(19)
(11)EP 3 647 427 A1

(12)EUROPEAN PATENT APPLICATION
published in accordance with Art. 153(4) EPC

(43)Date of publication:
06.05.2020 Bulletin 2020/19

(21)Application number: 18818407.1

(22)Date of filing:  12.06.2018
(51)Int. Cl.: 
C12N 15/82  (2006.01)
(86)International application number:
PCT/ES2018/070421
(87)International publication number:
WO 2018/229319 (20.12.2018 Gazette  2018/51)
(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
Designated Extension States:
BA ME
Designated Validation States:
KH MA MD TN

(30)Priority: 12.06.2017 ES 201730792

(71)Applicant: García Alvarez, Juan Antonio
28049 Madrid (ES)

(72)Inventors:
  • PASIN, Fabio
    28049 Madrid (ES)
  • GARCÍA ÁLVAREZ, Juan Antonio
    28016 Madrid (ES)
  • BEDOYA ROJAS, Leonor Cecilia
    28049 Madrid (ES)
  • SIMÓN MATEO, María del Carmen
    28049 Madrid (ES)
  • ORZÁEZ CALATAYUD, Diego Vicente
    46022 Valencia (ES)
  • BERNABÉ ORTS, Juan Miguel
    46022 Valencia (ES)

  


(54)BINARY VECTORS AND USES OF SAME


(57) The invention relates to binary vectors based on replication origins that are compatible and autonomous, specifically based on pBBR1 and RK2 replication origins, which are used for a wide range of hosts, for maintenance in Agrobacterium sp. and Escherichia coli as a novel plant synthetic biology tool, as well as a flexible framework for assembly of multiple DNA elements, transfer and characterisation of parts of DNA. The binary vectors disclosed are small, preferably less than 3.8 kb in size, stable, with an origin compatible with the most commonly used T-DNA binary vectors, comply with current plant synthetic biology standards, and allow the administration of multiple T-DNA cassettes by means of the multiplexing of the vectors. The present invention also relates to methods for transferring and expressing DNA sequences using said binary vectors, and to the uses of same.


Description

DESCRIPTION



[0001] The invention relates generally to the field of molecular biology and agents useful in the manipulation of eukaryotic organisms. More particularly, the present invention provides methods to assembly, transfer and express DNA sequences using binary vectors, the binary vectors itself, and uses thereof.

BACKGROUND ART



[0002] Plants are plastic organisms that sense and respond to environmental stimuli. These responses or specific plant features might not fit human needs, and their manipulation can be achieved by targeted use of plant-interacting microorganisms, or by plant genetic transformation. Plant biotechnology uses advanced tools to generate plants with new functions, better agronomic traits, or to produce new products. Synthetic biology applies engineering principles to facilitate the production of organisms with customized functions and for precise control of specific biological functions. Genetic components of complex biological systems are reduced to DNA parts with modular and defined assignments. Once characterized with the aid of computational tools, libraries of parts are assembled to yield pathways and networks with predictable outputs. Methods to analyze dynamic molecular devices have been used to genetically engineer plants with tunable functions.

[0003] Assembled DNA constructs are transferred directly to plants, or introduced into disarmed-pTi Agrobacterium tumefaciens strains which serve as a shuttle chassis for delivery of constructs maintained in T-DNA binary vectors to plants. From 1986 to 2000, T-DNA binary vectors were generated using diverse replication origins and parts (Murai N., Am. J. Plant Sci. 2013, 4, 932-939). A disadvantage of existing binary vectors is their size, which is difficult to handle, and their low-copy number, for example the 12-kb pBIN19 (Bevan M., Nucleic Acids Res. 1984, 12, 8711-8721). The low-copy number leads to low yields of the DNA plasmids, and makes the cloning procedures difficult. To improve the low yields of DNA and ease cloning procedures, plasmid backbones can be amplified by PCR and used in one-step DNA assembly reactions. Due to large sizes of many binary vectors, amplification of plasmid backbones by PCR is not practical. The pPZP (Hajdukiewicz P., et al., Plant Mol. Biol. 1994, 25, 989-994) and pGreen (Helles R., et al., Plant Molecular Biology. 2000, 42, 819-832) series of binary vectors include origins with high-copy number that give high yields of plasmid. Unstable replication origins may lead to variable plasmid losses during replication. While the pGreen vector, which is very small (4.6 kb in size), is not autonomous and lacks the elements required for stable multiplication in agrobacteria, so that it can only be used with specific Agrobacterium strains (Helles R., et al., Plant Molecular Biology. 2000, 42, 819-832). For certain applications, use of high-copy number origins is not desirable since it may promote deletions/alterations of large DNA inserts, of sequences that show bacterial toxicity, or of repeated sequence elements. Instability is particularly manifested when DNA components are employed multiple times within constructs. For example, it is not uncommon that a particular plant-expressible promoter may be used to drive the expression of different protein coding regions in a transgenic plant. Other genetic components such as 3' untranslated regions (i.e. sequences that determine transcription terminator and polyadenylation addition) and even highly similar protein-coding regions may be duplicated or present in multiple copies within a single T-DNA region. As mentioned above, these repeated sequence elements, which may exist in either inverted or directly repeated orientations, are targets for intramolecular recombinations that may lead to DNA deletions and other rearrangements.

[0004] Described binary vectors lack features that reduce undesired expression of T-DNA sequences in bacterial hosts. Undesired expression of exogenous sequences may lead to production of toxic products during vector propagation in bacteria, and may enhance insert and vector instability. In this sense, it is known that natural or synthetic transcription terminators can insulate against promoters active in bacteria (Chen Y.J., et al., Nat Methods. 2013, 10, 7, 659-664).

[0005] In early binary vector series, there is frequently also a lack of a sufficient number of restriction enzyme sites for cloning of desired sequences into T-DNA cassettes, or the vectors only permit the use of few selectable markers.

[0006] More recent versions of previously described vectors have been reported (Murai N., Am. J. Plant Sci. 2013, 4, 932-939). These versions generally adopt described backbones which were modified to include sequences to improve T-DNA cassette delivery to eukaryotic cells, and to ease insertion of exogenous sequences into T-DNA cassettes. For decades, the most common approaches to assemble DNA constructs into binary vectors have taken advantage of the specificity of restriction endonucleases to create compatible ends that can be joined using DNA ligases. Presence or lack of restriction sites in vector and insert sequences can constrain possible assemblies, in particular those involving multiple inserts. Cloning methods have been developed to overcome these constraints, therefore allowing for high-throughput assembly of DNA constructs. Recombinase-based technologies, such as Gateway, Creator, Echo, and Univector cloning, are highly efficient and rely on enzymes that specifically recombine insert and vector sequences. Recombinase-based technologies are limited to vectors with appropriate recombination sequences, they allow cloning of small number of inserts at a time, and they are not always scar-benign, as they leave > 20-bp scars between building blocks. New cloning strategies developed within the past decade use Type IIS restriction endonuclease- and overlap-based assembly methods (e.g., Golden Gate, and Gibson assembly) to overcome sequence requirements, and allow for assembly of multiple inserts in a given reaction. Only a small number of described binary vectors allow generation of T-DNA constructs by high-throughput DNA assembly based on Type IIS restriction endonuclease- and overlap-based assembly methods. The Golden Gate is a robust system used by many plant scientists (Patron N.J., et al., New Phytol. 2015, 208, 13-19) and the Gibson assembly is very versatile, since it requires no domestication of the parts, has the ability to join 2-10 fragments in a predetermined order, with no sequence restrictions or scars (Gibson D.G., et al., Nat. Methods 2009, 6, 343-345), but has not been widely adopted for building of plant constructs. To significantly reduce the background of unwanted vector-only colonies in Gibson assembly reactions, the vector should be a PCR product rather than a restriction fragment, and Dpnl-treated to remove the carry-over of templates. Due to large sizes of many binary vectors, backbone linearization by PCR is not practical, and therefore small-sized binary vectors are desirable for efficient construct cloning by Gibson assembly and other overlap-based assembly methods.

[0007] Multigene transfer is imperative in multiplexed gene editing and to genetically engineer complex traits, circuit designs, and metabolic pathways. In plants, conventional stacking methods require substantial breeding efforts that can be overcome by placing multiple genes within a single T-DNA, or simultaneous infections of plant cells with multiple A. tumefaciens strains, each harboring a different T-DNA binary vector. It is known that a single A. tumefaciens strain can deliver two unlinked T-DNA cassettes and transform them into the same eukaryotic cell; however, simultaneous use of compatible T-DNA binary vectors is a seldom-applied strategy in plant biotechnology. Moreover, the majority of existing binary vector systems lacks the possibility of removing selectable markers from the transgenic lines at a later point in time. Delivery of unlinked T-DNA cassettes allows the use of a selectable marker during plant regeneration and subsequent recovery of marker-free progeny.

[0008] In this sense, it is known binary vector systems wherein two T-DNA cassettes were delivered to plant by a single A. tumefaciens strain. Specifically, a single binary vector hosting two T-DNA cassettes (Komari T., et al., Plant J. 1996, 10, 165-174) or two T-DNA cassettes hosted in two compatible binary vectors (Daley M., et al., Plant Cell Rep. 1998, 17, 489-496) were delivered to plant by a single A. tumefaciens strain. Technical constrains of known systems include limited cloning flexibility due to large plasmid sizes (> 15 kb), incompatibility with high-throughput construct assembly methods, or lack of replication independence of the binary vectors used.

[0009] Another operational disadvantage of binary vectors is the use of common components in their backbone sequences, which hampers their simultaneous maintenance in a single bacterial cell. As is well known to those skilled in the field of molecular biology, use of origins that belong to identical incompatibility groups impedes vector replication and maintenance in the same cell. Moreover, large sequence repeats may lead to DNA deletions and other rearrangements, particularly when the repeats are a part of plasmid structure. Such rearrangements may lead to partial or complete loss of the T-DNA region, ultimately resulting in little or no transfer of intact desired foreign sequences into eukaryotic cells.

[0010] Another disadvantage of binary vectors is the presence of plasmid mobilization sequences required to mobilize the vectors into Agrobacterium by triparental mating. Presence of mobilization sequences in binary vectors contributes to increase their sizes and to reduce their biological safety. Moreover, it is known that the origin of transfer of certain plasmids can interfere with the desired T-DNA processing and its delivery to eukaryotic cells (Buchanan-Wollaston V., et a/., Nature 1987, 328, 172-175). In this sense it is known that Agrobacterium are transformed by plasmids by physical approaches, e.g., electroporation or freeze-thaw methods (Höfgen R. & Willmitzer L., Nucleic Acids Res. 1988, 16, 9877).

[0011] Taking into account the hereinabove disadvantages, it would be desirable to design improved binary vectors and binary vector systems that no longer have the above-mentioned limitations. Therefore, there exists a necessity to provide a binary vector having a reduced size and features that make them stable and limit their horizontal transfer. A further need exists for a binary vector compatible with advanced, high-throughput DNA cloning methods and that eases assembly of multiple components. It is also desirable to provide a binary vector system incorporating minimal, single and compatible broad-host range replication origins that allow simultaneous maintenance of multiple binary vectors in a single bacterial cell. Consequently, the required binary vectors, binary vector systems, compositions, uses and method comprising thereof, can be applied to improve the transformation process to integrate full length T-DNA constructs into the eukaryotic cell or organism that are free of any residual binary vector backbone sequence. In this sense, a further need exists for a binary vector system that facilitates multi T-DNA cassette delivery to eukaryotic cells. The development of a novel and improved plant transformation system provides significant benefits for cell biologists, agronomic uses, for production of pharmaceutical compound and recombinant protein.

SUMMARY OF THE INVENTION



[0012] The invention solves the problems mentioned above by generation of the pLX series, a set of T-DNA binary vectors that ease multicomponent construct assembly and delivery. The T-DNA binary vectors of the present invention are a new tool for plant synthetic biology as well as a flexible framework for multigene transfer and DNA parts characterization. The advantages of the T-DNA binary vectors of the present invention are: (i) reduced size, preferably below 3.8 kb; (ii) presence of a single, autonomous and broad-host range replication origin for maintenance in bacteria, preferably in Escherichia coli and Agrobacterium tumefaciens; (iii) the use of replication origin compatible with the most commonly used T-DNA binary vectors; (iv) presence of transcription terminators to reduce undesired expression of T-DNA sequences in bacterial hosts and promote plasmid stability; (v) incorporation of T-DNA cassettes with unique rare-cutting recognition sites; (vi) consistency with current plant synthetic biology standards to allow high-throughput T-DNA construct assembly of pre-made DNA elements by Type IIS restriction endonuclease-based cloning methods; (vii) the possibility to adopt overlap-dependent methods for high-throughput T-DNA construct assembly; (viii) the possibility to be amplified and linearized by PCR to improve efficiency of overlap-based cloning; (ix) incorporation of a binary vector pair with compatible origins and specifically engineered to have no backbone regions with > 28 nucleotide identity; and (x) the possibility to deliver multi T-DNA cassettes by a binary vector system that allows vector multiplexing in a single bacterial cell.

[0013] The T-DNA binary vectors of the present invention comprises a minimal replication origin derived from pBBR1 (pBBR1-based pLX) or RK2 (RK2-based pLX) plasmid, preferably, from the pBBR1 plasmid (Antoine R. & Locht C., Mol. Microbiol. 1992, 6, 1785-1799). The pBBR1-based backbone and the RK2-based backbone of pLX vectors of the invention is substantially smaller than the widely used pBIN19- and pCAMBIA-based vectors, and equals to pGreen-based vectors, the smallest available binary plasmids (Fig. 2A and Fig. 9). Replication of pGreen vectors in A. tumefaciens requires a co-resident plasmid that supplies the pSa-RepA gene (e.g., pSoup). In contrast, pLX binary vectors of the present invention facilitate flexible experimental designs since their replication is autonomous in both E coli and A. tumefaciens, and consequently, does not require additional factors for their maintenance in bacterial hosts. In this sense, the binary pLX vectors of the present invention are useful for their autonomous replication in diverse bacteria, and presence of T-DNA cassettes.

[0014] The pLX binary vectors of the invention also include diverse selectable markers (nptI, aadA, or aacC1 genes) for their selection in bacterial host cells, a T-DNA cassette with borders from an octopine- or succinamopine-type pTi from A. tumefaciens and a second left border sequence that was shown to reduce backbone transfer (Fig. 2A). Bacterial synthetic terminators based on different scaffolds (T1, T2, λT1 and/or λT2) were included to reduce undesired expression of T-DNA sequences in bacterial hosts and increase plasmid stability. A rare-cutting Ascl recognition site outside the T-DNA cassette was included to modify pLX vector backbone of the present invention for a given purpose, for example, without limitation, by inserting toxin-antitoxin, counter segregation systems, or virulence gene sequences to improve plasmid stability and/or enhance transformation efficiency, such as and without limitation, hok/sok, parD/parE, and virGgenes. Additionally, the pLX binary vectors of the present invention facilitate molecular cloning procedures since T-DNA cassettes comprise rare-cutting Pmll and Sbfl recognition sites useful for standard restriction endonuclease/DNA ligase cloning, and Bsal and BsmBI recognition sites compatible with high-throughput Type IIS restriction endonuclease-based method, such as Golden Gate and GolderBraid cloning. The Bsal- and BsmBI-produced produced overhangs meet proposed plant synthetic biology standards and ease assembly of pre-made DNA elements that are available in public libraries. T-DNA cassettes also include divergent primer annealing regions with no secondary structures and sequence similarity among them. Thus, the mini T-DNA binary vectors of the present invention can be easily linearized by PCR, Dpnl-treated and used in overlap-dependent methods with high efficiency and no background of unwanted vector-only colonies. Therefore, the binary pLX vectors of the present invention are a set of mini T-DNA binary plasmids suitable for standard restriction endonuclease/DNA ligase cloning, and advanced Type IIS restriction endonuclease- and overlap-based assembly methods, such as and without limitation, Golden Gate/Golden Braid and Gibson assembly.

[0015] Due to their small size pLX vectors might be directly delivered to eukaryotic cells, for example by cell/protoplast transfection. Alternatively, pLX vectors can use suitable bacterial strains, preferably Agrobacterium sp. strains, as shuttle chassis for transfer of their T-DNA cassette to eukaryotic cells. pLX vectors can be introduced into bacteria by physical methods (e.g., electroporation, heat shock), and unwanted horizontal transfer of pLX vector is less likely to occur since they do not include an origin of conjugative transfer or other plasmid mobilization regions. Transfer of pLX vector backbone sequences that flank T-DNA cassettes is predicted to be reduced by incorporation of double left borders. In this sense, Escherichia coli, Agrobacterium tumefaciens and plants have been used in the examples of the present invention, however, the binary vectors of the invention are suitable for use in alternative systems, such as in prokaryotic chassis other than E coli and A. tumefaciens, and to transform eukaryotic organisms other than superior plants, such as algae, fungi, and animal cells.

[0016] The binary pLX vectors of the present invention include the pBBR1 origin, which shows no incompatibility with known plasmids. A vector system that uses, without limitation, pBBR1- and RK2-based pLX binary vectors of the invention facilitates multiple T-DNA delivery to eukaryotic cells since it includes vectors with compatible replication origins, diverse selectable markers, and low sequence similarity to reduce homologous recombination events. Simultaneous use of pBBR1-and RK2-based pLX vectors as a transformation system, in a two-vector/one-Agrobacterium strain system allows multi T-DNA and multigene delivery to eukaryotic organisms, such as plants, fungi and animals.

[0017] Usage of alternative compatible replication origins might further expand the multigene delivery design to an "N-vector/one-strain" system. This system can be combined by co-infection with multiple A. tumefaciens strains, to further increase the number of delivered T-DNA cassettes.

[0018] Binary vectors disclosed in the present invention have been tested (see examples below) for transient and stable plant transformation, genome editing, agro-inoculation of a new viral infectious clone and delivery of exogenous sequences to plants by viral vectors. The inventors have used a two-vector/one-strain system to deliver multiple T-DNA cassettes to plant germ line cells, and to express in plants components of a simple buffer gate activated by a chemical inducer.

[0019] Applications of the binary pLX vectors of the present invention include, without limitation, their use for assembly of large T-DNA constructs and transcription units; for transient and stable transgene expression; for generation of transgenic plants free of drug-resistance markers; for launching viral infections by agro-inoculation; for exogenous sequence delivery and recombinant protein production using viral vectors; for genome editing; for delivery of Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system components; for delivery of chemically regulated expression systems; and for delivery of genetic circuit components.

