A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard

[EN] 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 con¿ struction 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 engineeringThis work has been funded by Grant BIO2013-42193-R from Plan Nacional I + D of the Spanish Ministry of Economy and Competitiveness. Vazquez-Vilar M. is a recipient of a Junta de Ampliacion de Estudios fellowship. Bernabe-Orts J.M. is a recipient of a FPI fellowship. We want to thank Nicola J. Patron and Mark Youles for kindly providing humanCas9 and U6-26 clones. We also want to thank Eugenio Gomez for providing Arabidopsis thaliana genomic DNA and Concha Domingo for providing rice genomic DNA. We also want to thank the COST Action FA1006 for the support in the development of the software tools.Vázquez-Vilar, M.; Bernabé-Orts, JM.; Fernández Del Carmen, MA.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.; Granell Richart, A.; Orzáez Calatayud, DV. (2016). A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods. 12. https://doi.org/10.1186/s13007-016-0101-2S12Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281–308. doi: 10.1038/nprot.2013.143 .Yang X. Applications of CRISPR-Cas9 mediated genome engineering. Mil Med Res. 2015;2:11. doi: 10.1186/s40779-015-0038-1 .Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, et al. 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Transfer of Xanthomonas campestris pv. arecae, and Xanthomonas campestris pv. musacearum to Xanthomonas vasicola (Vauterin) as Xanthomonas vasicola pv. arecae comb. nov., and Xanthomonas vasicola pv. musacearum comb. nov. and description of Xanthomonas vasicola pv. vasculorum pv. nov.

The "Ca2+ switch" model with cultured Madin-Darby canine kidney (MDCK) cells is useful in studying the biogenesis of epithelial polarity and junction formation and provides insight into early steps in the morphogenesis of polarized epithelial tissues. When extracellular Ca2+ in the medium is changed from less than 5 microM to 1.8 mM, MDCK cells rapidly change from a nonpolarized state exhibiting little cell-cell contact (with the apical membrane and junctional proteins largely within the cell) to a polarized state with well-formed tight junctions and desmosomes. To examine the role of intracellular Ca2+ in the development of polarity and junctions, we made continuous spectrofluorimetric measurements of intracellular Ca2+ during the "switch," using the fluorescent indicator fura-2. Intracellular Ca2+ increased greater than 10-fold during the switch and gave a complex pattern of increase, decrease, and stabilization. In contrast, intracellular pH [monitored with 29,79-bis(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF)] did not change during the period studied. When intracellular Ca2+ curves in several cells were compared, considerable heterogeneity in the rate of increase of intracellular Ca2+ levels and in peak levels was evident, perhaps reflecting the heterogeneity among cells in establishing junctions and polarity. The heterogeneity of the process was confirmed by digital imaging of intracellular Ca2+ and was present even in a "clonal" line of MDCK cells, indicating the heterogeneity was intrinsic to the process and not simply a function of slight genetic variation within the population of MDCK cells. In pairs of cells that had barely established cell-cell contact, often one cell exhibited a much greater increase in intracellular Ca2+ than the other cell in the pair. At the site of cell-cell contact, an apparent localized change (an increase over the basal level) in intracellular Ca2+ was frequently present and occasionally appeared to extend beyond the point of cell-cell contact. Since the region of cell-cell contact is also the site where junctions form and where vesicles containing apical membranes fuse during the development of polarity, we postulate a role for global and local changes in intracellular Ca2+ in these events

Genome sequence and rapid evolution of the rice pathogen Xanthomonas oryzae pv. oryzae PXO99A

