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US20150225734A1 - Gene targeting in plants using dna viruses - Google Patents

Gene targeting in plants using dna viruses Download PDF

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US20150225734A1
US20150225734A1 US14/409,148 US201314409148A US2015225734A1 US 20150225734 A1 US20150225734 A1 US 20150225734A1 US 201314409148 A US201314409148 A US 201314409148A US 2015225734 A1 US2015225734 A1 US 2015225734A1
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Daniel F. Voytas
Nicholas Baltes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8203—Virus mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/12011—Geminiviridae
    • C12N2750/12041—Use of virus, viral particle or viral elements as a vector
    • C12N2750/12043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2999/00—Further aspects of viruses or vectors not covered by groups C12N2710/00 - C12N2796/00 or C12N2800/00
    • C12N2999/007—Technological advancements, e.g. new system for producing known virus, cre-lox system for production of transgenic animals

Definitions

  • This document relates to materials and methods for gene targeting in plants, and particularly to methods for gene targeting that include using geminiviruses and customizable endonucleases.
  • GT gene targeting
  • HR homologous recombination
  • NHEJ non-homologous end joining
  • a targeted DNA double-strand break can stimulate recombination by a factor of 100 between transforming T-DNA and a native chromosomal locus (Puchta et al., Proc. Natl. Acad. Sci. USA, 93:5055-5060, 1996).
  • high-frequency GT may be achieved in plants (Townsend et al., Nature, 459:442-445, 2009).
  • Such methods are designed for use in protoplasts, which enables direct delivery of repair templates and nuclease-expressing plasmids to individual cells though PEG transformation or electroporation.
  • the ability to practice GT is limited to labs with the expertise and equipment for tissue culturing and plant regeneration.
  • Gene targeting in plant cells has been performed primarily by two techniques: (1) direct transfer of DNA into plant cells by either electroporation/PEG transformation of protoplasts, or by biolistic bombardment of DNA into various plant tissues; and (2) by Agrobacterium -mediated transformation.
  • the exogenously supplied DNA is either T-DNA, PCR-derived, or plasmid-derived.
  • This document is based in part on the development of a novel and effective in planta method for gene targeting that combines the use of geminiviral-based gene targeting vectors and a targeted DNA double strand break engineered by a co-delivered endonuclease.
  • This is the first account demonstrating concurrent use of these techniques as a gene targeting methodology, which is likely to have vast implications in all areas of plant biology.
  • this technology can be used to accelerate the rate of functional genetic studies in plants.
  • the technology also can be used to engineer plants with improved characteristics, including enhanced nutritional quality, increased resistance to disease and stress, and heightened production of commercially valuable compounds.
  • geminiviruses and endonucleases for gene targeting in plants, including (i) the ability of the virus to stably propagate the gene targeting vector from cell-to-cell within the plant, (ii) the ability of the virus to replicate the gene targeting vector to high copy numbers within plant cell nuclei (on average 1000 copies per cell, but numbers can reach up to 30,000), and (iii) the circular nature of the geminivirus genome, as circular DNA is thought to participate less frequently in illegitimate recombination. These properties contribute to an effective, reliable and reproducible procedure for gene targeting in plant cells.
  • the methods provided herein enable practitioners to achieve high frequency gene targeting by creating a chromosome break in a target locus while simultaneously using the viral replication machinery to make repair templates to achieve gene targeting.
  • the viral repair templates can be generated either by infecting plants with engineered viruses or by using deconstructed viral vectors. The latter vectors replicate viral DNA and thereby produce the repair template, but they do not generate a productive infection.
  • this disclosure features a method for modifying the genetic material of a plant cell.
  • the method can include (a) introducing into the cell a virus nucleic acid comprising a repair template that is heterologous to the virus and is targeted to a first sequence that is endogenous to the plant cell; and (b) inducing a double strand break at or near the sequence to which the repair template is targeted, wherein said double strand break is generated by an endonuclease targeted to a second endogenous plant sequence at or near the first sequence that is targeted by the repair template, wherein homologous recombination occurs between the first endogenous plant sequence and the repair template.
  • the virus nucleic acid can be a plant DNA virus nucleic acid.
  • the virus nucleic acid can be a geminivirus nucleic acid.
  • the endonuclease can be a zinc finger nuclease, a transcription activator-like effector nuclease, a meganuclease, or a CRISPR/Cas system endonuclease.
