title: A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard.
abstract: BACKGROUND
  The efficiency, versatility and multiplexing capacity of RNA-guided genome engineering using the CRISPR/Cas9 technology enables a variety of applications in plants, ranging from gene editing to the construction of transcriptional gene circuits, many of which depend on the technical ability to compose and transfer complex synthetic instructions into the plant cell. The engineering principles of standardization and modularity applied to DNA cloning are impacting plant genetic engineering, by increasing multigene assembly efficiency and by fostering the exchange of well-defined physical DNA parts with precise functional information.
  
  
  RESULTS
  Here we describe the adaptation of the RNA-guided Cas9 system to GoldenBraid (GB), a modular DNA construction framework being increasingly used in Plant Synthetic Biology. In this work, the genetic elements required for CRISPRs-based editing and transcriptional regulation were adapted to GB, and a workflow for gRNAs construction was designed and optimized. New software tools specific for CRISPRs assembly were created and incorporated to the public GB resources site.
  
  
  CONCLUSIONS
  The functionality and the efficiency of gRNA-Cas9 GB tools were demonstrated in Nicotiana benthamiana using transient expression assays both for gene targeted mutations and for transcriptional regulation. The availability of gRNA-Cas9 GB toolbox will facilitate the application of CRISPR/Cas9 technology to plant genome engineering.
authors.affiliation: Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Vazquez-Vilar
authors.affiliation: Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
authors.forename: Joan Miquel
authors.initials: JM
authors.lastname: Bernabé-Orts
authors.affiliation: Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
authors.forename: Asun
authors.initials: A
authors.lastname: Fernandez-Del-Carmen
authors.affiliation: Centro de Conservación y Mejora de la Agrodiversidad Valenciana (COMAV), Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
authors.forename: Pello
authors.initials: P
authors.lastname: Ziarsolo
authors.affiliation: Centro de Conservación y Mejora de la Agrodiversidad Valenciana (COMAV), Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
authors.forename: Jose
authors.initials: J
authors.lastname: Blanca
authors.affiliation: Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Granell
authors.affiliation: Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
authors.forename: Diego
authors.initials: D
authors.lastname: Orzaez
keywords: CRISPR/Cas9; GoldenBraid; Luciferase/renilla assay; Multigenic assemblies; Plant gene activation; Plant gene editing; Plant gene repression; gRNAs
references.citation: New Phytol. 2015 Oct;208(1):13-9
references.pmid: 26171760
references.citation: Nat Biotechnol. 2013 Aug;31(8):686-8
references.pmid: 23929338
references.citation: Plant J. 2014 Jul;79(2):348-59
references.pmid: 24836556
references.citation: PLoS One. 2011;6(7):e21622
references.pmid: 21750718
references.citation: Methods Mol Biol. 2015;1284:399-420
references.pmid: 25757784
references.citation: Plant Biotechnol J. 2015 May;13(4):578-89
references.pmid: 25400128
references.citation: Plant Mol Biol. 2015 Jan;87(1-2):99-110
references.pmid: 25344637
references.citation: Biotechnol J. 2014 Nov;9(11):1402-12
references.pmid: 25186301
references.citation: Biotechnol Adv. 2015 Jan-Feb;33(1):41-52
references.pmid: 25536441
references.citation: Nat Biotechnol. 2013 Aug;31(8):688-91
references.pmid: 23929339
references.citation: Curr Opin Biotechnol. 2015 Apr;32:76-84
references.pmid: 25437637
references.citation: PLoS One. 2009;4(5):e5553
references.pmid: 19436741
references.citation: BMC Plant Biol. 2014 Nov 29;14:327
references.pmid: 25432517
references.citation: Cell. 2013 May 9;153(4):910-8
references.pmid: 23643243
references.citation: Planta. 2013 Dec;238(6):991-1003
references.pmid: 23955146
references.citation: Plant Mol Biol. 2015 Aug;88(6):561-72
references.pmid: 26188471
references.citation: Science. 2011 Sep 30;333(6051):1843-6
references.pmid: 21960622
references.citation: Plant J. 2014 Dec;80(6):1139-50
references.pmid: 25327456
references.citation: Sci Rep. 2014 Jun 23;4:5400
references.pmid: 24954249
references.citation: Nat Biotechnol. 2002 Feb;20(2):135-41
references.pmid: 11821858
references.citation: G3 (Bethesda). 2013 Dec 09;3(12):2233-8
references.pmid: 24122057
references.citation: Nucleic Acids Res. 2015 Jul 27;43(13):e88
references.pmid: 25956650
references.citation: Mil Med Res. 2015 May 09;2:11
references.pmid: 25984354
references.citation: Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3570-5
references.pmid: 25733849
references.citation: Nucleic Acids Res. 2014 Dec 1;42(21):13440-51
references.pmid: 25378321
references.citation: Mol Syst Biol. 2014 Nov 24;10:763
references.pmid: 25422271
references.citation: Trends Plant Sci. 2015 May;20(5):309-17
references.pmid: 25825364
references.citation: Mol Plant. 2015 Aug;8(8):1274-84
references.pmid: 25917172
references.citation: PLoS One. 2011 Feb 18;6(2):e16765
references.pmid: 21364738
references.citation: Science. 2013 Feb 15;339(6121):823-6
references.pmid: 23287722
references.citation: Plant Physiol. 2015 Oct;169(2):971-85
references.pmid: 26297141
references.citation: Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):E2967-76
references.pmid: 25002478
references.citation: Nat Protoc. 2013 Nov;8(11):2281-308
references.pmid: 24157548
references.citation: Mol Plant. 2014 Sep;7(9):1494-6
references.pmid: 24719468
references.citation: Plant Physiol. 2013 Jul;162(3):1618-31
references.pmid: 23669743
references.citation: Nat Biotechnol. 2013 Aug;31(8):691-3
references.pmid: 23929340
references.citation: Nucleic Acids Res. 2013 Aug;41(15):7429-37
references.pmid: 23761437