[0020] In this sense, a first aspect to the present invention relates to a binary vector, hereinafter first binary vector of the invention (pBBR1-based pLX vector), comprising at least three modules: (a) a T-DNA cassette module comprising at least a right and a T-DNA left borders, (b) a replication origin module comprising a minimal pBBR1 origin, or a variant functionally equivalent thereof, and (c) at least a selectable marker module.

[0021] In a preferred embodiment, the pBBR1-based pLX vector of the invention comprises a T-DNA cassette comprising a right and two T-DNA left borders.

[0022] In a further preferred embodiment of the pBBR1-based pLX vector of the invention, the pBBR1 origin comprise pBBR1-oriV and -rep regions, or a variant functionally equivalent thereof. In a more preferred embodiment, the pBBR1 origin comprises the SEQ ID NO: 105.

[0023] In another preferred embodiment, the pBBR1-based pLX vector of the invention comprises a T-DNA cassette which is flanked by at least two transcription terminators, preferably selected from T1 (SEQ ID NO: 108), T2 (SEQ ID NO: 109), λT1 (SEQ ID NO: 110), λT2 (SEQ ID NO: 111), or any combinations thereof.

[0024] In a second aspect, the present invention further relates to another binary vector, named as RK2-based pLX vector, comprising at least three modules: (a) a T-DNA cassette module comprising at least a right and a T-DNA left borders, (b) a replication origin module comprising a minimal RK2 origin, or a variant functionally equivalent thereof, and (c) at least a selectable marker module.

[0025] In another aspect, the present invention relates to another binary vector that can preferably use in combination with the pBBR1-based pLX vector of the invention, comprising at least three modules: (a) a T-DNA cassette module comprising at least a right and a T-DNA left borders, (b) a replication origin module comprising an origin compatible with pBBR1 origin, preferably selecting form the list consisting of IncQ, IncW, IncU, pRi, pVS1, IncP-α plasmid incompatibility group origins, and (c) at least a selectable marker module.

[0026] In a preferred embodiment, the RK2-based pLX vector of the invention comprises a T-DNA cassette comprising a right and two T-DNA left borders.

[0027] In a more preferred embodiment, the replication origin module is an IncP-α plasmid incompatibility group origin, and more preferably is the RK2 origin. In a further preferred embodiment of the RK2 origin comprises the RK2-oriV and -trfA regions, or a variant functionally equivalent thereof. In a more preferred embodiment the RK2 origin comprises the SEQ ID NO: 106 or SEQ ID NO: 107.

[0028] In another preferred embodiment, the RK2-based pLX vector of the invention comprises a T-DNA cassette that is flanked by at least two transcription terminators, preferably, bacterial transcription terminators.

[0029] In another preferred embodiment, the selectable marker gene of the RK2-based pLX vector differs from the selectable marker gene of the pBBR1-based pLX vector.

[0030] In another preferred embodiment, the backbone of the RK2-based pLX vector has no backbone regions with > 28 nucleotide identity to the pBBR1-based pLX vector of the present invention.

[0031] In a third aspect, the present invention relates to a binary vector system comprising: (a) a first binary vector being the pBBR1-based pLX binary vector disclosed in the present invention and (b) a second binary vector selecting from the RK2-based pLX vector or the vector that can be used preferably in combination with the first binary vector of the invention wherein the pBBR1 origin module is replaced by any of the replication origin selected from the plasmid incompatibility group origins: IncQ, IncW, IncU, pRi, pVS1, IncP-α, and wherein each of the binary vectors of (a) and (b) has a replication and bacterial selection mechanism that enables a mutual and autonomous coexistence with each other in the same host cell.

[0032] In a preferred embodiment of the binary vector system of the invention, the origin module of the second binary vector is an IncP-α plasmid incompatibility group origin, and more preferably is the RK2 origin according to the present invention. In a more preferred embodiment, the second binary vector is the RK2-based pLX vector of the present invention.

[0033] Another aspect of the present invention relates to a host cell comprising the pBBR1-based pLX vector, the RK2-based pLX vector, or the binary vector system disclosed in the present invention.

[0034] Another aspect of the present invention relates to a culture cell comprising the host cell of the present invention.

[0035] Another aspect of the present invention relates to a method for delivering at least one nucleotide sequence of interest in at least one plant cell comprising: (a) inserting at least one nucleotide sequence of interest into the first or the second binary vectors, or into the binary vector system of the invention, (b) introducing the binary vectors or binary vector system of step (a) into at least one bacterial host cell, and (c) contacting the host cell of step (b) with at least one eukaryotic cell.

[0036] Another aspect of the present invention relates to a method for in vitro delivering at least one nucleotide sequence of interest in at least one eukaryotic organism, comprising: (a) inserting at least one nucleotide sequence of interest into the binary vector or into the binary vector system of the invention, and (b) introducing the binary vector or binary vector system of step (a) into at least one eukaryotic organism.

[0037] Another aspect of the present invention relates to a method for obtaining a genetically-engineered plant cell or plant comprising the step of introducing the binary vector, the vector system, or the bacterial host cell of the invention into an plant cell. Another aspect of the present invention relates to a genetically-engineered plant cell or plant obtainable by the method for obtaining a genetically-engineered plant cell or plant of the present invention.

[0038] Another aspect of the present invention relates to a method for in vitro obtaining a genetically-engineered eukaryotic cell or organism comprising the step of introducing the binary vector or the vector system of the present invention, into a eukaryotic cell. Another aspect of the present invention relates to a genetically-engineered eukaryotic cell or organism obtainable by the method for in vitro obtaining a genetically-engineered eukaryotic cell or organism according to the present invention.

[0039] As used herein the term "genetically-engineered" as used herein refers to a plant cell, plant, eukaryotic cell or individual which has been generated through the aforementioned methods.

[0040] The present invention furthermore relates to a genetically-modified, preferably transformed, mutant or modified plant system, to regenerated cells or a regenerated plant therefrom, to their progeny or seeds therefrom generated in accordance with the methods of the invention described hereinabove. In a particular embodiment of the present invention, this transformed plant system is characterized by single or multiple modifications of plant cell genome, epigenome, transcriptome or metabolome, and in that it may comprise or may not comprise any sequence segments of the abovementioned vector, vector system and their T-DNA cassettes.

[0041] Another aspect of the present invention relates to a method for transforming eukaryotic cells or eukaryotic organisms comprising the step of introducing into the eukaryotic cell or organism the binary vectors, the binary vector system, the host cell, the genetically-engineered plant cell or plant or the genetically-engineered eukaryotic cell or organism, disclosed in the present invention.

[0042] Another aspect of the present invention relates to methods to assemble synthetic, genomic, metagenomic, and/or cDNA sequences of interest into the binary vectors or the binary vector system disclosed in the present invention. According to the present invention, a variety of methods can be used for nucleic acid assembly. In a preferred embodiment sequences of interest are assembled by use of high-throughput restriction endonuclease-, preferably and without limitations Type IIS restriction endonucleases, or overlap-dependent assembly methods, such as and without limitation, Golden Gate/Golden Braid or Gibson assembly.

[0043] Another aspect of the present invention relates to the in vitro or ex vivo use of the binary vector, the binary vector system, the host cell, or the culture cell of the invention: (a) for site specific gene knockout, (b) for site-specific genome editing, (c) for DNA sequence-specific interference, (d) for site-specific epigenome editing, (e) for site-specific transcription modulation; or (f) for multiplex genome engineering, and provided the in vitro or ex vivo use does not comprise a process for modifying the germ line genetic identity of human beings.

[0044] Another aspect of the present invention relates to the kit comprising the binary vectors, the binary vector system, the host cell, or the culture cell of the invention.

DESCRIPTION OF THE DRAWINGS



[0045] 

FIG. 1. Construction of T-DNA binary vectors by modular parts assembly.
Module 1, 2, and 3 refer to T-DNA cassette, pBBR1 origin and selectable marker, respectively. Each module includes one or several DNA parts, which are flanked by two diverse assembly linkers (diamonds): Linker_1 (SEQ ID NO: 112), Linker_2 (SEQ ID NO: 113), Linker_3 (SEQ ID NO: 114). Parts from the three modules were obtained by PCR or chemically synthesized, and joined by one-step isothermal DNA assembly to generate pLX-B2 (SEQ ID NO: 3), pLX-B3 (SEQ ID NO: 4), pLX-B4 (SEQ ID NO: 5) binary vectors.

FIG. 2. Novel T-DNA binary vectors of the pLX series and their features.
(A) Organization of pBBR1-based pLX plasmids. Binary vectors are composed of three modules, (i) a T-DNA cassette that includes a right border, an Escherichia coli reporter gene, two left borders and is flanked by bacterial transcription terminators (T1 and T2); (ii) the broad host-range pBBR1 origin for suitable plasmid replication in E coli and Agrobacterium tumefaciens (oriV+rep); and (iii) a selectable marker such as antibiotic resistance (R) genes. Plasmid vectors are indicated by a letter that reflects their origin module (B, pBBR1-derived origin) and a digit according the R gene: 2, nptI, gene that confers resistance to kanamycin; 3, aadA, gene that confers resistance to spectinomycin/streptomycin; 4, aacC1, gene that confers resistance to gentamicin. (B) Cloning features of a pLX vector T-DNA cassette. The /acZα reporter is flanked by two divergent Bsal recognition sites (solid triangles), the nonpalindromic overhangs generated by Bsal digestion allow assembly of transcription units using one-step digestion-ligation Golden Gate cloning. Convergent BsmBI sites (open triangles) are included to build multiple transcription unit constructs by GoldenBraid assembly. Alternatively, pLX vectors can be linearized by inverse PCR using divergent primers (arrows), Dpnl-treated and used to join one or several overlapping inserts by one-step isothermal DNA assembly (Gibson assembly). (C) Diagrams of pLX vector cloning features. Parts or transcription units can be assembled into pLX vectors by using Bsal-based Golden Gate and GoldenBraid standards. Overlapping DNA fragments can be joined into linearized pLX vectors by Gibson assembly. pLX vectors can be multiplexed into Agrobacterium cells for multi T-DNA delivery.

FIG. 3. Plant transient transgene expression using pLX vector series.
(A) Scheme of transgene construct for transient transformation of Nicotiana benthamiana. The Cauliflower mosaic virus (CaMV) 35S promoter-driven TagRFP-T gene (RFP) was inserted into different pLX-derived backbones, which were delivered to plants by agro-infiltration. Data were collected at 6 days post-agro-infiltration (dpa); CTRL, empty control; B2-RFP, pLX-B2-TagRFP-T (SEQ ID NO: 13); B3-RFP, pLX-B3-TagRFP-T (SEQ ID NO: 14); B4-RFP, pLX-B4-TagRFP-T (SEQ ID NO: 15). (B) RFP fluorescence of infiltrated leaves was imaged under a fluorescence stereomicroscope. (C) Cell RFP fluorescence was imaged by confocal microscopy; scale bars, 100 µm. (D) RFP accumulation was assessed by immunoblot analysis. Ponceau red-stained blot as loading control.

FIG. 4. Plant stable transgene expression using pLX vector series.
(A) Transgene construct assembled in pLX-B2-PCRC:mTFP1 (SEQ ID NO: 23) for stable transformation of Arabidopsis thaliana. A cyan fluorescent protein gene (mTFP1) is driven by the A. thaliana cruciferin C promoter, which is active in seeds (PCRC). (B) Genomic DNA PCR was performed to confirm stable transgene integration, using transgene specific (mTFP1; 765bp) or control primers (PCRC; 1081bp). Each line represents a single plant sample; C, untransformed plant sample; T1, independent lines selected by cyan fluorescence of seed collected from Agrobacterium-treated plants. Fluorescence images are shown of untransformed seeds (Col-0), and seeds collected from a single T1 plant (T2).

FIG. 5. Stability of pLX vector series in Escherichia coli.
(A) The expression cassette of a GFP-tagged Plum pox virus cDNA clone (PPV) was subcloned from a pBIN19-derived vector (pSN-PPV) to a pLX plasmid, to generate the pLX-PPV vector (SEQ ID NO: 21). Schemes are not to scale. (B) Clones #A and #B of the new pLX-PPV vector were transformed in E colito evaluate plasmid stability, inputs (In). For each transformation, eight individual colonies were picked and subjected to six growth cycles (24 h, 37°C). Purified plasmids, outputs (Out), were EcoRI-digested and resolved by agarose gel electrophoresis. Fragments derived from the cDNA copy cassette of PPV genome are indicated (left); upper bands are backbone-specific fragments. (C) The expression cassette of a GFP-tagged Turnip mosaic virus cDNA clone (TuMV) was subcloned from a pUC-based vector (p35Tunos-vec01-NAT1) to a pLX-B2-derived plasmid, and generating the pLX-TuMV vector (SEQ ID NO: 28). (D) The pLX-TuMV (SEQ ID NO: 28) was transformed into E coli to evaluate plasmid stability, input (In). Ten individual colonies were picked and subjected to six growth cycles of 24h, at 37°C. Purified plasmids, outputs (Out), were EcoRI-digested, and subjected to agarose gel electrophoresis.

FIG. 6. Viral vector delivery and recombinant protein production in plants using pLX vector series.
The pLX-PPV (pLX) (SEQ ID NO: 21) and pSN-PPV (pSN) viral vectors were delivered to N. benthamiana plants by agro-infiltration (A-D); the pLX-TuMV viral vector was delivered to A. thaliana plants by agro-inoculation (E-G). (A) Recombinant GFP was expressed in plants using a chimeric PPV clone. (B) Viral accumulation was assessed by anti-PPV coat protein (CP) immunoblot analysis of agro-infiltrated and upper uninoculated leaf samples, at 6 and 14 dpa, respectively. Ponceau red-stained blots as loading control. (C) GFP fluorescence intensity (FI) of the agro-infiltrated leaf patches was quantified in a 96-well plate reader at 6 dpa. Bar graph shows mean ± SD (n = 4); * p < 0.001, Student's t-test. (D) Upper uninoculated leaves were imaged at 14 dpa on a blue light transilluminator; GFP fluorescence in light gray; scale bar, 2 cm. (E) Recombinant GFP was expressed in plants using a chimeric TuMV clone. (F) The pLX-TuMV (SEQ ID NO: 28) vector was delivered to A. thaliana plants by agro-inoculation, and data collected at 11 days post agro-inoculation. Viral accumulation was assessed by anti-TuMV coat protein (CP) immunoblot analysis of upper uninoculated leaves; Ponceau red-stained blots as loading control. (G) Upper uninoculated leaves were imaged; GFP fluorescence in light gray; scale bar, 1 cm.

FIG. 7. Assembly of DNA parts into pLX vectors using synthetic biology standards.
(A) Standardized units for plant delivery of kanamycin resistance (Nptll) and red fluorescent protein (DsRED) genes into the pLX-B2 vector (SEQ ID NO: 3), to generate the pLX-B2-Nptll-DsRED vector (SEQ ID NO: 20). (B) The pLX-B2-XT1-XT2-hCas9 vector (SEQ ID NO: 19) was assembled for delivery of standardized units: a kanamycin resistance gene (Nptll), human codon-optimized Streptococcus pyogenes Cas9 gene (hCas9), and sgRNA targeting the N. benthamiana Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) endogenous genes. (C) Scheme of pLX vectors that incorporate cloning cassettes compatible with GoldenBraid binary assembly. Alpha level kanamycin-resistant plasmids have divergent Bsal and convergent BsmBI sites; omega level spectinomycin-resistant plasmids have divergent BsmBI and convergent Bsal sites. All plasmids include the pBBR1 origin and the lacZα reporter.

FIG. 8. Assembly of large transcription units by overlap-based cloning methods, and virus agro-inoculation using pLX vector series.
(A) Use of a pLX vector to generate an RNA virus infectious cDNA clone. Three RT-PCR fragments (gray boxes) spanning the entire Ugandan cassava brown streak virus (UCBSV) genome were cloned in linearized pLX-B2-based vector by Gibson assembly. The pLX-UCBSV vector (SEQ ID NO: 22) obtained was delivered to N. benthamiana plants by agro-infiltration and data collected at 12 dpa. (B) Photographs of mock- and pLX-UCBSV-infiltrated plants (left and right, respectively). Plant relative height is plotted, mean ± SD (n = 4); * p = 0.0059, Student's t-test. (C) Transmission electron micrograph shows particles observed in infected plant sample; scale bar, 100 nm. (D) Viral accumulation was assessed by anti-UCBSV coat protein (CP) immunoblot analysis of upper uninoculated leaf samples. Ponceau red-stained blot as loading control.

FIG. 9. Relative size comparison of pLX-B2 backbone and selected T-DNA binary vectors.
Relative size comparison of pLX-B2 backbone and selected binary vectors (T-DNA cassette sequences were not considered). Graph bars are filled according plasmid replication origins shown on the right; pVS1- and pSa-based binary vectors include a narrow-host-range origin for maintenance in E coli; *, pSa origin in pGreen-based vectors is not autonomous, thus, size of the RK2-based pSoup plasmid required for pGreenll maintenance in A. tumefaciens is also included in the bar graph. Glyphs according to Synthetic Biology Open Language visual format.

FIG. 10. Comparison of pBBR1-based pLX, RK2 and pVS1 T-DNA binary vectors in plant expression assays.
(A) pBBR1 replication module of pLX vectors was replaced by a minimal RK2 origin to build pLX-R2 (SEQ ID NO: 6), -R3 (SE ID NO: 7) and -R4 (SEQ ID NO: 8) vectors. These were engineered to express the TagRFP-T gene (RFP) obtaining the pLX-R2-TagRFP-T (SEQ ID NO: 16), pLX-R3-TagRFP-T (SEQ ID NO: 17) and pLX-R4-TagRFP-T (SEQ ID NO: 18) vectors. (B) In transient expression assays, RFP vectors from the Figure 3 (B2-RFP: pLX-B2-TagRFP-T (SEQ ID NO: 13); B3-RFP: pLX-B3-TagRFP-T (SEQ ID NO: 14); B4-RFP: pLX-B4-TagRFP-T (SEQ ID NO: 15)) were compared to RK2-based pLX vectors (R2-RFP: pLX-R2-TagRFP-T (SEQ ID NO: 16); R3-RFP: pLX-R3-TagRFP-T (SEQ ID NO: 17); R4-RFP:, pLX-R4-TagRFP-T (SEQ ID NO: 18)); CTRL, empty control. RFP fluorescence intensity (FI) of bacterial suspensions and infiltrated plant samples (at 4 or 6 dpa) was measured in a plate reader. Bar graphs show plant FI values, mean ± SD (n ≥ 3); letters indicate p < 0.05, one-way Anova and Tukey's HSD test; * p = 0.00047, Student's t-test. Scatter plot shows linear regression analysis of plant and bacterial FI values; B3-RFP, R3-RFP, and empty control samples are filled in black, gray and white, respectively. (C) Expression of a standard DsRED cassette was compared in transient and stable expression assays. A pCAMBIA-derived (GB1686, SEQ ID NO: 27) and the pLX-B2-Nptll-DsRED (pLX, SEQ ID NO: 20) vectors were transformed into N. benthamiana plants; CTRL, control. In agro-infiltrated leaf samples, cell DsRED fluorescence was imaged by confocal microscopy (scale bars, 100 µm) and quantified in a plate reader. FI values were plotted, mean ± SD (n = 4); letters indicate p < 0.05, one-way Anova and Tukey's HSD test. In stable transformation assays, leaf samples were co-culture with indicated A. tumefaciens strains and transferred to kanamycin containing medium. Images show plantlets imaged under an epifluorescence microscope at 40 days post inoculation. Plot shows transformation efficiency defined as number of kanamycin-resistant plantlets that showed DsRED fluorescence, mean ± SD (n = 7); n.s., p = 0.91. Vector origins are indicated: pBBR1, solid circle/bars; pVS1, open circle/bars.