  • the endonuclease can be encoded by a transgene sequence stably integrated into the genetic material of the plant, or can be expressed transiently.
  • the transgene When the endonuclease is encoded by a transgene, the transgene can be operably linked to a promoter that is constitutive, cell specific, inducible, or activated by alternative splicing of a suicide exon.
  • the virus nucleic acid can include a sequence encoding the endonuclease.
  • the method can further include introducing into the plant cell an RNA virus nucleic acid comprising a nucleotide sequence encoding the endonuclease.
  • the RNA virus nucleic acid can be introduced into the plant cell after or simultaneous with step (a).
  • the RNA virus nucleic acid can be from a tobacco rattle virus, a potato virus X, a pea early browning virus, or a barley stripe mosaic virus.
  • the plant can be a monocotyledonous plant (e.g., wheat, maize, a grass such as purple false brome ( Brachypodium distachyon ), Haynaldia villosa , or Setaria ), or a dicotyledonous plant (e.g., tomato, soybean, tobacco, potato, or Arabidopsis ).
  • a monocotyledonous plant e.g., wheat, maize, a grass such as purple false brome ( Brachypodium distachyon ), Haynaldia villosa , or Setaria
  • a dicotyledonous plant e.g., tomato, soybean, tobacco, potato, or Arabidopsis
  • FIG. 1 is an illustration of the cabbage leaf curl virus (CaLCuV) genome.
  • CaLCuV contains a bipartite genome, with the DNA A component encoding proteins necessary for viral replication and encapsidation, and the DNA B component encoding proteins necessary for cell-to-cell movement.
  • the coat protein nucleotide sequence (CP) can be replaced by up to 800 nucleotides of repair template DNA sequence. See, Gutierrez, Physiol. Mol. Plant Pathol. 6060:219-230, 2002.
  • FIG. 2 is a schematic of an experimental approach for gene targeting using engineered geminiviruses and transgenic Arabidopsis plants encoding a stably integrated zinc finger nuclease (ZFN) transgene. Repair of the ZFN-induced DSB using a repair template on the CaLCuV A genome results in the stable incorporation of a unique 18 bp sequence into the ADH1 gene.
  • ZFN zinc finger nuclease
  • FIG. 3 is an illustration of a nested PCR method that can be used to detect gene-targeted ADH1 alleles.
  • Amplicons are gel purified and used as templates for a second PCR, with one primer specific for the GT modification. Dashed lines represent the outer limit of homology carried by the repair template.
  • FIG. 4A is a diagram of pCPCbLCVA.007, which contains the entire genome of the CaLCuV A component flanked by direct repeats of the common region for viral excision from the plasmid.
  • AR1 the coding region of the coat protein gene
  • AR1 was replaced with a polylinker.
  • the AR1 promoter, the translational start (ATG) and the putative polyadenylation sites are retained.
  • ATG translational start
  • Virus derived from these vectors moves from cell-to-cell within Arabidopsis plants but, without the coat protein gene, it is not transmissible.
  • FIG. 4B is a diagram of pCPCbLCVB.002, which contains the entire genome of the CaLCuV B component flanked by direct repeats of the common region for viral excision from the plasmid. Bombardment of the B component alone can be used as a negative control for DNA contamination (no virus should be replicated). See, Muangsan and Robertson, Meth. Mol. Biol. 265:101-15, 2004.
  • FIG. 5 is a picture of gels with amplicons generated from an enrichment PCR designed to detect ZFN-induced mutations at the ADH1 gene after induction by ⁇ -estradiol.
  • DNA was assessed for NHEJ mutations from (i) non-induced and non-infected plants ( ⁇ Estradiol, ⁇ Virus), (ii) induced and non-infected plants (+Estradiol, ⁇ Virus), (iii) non-induced and infected plants ( ⁇ Estradiol, +Virus), and (iv) induced and infected plants (+Estradiol, +Virus).
  • D digested; UD, undigested.
  • FIG. 6 is a diagram of the CaLCuV A plasmid (left panel) and a series of pictures of gels showing the stability of repair template sequences in infected plants (right panels).
  • Genomic DNA from infected plants was used as a template for PCR amplification of the repair template sequence.
  • Primers NB153 and NB158 (left panel) recognize sequences in the viral genome and amplify across the repair template.
  • Five differently sized repair templates were analyzed. Repair templates with sizes 400 nt, 600 nt, 800 nt, and 1000 nt contained ADH1 homology sequences, while 715 nt contained gus::nptII homology sequence.