Background: Xanthomonas oryzae pv. oryzae causes bacterial blight of rice (Oryza sativa L.), a major disease that constrains production of this staple crop in many parts of the world. We report here on the complete genome sequence of strain PXO99A and its comparison to two previously sequenced strains, KACC10331 and MAFF311018, which are highly similar to one another. Results: The PXO99 A genome is a single circular chromosome of 5,240,075 bp, considerably longer than the genomes of the other strains (4,941,439 bp and 4,940,217 bp, respectively), and it contains 5083 protein-coding genes, including 87 not found in KACC10331 or MAFF311018. PXO99A contains a greater number of virulence-associated transcription activator-like effector genes and has at least ten major chromosomal rearrangements relative to KACC10331 and MAFF311018. PXO99 A contains numerous copies of diverse insertion sequence elements, members of which are associated with 7 out of 10 of the major rearrangements. A rapidly-evolving CRISPR (clustered regularly interspersed short palindromic repeats) region contains evidence of dozens of phage infections unique to the PXO99A lineage. PXO99A also contains a unique, near-perfect tandem repeat of 212 kilobases close to the replication terminus. Conclusion: Our results provide striking evidence of genome plasticity and rapid evolution within Xanthomonas oryzae pv. oryzae. The comparisons point to sources of genomic variation and candidates for strain-specific adaptations of this pathogen that help to explain the extraordinary diversity of Xanthomonas oryzae pv. oryzae genotypes and races that have been isolated from around the world. © 2008 Salzberg et al; licensee BioMed Central Ltd

Rapid assembly of customized TALENs into multiple

Transcriptional activator-like effector nucleases (TALENs) have become a powerful tool for genome editing. Here we present an efficient TALEN assembly approach in which TALENs are assembled by direct Golden Gate ligation into Gateway® Entry vectors from a repeat variable di-residue (RVD) plasmid array. We constructed TALEN pairs targeted to mouse Ddx3 subfamily genes, and demonstrated that our modified TALEN assembly approach efficiently generates accurate TALEN moieties that effectively introduce mutations into target genes. We generated "user friendly" TALEN Entry vectors containing TALEN expression cassettes with fluorescent reporter genes that can be efficiently transferred via Gateway (LR) recombination into different delivery systems. We demonstrated that the TALEN Entry vectors can be easily transferred to an adenoviral delivery system to expand application to cells that are difficult to transfect. Since TALENs work in pairs, we also generated a TALEN Entry vector set that combines a TALEN pair into one PiggyBac transposon-based destination vector. The approach described here can also be modified for construction of TALE transcriptional activators, repressors or other functional domains. © 2013 Zhang et al

Acquisition and Evolution of Plant Pathogenesis–Associated Gene Clusters and Candidate Determinants of Tissue-Specificity in Xanthomonas

is a large genus of plant-associated and plant-pathogenic bacteria. Collectively, members cause diseases on over 392 plant species. Individually, they exhibit marked host- and tissue-specificity. The determinants of this specificity are unknown. lineage. genome and indicate that differentiation with respect to host- and tissue-specificity involved not major modifications or wholesale exchange of clusters, but subtle changes in a small number of genes or in non-coding sequences, and/or differences outside the clusters, potentially among regulatory targets or secretory substrates

Cisgenesis and intragenesis as new strategies for crop improvement

Cisgenesis and intragenesis are emerging plant breeding technologies which offer great promise for future acceptance of genetically engineered crops. The techniques employ traditional genetic engineering methods but are confined to transferring of genes and genetic elements between sexually compatible species that can breed naturally. One of the main requirements is the absence of selectable marker genes (such as antibiotic resistance genes) in the genome. Hence the sensitive issues with regard to transfer of foreign genes and antibiotic resistance are overcome. It is a targeted technique involving specific locus; therefore, linkage drag that prolongs the time for crop improvement in traditional breeding does not occur. It has great potential for crop improvement using superior alleles that exist in the untapped germplasm or wild species. Cisgenic and intragenic plants may not face the same stringent regulatory assessment for field release as transgenic plants which is a clear added advantage that would save time. In this chapter, the concepts of cis/intragenesis and the prerequisites for the development of cis/intragenesis plants are elaborated. Strategies for marker gene removal after selection of transformants are discussed based on the few recent reports from various plant species

Rational Diversification of a Promoter Providing Fine-Tuned Expression and Orthogonal Regulation for Synthetic Biology

Yeast is an ideal organism for the development and application of synthetic biology, yet there remain relatively few well-characterised biological parts suitable for precise engineering of this chassis. In order to address this current need, we present here a strategy that takes a single biological part, a promoter, and re-engineers it to produce a fine-graded output range promoter library and new regulated promoters desirable for orthogonal synthetic biology applications. A highly constitutive Saccharomyces cerevisiae promoter, PFY1p, was identified by bioinformatic approaches, characterised in vivo and diversified at its core sequence to create a 36-member promoter library. TetR regulation was introduced into PFY1p to create a synthetic inducible promoter (iPFY1p) that functions in an inverter device. Orthogonal and scalable regulation of synthetic promoters was then demonstrated for the first time using customisable Transcription Activator-Like Effectors (TALEs) modified and designed to act as orthogonal repressors for specific PFY1-based promoters. The ability to diversify a promoter at its core sequences and then independently target Transcription Activator-Like Orthogonal Repressors (TALORs) to virtually any of these sequences shows great promise toward the design and construction of future synthetic gene networks that encode complex “multi-wire” logic functions