FIG. 11. Delivery of CRISPR/Cas system components and comparison of pBBR1-based pLX and pVS1 T-DNA binary vectors in targeted genome mutagenesis assays.
Targeted mutagenesis using a GoldenBraid-based CRISPR/Cas9 system in transient expression assays. (A) Nicotiana benthamiana plants were infiltrated with a pCAMBIA-derived (GB1108) and the pLX-B2-Nptll-DsRED (pLX: SEQ ID NO: 20) vectors that bear transcription units for human codon-optimized Cas9 (hCas9), and sgRNA targeting the endogenous Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) genes. (B) Gels show PCR/digestion assays; asterisks mark cleavage-resistant DNA bands; CTRL, hCas9 delivered with no sgRNA sequences. Mutagenesis efficiency was estimated by quantifying ratio of uncleaved/cleaved bands and plotted, mean ± SD (n = 4); * p < 0.001. Vector origins are indicated: pBBR1, solid circle/bars; pVS1, open circle/bars.

FIG. 12. Sequence similarity of the pLX and reference T-DNA binary vectors.
(A) Representation of the new pLX binary vector compatible with pBBR1 origin. pLX-Z4 (SEQ ID NO: 9) shares the pLX modular organization and cloning cassette shown in Figure 2; it includes T-DNA border sequences from the succinamopine-type pTiBo542 plasmid, a second left border sequence, lambda phage terminators, a gentamicin resistance gene (aacC1), and a 2.2-kb minimal replicon from the broad host-range RK2 plasmid. (B) Percent identity plots show significant DNA local alignments between the pBBR1-based pLX-B2 and RK2-based pLX-Z4 (SEQ ID NO: 9), or pLX-R4 (SEQ ID NO: 8) vectors. Cloning cassette sequences were omitted in the comparison; plots were generated by PipMaker (Schwartz S., et al., Genome Res. 2000, 10, 577-586). (C) Sequence similarity of the new pLX and reference T-DNA binary vectors. The matrix shows outputs obtained by pairwise sequence analysis of vector backbones. Sequence similarity was classified according to BLASTN total score values: high, > 4100; partial, 800 - 4100; low, < 800. Matrix entries of pBBR1-based pLX vectors are boxed, and crossed entries mark vector pairs that show low sequence similarity but share selection antibiotics.

FIG. 13. Characterization of a disarmed octopine-type A. tumefaciens strain that shows sensitivity to several antibiotics and its usage for vector multiplexing.
(A) Antibiotic sensitivity of the disarmed A. tumefaciens strain C58C1-313. Bacteria were inoculated into LB supplemented with rifampicin plus indicated antibiotics: AMP (ampicillin), CL (chloramphenicol), GENT (gentamicin), TC (tetracycline), KAN (kanamycin), SP (spectinomycin) and ST (streptomycin). To monitor growth curves, absorbance (OD600) was measured in a plate reader. Plot shows mean ± SD (n = 6); h, hours. (B) C58C1-313 harbors a pTi of the octopine type. A fragment of pTi repB gene was PCR-amplified from C58C1-313 and sequenced. A phylogenetic tree was built from alignment of the 607-nt repB sequence from C58C1-313 strain and deposited Ti plasmid sequences (NCBI: DQ058764.1; AB016260.1; AE007871.2; M24529.1; CP011249.1; AF242881.1). C58C1-313 clusters with octopine-type pTi accessions. (C) Stability of pTi maintenance in A. tumefaciens strain C58C1-313. C58C1-313 was plated, and the presence of pTi in individual colonies was confirmed by PCR using pTi-specific primers (repB; 724bp); N, negative control. (D) Diagram of an A. tumefaciens strain (dashed hexagon) that simultaneously hosts pLX-B2- and pLX-Z4-derived vectors conferring kanamycin and gentamicin resistance, respectively. Growth curves of A. tumefaciens C58C1-313 that harbors no vectors (CTRL, gray), or pLX-B2- plus pLX-Z4-derived vectors (black). Kanamycin- and gentamicin-supplemented LB was inoculated with the indicated strains, and absorbance measured in a plate reader. The plot shows mean ± SD (n = 6); h, hours.

FIG. 14. Usage of pLX vector series for multiple T-DNA delivery to plants.
(A) Diagram of an A. tumefaciens strain (dashed hexagon) that simultaneously hosts pLX-B2- and pCAMBIA-derived vectors conferring kanamycin and spectinomycin resistance, respectively; vector origins are indicated: pBBR1, solid circle; pVS1, open circle. Components of pLX-B2-PCRC:mTFP1 were described in Figure 4; in pSN.5-PPAP85:RFP, the TagRFP-T gene (RFP) is driven by the A. thaliana PAP85 promoter (PPAP85). The used PCRC and PPAP85 promoters are active in seeds. (B) Arabidopsis plants were treated with the A. tumefaciens pLX-B2-PCRC:mTFP1 plus pSN.5-PPAP85:RFP strain by floral dip. Collected T1 seeds were visualized under a fluorescence stereomicroscope. Pictures show seeds that express mTFP1 only (Single T-DNA), or mTFP1 plus RFP (Double T-DNA); for each condition, number and percentage of obtained seeds is indicated.

FIG. 15. Experimental design for synthetic circuit component delivery to plants by pLX vector multiplexing.
(A) Sequence of the PEtOH synthetic promoter (SEQ ID NO: 35). The Cauliflower mosaic virus (CaMV) 35S terminator was included to insulate against promoters that might flank T-DNA integration sites; AlcR DNA-binding sites (triangles) derived from Aspergillus nidulans a/cM, alcR, aldA, alcA promoters are placed upstream a minimal Figwort mosaic virus 34S promoter (arrow); open box, starting codon of coding sequence. (B) Buffer gate truth table. Symbol of a buffer gate that uses ethanol (EtOH) as input and mNeonGreen (NEON) fluorescence as output. (C) Genetic circuit that implements the previous panel gate. The dashed hexagon represents a single A. tumefaciens strain (R-AlcR + PEtOH:NEON) that hosts two compatible T-DNA binary vectors, pLX-Z4-Pmas:RFP-AlcR (SEQ ID NO: 24) and pLX-B2-PEtOH:NEON (SEQ ID NO: 25), which confer gentamicin and kanamycin resistance, respectively. Once delivered to plants, the constitutive mannopine synthase promoter (Pmas) drives expression of RFP and AlcR proteins. In the presence of EtOH (star), AlcR binds to, and activates an otherwise silent synthetic promoter (PEtOH). NEON accumulation results from activation of the gate.

FIG. 16. Gene expression control and synthetic circuit component delivery to plants by pLX vector.
Evaluation of an ethanol buffer gate in plants. (A) Nicotiana benthamiana plants were infiltrated with an Agrobacterium strain that harbors both pLX-Z4-Pmas:RFP-AlcR (SEQ ID NO: 24) and pLX-B2-PEtOH:NEON (SEQ ID NO: 25) binary vectors (R-AlcR + PEtOH:NEON), treated twice with water or EtOH. At 4 dpa, RFP and NEON fluorescence was imaged by leaf laser scanning. Scale bar, 3 cm. (B) Nicotiana benthamiana leaves were untreated (N), or infiltrated with the A. tumefaciens R-AlcR + PEtOH:NEON strain. Leaf disks were collected, placed in a 96-well plate and supplied with or without EtOH. Cell RFP and NEON fluorescence was imaged by confocal microscopy at 24 h post-treatment (hpt). (C) Leaf disks from agro-infiltrated patches were placed in a 96-well plate and different amounts of inducer were added. Fluorescence intensities (FI) were measured in a plate reader at 22 hpt, and relative NEON/RFP FI value of the non-inducer condition (None) was set to 1. Bar graph shows mean ± SD (n = 18). Letters indicate p <0.01, one-way Anova and Tukey's HSD test. (D) Kinetics of the EtOH-responsive buffer gate. Leaf disks from agro-infiltrated patches were treated with water (gray, minus) or 0.1% EtOH (black, plus), and fluorescence intensity measured in a plate reader. Relative NEON/RFP FI value of the water condition was set to 1. The plot shows mean ± SD (n = 5).


DETAILED DESCRIPTION OF THE INVENTION



[0046] In a first aspect, the present invention relates to a binary vector (pBBR1-based pLX vector) comprising at least three modules: (a) a T-DNA cassette module comprising at least a right and a T-DNA left borders sequences, (b) a replication origin module comprising a pBBR1 origin or a variant functionally equivalent thereof, and (c) at least a selectable marker module.

[0047] The terms "plasmid" and "vector", as used herein are interchangeable, and refer to an extra chromosomal element that may carry one or more gene(s). Plasmids and vectors typically are circular double-stranded DNA molecules. However, plasmids and vectors may be linear or circular nucleic acids, of a single- or double-stranded DNA or RNA, and may be derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construct that is capable of introducing a promoter fragment and a coding polynucleotide sequence along with any appropriate 3' untranslated sequence into a cell. In examples, plasmids and vectors may comprise autonomously replicating sequences, genome integrating sequences, and/or phage or nucleotide sequences.

[0048] The term "viral vector" refers to a vector that hosts viral genome sequences that can launch viral infections, and are useful for rapid, high-level delivery of exogenous sequences to eukaryotic cells.

[0049] The terms "Ti plasmid", "Ri plasmid", "pTi" and "pRi" as used herein are interchangeable, and refer to a large plasmid contained in the wild-type Agrobacterium sp., which comprises T-DNA (transfer DNA) that are introduced into plants, virulence region (vir region), etc. T-DNA is a DNA fragment inserted into the genome of a plant cell, and in wild-type Agrobacterium sp. comprises the genes for synthesis of opines and plant growth regulators. The vir region is a region encoding virulence proteins, a protein group required for integration of T-DNA into plants, and it comprises genes such as the virA, virB, virC, virD1, virD2, virD3, virG and virJ genes.

[0050] The term "disarmed Ti plasmid" refer to a plasmid produced by removing the T-DNA region from a wild-type Ti plasmid and encoding virulence proteins, or a functionally equivalent artificial or natural plasmid, such as and without limitation, the p42a plasmid of Rhizobium etli (Lacroix B. & Citovsky V., PLoS Pathog. 2016, 12, 3, e1005502). Thus, a disarmed Ti plasmid lacks the T-DNA region, and is able to mediate DNA transfer to eukaryotic cells and their subsequent genetic modification.

[0051] The term "border sequence", e.g. right border (RB) or left border (LB), refers to a directly repeated nucleic acid sequence defining an end of the T-DNA region. Border sequences may be from a Ti plasmid, or may be other bacterial, plant derived or synthetic sequences that function similarly. In a preferred embodiment of the pBBR1-based pLX vector of the invention, the LB and RB are independently selected from the group consisting of a T-DNA border from a nopaline-, an octopine-, a succinamopine-type Ti plasmid, or any combination thereof. In a preferred embodiment the T-DNA borders are selected form octopine- or succinamopine-type Ti plasmids from A. tumefaciens, and include a second left border of the nopaline type.

[0052] The terms "binary vector" and "T-DNA binary vector", as used herein, are interchangeable. They refer to a plasmid that has an origin of replication (ori) that permits the maintenance of the vector in a wide range of bacteria including E coli and Agrobacterium sp., and that comprises a T-DNA cassette; markers for selection and maintenance in bacteria; and, in some embodiments, the binary vector may include a selectable marker for selection in eukaryotic organisms, preferably for selection in plants.

[0053] The terms "T-DNA cassette" and "T-DNA cloning cassette", as used herein are interchangeable, refer to a T-DNA region that comprises at least the RB and LB sequences, and features that allow insertion of the sequence of interest between RB and LB sequences in a way that the sequence of interest can be transferred to eukaryotic cells.

[0054] In a more preferred embodiment, the pBBR1-based pLX binary vector of the present invention is characterized in that the T-DNA cassette comprises a right and two T-DNA left borders sequences. In a more preferred embodiment, the right border comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 102 or SEQ ID NO: 115. In a more preferred embodiment the left border comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, identical to SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 116. In a more preferred embodiment, the right border comprises the SEQ ID NO: 102 and the left borders comprises the SEQ ID NO: 103 and SEQ ID NO: 104. In a more preferred embodiment, the right border consists of SEQ ID NO: 102 and the left borders consists of SEQ ID NO: 103 and SEQ ID NO: 104.

[0055] In a further preferred embodiment of the pBBR1-based pLX binary vector is characterized in that the T-DNA region also comprises at least two transcription terminators. Transcription terminators useful in the present invention are known in the art (i.e., in Chen Y.J., et al., Nat Methods. 2013, 10, 7, 659-664). In a more preferred embodiment the transcription terminators comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any of the sequences selected from the list consisting of: SEQ ID NO: 108, 109, 110, 111, or any combinations thereof, more preferably, SEQ ID NO: 108 and 109. In a more preferred embodiment, the transcription terminators are selected from the sequences comprising the SEQ ID NO: 108, 109, 110, 111 or any combinations thereof, more preferably, SEQ ID NO: 108 and 109. In a more preferred embodiment, the transcription terminators consist of any of the sequences selected from SEQ ID NO: 108, 109, 110, 111, or any combinations thereof, more preferably, SEQ ID NO: 108 and 109.

[0056] "Homology" or "identity" or "similarity" refers to sequence similarity between two nucleic or amino acid sequences. Homology can be determined by comparing a position in each sequence, which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of homology or similarity or identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. A degree of identity of amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences. A degree of homology or similarity of amino acid sequences is a function of the number of amino acids, i.e. structurally related, at positions shared by the amino acid sequences. The degree of homology, identity, and/or similarity can be determined by use of algorithms, programs and methods, such as and without limitations Clustal, Wilbur-Lipman, GAG, GAP, BLAST, BLASTN, BLASTP, EMBOSS Needle, FASTA, Smith Waterman or BLOSUM.

[0057] In a more preferred embodiment, the pBBR1-based pLX vector of the invention is characterized in that the T-DNA borders flank a sequence of interest. The nucleic acid sequence(s) of interest is operatively linked to sequences required for DNA transfer to the target eukaryotic cell.

[0058] The term "operatively linked" or "operably associated" refers to a functional linkage between the regulatory sequence and a coding sequence or a functional linkage between two regulatory sequences. The term "construct" refers to units or components so described that are assembled and operatively linked thus in a relationship permitting them to function in their intended manner. By placing a coding sequence under regulatory control of a promoter or another regulatory sequence means positioning the coding sequence such that the expression of the coding sequence is controlled by the regulatory sequence. The term "transcription unit" refers to a construct including promoter, coding and terminator sequences that are operatively linked to permit the expression or delivery of the sequence of interest in intended manner.

[0059] The sequence of interest, although often a gene sequence, can actually be any nucleic acid sequence whether or not it produces a protein, a RNA, an antisense molecule or regulatory sequence or the like.

[0060] A "transgene" refers to a sequence of interest that independently of whether this sequence has been introduced exogenously or has been manipulated; in both cases, the sequence defined as "transgene" has not been shown to be naturally occurring. The term "endogenous gene", "endogenous sequence", "wild-type gene" or "wild-type sequence" refers to a native gene in its natural location in the genome of an organism.

[0061] Sequences of interest or transgenes may include functional elements that affect developmental processes, fertility, abiotic and biotic stress resistance that affect or confer new phenotypes, and like. Other transgenes that are useful include sequences to make edible vaccines (e.g. United States Patent No: U.S. Pat. No. 6,136,320; U.S. Pat. No. 6,395,964) for humans or animals, alter fatty acid content, change amino acid composition of food crops (e.g. U.S. Pat. No. 6,664,445), introduce enzymes in pathways to synthesize metabolites such as vitamin A and vitamin E, increase iron concentration, control fruit ripening, reduce allergenic properties of e.g., wheat and nuts, absorb and store toxic and hazardous substances to assist in cleanup of contaminated soils, alter fibre content of woods, enhance resistance to diseases, bacteria, fungi, nematodes, herbicides, viruses and insects, increase salt tolerance and drought resistance, amongst others.

[0062] In a typical vector, the sequence of interest is operatively linked to a promoter. A "promoter" is a sequence of nucleotides from which transcription may be initiated of DNA operatively linked downstream. The product of the sequence of interest may be expressed constitutively, after induction, in selective tissues or at certain stages of development. Regulatory elements to effect such expression are well known in the art. Many examples of regulatory elements may be found in the Patent Lens document "Promoters used to regulate gene expression" version 1.0, October 2003 (incorporated in its entirety). Other promoters can be identified through a variety of assays. Enhancer elements or other regulatory elements can be included in addition to a promoter. "Minimal promoter" sequences, such as the so-called minimal 35S promoter from Cauliflower mosaic virus (CaMV), or minimal 34S promoter from Figwort mosaic virus, usually require an enhancer element for activity.

[0063] In a more preferred embodiment, the pBBR1-based vector of the invention is characterized in that the T-DNA cassette module also comprises a cloning cassette, more preferably, the T-DNA cloning cassette comprises restriction endonuclease and primer annealing sites, in a more preferred embodiment, these sites are compatible with high-throughput Type IIS restriction endonuclease- and/or overlap-based DNA assembly methods, such as and without limitations, Golden Gate, GoldenBraid, Modular Cloning (MoClo), one- or two-steps Gibson assembly (Gibson D.G., et al., Nat. Methods 2009, 6, 343-345), Sequence and Ligation Independent Cloning (SLIC), GeneArt seamless cloning and assembly (Thermo Fisher Scientific), NEBuilder HiFi DNA assembly (New England BioLabs), Cold Fusion cloning (System Biosciences), In-fusion cloning (Clontech).

