a bioinformatics approach and then validated in planta the activity for a few of those systems, demonstrating the potential diversity of such a two-component repressor system.

Authors:
 [1]; ORCiD logo [1];  [1];  [1];  [1];  [1]; ORCiD logo [1];  [1]; ORCiD logo [2];  [3];  [4]
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Environmental Genomics and Systems Biology Division
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Environmental Genomics and Systems Biology Division; Univ. of California, Berkeley, CA (United States). Dept. of Bioengineering, Dept. of Chemical and Biomolecular Engineering
  3. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Environmental Genomics and Systems Biology Division; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology
  4. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Environmental Genomics and Systems Biology Division; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology; National Inst. of Applied Sciences (INSA), Lyon (France)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1361519
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 6; Journal Issue: 5; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; CRISPR/Cas9; Csy4; endoribonuclease; gene regulation; synthetic biology; two-component repressor system

Citation Formats

Liang, Yan, Richardson, Sarah, Yan, Jingwei, Benites, Veronica T., Cheng-Yue, Clarabelle, Tran, Thu, Mortimer, Jenny, Mukhopadhyay, Aindrila, Keasling, Jay D., Scheller, Henrik V., and Loqué, Dominique. Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants. United States: N. p., 2017. Web. doi:10.1021/acssynbio.6b00295.
Liang, Yan, Richardson, Sarah, Yan, Jingwei, Benites, Veronica T., Cheng-Yue, Clarabelle, Tran, Thu, Mortimer, Jenny, Mukhopadhyay, Aindrila, Keasling, Jay D., Scheller, Henrik V., & Loqué, Dominique. Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants. United States. https://doi.org/10.1021/acssynbio.6b00295
Liang, Yan, Richardson, Sarah, Yan, Jingwei, Benites, Veronica T., Cheng-Yue, Clarabelle, Tran, Thu, Mortimer, Jenny, Mukhopadhyay, Aindrila, Keasling, Jay D., Scheller, Henrik V., and Loqué, Dominique. Tue . "Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants". United States. https://doi.org/10.1021/acssynbio.6b00295. https://www.osti.gov/servlets/purl/1361519.
@article{osti_1361519,
title = {Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants},
author = {Liang, Yan and Richardson, Sarah and Yan, Jingwei and Benites, Veronica T. and Cheng-Yue, Clarabelle and Tran, Thu and Mortimer, Jenny and Mukhopadhyay, Aindrila and Keasling, Jay D. and Scheller, Henrik V. and Loqué, Dominique},
abstractNote = {Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits. In meeting these challenges we will facilitate transgene expression regulation and support the fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5'UTR of mRNA, we developed a two-component expression–repression system to tightly control synthesis of transgene products. We demonstrated that this regulatory device was functional in monocotyledonous and dicotyledonous plant species, and showed that it can be used to repress transgene expression by >400-fold and to synchronize transgene repression. In addition to tissue-specific transgene repression, this system offers stimuli-dependent expression control. Here, we identified 54 orthologous systems from various bacteria, using a bioinformatics approach and then validated in planta the activity for a few of those systems, demonstrating the potential diversity of such a two-component repressor system.},
doi = {10.1021/acssynbio.6b00295},
journal = {ACS Synthetic Biology},
number = 5,
volume = 6,
place = {United States},
year = {2017},
month = {1}
}

Works referenced in this record:

Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein
journal, December 2011


Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency
journal, January 2015

  • Eudes, Aymerick; Sathitsuksanoh, Noppadon; Baidoo, Edward E. K.
  • Plant Biotechnology Journal, Vol. 13, Issue 9
  • DOI: 10.1111/pbi.12310

An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3
journal, May 2011

  • Sashital, Dipali G.; Jinek, Martin; Doudna, Jennifer A.
  • Nature Structural & Molecular Biology, Vol. 18, Issue 6
  • DOI: 10.1038/nsmb.2043

Sequence- and Structure-Specific RNA Processing by a CRISPR Endonuclease
journal, September 2010

  • Haurwitz, R. E.; Jinek, M.; Wiedenheft, B.
  • Science, Vol. 329, Issue 5997, p. 1355-1358
  • DOI: 10.1126/science.1192272

ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering
journal, July 2013


Detecting microRNA binding and siRNA off-target effects from expression data
journal, November 2008

  • van Dongen, Stijn; Abreu-Goodger, Cei; Enright, Anton J.
  • Nature Methods, Vol. 5, Issue 12
  • DOI: 10.1038/nmeth.1267

