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

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:
Grant/Contract Number:
AC02-05CH11231
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)
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
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
OSTI Identifier:
1361519
Alternate Identifier(s):
OSTI ID: 1393235

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., 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. 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., and Loqué, Dominique. 2017. "Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants". United States. doi: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}
}