[0064] In another preferred embodiment, a T-DNA cloning cassette also comprises a selectable, screenable marker or reporter elements for identifying insertion of the sequence of interest. The marker or reporter element is a gene or an operon that confers a visual phenotype or negative selection, such as and without limitations the lacZα, ccdB, sacB, a luciferase, a fluorescent protein genes, or a canthaxanthin biosynthesis operon. Additionally, the screenable marker or reporter element included in the T-DNA cassette can be selected from the list mentioned below for the selectable marker module of the binary vector of the present invention.

[0065] In a further preferred embodiment, the replication origin module of the pBBR1-based pLX vector of the invention comprises a pBBR1 origin comprising pBBR1-oriV and -rep regions, or a variant functionally equivalent thereof. In a further preferred embodiment, the pBBR1 origin comprising a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 105, more preferably the pBBR1 origin comprises the SEQ ID NO: 105, and more preferably the pBBR1 origin consists of SEQ ID NO: 105.

[0066] As used herein, the term "functionally equivalent variant" refers to any variant in which the nucleotide sequence encodes an amino acid sequence comprising conservative or non-conservative alterations in the amino acid sequence that result in silent changes that preserve the functionality of the molecule including, for example, deletions, additions, and substitutions. Such altered molecules may be desirable where they provide certain advantages in their use. As used herein, conservative substitutions involves the substitution of one or more amino acids within the sequence of the corresponding peptide with another amino acid having similar polarity and hydrophobicity/hydrophilicity characteristics resulting in a functionally equivalent molecule. Such conservative substitutions include but are not limited to substitutions within the following groups of amino acids: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine; and methionine, norleucine. The skilled person in the art will understand that mutations in the nucleotide sequence encoding a peptide, which give rise to conservative amino acid substitutions in positions that are non-critical for the functionality of the peptide, are evolutionarily neutral mutations that do not affect its global structure or its functionality.

[0067] The term "replication origin" (ori) refers to a cis-acting sequence essential for replication. Origin sequences that permit the plasmid replication or maintenance in a wide range of bacteria have been described (U.S. Pat. Nos. 4,940,838; 5,149,645; 6,165,780; 6,265,638, incorporated in its entirety). In a preferred embodiment, the origin of replication is a wide-host-range or broad-host-range origin, used interchangeable in the present invention. As used herein, "wide-host-range" or "broad-host-range" means that the vector replicates in at least two bacterial species, preferably in Agrobacteriumsp. and E coli. Host range is conferred by an origin of replication. When the nucleic acid molecule is integrated into the bacterial chromosome or other self-replicating bacterial DNA molecule, an origin is not necessary. Thus, when suitably modified and engineered, these bacteria may be used for transferring nucleic acid sequences into eukaryotic cells, and especially into plant cells.

[0068] In another preferred embodiment, the pBBR1-based pLX vector also comprises a selectable or a screenable marker module for identifying host cell transformants, preferably bacterial transformants. Well-known selectable markers are genes that confer resistance to drugs, such as antibiotics selected from the list consisting of: neomycin, ampicillin, carbenicillin, chloramphenicol, kanamycin, tetracycline, gentamicin, spectinomycin, bleomycin, phleomycin, streptomycin, erythromycin, blasticidin and hygromycin; herbicide resistance genes, and the like. Other selection systems, including genes encoding resistance to other toxic compounds, such as potassium tellurite resistance genes, genes encoding products required for growth of the cells, such as in positive selection, can alternatively be used. Examples of these "positive selection" systems are abundant (see for example, U.S. Pat. No. 5,994,629). Otherwise, "negative selection" systems can also be used. Alternatively, a screenable marker or reporter gene may be employed to allow selection of transformed cells based on a visual phenotype, e.g. a β-glucuronidase, a luciferase, or a fluorescent protein gene. The selectable marker also typically has operably linked regulatory elements necessary for transcription of the genes, e.g., a constitutive or inducible promoter and a terminator sequence. Elements that enhance efficiency of transcription are optionally included. In a preferred embodiment, the selectable marker module comprises a gene that confers resistance to a drug selected from the group consisting of neomycin, ampicillin, carbenicillin, chloramphenicol, kanamycin, tetracycline, gentamicin, spectinomycin, bleomycin, phleomycin, streptomycin, erythromycin, blasticidin and hygromycin resistance genes.

[0069] In a more preferred embodiment, the pBBR1-based pLX vector is selected from the list consisting of: SEQ ID NO: 3 (pLX-B2), SEQ ID NO: 4 (pLX-B3), SEQ ID NO: 5 (pLX-B4), SEQ ID NO: 10 (pLX-B2α2), SEQ ID NO: 11 (pLX-B3Ω1), SEQ ID NO: 12 (pLX-B3Ω2), SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 28.

[0070] Furthermore, a pBBR1-based pLX vector of the present invention can be used either as a single binary vector, which has autonomous replication, or in a binary vector system, that includes a combination of binary vectors which have replication and bacterial selection mechanisms that enable a mutual and autonomous coexistence with each other.

[0071] As used herein, the phrase "binary vector system" refers to binary vectors that are capable of replicating in both E coli and A. tumefaciens, and host unlinked T-DNA cassettes. In a binary vector system, vectors are multiplexed and employed for delivery of multiple T-DNA cassettes to eukaryotic cells or organisms, preferably to plants.

[0072] In a more preferred embodiment, the binary vectors and vectors of the binary vector system of the present invention have a minimal size between 2 to 20 kb, preferably between 2.5 to 3.8 kb, more preferably have a size below 3.8 kb.

[0073] Another aspect of the present invention refers to a binary vector system comprising the pBBR1-based pLX plasmid according to the present disclosure and another binary vector (second binary vector) described in state of art and compatible with a first binary vector of the present invention. In a more preferred embodiment of the binary vector system of the present invention, the second binary vector is a RK2-based pLX plasmid as described herein.

[0074] Another aspect of the present invention, the RK2-based pLX plasmid according to the present disclosure refers to a binary vector comprising at least three modules: (a) a T-DNA cassette module comprising at least a right and a T-DNA left borders, (b) a replication origin module comprising an origin compatible with pBBR1 origin, preferably selected from the list consisting of IncQ, IncW, IncU, pRi, pVS1 and IncP-α plasmid incompatibility group origins, wherein more preferably is an IncP-α plasmid incompatibility group origin, and wherein more preferably, the replication origin is the RK2 origin, or a variant functionally equivalent thereof, and (c) at least a selectable marker module.

[0075] In a preferred embodiment, the RK2-based pLX vector of the invention comprises a T-DNA cassette comprising a right and two T-DNA left borders, preferably comprising the T-DNA border sequences mentioned above. In a more preferred embodiment, the right border comprises the SEQ ID NO: 115 and the left borders comprise the SEQ ID NO: 116 and SEQ ID NO: 104. In a more preferred embodiment, the right border consists of SEQ ID NO: 115, and the left borders consist of the SEQ ID NO: 104 and SEQ ID NO: 116.

[0076] In a further preferred embodiment of the RK2-based pLX vector is characterized in that the T-DNA cassette is flanked by at least two transcription terminators, preferably the transcription terminators that are disclosed above. In a more preferred embodiment, the transcription terminators comprise the SEQ ID NO: 110 and 111, more preferably the transcription terminators consist of SEQ ID NO: 110 and 111.

[0077] In a more preferred embodiment, the RK2-based pLX vector of the invention is characterized in that the T-DNA borders flank a sequence of interest. The nucleic acid sequence(s) of interest is operatively linked to sequences required for DNA transfer to the target eukaryotic cell. In a more preferred embodiment, the sequences of interest are mentioned above.

[0078] In a more preferred embodiment, the RK2-based vector of the invention is characterized in that the T-DNA cassette module also comprises a cloning cassette, more preferably, the T-DNA cloning cassette comprises the selectable, screenable marker or reporter elements mentioned above. In a further preferred embodiment, the T-DNA cassette comprises restriction endonuclease and primer annealing sites, in a more preferred embodiment, these sites are compatible with high-throughput Type IIS restriction endonuclease- and overlap-based assembly methods as mentioned above.

[0079] In a further preferred embodiment, the replication origin of the RK2-based pLX vector of the invention comprises the RK2 replication origin comprising the RK2-oriV and -trfA regions, or a variant functionally equivalent thereof. In a more preferred embodiment the RK2 origin comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 106 or SEQ ID NO: 107, more preferably the RK2 origin comprises the SEQ ID NO: 106 or SEQ ID NO: 107, and more preferably the RK2 origin consists of SEQ ID NO: 106 or SEQ ID NO: 107.

[0080] In another preferred embodiment, the selectable marker module of the RK2-based pLX binary vector comprises a selectable or a screenable marker gene mentioned above.

[0081] In a more preferred embodiment, the selectable marker gene of the RK2-based pLX binary vector differs from the selectable marker gene of the pBBR1-based pLX vector so as to facilitate simultaneous selection of both plasmids.

[0082] In a more preferred embodiment, the RK2-based pLX vector is selected from the list consisting of: SEQ ID NO: 6 (pLX-R2), SEQ ID NO: 7 (pLX-R3), SEQ ID NO: 8 (pLX-R4), SEQ ID NO: 9 (pLX-Z4), SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 24.

[0083] In another preferred embodiment, the backbone of the RK2-based pLX vector has no regions with > 28 nucleotide identity to the pBBR1-based pLX vector of the present invention.

[0084] Consequently, the binary vector system of the present invention comprises the pBBR1-based pLX plasmid and preferably, the RK2-based pLX plasmid according to the present disclosure.

[0085] Another aspect of the present invention relates to methods to assemble synthetic, genomic, metagenomic, and/or cDNA sequences of interest into the binary vectors or the vector system disclosed in the present invention.

[0086] In another aspect of the present invention, it is related to a host cell comprising the pBBR1-based pLX, RK2-based pLX, or the binary vector system of the present invention.

[0087] In accordance with the present invention, the term "host cell" refers to a cell which has been transformed, or is capable of been transformed, by an exogenous DNA sequence, preferably by the binary vectors or the binary vector system of the present invention. A host cell can be used, for example, for expression of a nucleic acid of interest, propagation of plasmid vectors and/or delivery of a sequence of interest to eukaryotic cells.

[0088] In a preferred embodiment, the host cell of the present invention is a bacterial cell, preferably selected from Agrobacterium sp. and E coli. In a more preferred embodiment, the host cell is preferably a species of the Rhizobiaceae family, more preferably an Agrobacterium sp. bacterium, especially preferably an Agrobacterium strain that comprises a disarmed Ti plasmid.

[0089] Alternatively, genome sequences of Agrobacterium sp. and other bacterial species can be compared; missing genes in the latter bacteria that are important for T-DNA delivery and transformation into eukaryotic cells may be individually picked from the Agrobacterium genome and inserted into the desired bacterial genome by any means or expressed on a plasmid. Similarly, the bacteria can be used to transform a eukaryotic organism or cell under a variety of test conditions, such as temperature, pH, nutrient additives and the like. The best conditions can be quickly determined and then tested in transformation of plant cells or other eukaryotic cells as mentioned above. Furthermore, host bacterial species may naturally interact in specific ways with a number of eukaryotic organisms such as plants. These bacterial-plant interactions are very different from the way Agrobacterium naturally interacts with plants. Thus, the tissues and cells that have are transformable by Agrobacterium sp. may be different in the case of the employment of other bacteria.

[0090] In general, the plasmids are transferred through a direct transfer method to the bacteria (host cell) of this invention. By transferring either single or multiple binary vectors as described herein, transformation competent bacteria are generated. These bacteria can be used to transform a eukaryotic organism or a eukaryotic cell, such as a yeast, a fungi, a plant, an insect and an animal.

[0091] The term "eukaryotic cell" refers to either individual cells or aggregations of cells, such as tissues or organs, parts of tissues or organs, and entire organisms, comprising a yeast, a fungus, an alga, a plant, an insect and an animal.

[0092] In a more preferred embodiment, the term "plant cell" refers to individual cells or aggregations of cells, organized plant tissues, organs, or entire plants, such as and without limitation, protoplasts, calli, cell cultures, meristems and meristematic tissues, leaves, shoots, roots, flowers, ovules, pollen and pollen tubes, seeds, embryos, hypocotyls, cotyledons, seedlings and mature plants.

[0093] Eukaryotic cells may be transformed within the context of this invention. Generally, eukaryotic cells to be transformed are cultured before transformation, or cells may be transformed in situ. In some embodiments, the cells are cultured in the presence of additives to render them more susceptible to transformation. Transformants can be easily detected by their changed phenotype, e.g., growth on a medium including drugs/herbicides/toxic compounds or lacking an essential growth component on which the untransformed cells cannot grow. In other embodiments, the cells are transformed without prior culturing.

[0094] Briefly, in an exemplary transformation protocol, to generate transformed plants, plant cells are transformed by their co-cultivation with a culture of bacteria containing the binary vectors or the binary vector system described herein. After co-cultivation for a few days, bacteria are removed, for example by washing and treatment with antibiotics, and plant cells are transferred to post-cultivation medium plates generally containing an antibiotic to inhibit or kill bacterial growth and optionally a selective agent, such as described in U.S. Pat. No. 5,994,629. Plant cells are further incubated for several days. The expression of the transgene may be tested at this time. After further incubation for several weeks in selecting medium, plant cells are transferred to regeneration medium and placed in the light. Shoots obtained are transferred to rooting medium and resulting plants are further propagated.

[0095] Alternative methods of plant cell transformation include dipping whole flowers into a suspension of bacteria, growing the plants further into seed formation, harvesting the seeds and germinating them in the presence of a selection agent that allows the growth of the transformed seedlings only. Alternatively, germinated seeds may be treated with a selection agent that only the transformed plants tolerate. Alternatively, seeds may be visually selected by detection of fluorescent proteins that only the transformed seeds accumulate.

[0096] Cell transformation by Agrobacterium occurs independent of stable transgene integration into host genomes, and use of transient expression systems or autonomously replicating RNA/DNA units (viral vectors), can avoid the need for gene integration, if desired. In this sense, the terms "infiltration" and "agro-infiltration" refer to a transient transformation method that relies on mechanical introduction of cultures of host cells comprising at least one binary vector, into eukaryotic organisms or their organs, preferably entire plants, seedlings or leaves. Scale-up is achieved through, for example, the use of vacuum infiltration. The term "agro-inoculation" refers to delivery of viral vectors by Agrobacterium-mediated transient transformation.

[0097] Plants that are especially desirable to transform include corn, rice, wheat, soybean, alfalfa and other leguminous plants, potato, tomato, tobacco, Nicotiana benthamiana, and so on.

[0098] Another aspect of the present invention refers to a cell culture comprising the host cells of the present invention.

[0099] Another aspect of the present invention relates to a method for delivering at least one nucleotide sequence of interest in at least one plant cell, comprising: (a) inserting the nucleotide sequence of interest into the T-DNA cassette of the pBBR1-based pLX vector, the RK2-based pLX vector, or the binary vector system of the present invention, (b) introducing the pBBR1-based pLX vector, the RK2-based pLX vector, or the binary vector system of step (a) into at least one bacterial host cell according to the present invention, and (c) contacting the host cell of step (b) with a plant cell.

[0100] In a preferred embodiment, the method for delivering at least one nucleotide sequence of interest in at least one plant cell it is characterized in that the bacterial host cell is an Agrobacterium sp. cell, more preferably, the Agrobacterium cell comprises a disarmed Ti plasmid.

[0101] In addition to numerous technologies for transforming plants or plant cells, the type of cell, tissue, organ that is contacted with the foreign constructs may vary as well. Almost all plant tissues may be transformed during dedifferentiation using appropriate techniques within the skill of the art. One skilled in the field of plant transformation will understand that multiple methodologies are available for the production of transformed plants, and that they may be modified and specialized to accommodate biological differences between various plant species. Regardless of the particular transformation technique employed, the nucleotide of interest can be incorporated into the binary vectors or the binary vector system of the present invention adapted to express the nucleotide sequence of interest in a plant cell by including in the vector a plant promoter. In addition to plant promoters, promoters from a variety of sources can be used efficiently in plant cells to express foreign genes. For example, promoters of bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promoter; promoters of viral origin, such as the 35S and 19S promoters of CaMV, a promoter from sugarcane bacilliform virus, and the like may be used. Plant-derived promoters include, but are not limited to, ribulose-1,5-bisphosphate carboxylase (RuBisCO) small subunit promoter, beta-conglycinin promoter, cruciferin promoter, phaseolin promoter, alcohol dehydrogenase promoter, heat-shock promoters, actin depolymerization factor promoter, and tissue specific promoters. Promoters may also contain certain enhancer sequence elements that may improve the transcription efficiency. Typical enhancers include, but are not limited to, alcohol dehydrogenase 1 (ADH1-intron 1 and ADH1-intron 6). Constitutive promoters may be used. Constitutive promoters direct continuous gene expression in nearly all cell types and at nearly all times (e.g., actin promoter, ubiquitin promoter, CaMV 35S promoter). Tissue specific promoters are responsible for gene expression in specific cell, tissue, or organ types. Examples of other promoters that may be used include those that are active during a certain stage of the plant's development, as well as active in specific plant tissues and organs. Examples of such promoters include, but are not limited to, promoters that are root-, pollen-, embryo-, corn silk-, cotton fiber-, seed endosperm-, and phloem-specific promoters. In a further embodiment, the promoter is an inducible promoter. An inducible promoter is "switched on" or increases expression of genes in response to a specific signal, such as physical stimuli (e.g., temperature, heat shock gene promoters; light, RuBisCO promoter); hormones (e.g., glucocorticoid); antibiotics (e.g., tetracycline); metabolites or chemical compounds (e.g., ethanol); and stresses (e.g., drought). Other desirable transcription and translation elements that function in plants also may be used, such as, for example, 5' untranslated leader sequences, RNA transcription termination sequences and poly-adenylate addition signal sequences. Any additional element known in the art and functional in plants may be used.

[0102] The biological transformation method described here can be used to introduce one or more sequences of interest (transgene) into eukaryotic cells, wherein the eukaryotic cell is selected from the group consisting of a yeast cell, a fungal cell, a plant cell, an insect cell and an animal cell, preferably the eukaryotic cell is a plant cell.

[0103] Agrobacterium is an extremely advantageous method for eukaryotic transformation, alternatively the binary vectors or the vector system disclosed in the present invention can be introduced into eukaryotic cells using any physical methods, such as particle or microprojectile bombardment, electroporation, or other forms of direct DNA uptake such as liposome mediated DNA uptake, or the vortexing method. In a preferred embodiment, physical methods for the transformation of plant cells are reviewed in Oard J.H., Biotech. Adv. 1991, 9, 1-11.