  • PCR amplicons (right panel) were run out on a 1% agarose gel. Controls for 1000 nt and 800 nt used plasmid DNA as a template for PCR (CaLCuVA.ADH1-1000 and CaLCuVA.ADH1-800, respectively).
  • FIG. 7 is a series of pictures of agarose gels showing PCR detection of amplicons from modified ADH1 loci. Genomic DNA from infected plants exposed to ⁇ -estradiol (left panels; +Virus, +Estradiol) or not exposed to ⁇ -estradiol (right panel; +Virus, ⁇ Estradiol) was subjected to nested PCR using primers designed to detect the 5′ modification junction (5′ check), the 3′ modification junction (3′ check), and amplification of the starting template (input).
  • 5′ check 5′ modification junction
  • 3′ check 3′ modification junction
  • FIG. 8 is a series of pictures showing evidence of GT at the gus::nptII gene.
  • Co-infected plants (CaLCuVA.GUS-FIX and CaLCuVB with TRV-Zif268) were stained in X-Gluc and chlorophyll was removed. Images of selected plants are shown. Arrows point to blue-staining cells.
  • FIG. 9 is an illustration of a strategy for creating a geminivirus replicon (GVR) system for transient protein expression, and subsequently transient genome editing, in plants.
  • LSL T-DNA functions as a template for Rep-assisted replicative release of replicons (top).
  • LIR, SIR, and Rep/RepA nucleotide sequences were derived from Bean yellow dwarf virus (BeYDV, GenBank accession number DQ458791.1).
  • Rep protein mediates replicational release of single-stranded DNA (ssDNA) replicons (middle).
  • dsDNA double-stranded DNA
  • SD DEM2 splice donor
  • SA DEM2 splice acceptor
  • FIG. 10A is an illustration of an approach for cloning customizable endonucleases into pLSL.
  • the pZHY013 entry vector encodes unique restriction enzyme sites (XbaI, BamHI, NheI and BglII) for sequential cloning of nucleotide sequences for TALE or ZF binding domains.
  • FIG. 10B is an illustration of vectors for Gateway cloning of customizable endonucleases and repair templates into pLSL.
  • FokI nucleotide sequences encode obligate heterodimeric proteins (EL-KK).
  • EL-KK obligate heterodimeric proteins
  • an AatII enzyme site permits cloning of Cas9 or MN nucleotide sequences upstream of Nos terminator sequence (Nos-T).
  • FIG. 10C is the full sequence of the LSL region (SEQ ID NO:78) located between the left and right T-DNA borders in pLSL.
  • the hygromycin resistance gene located between the left border and the upstream LIR, is not shown.
  • the highly-conserved nonanucleotide sequence (TAATATTAC), required for Rep-initiated rolling circle replication, is underlined in both LIR elements.
  • FIG. 11 is an illustration showing the general structure of the replicase expressing T-DNA plasmids used in the experiments described herein.
  • Rep/RepA nucleotide sequences (both wild type and LxCxQ) were cloned into pMDC32 (2 ⁇ 35S promoter) or pFZ19 (XVE promoter).
  • FIG. 12 is an image of plant tissue expressing GUS enzyme.
  • LSL T-DNA encoding NLS-tagged beta-glucuronidase (pLSLGUS)
  • pLSLGUS NLS-tagged beta-glucuronidase
  • FIG. 13 is a series of images of plant tissue expressing GFP.
  • FIG. 14 is an image of a representative leaf seven dpi, demonstrating tissue health.
  • Leaf tissue from WT Nicotiana tabacum plants was syringe infiltrated with Agrobacterium containing pLSLGUS (right), or coinfiltrated with Agrobacterium containing pLSLGUS and p35SREP.
  • Leaf tissue was removed from the plant seven dpi and imaged. Slight browning in tissue transformed with p35SREP was observed.
  • FIG. 15 is an illustration (top) and example (bottom) of detecting GVRs encoding GUS and GFP nucleotide sequences in plant cells.
  • genomic DNA was extracted three dpi and used as template for PCR. Primers were designed to amplify LIR sequence contained on the replicon. Amplicons were present only when p35SREP was co-transformed with pLSL, suggesting the presence of GVRs.
  • FIG. 16 is an illustration of target loci for Zif268::FokI, the T30 TALE nuclease pair, and the CRISPR/Cas system.