Genome-Wide Identification of HrpL-Regulated Genes in the Necrotrophic Phytopathogen Dickeya dadantii 3937

BACKGROUND: Dickeya dadantii is a necrotrophic pathogen causing disease in many plants. Previous studies have demonstrated that the type III secretion system (T3SS) of D. dadantii is required for full virulence. HrpL is an alternative sigma factor that binds to the hrp box promoter sequence of T3SS genes to up-regulate their expression. METHODOLOGY/PRINCIPAL FINDINGS: To explore the inventory of HrpL-regulated genes of D. dadantii 3937 (3937), transcriptome profiles of wild-type 3937 and a hrpL mutant grown in a T3SS-inducing medium were examined. Using a cut-off value of 1.5, significant differential expression was observed in sixty-three genes, which are involved in various cellular functions such as type III secretion, chemotaxis, metabolism, regulation, and stress response. A hidden Markov model (HMM) was used to predict candidate hrp box binding sites in the intergenic regions of 3937, including the promoter regions of HrpL-regulated genes identified in the microarray assay. In contrast to biotrophic phytopathgens such as Pseudomonas syringae, among the HrpL up-regulated genes in 3937 only those within the T3SS were found to contain a hrp box sequence. Moreover, direct binding of purified HrpL protein to the hrp box was demonstrated for hrp box-containing DNA fragments of hrpA and hrpN using the electrophoretic mobility shift assay (EMSA). In this study, a putative T3SS effector DspA/E was also identified as a HrpL-upregulated gene, and shown to be translocated into plant cells in a T3SS-dependent manner. CONCLUSION/SIGNIFICANCES: We provide the genome-wide study of HrpL-regulated genes in a necrotrophic phytopathogen (D. dadantii 3937) through a combination of transcriptomics and bioinformatics, which led to identification of several effectors. Our study indicates the extent of differences for T3SS effector protein inventory requirements between necrotrophic and biotrophic pathogens, and may allow the development of different strategies for disease control for these different groups of pathogens

The TAL Effector PthA4 Interacts with Nuclear Factors Involved in RNA-Dependent Processes Including a HMG Protein That Selectively Binds Poly(U) RNA

Plant pathogenic bacteria utilize an array of effector proteins to cause disease. Among them, transcriptional activator-like (TAL) effectors are unusual in the sense that they modulate transcription in the host. Although target genes and DNA specificity of TAL effectors have been elucidated, how TAL proteins control host transcription is poorly understood. Previously, we showed that the Xanthomonas citri TAL effectors, PthAs 2 and 3, preferentially targeted a citrus protein complex associated with transcription control and DNA repair. To extend our knowledge on the mode of action of PthAs, we have identified new protein targets of the PthA4 variant, required to elicit canker on citrus. Here we show that all the PthA4-interacting proteins are DNA and/or RNA-binding factors implicated in chromatin remodeling and repair, gene regulation and mRNA stabilization/modification. The majority of these proteins, including a structural maintenance of chromosomes protein (CsSMC), a translin-associated factor X (CsTRAX), a VirE2-interacting protein (CsVIP2), a high mobility group (CsHMG) and two poly(A)-binding proteins (CsPABP1 and 2), interacted with each other, suggesting that they assemble into a multiprotein complex. CsHMG was shown to bind DNA and to interact with the invariable leucine-rich repeat region of PthAs. Surprisingly, both CsHMG and PthA4 interacted with PABP1 and 2 and showed selective binding to poly(U) RNA, a property that is novel among HMGs and TAL effectors. Given that homologs of CsHMG, CsPABP1, CsPABP2, CsSMC and CsTRAX in other organisms assemble into protein complexes to regulate mRNA stability and translation, we suggest a novel role of TAL effectors in mRNA processing and translational control