[0104] The present invention furthermore relates to a transformed plant system, to regenerated cells or a regenerated plant therefrom, to their progeny or seeds therefrom generated in accordance with the method described hereinabove.

[0105] In a particular embodiment of the present invention, this transformed plant system is characterized by single or multiple modifications of plant cell genome, epigenome, transcriptome or metabolome, and in that it may comprise or may not comprise any sequence segments of the abovementioned vector, vector system and their T-DNA cassettes. In this sense, a component of Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems from bacteria and archaea can be used to target specific sequences in eukaryotic genomes, and in plant genomes (Murovec J., et al., Plant Biotechnol. J. 2017, doi:10.1111/pbi.12736). This document features a method for modifying the genomic material in a eukaryotic cells, preferably in a plant cell, based on the use of binary vectors of the invention together with components of CRISPR/Cas systems which provides a relatively simple, effective tool for generating modifications in genomic DNA at selected sites, without need of transgene integration or maintenance into eukaryotic cell genomes. CRISPR/Cas systems and their derivatives can be used for, without limitation, targeted mutagenesis, gene targeting, gene replacement, targeted deletion, targeted inversion, targeted translocation, and/or targeted insertion at single or multiple genome site(s). CRISPR/Cas system applications also include epigenetic and transcription regulation, cellular imaging and pathogen targeting. This technology can be used to accelerate the rate of functional genetic studies in eukaryotes, preferably in plants, and to engineer plants with improved characteristics, including enhanced nutritional quality, increased resistance to disease and stress, and heightened production of commercially valuable compounds.

[0106] In another aspect, the present invention relates to a method for in vitro for delivering at least one nucleotide sequence of interest in at least one eukaryotic cell or organism, comprising: (a) inserting at least one nucleotide sequence of interest into the binary vectors or into the binary vector system of the invention, (b) introducing the binary vectors or the binary vector system, of step (a) into at least one eukaryotic cell or organism.

[0107] In a preferred embodiment of the method for in vitro for delivering at least one nucleotide sequence of interest in at least one eukaryotic cell or organism, the eukaryotic organism is selected from the group consisting of yeasts, fungi, insects and animals.

[0108] Another aspect of the present invention relates to a method for transforming eukaryotic cells comprising the step of introducing into the eukaryotic cell the pBBR1-based pLX vector, the RK2-based pLX vector, the binary vector system or the host cell disclosed in the present invention.

[0109] Another aspect the present invention relates to a method for obtaining a genetically-engineered plant cell or plant comprising the step of introducing the binary vectors, preferably the pBBR1-based pLX vector, RK2-based pLX vector, the vector system, or the bacterial host cell of the invention, into a plant cell

[0110] In another aspect, the present invention relates to a genetically-engineered plant cell or plant obtainable by the method disclosed above.

[0111] Another aspect the present invention relates to a method for in vitro obtaining a genetically-engineered eukaryotic cell or organism, comprising the step of introducing the binary vectors, preferably the pBBR1-based, or the RK2-based binary vectors or the binary vector system into a eukaryotic cell or organism. In a preferred embodiment, the eukaryotic cell or organism is selected from the group consisting of a yeast, a fungal, an insect and an animal.

[0112] In another aspect, the present invention relates to a genetically-engineered eukaryotic cell or organism obtainable by the method disclosed above.

[0113] Another aspect of the present invention relates to the use in vitro or ex vivo of the binary vectors, preferably the pBBR1-based pLX binary vector, the RK2-based pLX binary vector, the binary vector system, the bacterial host cell, the culture cells, the genetically-engineered plant cell or plant obtainable by the method disclosed above, or the genetically-engineered eukaryotic cell or organism obtainable by the method disclosed above: (a) for site specific gene knockout, (b) for site-specific genome editing, (c) for DNA sequence-specific interference, (d) for site-specific epigenome editing, (e) for site-specific transcription modulation, or (f) for multiplex genome engineering, and provided that the use does not comprise a process for modifying the germ line genetic identity of human beings.

[0114] In another aspect, the disclosure provides a kit comprising one or more of the components described herein. In some embodiments, the kit comprises the binary vector, the binary vector system, the host cell or the culture cell disclosed herein, and instructions for using the kit. The components or elements may be provided individually or in combinations, and may be provided in any suitable container, such as a vial, a bottle, or a tube. By "kit" as used herein, it refers to a product containing the different reagents necessary to carry out the methods of the invention packaged allowing transport and storage. Suitable materials for packaging kit components include glass, plastic (polyethylene, polypropylene, polycarbonate and the like), bottles, vials, paper, envelopes and the like. Additionally, kits invention may contain instructions for simultaneous, sequential or separate use of the different components found in the kit use. Such instructions may be in the form of printed material or in the form of an electronic device capable of storing instructions so that they can be read by a subject, such as electronic storage media (magnetic disks, tapes and the like), optical media (CD- ROM, DVD) and the like. Additionally or alternatively, the media can contain Internet addresses that provide such instructions.

[0115] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the description and claims the word "comprise", "include" and their variations are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples, drawings and sequence listing are provided by way of illustration and are not intended to be limiting of the present invention.

EXAMPLES



[0116] The following examples are offered to illustrate, but not to limit, the claimed invention. It is understood that the examples and embodiments described herein are for illustrative purposes only, and persons skilled in the art will recognize various reagents or parameters that can be altered without departing from the spirit of the invention or the scope of the appended claims.

MATERIALS AND METHODS


DNA constructs



[0117] Unless otherwise indicated, standard molecular cloning methods were used (Sambrook J. & Russel D.W., Molecular cloning: a laboratory manual - 3rd edition. Cold Spring Harbor Laboratory Press. 2001). DNA constructs were generated using chemically synthesized and available parts (Table 1). The Ugandan cassava brown streak virus isolate Ke_125 was obtained from DSMZ (PV-0912). Nucleic acids were purified using silica column-based purification kits. Alternatively, genomic DNA from plant samples was extracted following the procedure described by Edwards and collaborators (Edwards K., et al., Nucleic Acids Res. 1991, 19, 1349). PCR reactions were performed with Phusion High-Fidelity DNA Polymerase (Fermentas or New England BioLabs), and Dnpl-treated to remove plasmid templates, if required. Synthetic T-DNA cassettes T-DNA_1 (SEQ ID NO: 1) for pLX-B-series and pLX-R series, and T-DNA_2 (SEQ ID NO: 2) for pLX-Z4 were obtained from GeneArt. Overlapping DNA fragments were gel-purified and joined using homemade one-step isothermal (Gibson D.G., et al., Nat. Methods. 2009, 6, 343-345) or NEBuilder HiFi (New England BioLabs) DNA assembly master mixes. One-step digestion-ligation reactions were done using Type IIS restriction endonucleases (Bsal or BsmBI, New England BioLabs) and T4 Ligase (Promega) as described (Sarrion-Perdigones A., et al., Plant Physiol. 2013, 162, 1618-1631).

[0118] The complete plasmid details disclosed in the present invention are reported in Table 1.
Table 1
Plasmid nameOrigin(s)Reference
pSEVA431 pBBR1 http://seva.cnb.csic.es/
pSEVA631 pBBR1 http://seva.cnb.csic.es/
pSEVA221 RK2 http://seva.cnb.csic.es/
pSN.5-TagRFP-T pVS1+CoIE1 Pasin F., et al., Plant Methods. 2014, 10, 22
pSN.5-mTFP1 pVS1+CoIE1 Pasin F., et al., Plant Methods. 2014, 10, 22
pSN.5-mNeon pVS1+CoIE1 Pasin F., et al., Plant Methods. 2014, 10, 22
pGGF003 pUC Lampropoulos A., et al., PLoS One. 2013, 8, e83043
pGGC011 pUC Lampropoulos A., et al., PLoS One. 2013, 8, e83043
p35Tunos-vec01-NAT1 pUC Touriño A., et al., Span. J. Agric. Res. 2008, 6, 48-58
pSN-PPV RK2 Pasin F., et al., PLoS Pathog. 2014, 10, e1003985
pSN-PPV-TagRFP-T2A RK2 Pasin F., et al., PLoS Pathog. 2014, 10, e1003985
pSN2-ccdB pVS1+CoIE1 Pasin F., et al., PLoS Pathog. 2014, 10, e1003985
GB0639 pVS1+CoIE1 Vazquez-Vilar M., et al., Plant Methods. 2016, 12,1-12
GB1108 pVS1+CoIE1 Vazquez-Vilar M., et al., Plant Methods. 2016, 12,1-12
GB1181 pVS1+CoIE1 Vazquez-Vilar M., et al., Plant Methods. 2016, 12,1-12
GB0460 pSa+pUC Sarrion-Perdigones A., et al., Plant Physiol. 2013, 162, 1618-1631.
pDGB3_alpha1 pVS1+CoIE1 Vazquez-Vilar M., et al., Nucleic Acids Res. 2017,45,
    2196-2209
pLX-B2 (SEQ ID NO: 3) pBBR1 Present disclosure
pLX-B3 (SEQ ID NO: 4) pBBR1 Present disclosure
pLX-B4 (SEQ ID NO: 5) pBBR1 Present disclosure
pLX-R2 (SEQ ID NO: 6) RK2 Present disclosure
pLX-R3 (SEQ ID NO: 7) RK2 Present disclosure
pLX-R4 (SEQ ID NO: 8) RK2 Present disclosure
pLX-Z4 (SEQ ID NO: 9) RK2 Present disclosure
pLX-B2α2 (SEQ ID NO: 10) pBBR1 Present disclosure
pLX-B3Ω1 (SEQ ID NO: 11) pBBR1 Present disclosure
pLX-B3Q2 (SEQ ID NO: 12) pBBR1 Present disclosure
pLX-B2-TagRFP-T (SEQ ID NO: 13) pBBR1 Present disclosure
pLX-B3-TagRFP-T (SEQ ID NO: 14) pBBR1 Present disclosure
pLX-B4-TagRFP-T pBBR1 Present disclosure
(SEQ ID NO: 15)    
pLX-R2-TagRFP-T (SEQ ID NO: 16) RK2 Present disclosure
pLX-R3-TagRFP-T (SEQ ID NO: 17) RK2 Present disclosure
pLX-R4-TagRFP-T (SEQ ID NO: 18) RK2 Present disclosure
pLX-B2-XT1-XT2-hCas9 (SEQ ID NO: 19) pBBR1 Present disclosure
pLX-B2-Nptll-DsRED (SEQ ID NO: 20) pBBR1 Present disclosure
LX-PPV (SEQ ID NO: 21) pBBR1 Present disclosure
pLX-UCBSV (SEQ ID NO: 22) pBBR1 Present disclosure
pLX-B2-PCRC:mTFP1 (SEQ ID NO: 23) pBBR1 Present disclosure
pLX-Z4-Pmas:RFP-ALCR (SEQ ID NO: 24) RK2 Present disclosure
pLX-B2-PEtOH:mNEON (SEQ ID NO: 25) pBBR1 Present disclosure
pSN.5-PPAP85:RFP (SEQ ID NO: 26) pVS1+CoIE1 Present disclosure
GB1686 pVS1+CoIE1 Present disclosure
(SEQ ID NO: 27)    
pLX-TuMV (SEQ ID NO: 28) pBBR1 Present disclosure