Cell-to-cell and long-distance siRNA movement in plants: mechanisms and biological implications
journal, October 2011

  • Brosnan, Christopher Andrew; Voinnet, Olivier
  • Current Opinion in Plant Biology, Vol. 14, Issue 5
  • DOI: 10.1016/j.pbi.2011.07.011

PlantGDB: a resource for comparative plant genomics
journal, December 2007

  • Duvick, J.; Fu, A.; Muppirala, U.
  • Nucleic Acids Research, Vol. 36, Issue Database
  • DOI: 10.1093/nar/gkm1041

Isolation of a strong Arabidopsis guard cell promoter and its potential as a research tool
journal, January 2008

  • Yang, Yingzhen; Costa, Alex; Leonhardt, Nathalie
  • Plant Methods, Vol. 4, Issue 1
  • DOI: 10.1186/1746-4811-4-6

Chemically regulated gene expression in plants
journal, April 2003


RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors
journal, November 2014

  • Piatek, Agnieszka; Ali, Zahir; Baazim, Hatoon
  • Plant Biotechnology Journal, Vol. 13, Issue 4
  • DOI: 10.1111/pbi.12284

Csy4 relies on an unusual catalytic dyad to position and cleave CRISPR RNA: Mechanism of CRISPR RNA cleavage
journal, April 2012

  • Haurwitz, Rachel E.; Sternberg, Samuel H.; Doudna, Jennifer A.
  • The EMBO Journal, Vol. 31, Issue 12
  • DOI: 10.1038/emboj.2012.107

Synthetic biology to access and expand nature's chemical diversity
journal, February 2016

  • Smanski, Michael J.; Zhou, Hui; Claesen, Jan
  • Nature Reviews Microbiology, Vol. 14, Issue 3
  • DOI: 10.1038/nrmicro.2015.24

Tight regulation of plant immune responses by combining promoter and suicide exon elements
journal, July 2015

  • Gonzalez, Tania L.; Liang, Yan; Nguyen, Bao N.
  • Nucleic Acids Research, Vol. 43, Issue 14
  • DOI: 10.1093/nar/gkv655

Identification of an essential upstream element in the nopaline synthase promoter by stable and transient assays
journal, August 1987

  • Ebert, P. R.; Ha, S. B.; An, G.
  • Proceedings of the National Academy of Sciences, Vol. 84, Issue 16
  • DOI: 10.1073/pnas.84.16.5745

CRISPR-Cas systems for editing, regulating and targeting genomes
journal, March 2014

  • Sander, Jeffry D.; Joung, J. Keith
  • Nature Biotechnology, Vol. 32, Issue 4
  • DOI: 10.1038/nbt.2842

Transgene regulation in plants by alternative splicing of a suicide exon
journal, February 2012

  • Hickey, Scott F.; Sridhar, Malathy; Westermann, Alexander J.
  • Nucleic Acids Research, Vol. 40, Issue 10
  • DOI: 10.1093/nar/gks032

Gene silencing in plants using artificial microRNAs and other small RNAs: Engineering small RNA-mediated gene silencing
journal, February 2008


The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats
journal, January 2007

  • Grissa, Ibtissem; Vergnaud, Gilles; Pourcel, Christine
  • BMC Bioinformatics, Vol. 8, Issue 1
  • DOI: 10.1186/1471-2105-8-172

KEGG as a reference resource for gene and protein annotation
journal, October 2015

  • Kanehisa, Minoru; Sato, Yoko; Kawashima, Masayuki
  • Nucleic Acids Research, Vol. 44, Issue D1
  • DOI: 10.1093/nar/gkv1070

A transcription activation system for regulated gene expression in transgenic plants
journal, January 1998

  • Moore, I.; Galweiler, L.; Grosskopf, D.
  • Proceedings of the National Academy of Sciences, Vol. 95, Issue 1
  • DOI: 10.1073/pnas.95.1.376

Computational discovery of soybean promoter cis -regulatory elements for the construction of soybean cyst nematode-inducible synthetic promoters
journal, June 2014

  • Liu, Wusheng; Mazarei, Mitra; Peng, Yanhui
  • Plant Biotechnology Journal, Vol. 12, Issue 8
  • DOI: 10.1111/pbi.12206

Transactivated and chemically inducible gene expression in plants
journal, February 2006


Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor
journal, March 1999

  • Kasuga, Mie; Liu, Qiang; Miura, Setsuko
  • Nature Biotechnology, Vol. 17, Issue 3
  • DOI: 10.1038/7036