  • ZFN target sequence is present within a stably integrated, and defective gus::nptII reporter gene (top).
  • the T30 TALE nuclease and CRISPR/Cas target sequences are present within the endogenous acetolactate synthase genes (ALS), SuRA (middle) and SuRB (bottom).
  • AI artificial intron IV of ST-LS1 gene from Solanum tuberosum.
  • FIG. 17 is an image of a gel from a PCR designed to detect GVRs containing ZFN (pLSLZ.D), TALE nuclease (pLSLT), and CRISPR/Cas (pLSLC) sequences.
  • FIG. 18 is an image of a gel (middle) from a PCR-digest (top) designed to detect ZFN-induced mutations at the gus::nptII gene.
  • Plant DNA was isolated from leaf tissue seven dpi. Amplicons encompassing the ZFN target site were digested overnight with MseI and separated on an agarose gel. Cleavage-resistant bands were cloned into pJet1.2 and sequenced (bottom).
  • FIG. 19 is an image of a gel (middle) from an enrichment PCR (top) designed to detect TALE nuclease-induced mutations at the ALS loci.
  • Plant DNA was pre-digested overnight with AluI before PCR amplification of SuRA and SurB loci. Amplicons were digested overnight with AluI, separated on an agarose gel, and cleavage-resistant bands were cloned into pJet1.2 and sequenced (bottom).
  • FIG. 20 is an image of a gel (middle) from a PCR-digest (top) designed to detect Cas9-included mutations at the ALS loci.
  • Plant DNA was isolated from leaf tissue five dpi and the CRISPR/Cas target site was amplified by PCR. The resulting amplicons were digested with AlwI, separated on an agarose gel, and cleavage resistant bands were cloned and sequenced (bottom).
  • FIG. 21 is a schematic outlining the approach to correct a non-functional gus::nptII reporter.
  • Repair template sequence present within pLSLZ.D, encodes 1 kb homology arms isogenic to gus::nptII sequence, as well as 600 bp of sequence designed to restore gus::nptII protein function.
  • FIG. 22 shows selected images leaf tissue with GUS-expressing cells.
  • leaf tissue was stained in X-Gluc solution for 24 to 48 hours at 37° C., and chlorophyll was removed. Images shown are selected examples from tissue transformed with p35SZ.D (left), pLSLZ.D (center), and both pLSLZ.D and p35SREP (right).
  • FIG. 23 is an image of a gel (bottom) from a PCR (top) designed to detect GUS::NPTII genes. PCR was performed on genomic DNA extracted from leaf tissue seven dpi. Primers were designed to be complementary to sequence downstream of the NPTII coding sequence and homologous to the sequence within the repair template (top). A high number of amplicons of the expected size (1.078 kb) were observed only from genomic DNA isolated from tissue transformed with pLSLZ.D and p35SREP.
  • FIG. 24 is a graph plotting the density of GUS-expressing cells across multiple transgenic lines (identified as 1.7, 4.3, 9.1, and 11.3). Error bars represent SEM of at least three biological replicates.
  • FIG. 25 is a series of graphs plotting the density of GUS-expressing cells with different transformed vectors. Error bars represent SEM of at least three biological replicates.
  • FIG. 26 is a series of images of leaf tissue with GUS-expressing cells following Agrobacterium -mediated delivery of pLSLZ.D and p35SREP to transgenic lines 1.7, 4.3, and 11.3, as indicated.
  • FIG. 27 is a series of images of leaf tissue with GUS-expressing cells following Agrobacterium -mediated delivery of pLSLZ.D to transgenic lines 1.7 and 11.3, as indicated.
  • FIG. 28 is a series of images of leaf tissue with GUS-expressing cells following Agrobacterium -mediated delivery of p35SREP to transgenic lines 1.7, 4.3, and 11.3, as indicated.
  • FIG. 29 is a series of images of leaf tissue with GUS-expressing cells following Agrobacterium -mediated delivery of pLSLD and p35SREP to transgenic lines 1.7, 4.3, and 11.3, as indicated.
  • FIG. 30 is a series of images of leaf tissue with GUS-expressing cells following Agrobacterium -mediated delivery of p35SZ.D and p35SREP to transgenic lines 1.7, 4.3, and 11.3, as indicated.