[0119] The details of the plasmid of the present invention are the followings:
  • pLX-B2 (SEQ ID NO: 3) is a T-DNA binary vector according to the present invention (pBBR1-based pLX vector) and comprises the replication origin from pBBR1 plasmid (SEQ ID NO: 105). The following parts were joined by Gibson assembly: (i) pBBR1 origin (SEQ ID NO: 105), amplified from pSEVA631, using X198_F/X199_R ((SEQ ID NO: 42)/X199_R (SEQ ID NO: 43)) primers; (ii) nptI gene, from pSEVA221, using X192_F (SEQ ID NO: 36)/X193_R SEQ ID NO: 37); (iii) synthetic T-DNA_1 cassette (SEQ ID NO: 1).
  • pLX-B3 (SEQ ID NO: 4) is a T-DNA binary vector according to the present invention (pBBR1-based pLX vector) and comprises the replication origin from pBBR1 plasmid (SEQ ID NO: 105). The following parts were joined by Gibson assembly: (i) pBBR1 origin (SEQ ID NO: 105) amplified from pSEVA631, using X198_F/X199_R primers (SEQ ID NO: 42/SEQ ID NO: 43); (ii) aadA gene, from pSEVA431, using X194_F/X195_R primers (SEQ ID NO: 38/SEQ ID NO: 39); (iii) synthetic T-DNA_1 cassette (SEQ ID NO: 1).
  • pLX-B4 (SEQ ID NO: 5) is a T-DNA binary vector according to the present invention (pBBR1-based pLX vector) and comprises the replication origin from pBBR1 plasmid (SEQ ID NO: 105). The following parts were joined by Gibson assembly: (i) pBBR1 origin (SEQ ID NO: 105), amplified from pSEVA631, using X198_F/X199_R primers (SEQ ID NO: 42/SEQ ID NO: 43); (ii) aacC1 gene, from pSEVA631, using X196_F/X197_R (SEQ ID NO: 40/SEQ ID NO: 41); (iii) synthetic T-DNA_1 cassette (SEQ ID NO: 1).
  • pLX-R2 (SEQ ID NO: 6) is a T-DNA binary vector according to the present invention (RK2-based pLX vector) and comprises the replication origin from RK2 plasmid (SEQ ID NO: 106). The following parts were joined by Gibson assembly: (i) RK2 origin (SEQ ID NO: 106), amplified from pSEVA221, using X200_F/X201_R primers (SEQ ID NO: 44/SEQ ID NO: 45); (ii) nptI gene, from pSEVA221, using X192_F/X193_R (SEQ ID NO: 36/SEQ ID NO: 37); (iii) synthetic T-DNA_1 cassette (SEQ ID NO: 1).
  • pLX-R3 (SEQ ID NO: 7) is a T-DNA binary vector according to the present invention (RK2-based pLX vector) and comprises the replication origin from RK2 plasmid (SEQ ID NO: 106). The following parts were joined by Gibson assembly: (i) RK2 origin (SEQ ID NO: 106), amplified from pSEVA221, using X200_F/X201_R primers (SEQ ID NO: 44/SEQ ID NO: 45); (ii) aadA gene, from pSEVA431, using X194_F/X195_R (SEQ ID NO: 38/SEQ ID NO: 39); (iii) synthetic T-DNA_1 cassette (SEQ ID NO: 1).
  • pLX-R4 (SEQ ID NO: 8) is a T-DNA binary vector according to the present invention (RK2-based pLX vector) and comprises the replication origin from RK2 plasmid (SEQ ID NO: 106). The following parts were joined by Gibson assembly: (i) RK2 origin (SEQ ID NO: 106), amplified from pSEVA221, using X200_F/X201_R primers (SEQ ID NO: 44/SEQ ID NO: 45); (ii) aacC1 gene, from pSEVA631, using X196_F/X197_R (SEQ ID NO: 40/SEQ ID NO: 41); (iii) synthetic T-DNA_1 cassette (SEQ ID NO: 1).
  • pLX-Z4 (SEQ ID NO: 9) is a T-DNA binary vector according to the present invention (pLX-R4 derivative with T-DNA_2 cassette (SEQ ID NO: 2), and no BsmBI sites in RK2-trfA and aacC1 genes) and comprises the replication origin from RK2 plasmid (SEQ ID NO: 107). The following parts were joined by Gibson assembly: (i) aacC1_3', amplified from pLX-R4 (SEQ ID NO: 8), using X295_F/X296_R primers (SEQ ID NO: 73/SEQ ID NO: 74); (ii) aacC1_RK2, from pLX-R4 (SEQ ID NO: 8), using X297_F/X298_R (SEQ ID NO: 75/SEQ ID NO: 76);(iii) RK2_5', from pLX-R4 (SEQ ID NO: 8), using X299_F/X300_R (SEQ ID NO: 77/SEQ ID NO: 78); (iv) synthetic T-DNA_2 cassette (SEQ ID NO: 2).
  • pLX-B2α2 (SEQ ID NO: 10) is a pLX-B2 derivative with GoldenBraid alpha2 cloning cassette (Sarrion-Perdigones A., et al., Plant Physiol. 2013, 162, 1618-1631) and comprises the replication origin from pBBR1 plasmid (SEQ ID NO: 105). The following parts were joined by Gibson assembly: (i) backbone, amplified from pLX-B2 (SEQ ID NO: 3), using X210_R/X321_F primers (SEQ ID NO: 46/SEQ ID NO: 89); (ii) lacZα cloning cassette, amplified using X322_F/X323_R (SEQ ID NO: 90/SEQ ID NO: 91).
  • pLX-B3Ω1 (SEQ ID NO: 11) is a pLX-B3 derivative with GoldenBraid omega1 cloning cassette (Sarrion-Perdigones A., et al., Plant Physiol. 2013, 162, 1618-1631) and comprises the replication origin from pBBR1 plasmid (SEQ ID NO: 105). The following parts were joined by Gibson assembly: (i) backbone, amplified from pLX-B3 (SEQ ID NO: 4), using X324_R/X325_F primers (SEQ ID NO: 92/SEQ ID NO: 93); (ii) lacZα cloning cassette, amplified using X326_F/X327_R (SEQ ID NO: 94/SEQ ID NO: 95).
  • pLX-B3Ω2 (SEQ ID NO: 12) is a pLX-B3 derivative with GoldenBraid omega2 cloning cassette (Sarrion-Perdigones A., et al., Plant Physiol. 2013, 162, 1618-1631) and comprises the replication origin from pBBR1 plasmid (SEQ ID NO: 105). The following parts were joined by Gibson assembly: (i) backbone, amplified from pLX-B3 (SEQ ID NO: 4), using X324_R/X325_F primers (SEQ ID NO: 92/SEQ ID NO: 93); (ii) lacZα cloning cassette, amplified using X328_F/X329_R (SEQ ID NO: 96/SEQ ID NO: 34).
  • pLX-B2-TagRFP-T (SEQ ID NO: 13) is a pLX-B2 derivative with CaMV 35S promoter, TagRFP-T and nopaline synthase terminator transcription unit (P35s:RFP:Tnos). The following parts were joined by Gibson assembly: (i) backbone, amplified from pLX-B2 (SEQ ID NO: 3), using X210_R/X211_F primers (SEQ ID NO: 46/SEQ ID NO: 47); (ii) P35S:RFP:Tnos, from pSN.5-TagRFP-T, using X218_F/X219_R (SEQ ID NO: 51/SEQ ID NO: 52).
  • pLX-B3-TagRFP-T (SEQ ID NO: 14) is a pLX-B3 derivative with P35S:RFP:Tnos. The following parts were joined by Gibson assembly: (i) backbone, amplified from pLX-B3 (SEQ ID NO: 4), using X210_R/X211_F primers (SEQ ID NO: 46/SEQ ID NO: 47); (ii) P35S:RFP:Tnos, from pSN.5-TagRFP-T, using X218_F/X219_R (SEQ ID NO: 51/SEQ ID NO: 52).
  • pLX-B4-TagRFP-T (SEQ ID NO: 15) is a pLX-B4 derivative with P35S:RFP:Tnos. The following parts were joined by Gibson assembly: (i) backbone, amplified from pLX-B4 (SEQ ID NO: 5), using X210_R/X211_F primers (SEQ ID NO: 46/SEQ ID NO: 47); (ii) P35S:RFP:Tnos, from pSN.5-TagRFP-T, using X218_F/X219_R (SEQ ID NO: 51/SEQ ID NO: 52).
  • pLX-R2-TagRFP-T (SEQ ID NO: 16) is a pLX-R2 derivative with P35S:RFP:Tnos. The following parts were joined by Gibson assembly: (i) backbone, amplified from pLX-R2 (SEQ ID NO: 6), using X210_R/X211_F primers (SEQ ID NO: 46/SEQ ID NO: 47); (ii) P35S:RFP:Tnos, from pSN.5-TagRFP-T, using X218_F/X219_R (SEQ ID NO: 51/SEQ ID NO: 52).
  • pLX-R3-TagRFP-T (SEQ ID NO: 17) is a pLX-R3 derivative with P35S:RFP:Tnos. The following parts were joined by Gibson assembly: (i) backbone, amplified from pLX-R3 (SEQ ID NO: 7), using X210_R/X211_F primers (SEQ ID NO: 46/SEQ ID NO: 47); (ii) P35S:RFP:Tnos, from pSN.5-TagRFP-T, using X218_F/X219_R (SEQ ID NO: 51/SEQ ID NO: 52).
  • pLX-R4-TagRFP-T (SEQ ID NO: 18) is a pLX-R4 derivative with P35S:RFP:Tnos. The following parts were joined by Gibson assembly: (i) backbone, amplified from pLX-R4 (SEQ ID NO: 8), using X210_R/X211_F primers (SEQ ID NO: 46/SEQ ID NO: 47); (ii) P35S:RFP:Tnos, from pSN.5-TagRFP-T, using X218_F/X219_R (SEQ ID NO: 51/SEQ ID NO: 52).
  • pLX-B2-XT1-XT2-hCas9 (SEQ ID NO: 19) is a pLX-B2 derivative with XT1 sgRNA, XT2 sgRNA, and hCas9 transcription units. Transcription units were transferred from GB1108 vector to pLX-B2 performing a restriction-ligation reaction that included BsmBI (New England BioLabs) and T4 Ligase (Promega). The reaction mixture was subjected to 30 cycles of 7 min each (3 min at 37°C and 4 min at 16°C). Clones were selected onto LB kanamycin plates, and by restriction enzyme assay.
  • pLX-B2-NptII-DsRED (SEQ ID NO: 20) is a pLX-B2 derivative with Pnos:NptII:Tnos and P35S:DsRED:T35S transcription units. Transcription units were transferred from GB0460 and GB1181 vectors to pLX-B2 performing a restriction-ligation reaction that included Bsal (New England BioLabs) and T4 Ligase (Promega). The reaction mixture was subjected to 30 cycles of 7 min each (3 min at 37°C and 4 min at 16°C). Clones were selected onto LB kanamycin plates, and by restriction enzyme assay.
  • pLX-PPV (SEQ ID NO: 21) is a pLX-B2 derivative with a GFP-tagged Plum pox virus cDNA clone cassette (P35S:PPV:Tnos). Scal/Xbal-digested pSN-PPV was mixed with Scal/Nhel-digested pLX-B2. Fragments were ligated using T4 DNA ligase (New England BioLabs).
  • pLX-UCBSV (SEQ ID NO: 22) is a pLX-B2 derivative with an Ugandan cassava brown streak virus cDNA clone cassette (P35S:UCBSV:Tnos). Total RNA purified from plants infected with UCBSV isolate Ke_125 (PV-0912, DSMZ) was used in a cDNA synthesis reaction. This included X122_R, X123_R (SEQ ID NO: 32/SEQ ID NO: 33), random primers and commercial kit components (High Capacity cDNA reverse transcription kit, Applied Biosystems). The cDNA sample was used in PCR reactions: (i) 5UTR-P3, using X240_F/X241_R primers (SEQ ID NO: 59/SEQ ID NO: 60); (ii) P3-Nlb, using X242_F/X243_R (SEQ ID NO: 61/SEQ ID NO: 62); (iii) Nlb-3UTR, using X244_F/X245_R (SEQ ID NO: 63/SEQ ID NO: 64). pLX-B2 backbone with P35S and Tnos was amplified from pLX-PPV using X238_R/X239_F (SEQ ID NO: 57/SEQ ID NO: 58). RT- and PCR fragments were joined by Gibson assembly. UCBSV cDNA clone sequence was determined by Sanger sequencing using 1989_F (SEQ ID NO: 29), X241_R (SEQ ID NO: 60), X244_F (SEQ ID NO: 63), X245_R (SEQ ID NO: 64), X253_R (SEQ ID NO: 65), X254_F (SEQ ID NO: 66), X255_R (SEQ ID NO: 67), X256_F (SEQ ID NO: 68), X257_F (SEQ ID NO: 69), X258_F (SEQ ID NO: 70), X259_R (SEQ ID NO: 71), X260_R (SEQ ID NO: 72) primers.
  • pLX-B2-PCRC:mTFP1 (SEQ ID NO: 23) is a pLX-B2 derivative with A. thaliana AT4G28520 seed promoter of cruciferin C gene, a cyan fluorescent protein (mTFP1) and AT4G28520 terminator transcription unit (PCRC:mTFP1:TCRC). The following parts were joined by Gibson assembly: (i) backbone, amplified from pLX-B2 using X210_R/X211_F primers (SEQ ID NO: 46/SEQ ID NO: 47); (ii) PCRC, from A. thaliana Col-0 genomic DNA using X220_F/X221_R (SEQ ID NO: 53/SEQ ID NO: 54); (iii) mTFP1, from pSN.5-mTFP1, using X212_F/X213_R (SEQ ID NO: 48/SEQ ID NO: 49); (iv) TCRC, from A. thaliana Col-0 genomic DNA using X222_F/X223_R (SEQ ID NO: 55/SEQ ID NO: 56).
  • pLX-Z4-Pmas:RFP-ALCR (SEQ ID NO: 24) is a pLX-Z4 derivative with the TagRFP-T, Thosea asigna virus 2A peptide (Donnelly M.L.L., et al., J. Gen. Virol. 2001, 82, 1027-1041), A. nidulans AlcR coding sequence flanked by mannopine synthase promoter and terminator (Pmas:RFP-2A-ALCR:Tmas). The following parts were joined by Gibson assembly: (i) backbone, amplified from pLX-Z4, using X210_R/X211_F primers (SEQ ID NO: 46/SEQ ID NO: 47); (ii) Pmas, from pGGF003, using X301_F/X302_R (SEQ ID NO: 79/SEQ ID NO: 80); (iii) RFP-2A, from pSN-PPV-TagRFP-T2A, using X216_F/X303_R (SEQ ID NO: 50/SEQ ID NO: 81); (iv) AlcR_5', from pGGC011, using X304_F/X305_R (SEQ ID NO: 82/SEQ ID NO: 83); (v) AlcR_3', from pGGC011, using X306_F/X307_R (SEQ ID NO: 84/SEQ ID NO: 85); (vi) Tmas, from pGGF003, using X308_F/X309_R (SEQ ID NO: 86/SEQ ID NO: 87).
  • pLX-B2-PEtOH:mNEON (SEQ ID NO: 25) is a pLX-B2 derivative with mNeonGreen sequence under a synthetic ethanol-responsive promoter (PEtOH:NEON:Tnos). The following parts were joined by Gibson assembly: (i) backbone, amplified from pLX-B2 using X210_R/X211_F primers (SEQ ID NO: 46/SEQ ID NO: 47); (ii) NEON:Tnos, from pSN.5-mNeon, using X310_F/X219_R (SEQ ID NO: 88/SEQ ID NO: 52); (iii) synthetic PEtOH fragment (SEQ ID NO: 35).
  • pSN.5-PPAP85:RFP (SEQ ID NO: 26) is a pSN2-ccdB derivative with A. thaliana AT3G22640 seed promoter, RFP and nopaline synthase terminator transcription unit (PPAP85:RFP:Tnos). To generate the pSN.5-PPAP85:RFP vector the following parts were joined by Gibson assembly: (i) backbone, Xbal/Pmll-digested pSN2-ccdB; (ii) PPAP85, from A. thaliana Col-0 genomic DNA using X228_F/X229_R (SEQ ID NO: 98/SEQ ID NO: 99); (iii) RFP:Tnos, from pSN.5-TagRFP-T, using X216_F/X80_R (SEQ ID NO: 50/SEQ ID NO: 97).
  • GB1686 (SEQ ID NO: 27) is a pDGB3_alpha1 derivative with Pnos:NptII:Tnos and P35S:DsRED:T35S transcription units. Transcription units were transferred from GB0460 and GB1181 vectors to pDGB3_alpha1 performing a restriction-ligation reaction that included Bsal (New England BioLabs) and T4 Ligase (Promega). The reaction mixture was subjected to 30 cycles of 7 min each (3 min at 37°C and 4 min at 16°C). Clones were selected onto LB kanamycin plates, and by restriction enzyme assay.
  • pLX-TuMV (SEQ ID NO: 28) is a pLX-B2 derivative with the P35S:TuMV:Tnos cassette from p35Tunos-vec01-NAT1. To generate the pLX-TuMV vector the following parts were joined by Gibson assembly: (i) backbone, amplified from pLX-PPV using X333_R/X334_F primers (SEQ ID NO: 100/SEQ ID NO: 101); (ii) Xmal/Sall-digested p35Tunos-vec01-NAT1.


[0120] The primers were synthesized by Sigma-Aldrich, and their sequences are listed in Table 2.

Table 2



[0121] 
Table 2. List of the primers, and assembly linkers
IDSequenceSEQ ID NO:
1989_F GATTGATGTGATTTCTCCACTGACG 29
2050_F GCCATTGTCCGAAATCTCACG 30
2051_R CTGGAAATGCGATTCTCTTAGC 31
X122_R CGTCAATCGTTAGAGC 32
X123_R CGACCTTGCACTTCA 33
X329_R

 
34
X192_F

 
36
X193_R

 
37
X194_F

 
38
X195_R

 
39
X196_F

 
40
X197 _R

 
41
X198_F

 
42
X199_R

 
43
X200_F

 
44
X201_R

 
45
X210_R TGAGACGGTTTCGACCAGG 46
X211_F GTCAGGAGACGGGACAAGGA 47
X212_F ATGGTTTCTAAAGGTGAAGAGAC 48
X213_R TTATGCTCCTTTATCGTCGTC 49
X216_F ATGGTTTCAAAGGGAGAAGAG 50
X218_F

 
51
X219_R

 
52
X220_F

 
53
X221_R

 
54
X222_F

 
55
X223_R

 
56
X238_R GTCATATTTATTTTTCCTCTCCAAATGAAATGAACTTCC 57
X239_F

 
58
X240_F

 
59
X241_R CTCTTCCTTTCGACCTTGCACTTCA 60
X242_F TTGAAGTGCAAGGTCGAAAGGAAGAG 61
X243_R AAAGAAGTATCAAACCTACTACCATCACAATC 62
X244_F GA TTGTGA TGGT AGTAG GTTT GATACTTCTT 63
X245_R

 
64
X253_R CTTTCGTAACAGCTTGCTTTCTCA 65
X254_F CTTTGGTTTAGACAAGCAATGTGTG 66
X255_R CCACTATTATTTCCACGATGCTTC 67
X256_F CAGAGGTGAAGTCTATTCTTGGCAT 68
X257_F AGTTTGGTGGAGTTTTGGATAGC 69
X258_F ATACACACGCTTGAGATAATGGATG 70
X259_R ATCGCCACTGATACAATTCAAAAG 71
X260_R AGGACCAAAATTCTCATAAGTCTCTCT 72
X295_F CAATTTACCCAACAACTCCGC 73
X296_R TGAGTTCGGCGATGTAGCCACCT 74
X297_F GGTGGCTACATCGCCGAACTCA 75
X298_R CGTTCGCGTCGGCTAGAACAGGAG 76
X299_F TGTTCTAGCCGACGCGAACGCT 77
X300_R GTAGAAAAGATCAAAGGATCTTCTTG 78
X301_F

 
79
X302_R

 
80
X303_R TGGCCCTGGATTTTCCTCAA 81
X304_F

 
82
X305_R TCCAGCACAGATTGCGTGAGAGAA 83
X306_F CTCTCACGCAATCTGTGCTGGATG 84
X307_R AGCTACAAGAAGCTGTCAACTTTCCCA 85
X308_F

 
86
X309_R

 
87
X310_F

 
88
X321_F GAGACGGGACAAGGATGCG 89
X322_F

 
90
X323_R

 
91
X324_R TGAGACCGTTTCGACCAGG 92
X325_F GAGACCGGACAAGGATGCG 93
X326_F

 
94
X327_R

 
95
X328_F

 
96
 

 
 
X80_R CTCAATGCTGCTGCCTTCATCTGGATATGAGCTTCAC 97
X228_F

 
98
X229_R

 
99
X333_R CGTGTCGTGCTCCACCATGTTCACGAAGATT 100
X334_F

 
101
Linker_1 CCATCATCAGTTCGGTGGTCTTCC 112
Linker_2 CGACTTGCGACATGCGGTCCTTTG 113
Linker_3 GATCGGATTGGCGGTTATGCGGTT 114

Bacterial growth conditions



[0122] E. coli DH10B strain was used for cloning and plasmid propagation. To increase plasmid miniprep yields, 10 mL cultures were grown in 50 mL tubes at 30 or 37°C. Overnight cultures were pelleted by centrifugation and processed using commercial minicolumn kits (FavorPrep Plasmid Extraction Mini Kit, Favorgen; Wizard Plus SV Minipreps, Promega). Double volumes of resuspension (50 mM Tris-HCI pH 7.5, 10 mM EDTA, 100 µg/mL RNase A), lysis (0.2 M NaOH, 1% SDS) and neutralization (4.09 M guanidine hydrochloride, 0.759 M potassium acetate, 2.12 M glacial acetic acid) kit solutions were used to improve clearing of bacterial lysates and final plasmid yields. Bacteria were grown in Luria-Bertani medium and antibiotics used at final concentrations of 100 mg/L ampicillin, 15 mg/L chloramphenicol, 20 mg/L gentamicin, 50 mg/L kanamycin, 50 mg/L rifampicin, 100 mg/L spectinomycin, 100 mg/L streptomycin, and 10 mg/L tetracycline. Growth curves were measured in 96-well plates, by recording OD600 absorbance values at 10-minute intervals in a plate reader (Infinite M200, Tecan). Maintenance of pTi in A. tumefaciens C58C1-313 strain was evaluated by PCR amplification of a repB fragment using 2050_F/2051_R primers (SEQ ID NO: 30/SEQ ID NO: 31).

Plant transformation and agro-inoculation



[0123] T-DNA binary vectors (See Table 1) were transformed into A. tumefaciens cells by freeze-thawing or electroporation methods. In transient expression and agro-inoculation assays, A. tumefaciens suspensions were mechanically infiltrated into N. benthamiana and A. thaliana leaves as described (Pasin F., et al., Plant Methods. 2014, 10, 22). The floral dip method was used to stably transform A. thaliana germ line cells (Clough S.J. & Bent A.F., Plant J. 1998, 16, 735-743). Stable transformation of N. benthamiana leaf disks was carried as described (Horsch R.B. & Klee H.J., Proc. Natl. Acad. Sci. U. S. A. 1986, 83, 4428-4432).

Protein detection



[0124] Plant samples that express fluorescent proteins were visualized under an epifluorescence stereoscope, confocal microscope, or imaged in a laser scanner (Pasin F., et al., Plant Methods. 2014, 10, 22). Fluorescence was measured by placing leaf discs in 96-well flat-bottom plates; in kinetics studies, plates were sealed with optical adhesive films (4311971, Applied Biosystems). The fluorescence signal was acquired in a filter- (VICTOR X5, PerkinElmer) or monochromator-based plate readers (Infinite M200, Tecan), as reported (Pasin F., et al., Plant Methods. 2014, 10, 22). Total protein extracts were resolved by SDS-PAGE, and immunodetection was done using rabbit anti-tRFP (AB234, Evrogen), -UCBSV CP (AS-0912, DSMZ), -PPV CP and -TuMV CP sera as primary antibodies. For electron microscopy, plant extracts were incubated with collodion-coated carbon-stabilized copper grids, and negative-stained with 2% uranyl acetate. Grids were observed in a transmission electron microscope (JEM 1011, Jeol).

Targeted genome mutagenesis



[0125] CRISPR/Cas constructs were transiently expressed in N. benthamiana leaves. To estimate mutagenesis efficiency PCR/restriction enzyme assay were done as described (Vazquez-Vilar M., et al., Plant Methods. 2016, 12, 1-12). Briefly, genomic DNA was purified from infiltrated leave samples and used in PCR reaction to amplify DNA fragments covering target sites of CRISPR/Cas constructs. The resulting PCR products were purified, and used in DNA digestion reactions that included restriction enzymes whose target sequences overlap predicted editing sites. Intensities of cleaved and cleavage-resistant bands were estimated using the ImageJ software (https://imagej.nih.gov/ij/).

Example 1. Construction of T-DNA binary vectors by modular parts assembly, and cloning features of a pBBR1-based pLX vector



[0126] In the design of new T-DNA binary vectors, the inventors chose basic principles: (i) reduced size, (ii) stability, (iii) broad host range replication origin for maintenance in E. coli and A. tumefaciens, (iv) origin compatible with the most commonly used T-DNA binary vectors, (v) consistency with current plant synthetic biology standards, and (vi) the possibility to adopt overlap-dependent methods for construct assembly.