Antisense Down-Regulation of 4CL Expression Alters Lignification, Tree Growth, and Saccharification Potential of Field-Grown Poplar
journal, August 2010

  • Voelker, Steven L.; Lachenbruch, Barbara; Meinzer, Frederick C.
  • Plant Physiology, Vol. 154, Issue 2
  • DOI: 10.1104/pp.110.159269

Cinnamate-4-Hydroxylase Expression in Arabidopsis (Regulation in Response to Development and the Environment)
journal, March 1997

  • Bell-Lelong, D. A.; Cusumano, J. C.; Meyer, K.
  • Plant Physiology, Vol. 113, Issue 3
  • DOI: 10.1104/pp.113.3.729

A glucocorticoid-mediated transcriptional induction system in transgenic plants
journal, March 1997


Multiplexed and Programmable Regulation of Gene Networks with an Integrated RNA and CRISPR/Cas Toolkit in Human Cells
journal, May 2014


Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4
journal, September 2015

  • Borchardt, Erin K.; Vandoros, Leonidas A.; Huang, Michael
  • RNA, Vol. 21, Issue 11
  • DOI: 10.1261/rna.051227.115

A 28 nt long synthetic 5′UTR (synJ) as an enhancer of transgene expression in dicotyledonous plants
journal, November 2012


A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation
journal, August 2015

  • Lowder, Levi G.; Zhang, Dengwei; Baltes, Nicholas J.
  • Plant Physiology, Vol. 169, Issue 2
  • DOI: 10.1104/pp.15.00636

Characteristics of Plant Essential Genes Allow for within- and between-Species Prediction of Lethal Mutant Phenotypes
journal, August 2015

  • Lloyd, John P.; Seddon, Alexander E.; Moghe, Gaurav D.
  • The Plant Cell, Vol. 27, Issue 8
  • DOI: 10.1105/tpc.15.00051

Mechanism of substrate selection by a highly specific CRISPR endoribonuclease
journal, February 2012


Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana
journal, December 1998


Plant synthetic biology
journal, May 2015


Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants
journal, November 2006

  • Sparkes, Imogen A.; Runions, John; Kearns, Anne
  • Nature Protocols, Vol. 1, Issue 4
  • DOI: 10.1038/nprot.2006.286

MicroRNA-mediated gene regulation: potential applications for plant genetic engineering
journal, June 2013


Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants
journal, April 2014

  • Dugdale, Benjamin; Mortimer, Cara L.; Kato, Maiko
  • Nature Protocols, Vol. 9, Issue 5
  • DOI: 10.1038/nprot.2014.068

Control of gene expression in tobacco cells using a bacterial operator-repressor system.
journal, April 1992


Design, implementation and practice of JBEI-ICE: an open source biological part registry platform and tools
journal, June 2012

  • Ham, T. S.; Dmytriv, Z.; Plahar, H.
  • Nucleic Acids Research, Vol. 40, Issue 18
  • DOI: 10.1093/nar/gks531

Standards for plant synthetic biology: a common syntax for exchange of DNA parts
journal, July 2015

  • Patron, Nicola J.; Orzaez, Diego; Marillonnet, Sylvestre
  • New Phytologist, Vol. 208, Issue 1
  • DOI: 10.1111/nph.13532

Biotechnology and synthetic biology approaches for metabolic engineering of bioenergy crops
journal, June 2016

  • Shih, Patrick M.; Liang, Yan; Loqué, Dominique
  • The Plant Journal, Vol. 87, Issue 1
  • DOI: 10.1111/tpj.13176

A library of synthetic transcription activator‐like effector‐activated promoters for coordinated orthogonal gene expression in plants
journal, April 2015

  • Brückner, Kathleen; Schäfer, Petra; Weber, Ernst
  • The Plant Journal, Vol. 82, Issue 4
  • DOI: 10.1111/tpj.12843

Initiation factors that bind mRNA. A comparison of mammalian factors with wheat germ factors.
journal, April 1988


Works referencing / citing this record:

Agrobacterium-mediated transient transformation of sorghum leaves for accelerating functional genomics and genome editing studies
journal, February 2020


A screening method to identify efficient sgRNAs in Arabidopsis, used in conjunction with cell-specific lignin reduction
journal, May 2019


Plant synthetic biology could drive a revolution in biofuels and medicine
journal, September 2018


Current approaches for RNA-labelling to identify RNA-binding proteins
journal, March 2019

  • Gemmill, Darren; D’souza, Simmone; Meier-Stephenson, Vanessa
  • Biochemistry and Cell Biology
  • DOI: 10.1139/bcb-2019-0041