  • FIG. 31 is an illustration of the approach used to create a SuRB::NPTII fusion protein (top) and an image of two gels from PCRs designed to genotype candidate recombinant plants (bottom). Primers were designed to detect the 5′ modification junction (5′ check) and the 3′ modification junction (3′ check).
  • FIG. 32 is an image of a gel from a PCR designed to detect BeYDV-based GVRs in potato cells. Genomic DNA from plants co-transformed with p35SREP and pLSLGFP was evaluated for replicational release (top), and for the presence of Rep/RepA nucleotide sequence (bottom).
  • FIG. 33 is an image of a gel from a PCR designed to detect Rep/RepA RNA transcripts in potato plants transformed with p35SREP.
  • FIG. 34 is a pair of images of potato leaves expressing GUS enzyme. Potato leaves were transformed with Agrobacterium containing pLSLGUS (left) or a mixture of Agrobacterium containing pLSLGUS and p35SREP (right). Leaf tissue was stained in X-Gluc solution and chlorophyll was removed.
  • FIG. 35 is a series of images of tomato leaf tissue with GUS-expressing cells.
  • Tomato leaf tissue was infiltrated with Agrobacterium containing pLSLGUS (right) or a mixture of Agrobacterium containing pLSLGUS and p35SREP (left and middle).
  • Agrobacterium containing pLSLGUS right
  • pLSLGUS pLSLGUS
  • p35SREP left and middle
  • FIG. 36 is an illustration showing the general structure of the Wheat dwarf virus LSL T-DNA. Rep/RepA nucleotide sequence is present within the LIR elements. Rep/RepA gene expression is initiated from the complementary sense LIR promoter.
  • FIG. 37 is a pair of images of wheat calli tissue expressing GFP.
  • GFP sequence was delivered to calli by particle bombardment of plasmid DNA containing BeYDV LSL sequences (left) or WDV LSL sequences (right). Images were taken three dpi.
  • FIG. 38 is a set of images of Setaria calli expressing GFP.
  • GFP sequence was delivered to calli by particle bombardment of plasmid DNA containing BeYDV LSL sequences (left) or WDV LSL sequences (right). Images were taken three dpi.
  • FIG. 39 is a set of images of corn embryos expressing GFP.
  • GFP sequence was delivered to calli by particle bombardment of plasmid DNA containing BeYDV LSL sequences (left), WDV LSL sequences (middle), or control (right). Images were taken three dpi.
  • FIG. 40 is an illustration describing an approach to correct a non-functional gus::nptII reporter gene in rice (top) and pictures of GUS activity in rice leaves (bottom).
  • the in planta system and methods for GT include the use of customizable endonucleases in combination with plant DNA viruses.
  • Plant DNA viruses including geminiviruses, have many attributes that may be advantageous for in planta GT, including their ability to replicate to high copy numbers in plant cell nuclei.
  • these viruses can be modified to encode a desired nucleotide sequence, such as a repair template sequence targeted to a particular sequence in a plant genome.
  • First generation geminiviruses or “full viruses” (viruses that retain only the useful “blocks” of sequence), can carry up to about 800 nucleotides (nt), while deconstructed geminiviruses (viruses that encode only the proteins needed for viral replication) have a much larger cargo capacity.
  • This document describes how customizable nucleases and plant DNA viruses enable in planta GT, and provides materials and methods for achieving such GT.
  • the methods can be used with both monocotyledonous plants (e.g., banana, grasses (e.g., Brachypodium distachyon ), wheat, oats, barley, maize, Haynaldia villosa , palms, orchids, onions, pineapple, rice, and sorghum) and dicotyledonous plants (e.g., Arabidopsis , beans, Brassica , carnations, chrysanthemums, citrus plants, coffee, cotton, eucalyptus, impatiens , melons, peas, peppers, Petunia, poplars, potatoes, roses, soybeans, squash, strawberry, sugar beets, tobacco, tomatoes, and woody tree species).
  • monocotyledonous plants e.g., banana, grasses (e.g., Brachypodium distachyon ), wheat, oats, barley, maize, Haynaldia villosa
  • the system and methods described herein include two components: a plant DNA virus (e.g., geminivirus) vector containing a repair template targeted to an endogenous plant sequence, and an endonuclease that also is targeted to a site near or within the target sequence.
  • a plant DNA virus e.g., geminivirus
  • the endonuclease can be activated to create targeted DNA double-strand breaks at the desired locus, and the plant cell can repair the double-strand break using the repair template present in the geminivirus, thereby incorporating the modification stably into the plant genome.