[0127] Therefore, to construct the pLX binary vectors of the present invention, modular parts were assembled by overlap-based cloning methods. Sequences of synthetic orthogonal overlapping junctions known as assembly linker were designed to allow combinatorial assembly of DNA modules (Table 2). Module 1, 2, and 3 refer to T-DNA cassette, pBBR1 origin and selectable marker (resistance (R) genes, such as nptI, aadA, and aacC1), respectively (Fig. 1). Each module includes one or several DNA parts, which are flanked by two diverse assembly linkers that are shown as diamonds in Fig. 1. Parts from the three modules were obtained by PCR or chemically synthesized, and joined by one-step isothermal DNA assembly to generate pLX-B2 (SEQ ID NO: 3), pLX-B3 (SEQ ID NO: 4) and pLX-B4 (SEQ ID NO: 5) binary vectors (Fig. 1, Table 3). The details for the generation of pLX-B2, pLX-B3 and pLX-B4 plasmid are disclosed above.

Table 3



[0128] 
Table 3. Binary T-DNA vectors of the present invention
VectorSize bpOriginT-DNA*Cloning features§
Bacterial selectionCassetteGolden GateGolden BraidGibson assemblyMultiplexing
pLX-B2 3287 pBBR1 octopine KAN alpha1
pLX-B3 3349 pBBR1 octopine SP alpha1 - n.t.
pLX-B4 3165 pBBR1 octopine GENT alpha1 - n.t.
pLX-B2α2 3287 pBBR1 octopine KAN alpha2 n.t.
pLX-B3Ω1 3349 pBBR1 octopine SP omega 1 - n.t.
pLX-B3Ω2 3349 pBBR1 octopine SP omega2 - n.t.
pLX-Z4 3740 RK2 succinamo pine GENT alpha1 -
* pTi type of right and left border source, all vectors include a second left border of the nopaline type; § cloning cassette nomenclature according to GoldenBraid standards (Sarrion-Perdigones A., et al.. Plant Physiol. 2013, 162, 1618-1631): solid square, suitable; open square, not suitable; n. t., not tested.


[0129] The pLX binary vectors of the present invention faciliate flexible experimental designs since their replication is autonomous in both E. coli and A. tumefaciens. Additional features of pLX binary vectors include diverse selectable markers, a T-DNA with borders from an actopine-type pTi and second left border sequence that was shown to reduce backbone transfer (Fig. 2A). Bacterial synthetic terminators based on different scaffolds (T1, SEQ ID NO: 108; and T2, SEQ ID NO: 109) were included to increase plasmid stability.

[0130] For cloning purposes, the T-DNA cassette hosts the E. coli lacZα reporter gene flanked by Type IIS restriction endonuclease sites (Fig. 2B). Sequences of Bsal- or BsmBI-produced overhangs agree with proposed syntax for plant synthetic biology; pLX vectors are thus suitable for assembly of single and multiple eukaryotic transcription units from standard DNA parts libraries. Parts or transcription units can be assembled from plasmid libraries into pLX vectors using BsaI-based Golden Gate, and GoldenBraid standards (Fig. 2C). The T-DNA cassette hosts divergent primer annealing regions with no sequence similarity and secondary structures (arrows, Fig. 2B). These allow linearization of the small pLX backbones by inverse PCR, and subsequent use in multiple overlapping fragments cloning by Gibson assembly (Fig. 2C). pLX vectors with compatible replicons can be multiplexed into Agrobacterium cells for multi T-DNA delivery (Multiplexing; Fig. 2C). Therefore, the binary vectors of the present invention comprises features that make them compatible with Type IIS restriction endonuclease- and overlap-based assembly methods, and delivery of multi T-DNA cassettes by multiplexing of binary vectors with compatible origins (Table 3).

Example 2. Transgene expression in plants using pLX vector series



[0131] In order to demonstrate that binary vectors of the present invention can be used to deliver DNA constructs to eukaryotic cells, specifically plant cells by Agrobacterium tumefaciens-mediated transformation, a transcription unit that comprises the Cauliflower mosaic virus 35S promoter, the red fluorescent protein RFP (Fig. 3A) as a reporter, and nopaline synthase terminator (P35S:RFP:Tnos) sequences was assembled in pLX vectors of the present invention to obtain pLX-B2-TagRFP-T (SEQ ID NO: 13), pLX-B3-TagRFP-T (SEQ ID NO: 14) and pLX-B4-TagRFP-T (SEQ ID NO: 15), respectively (Fig. 3A). The details for the generation of the vectors are disclosed above. Transient expression of RFP in Nicotiana benthamiana leaves was evaluated by A. tumefaciens-mediated delivery. At 6 dpa, leaves infiltrated with pLX-B2-TagRFP-T (SEQ ID NO: 13), pLX-B3-TagRFP-T (SEQ ID NO: 14) and pLX-B4-TagRFP-T (SEQ ID NO: 15) showed bright RFP fluorescence, which was absent in the control sample (Fig. 3B). Confocal images showed that RFP fluorescent protein signal distributes in cytosol and nucleoplasm of plant cells (Fig. 3C), consistent with genuine plant expression. RFP accumulation in total protein extracts of leaf samples was confirmed by immunoblot analysis (Fig. 3D).

[0132] For some applications, stable integration of T-DNA cassettes into eukaryotic cell genomes is desirable. To prove the suitability of pLX vectors to mediate stable transgene integration into plant genomes the inventors used Arabidopsis thaliana as model plant and the pLX-B2-PCRC:mTFP1 vector (SEQ ID NO: 23) (Fig. 4A). The details for its synthesis are disclosed above.

[0133] The construct was inserted in A. tumefaciens and transformed into plants by floral dipping. Consistent with mTFP1 expression, bright cyan fluorescence was detectable in seed collected from Agrobacterium-treated plants (T1 seeds). The mTFP1-expressing seeds were selected under an epifluorescence stereoscope, and sown to soil. Stable transgene integration in germ-line cells was confirmed by PCR analysis of T1 plants: the endogenous promoter of cruciferin C gene was amplified (PCRC) from transformed and untransformed plants, whereas the mTFP1 sequence could be amplified only from plants derived from cyan fluorescent seeds. Diverse fluorescence phenotypes of T2 seeds are consistent with transgene integration into plant genome and its segregation across generations (Fig. 4B).

[0134] The present invention shows that T-DNA cassettes from pLX vectors can be delivered to plants, and pLX vectors can be used to transiently express or stably integrate transgenes into eukaryotic cell genomes. The inventors generated transgenic plants that are "marker-free" since they do not include genes that confer resistance to antibiotics, herbicides or other chemical compounds used in transgenic plant selection.

Example 3. Stable maintenance of T-DNA cassettes in pLX binary plasmid series of the invention



[0135] cDNA copies of RNA virus genomes can be inserted into plasmids to generate viral infectious clones but often shown instability problems, and sequence deletions arise in these clones during their propagation in bacteria. To test the stability of pLX binary vectors of the present invention in challenging conditions, the inventors transferred the whole cDNA sequence of potyvirus genomes into a pLX vector. The vectors generated were propagated in Escherichia coli, and bacteria were subjected to several growth cycles. Vector stability was evaluated by restriction enzyme digestion assays.

[0136] Thus, the whole cDNA sequence of an RNA virus was obtained from a pBIN19-based vector, pSN-PPV (Pasin F., et al., PLoS Pathog. 2014, 10, e1003985), which contains Cauliflower mosaic virus 35S promoter, a cDNA copy of Plum pox virus (PPV) genome and nopaline synthase terminator (P35S:PPV:Tnos) sequences. As disclosed above the inventors generated pLX-PPV (SEQ ID NO: 21), a pLX-B2 derivative with the P35S:PPV:Tnos cassette from pSN-PPV (Fig. 5A). The pLX-PPV vector obtained (SEQ ID NO: 21) has the pBBR1 origin (SEQ ID NO: 105), and is 38% and 9.3 kb smaller than the pSN-PPV vector. Purified plasmids from two independent clones of pLX-PPV (In, #A and #B) were EcoRI-digested and resolved by agarose gel (Fig. 5B). Compared to the pSN-PPV digestion profile, pLX-PPV clones showed all the bands corresponding to the viral cDNA cassette of pSN-PPV. High molecular weight DNA bands are consistent with differences in pSN-PPV and pLX-PPV backbones, pBIN19 and pLX-B2, respectively. The new pLX-PPV #A and #B clones (pLX-PPV, In) were transformed in E. coli to evaluate plasmid stability. For each transformation, eight individual colonies were picked and subjected to six growth cycles (24 h, 37°C). Purified plasmids were digested with EcoRI and resolved by agarose gel electrophoresis (pLX-PPV, Out; Fig. 5B). The pLX-PPV plasmid showed no instability, since digestion profiles of input and output plasmids were identical.

[0137] To further confirm the results, the whole cDNA sequence from a different RNA virus was obtained from a pUC-based vector, p35Tunos-vec01-NAT1, which contains Cauliflower mosaic virus 35S promoter, a cDNA copy of Turnip mosaic virus (TuMV) genome and nopaline synthase terminator (P35S:TuMV:Tnos) sequences (Touriño A., et al., Span. J. Agric. Res. 2008, 6, 48-58). The p35Tunos-vec01-NAT1 vector cannot replicate in A. tumefaciens, and does not include T-DNA borders for its transformation to plants. The inventors generated pLX-TuMV (SEQ ID NO: 28), a pLX-B2 derivative with the P35S:TuMV:Tnos cassette from p35Tunos-vec01-NAT1 (Fig. 5C). The pLX-TuMV vector (SEQ ID NO: 28) obtained as disclosed above, is only slightly bigger (3%, and 0.4 kb) than the p35Tunos-vec01-NAT1 vector, but includes the pBBR1 origin (SEQ ID NO: 105) and T-DNA borders suitable for its delivery to plants by Agrobacterium-mediated transformation. The new pLX-TuMV vector (SEQ ID NO: 28) (pLX-TuMV, In) was transformed in E. colito evaluate plasmid stability. For each transformation, ten individual colonies were picked and subjected to six growth cycles (24 h, 37°C). Purified plasmids were EcoRI-digested and resolved by agarose gel electrophoresis (pLX-TuMV, Out; Fig. 5D). In agreement with pLX-PPV results, the newly generated pLX-TuMV plasmid showed no instability, since digestion profiles of input and output plasmids were identical.

[0138] The present example shows that pLX vectors of the present invention can host >10 kb T-DNA cassettes, and that they can be used to generate clones that contain viral genome sequences. cDNA copies of RNA virus genomes have been reported to cause plasmid instability and loss of partial or entire insert sequences. In contrast, pLX vectors that host cDNA genome copies of plant RNA viruses showed no instability when propagated in the bacterium E. coli.

Example 4. Viral agro-inoculation and exogenous sequence delivery to plants using a pLX-based viral vectors



[0139] pLX-PPV (SEQ ID NO: 21) and pLX-TuMV (SEQ ID NO: 28) binary vectors from Example 3, if properly expressed in plants would start an infection of a chimeric Plum pox virus (PPV) or Turnip mosaic virus (TuMV), respectively. These chimeric viruses would host in their genome the GFP coding sequence (Fig. 6A, 6E), and GFP fluorescence could be measured and visualized to prove exogenous sequence expression into plants by viral expression vectors.

[0140] An A. tumefaciensstrain that contains pLX-PPV (pLX) (SEQ ID NO: 21) was infiltrated to N. benthamiana plants, a pSN-PPV strain (pSN) (Pasin F., et al., PLoS Pathog. 2014, 10, e1003985) was used as positive control. PPV infection and viral accumulation were confirmed by coat protein immunoblot analyses of agro-infiltrated and upper uninoculated leaf samples (6 dpa and 14 dpa, respectively; Fig. 6B). Accumulation of recombinant GFP in infected plant samples was confirmed by measuring of fluorescence intensity (Fig. 6C), and imaging of upper uninoculated leaves (Fig. 6D). To further confirm the results, an A. tumefaciens strain that contains pLX-TuMV (SEQ ID NO: 28) was agro-inoculated to A. thaliana plants. In agreement with pLX-PPV results, TuMV infection and viral accumulation in upper uninoculated leaf samples, were confirmed by immunoblot analysis of TuMV coat protein (Fig. 6F). Bright green fluorescence signal was detectable in inoculated plants (Fig. 6G), confirming the accumulation of recombinant GFP.

[0141] Therefore, the present example shows that the pLX binary vectors of the present invention can be used to engineer viral infectious clones, and viral vectors. These can be delivered by agro-inoculation, and used to introduce exogenous sequences and express recombinant proteins into plants.

Example 5. Transcription unit assembly into pLX vectors of the invention using plant synthetic biology standards.



[0142] To demonstrate pLX vector compatibility with plant synthetic biology standards, DNA parts from published libraries were assembled into pLX binary vectors of the present invention.

[0143] The GB1181 and GB0460 plasmids that contain standardized units for plant delivery of kanamycin resistance (Nptll) and red fluorescent protein (DsRED) genes, respectively, were obtained from a public repository (https://gbcloning.upv.es/). As disclosed above, the inventors assembled the standardized units into the pLX-B2 vector (SEQ ID NO: 3) to generate pLX-B2-Nptll-DsRED vector (SEQ ID NO: 20) (Fig. 7A). The pLX-B2-Nptll-DsRED vector (SEQ ID NO: 20) obtained is a pLX-B2 derivative with the pBBR1 origin (SEQ ID NO: 105) and two transcription units for plant expression of Nptll and DsRED.

[0144] To further confirm the results, the inventors transferred the GB1108 (https://gbcloning.upv.es/) standardized units into the pLX-B2 vector (SEQ I NO: 3) to generate pLX-B2-XT1-XT2-hCas9 vector (SEQ ID NO: 19) (Fig. 7B). The pLX-B2-XT1-XT2-hCas9 vector (SEQ ID NO: 19) obtained is a pLX-B2 derivative with the pBBR1 origin (SEQ ID NO: 105) and four transcription units for plant expression of Nptll, a human codon-optimized Streptococcus pyogenes Cas9 gene (hCas9), and single-guide RNA targeting the N. benthamiana Niben101Scf04205Ctg025 (XT1) and Niben101Scf04551Ctg021 (XT2) endogenous genes. The details for the generation of pLX-B2-XT1-XT2-hCas9 vector are disclosed above. To improve flexibility of the binary vectors of the present invention and facilitate the reuse of assembled DNA parts, the inventors generated pLX-B2α2 (SEQ ID NO: 10), pLX-B3Ω1 (SEQ ID NO: 11) and pLX-B3Ω2 vectors (SEQ ID NO: 12) with GoldenBraid cloning cassettes (Sarrion-Perdigones A., et al., Plant Physiol. 2013, 162, 1618-1631) (Fig. 7C, Table 3). The pLX-B2α2 (SEQ ID NO: 10) vector is a pLX-B2 derivative with alpha2 cloning cassette; the pLX-B3Ω1 (SEQ ID NO: 11) vector is a pLX-B3 derivative with omega1 cloning cassette; and the pLX-B3Ω2 (SEQ ID NO: 12) is a pLX-B3 derivative with omega2 cloning cassette, the details for their generation are disclosed above. Together with pLX-B2 plasmids of the present invention, pLX-B2α2, pLX-B3Ω1 and pLX-B3Ω2 vectors include the replication origin from pBBR1 plasmid (SEQ ID NO: 105), and a minimal set of two alpha and two omega level cloning cassettes with convergent and divergent Bsal and BsmBI sites (Table 3). Following GoldenBraid standards (Sarrion-Perdigones A., et al., Plant Physiol. 2013, 162, 1618-1631), these cloning cassettes allow to reuse assembled parts and to build large multigenic constructs.

[0145] Therefore, based on pLX binary vectors described in the present invention, the inventors generated pLX-B2α2 (SEQ ID NO: 10), pLX-B3Ω1 (SEQ ID NO: 11) and pLX-B3Ω2 (SEQ ID NO: 12) vectors (Table 3) that include cloning cassettes to facilitate the combinatorial assembly of pre-made DNA elements, transcription units into multigene constructs.

Example 6. Direct cloning and assembly of large T-DNA constructs into pLX vector series without intermediate plasmids.



[0146] This example demonstrates that pLX binary vectors of the present invention can be used to assembly of large T-DNA constructs with no intermediate subcloning steps.

[0147] The inventors sought to use vectors of the present invention to generate an infectious clone of the plant virus Ugandan cassava brown streak virus (UCBSV) since: (i) UCBSV genome is a large RNA molecule of 9.1 kb; (ii) a cDNA copy of UCBSV genome is not available in public parts libraries; (iii) the cDNA copy of UCBSV genome would contain several Type IIS restriction endonuclease sites, whose removal is required for parts domestication and Golden Gate/GoldenBraid cloning; (iv) mutagenesis of UCBSV genome sequence (e.g., to remove Bsal/BsmBI sites) is not desirable as its effects on virus viability are unknown; (v) correct assembly of UCBSV genome into a pLX vector can be easily evaluated in plants; (vi) UCBSV is a major threat to the staple food crop cassava, and an UCBSV infectious clone would have commercial applications as it facilitates plant genetic resistance screens.

[0148] The inventors generated the pLX-UCBSV vector (SEQ ID NO: 22), a pLX-B2 derivative with an Ugandan cassava brown streak virus cDNA clone cassette (P35S:UCBSV:Tnos), by one-step assembly of three RT-PCR fragments that span the entire 9.1-kb UCBSV genome (Fig. 8A). The details for the generation of pLX-UCBSV vector are disclosed above.

[0149] An A. tumefaciens strain that contains pLX-UCBSV (SEQ ID NO: 22) was infiltrated to N. benthamiana plants. At 12 dpa, agro-inoculated plants showed reduced height (Fig. 8B). In upper uninoculated leaves, the inventors detected filamentous particles typical of potyvirid virions (Fig. 8C), and confirmed UCBSV coat protein accumulation in immunoblot analysis (Fig. 8D). These results demonstrated that a cDNA copy of UCBSV was assembled into a pLX vectors to obtain pLX-UCBSV (SEQ ID NO: 22), which is an infectious clone of UCBSV that can be delivered to plants by Agrobacterium-mediated inoculation.

[0150] Thus, the inventors assembled large T-DNA constructs into pLX binary vectors of the present invention and this assembly did not require the use of restriction enzymes, parts domestication, intermediate plasmids and subcloning steps.

Example 7. Comparison of a pBBR1-based pLX vector of the present invention versus RK2 and pVS1 binary vectors.



