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. Meeting these challenges 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. Using a bioinformatics approach, we identified 54 orthologous systems from various bacteria, 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.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis Street, 4th Floor, Emeryville, California 94608, United States.|Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , 1 Cyclotron Road, Berkeley, California 94720, United States.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liang
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis Street, 4th Floor, Emeryville, California 94608, United States.|Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , 1 Cyclotron Road, Berkeley, California 94720, United States.
authors.forename: Sarah
authors.identifier: 0000-0002-9820-9421
authors.initials: S
authors.lastname: Richardson
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis Street, 4th Floor, Emeryville, California 94608, United States.|Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , 1 Cyclotron Road, Berkeley, California 94720, United States.
authors.forename: Jingwei
authors.initials: J
authors.lastname: Yan
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis Street, 4th Floor, Emeryville, California 94608, United States.|Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , 1 Cyclotron Road, Berkeley, California 94720, United States.
authors.forename: Veronica T
authors.initials: VT
authors.lastname: Benites
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis Street, 4th Floor, Emeryville, California 94608, United States.|Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , 1 Cyclotron Road, Berkeley, California 94720, United States.
authors.forename: Clarabelle
authors.initials: C
authors.lastname: Cheng-Yue
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis Street, 4th Floor, Emeryville, California 94608, United States.|Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , 1 Cyclotron Road, Berkeley, California 94720, United States.
authors.forename: Thu
authors.initials: T
authors.lastname: Tran
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis Street, 4th Floor, Emeryville, California 94608, United States.|Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , 1 Cyclotron Road, Berkeley, California 94720, United States.
authors.forename: Jenny
authors.identifier: 0000-0001-6624-636X
authors.initials: J
authors.lastname: Mortimer
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis Street, 4th Floor, Emeryville, California 94608, United States.|Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , 1 Cyclotron Road, Berkeley, California 94720, United States.
authors.forename: Aindrila
authors.initials: A
authors.lastname: Mukhopadhyay
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis Street, 4th Floor, Emeryville, California 94608, United States.|Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , 1 Cyclotron Road, Berkeley, California 94720, United States.
authors.forename: Jay D
authors.identifier: 0000-0002-4910-2222
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis Street, 4th Floor, Emeryville, California 94608, United States.|Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , 1 Cyclotron Road, Berkeley, California 94720, United States.
authors.forename: Henrik V
authors.initials: HV
authors.lastname: Scheller
authors.affiliation: Joint BioEnergy Institute, EmeryStation East, 5885 Hollis Street, 4th Floor, Emeryville, California 94608, United States.|Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory , 1 Cyclotron Road, Berkeley, California 94720, United States.|INSA de Lyon, CNRS, UMR5240, Microbiologie, Adaptation et Pathogénie, Université Claude Bernard Lyon 1 , 10 rue Raphaël Dubois, F-69622, Villeurbanne, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Loqué
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D008954:Models, Biological; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D010944:Plants / Q000235:genetics / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods
chemical_list: D020121:5' Untranslated Regions; D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D010940:Plant Proteins; C552242:Csy4 endoribonuclease, Pseudomonas aeruginosa; D004722:Endoribonucleases
keywords: CRISPR/Cas9; Csy4; endoribonuclease; gene regulation; synthetic biology; two-component repressor system