[0151] Briefly, vectors that use the replicon from RK2 plasmid include pBIN19, and its smaller derivatives pEAQ and pCB301 (Fig. 9). Due to the reduced size, pCB301 is classified as mini binary vector. pVS1 origin is used in the pPZP series, and their derivatives of the pCAMBIA and pLSU series. pCAMBIA vectors are ones of the most common vectors used by plant scientist (http://www.cambia.org/daisy/cambia/585.html). A minimal pSa replicon includes the ori and RepA regions. These were split and used in the pGreen/pSoup dual plasmid system: pGreen is a T-DNA binary vector that hosts the pSa-ori sequence, and its replication in A. tumefaciens is not autonomous since it lacks the pSa-RepA gene. pGreen maintenance in A. tumefaciens requires the simultaneous presence of the helper plasmid pSoup, which provides pSa-RepA gene and allows replication of the pGreen binary vector (Fig. 9). Removal of RepA gene allowed, on one hand, to keep pGreen size to a minimum, on the other, it sacrificed plasmid replication autonomy and promoted instability under non-selective conditions.

[0152] In order to demonstrate that the pLX-binary vectors of the present invention can be classified as mini binary vectors and additionally are useful for driving high transient expression in plants, the inventors compared the pLX binary vectors to the binary vectors mentioned above which are known in the art.

[0153] Firstly, backbone size of pLX-B2 (SEQ ID NO: 3) and mentioned binary vectors known in the art was compared (Fig. 9). The pBBR1-based backbone of pLX vectors of the present invention is substantially smaller than the widely used pBIN- and pCAMBIA-based vectors (pBIN19, pCAMBIA-2300; Fig. 9). pLX-B2 (SEQ ID NO: 3) equals to pGreen-based vectors, which are not autonomous and require pSoup for their replication in A. tumefaciens. In contrast, pLX vectors facilitate flexible experimental designs since their replication is autonomous in both E. coli and A. tumefaciens.

[0154] pLX-B2 (SEQ ID NO: 3) can be classified as mini binary vector since its size is below the one of pCB301, a RK2-based vector. Although larger than pBBR1, the RK2 replicon is relatively small and has previously been used to generate autonomous mini binary vectors. To compare the performance of pBBR1 and RK2 replicons, the inventors replaced the pBBR1 replication module (SEQ ID NO: 105) of pLX vectors by a minimal RK2 origin (SEQ ID NO: 106) to build pLX-R2 (SEQ ID NO: 6), -R3 (SEQ ID NO: 7) and -R4 (SEQ ID NO: 8) vectors. A transcription unit that contains Cauliflower mosaic virus 35S promoter, RFP and nopaline synthase terminator (P35S:RFP:Tnos) sequences was inserted into pLX-R2, pLX-R3 and pLX-R4 vectors to obtain pLX-R2-TagRFP-T (SEQ ID NO: 16), pLX-R3-TagRFP-T (SEQ ID NO: 17) and pLX-R4-TagRFP-T (SEQ ID NO: 18), respectively (Fig. 10A). The details for the generation of these vectors are disclosed above.

[0155] Transient expression of RFP in Nicotiana benthamiana leaves was evaluated by A. tumefaciens-mediated delivery of pLX vectors that include the pBBR1 (Fig. 3A) or RK2 origins (Fig. 10A). Compared to RK2, the use of pBBR1-based pLX vectors led to significantly higher RFP accumulation in plant transient expression assays (Fig. 10B). The result was independent of resistance genes used for plasmid selection, and did not correlate significantly with the A. tumefaciens fluorescence that might derive from undesired RFP accumulation in bacteria (Fig. 10B).

[0156] pCAMBIA plasmids have the pVS1 origin, and are ones of the most commonly used T-DNA binary vectors. To compare pLX and pCAMBIA vectors, the inventors assembled standardized units for plant delivery of kanamycin resistance (Nptll) and red fluorescent protein (DsRED) genes into the pLX-B2 (SEQ ID NO: 3) and a pCAMBIA-derived vectors to obtain pLX-B2-Nptll-DsRED (SEQ ID NO: 20) and GB1686 (SEQ ID NO: 27), respectively (Fig. 7A, 10C). Agrobacterium strains that contain pLX-B2-Nptll-DsRED (pLX) (SEQ ID NO: 20) or GB1686 (SEQ ID NO: 27) were used in transient and stable transformation of N. benthamiana plants. Compared to GB1686, the pLX-B2 backbone significantly enhanced DsRED accumulation in transient expression assay. In stable transformation assays, a similar number of kanamycin-resistant plantlets that showed DsRED fluorescence was obtained (Fig. 10C). The result indicates that the pLX- and pCAMBIA-based vectors tested have equal stable transformation efficiency (Fig. 10C).

[0157] Therefore, whereas stable transformation efficiency of the present invention and commercially available vectors is similar, transient expression yields obtained by use of the pBBR1-based binary vectors of the present invention are higher than the ones obtained by use of RK2- and pVS1-based binary vectors.

Example 8. CRISPR/Cas delivery and high efficiency of plant genome editing using pLX vector series of the present invention.



[0158] In order to demonstrate that the binary vectors of the present invention can be used to deliver CRISPR/Cas constructs and also to induce targeted genome mutagenesis, transient expression of components of a CRISPR/Cas system in N. benthamiana leaves was evaluated by A. tumefaciens-mediated delivery of the pLX-B2-XT1-XT2-hCas9 (pLX) (SEQ ID NO: 19) vector and GB1108, a pCAMBIA-derived vector that has the pVS1 origin and also comprises the identical transcription units than pLX-B2-XT1-XT2-hCas9 (SEQ ID NO: 19) (Fig. 7C, 11A). The hCas9 gene was delivered with no sgRNA sequences as control (CTRL). In infiltrated samples, BsmBI and Spel site loss was predicted to occur in edited XT1 and XT2 loci, respectively. Mutagenesis was confirmed by the appearance of cleavage-resistant bands in PCR/digestion assays (Fig. 11B). Compared to pDGB3 and consistent with DsRED transient expression results, the pLX vector showed greater mutagenesis efficiency (Fig. 11B).

[0159] Therefore, genome mutagenesis obtained by the binary vectors of the present invention is higher than the one obtained by the use of a pVS1-based binary vector.

Example 9. T-DNA binary vector multiplexing using the pLX vector series of the present invention



[0160] In order to demonstrate that the vectors generated in Example 1 can be multiplexed with compatible T-DNA binary vectors into A. tumefaciens cells and delivered to plants, the inventors designed the pLX-Z4 plasmid (SEQ ID NO: 9). The pLX-Z4 is a novel T-DNA vector of low sequence similarity, and compatible with the pLX-B2 (SEQ ID NO: 3) and pLX-B3 (SEQ ID NO: 4) plasmids (Fig. 12, Table 3). pLX-B4 (SEQ ID NO: 5) and pLX-Z4 (SEQ ID NO: 9) are not compatible since their selection relies on the same antibiotic, gentamicin. Additional features of pLX-Z4 (SEQ ID NO: 9) include small size, autonomous replication, and compatibility with Type IIS endonuclease-based and overlap-dependent cloning. The pLX-Z4 obtained as disclosed above is an improved pLX-R4 derivative with T-DNA_2 cassette (SEQ ID NO: 2), and no BsmBI sites in RK2-trfA and aacC1 genes. It incorporates the RK2 replication origin (SEQ ID NO: 107), lambda phage terminators (λ T1, SEQ ID NO: 110; and λ T2, SEQ ID NO: 111), and T-DNA border sequences from a succinamopine-type pTi, pTiBo542, and a second left border sequence (Fig. 12A). For cloning purposes, pLX-Z4 T-DNA cassette includes the lacZα reporter, divergent Bsal and convergent BsmBI sites, and primer annealing regions with no sequence similarity and secondary structures to allow backbone linearization by inverse PCR. pLX-B2 (SEQ ID NO: 3) shows minimal sequence similarity with pLX-Z4 backbone (SEQ ID NO: 9) (Fig. 12B). More extensive sequence analyses predicted that pBBR1-based pLX vectors described in Examples 1 and 5 could be multiplexed with pLX-Z4, and a wide array of binary vectors commonly used by plant scientists (Fig. 12C).

[0161] To facilitate vector multiplexing, the inventors characterized a disarmed A. tumefaciens strain (C58C1-313) that is sensitive to antibiotics commonly used in plasmid selection: C58C1-313 growth is inhibited by the presence of ampicillin, chloramphenicol, gentamicin, tetracycline, kanamycin or spectinomycin (Fig. 13A). A pTi-repB fragment was amplified from C58C1-313 cells using 2050_F (SEQ ID NO: 30)/2051_R (SEQ ID NO: 31) primers, and sequenced. Phylogenetic analysis showed that C58C1-313 hosts an octopine-type Ti plasmid, which is stably retained (Fig. 13B, C). Thus, C58C1-313 is a disarmed A. tumefaciens strain of the octopine type that is suitable for simultaneous use of multiple plasmids, since it shows sensitivity to several antibiotics. To confirm the results, the C58C1-313 strain was sequentially transformed with pLX-B2 and pLX-Z4 derivatives disclosed in the present invention which include, respectively, pBBR1 origin (SEQ ID NO: 105) and kanamycin resistance gene or RK2 origin (SEQ ID NO: 107) and gentamicin resistance gene (Fig. 13D). A C58C1-313 strain that simultaneous hosts pLX-B2 and pLX-Z4 derivatives showed resistance to kanamycin and gentamicin, and grew in a medium supplemented with these antibiotics (Fig. 13D). In the same conditions, growth of C58C1-313, strain that harbors no vectors, was inhibited (CTRL; Fig. 13D).

[0162] In Example 2, the pLX-B2-PCRC:mTFP1 vector (SEQ ID NO: 23) with pBBR1 origin (SEQ ID NO: 105) and kanamycin resistance was used to drive seed expression of the cyan fluorescent mTFP1. Under epifluorescence stereoscopes, cyan and red fluorescence can be imaged with no signal overlap. The inventors generated the pSN.5-PPAP85:RFP vector (SEQ ID NO: 26) (Fig. 14A), a pCAMBIA derivative with pVS1 origin, spectinomycin resistance and a transcription unit that contains an A. thaliana seed-specific promoter from a seed storage protein gene (PAP85; AT3G22640), RFP and nopaline synthase terminator sequences (PPAP85:RFP:Tnos). The details for the generation of pSN.5-PPAP85:RFP vector are disclosed above.

[0163] To show that vectors of the present invention (Example 1) can be multiplexed with commercially available binary vectors, the inventors used a two-vector/one-strain system to transform A. thaliana. The pLX-B2-PCRC:mTFP1 (SEQ ID NO: 23) and pSN.5-PPAP85:RFP (SEQ ID NO: 26) T-DNA binary vectors were inserted in A. tumefaciens C58C1-313 (Fig. 14A), and transformed into plants by floral dipping. Consistent with Example 2 results and mTFP1 expression, cyan fluorescence was detectable in seed collected from Agrobacterium-treated plants (Fig. 14B). The 53% of the mTFP1-expressing seeds also showed red fluorescence derived by the RFP expression (Fig. 14B).

[0164] These results indicate that the binary vectors of the present invention can be multiplexed with compatible vectors, and used in a two-vector/one-strain system to deliver multiple and diverse T-DNA cassettes to plant cells.

Example 10. Gene expression control and delivery of synthetic circuit components to plants using binary vector multiplexing



[0165] The inventors used a chemical expression switch, in order to test whether the binary vectors of the present invention and the multiplexing strategy described in Example 9 could be applied to deliver synthetic circuit components to plants and to regulate gene expression. Ethanol was chosen as chemical inducer of the expression switch because of its potential in fundamental research and commercial biotechnology applications.

[0166] A novel synthetic promoter PEtOH (SEQ ID NO: 35) that is activated by Aspergillus nidulans AlcR in presence of ethanol was designed. The PEtOH promoter (SEQ ID NO: 35) includes multiple AlcR DNA-binding sites derived from A. nidulans alcM, alcR, aldA, alcA promoters, and a minimal Figwort mosaic virus 34S promoter (Fig. 15A). An ethanol-responsive buffer gate was designed to sense ethanol as input, and to produce a bright green fluorescent protein (NEON, output; Fig. 15B). To evaluate the two-vector/one-bacterial strain system to deliver synthetic circuit components, gate elements were distributed into the gentamicin-selectable pLX-Z4-Pmas:RFP-AlcR (SEQ ID NO: 24) and the kanamycin-selectable pLX-B2-PEtOH:NEON (SEQ ID NO: 25) vectors, which have RK2 and pBBR1 origins respectively (Fig. 15C). pLX-Z4-Pmas:RFP-AlcR (SEQ ID NO: 24) codes for RFP (used as expression control) and A. nidulans AlcR transcription factor under the mannopine synthase promoter (Pmas), which directs constitutive expression in plants; whereas the pLX-B2-PEtOH:NEON (SEQ ID NO: 25) encodes NEON sequence under PEtOH, a synthetic promoter activated by AlcR in the presence of the inducer (Fig. 15C). The details for the vector generation are disclosed above.

[0167] The plasmids were introduced sequentially into A. tumefaciens C58C1-313, and selected using gentamicin plus kanamycin media to obtain the R-AlcR + PEtOH:NEON strain. The R-AlcR + PEtOH:NEON strain was infiltrated into N. benthamiana leaves, and plants were treated with water or ethanol. As anticipated, while RFP fluorescence was visible in both conditions, NEON fluorescence was significantly higher in presence of the gate inducer (Fig. 16A). Circuit modeling requires quantitative characterization of genetic parts. To test whether the two-vector/one-strain expression system is compatible with medium-throughput analyses, leaf disks were collected from R-ALCR + PEtOH:NEON-infiltrated leaves, and placed in 96-well plates to evaluated gate responses. At 24 h post-treatment (hpt), gate function was maintained in leaf disks, since NEON fluorescence was detected only in the presence of gate input (Fig. 16B). Output fluorescence intensity quantification in intact leaf disks showed appropriate gate responsiveness and sensitivity, since 0.1% ethanol was sufficient to trigger > 200-fold induction (Fig. 16C). NEON detection requires no lysis or substrate addition step, which allowed to measure gate kinetics in a continuous-read assay. In the conditions tested, the NEON/RFP fluorescence intensity ratio was significantly higher than the water control at 1.5 hpt and reached a plateau at 15 hpt (Fig. 16D).

[0168] The results show that the pBBR1-based pLX and RK2-based pLX binary vectors of the present invention can be used to control gene expression in plants, and coupled to allow multi T-DNA delivery from A. tumefaciens in a two-plasmid/one-strain system. The binary vector system of the present invention is suitable for genetic circuit component delivery, and their quantitative characterization in a medium-throughput scale.


















































































































































































































Claims

1. A binary vector comprising a pBBR1 origin or a variant functionally equivalent thereof, and selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 28.
 
2. A binary vector system comprising: (a) a first binary vector according to claim 1, and (b) a second binary vector according to claim 1, wherein the pBBR1 origin is replaced by any of the replication origins selected from the list consisting of: IncQ, IncW, IncU, pRi, pVS1 and IncP-α plasmid incompatibility group origins.
 
3. A binary vector system according to claim 2 wherein the origin of replication of the second binary vector is selected from an IncP-α plasmid incompatibility group origin.
 
4. A binary vector system according to any of claims 2 to 3, wherein the origin of replication of the second binary vector is the RK2 origin.
 
5. A binary vector system according to claim 4 wherein the RK2 origin comprises the SEQ ID NO: 106 or the SEQ ID NO: 107.
 
6. A binary vector system according to any of claims 2 to 5 wherein the second binary vector is selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 24.
 
7. A bacterial host cell comprising the binary vector according to claim 1 or the binary vector system according to any of claims 2 to 6.
 
8. A bacterial host cell according to claim 7 wherein is Agrobacterium sp., or Escherichia coli.
 
9. A cell culture comprising the bacterial host cell according to any of claims 7 to 8.
 
10. A method for delivering at least one nucleotide sequence of interest in to at least one plant cell comprising: (a) inserting at least one nucleotide sequence of interest into the binary vector according to claim 1 or into the binary vector system according to any of claims 2 to 6, (b) introducing the binary vector or binary vector system of step (a) into at least one bacterial host cell, and (c) contacting the host cell of step (b) with at least one plant cell.
 
11. The method according to claim 10, wherein the bacterial host cell is an Agrobacterium sp. cell.
 
12. The method according to claim 11, wherein the Agrobacterium cell comprises a disarmed Ti plasmid.
 
13. A method for in vitro delivering at least one nucleotide sequence of interest into at least one eukaryotic cell or organism, comprising: (a) inserting at least one nucleotide sequence of interest into the binary vector according to claim 1 or into the binary vector system according to any of claims 2 to 6, (b) introducing the binary vector or binary vector system of step (a) into at least one eukaryotic cell or organism.
 
14. The method according to claim 13 wherein the eukaryotic cell or organism is selected from the group consisting of yeasts, fungi, insects and animals.
 
15. A method for obtaining a genetically-engineered plant or plant cell comprising the step of introducing the binary vector according to claim 1, the vector system according to any of claims 2 to 6, or the bacterial host cell according to any of claims 7 to 8, into a plant cell.
 
16. The method according to claim 15, wherein the bacterial host cell is an Agrobacterium sp. cell.
 
17. The method according to claim 16, wherein the Agrobacterium cell comprises a disarmed Ti plasmid.
 
18. A genetically-engineered plant cell or plant obtainable by the method according to any of claims 15 to 17.
 
19. A method for in vitro obtaining a genetically-engineered eukaryotic cell or organism comprising the step of introducing the binary vector according to claim 1, or the vector system according to any of claims 2 to 6, into a eukaryotic cell or organism.
 
20. An in vitro method according to claim 19, wherein the genetically-engineered eukaryotic cell or organism is selected from the group consisting of a yeast cell, a fungal cell, an insect cell, and an animal cell.
 
21. A genetically-engineered eukaryotic cell or organism obtainable by the method according to any of claims 19 to 20.
 
22. An in vitro or ex vivo use of the binary vector according to claim 1, the binary vector system according to any of claims 2 to 6, the host cell according to any of claims 7 to 8, the culture cell according to claim 9, the genetically-engineered plant or plant cell according to claim 18, or the genetically-engineered eukaryotic cell or organism according to claim 21: (a) for site-specific gene knockout; (b) for site-specific genome editing; (c) for sequence-specific DNA interference; (d) for site-specific epigenome editing; (e) for site-specific transcription modulation; or (f) for multiplexed genome engineering.
 
23. A kit comprising the binary vector according to claim 1, the binary vector system according to any of claims 2 to 6, the bacterial host cell according to any of claims 7 to 8, the cell culture according to claim 9, the genetically-engineered plant cell or plant according to claim 18, or the genetically-engineered eukaryotic cell or organism according to claim 21.
 




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REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description