  • Geminiviruses are a large family of plant viruses that contain circular, single-stranded DNA genomes.
  • Examples of geminiviruses include the cabbage leaf curl virus, tomato golden mosaic virus, bean yellow dwarf virus, African cassava mosaic virus, wheat dwarf virus, miscanthus streak mastrevirus, tobacco yellow dwarf virus, tomato yellow leaf curl virus, bean golden mosaic virus, beet curly top virus, maize streak virus, and tomato pseudo-curly top virus.
  • geminivirus sequences can be used as gene targeting vectors.
  • the geminivirus genome can be engineered to contain a desired modification flanked by sequences of homology to a target locus.
  • this can be accomplished by replacing non-essential geminivirus nucleotide sequence (e.g., CP sequence) with a desired repair template.
  • non-essential geminivirus nucleotide sequence e.g., CP sequence
  • Other methods for adding sequence to viral vectors include, without limitation, those discussed in Peretz et al. ( Plant Physiol., 145:1251-1263, 2007).
  • the repair template contains homology to a particular sequence within the genome of a plant.
  • a repair template includes a nucleic acid that will replace an endogenous target sequence within the plant, flanked by sequences homologous to endogenous sequences on either side of the target.
  • a non-essential (e.g., CP) sequence within a geminivirus vector is replaced with a repair template, the repair template can have a length up to about 800 nt (e.g., 100 nt, 200 nt, 300 nt, 400 nt, 500 nt, 600 nt, 700 nt, 800 nt, or any length between about 100 nt and about 800 nt).
  • flanking homologous sequences can have any suitable length (e.g., about 25 nt, 50 nt, 75 nt, 100 nt, 150 nt, 200 nt, 250 nt, 300 nt, 350 nt, 400 nt, or any length between about 25 nt and about 400 nt).
  • Repair templates and DNA virus plasmids can be prepared using techniques that are standard in the art, including those described below.
  • the second component of the system and methods described herein is an endonuclease that can be customized to target a particular nucleotide sequence and generate a double strand break at or near that sequence.
  • endonucleases include ZFNs, MNs, and TALE nucleases, as well as Clustered Regularly Interspersed Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas) systems. See, for example, Sander et al., Nature Methods, 8:67-69, 2011; Jacoby et al., Nucl. Acids Res., 10.1093/nar/gkr1303, 2012); Christian et al., Genetics, 186:757-761, 2010; U.S.
  • CRISPR/Cas molecules are components of a prokaryotic adaptive immune system that is functionally analogous to eukaryotic RNA interference, using RNA base pairing to direct DNA or RNA cleavage.
  • Directing DNA DSBs requires two components: the Cas9 protein, which functions as an endonuclease, and CRISPR RNA (crRNA) and tracer RNA (tracrRNA) sequences that aid in directing the Cas9/RNA complex to target DNA sequence (Makarova et al., Nat Rev Microbiol, 9(6):467-477, 2011).
  • the modification of a single targeting RNA can be sufficient to alter the nucleotide target of a Cas protein.
  • crRNA and tracrRNA can be engineered as a single cr/tracrRNA hybrid to direct Cas9 cleavage activity (Jinek et al., Science, 337(6096):816-821, 2012).
  • the components of a CRISPR/Cas system can be delivered to a cell in a geminivirus construct.
  • the sequence encoding the endonuclease can be stably integrated into the plant genome that will be infected with a geminivirus containing a repair template. See, for example, FIG. 2 , which depicts a plant genome into which a sequence encoding an ADH1 targeted ZFN has been stably integrated.
  • the coding sequence can be operably linked to a promoter that is inducible, constitutive, cell specific, or activated by alternative splicing of a suicide exon.
  • the ADH1 ZFN coding sequence is operably linked to an XVE promoter, which can be activated by estradiol.
  • the plant can be infected with a geminivirus containing a repair template (indicated by the black bar flanked by white bars in the “CaLCuV”), and expression of the ZFN can be activated by treating the plant with estradiol.
  • the ZFN protein then can cleave the DNA at the target sequence, facilitating HR on either side of the repair template to be integrated.
  • the endonuclease coding sequence can be contained in the same geminivirus construct as the repair template, or can be present in a second plasmid that is separately delivered to the plant, either sequentially or simultaneously with the geminivirus construct.
  • plants can be transfected or infected with a second viral vector, such as an RNA virus vector (e.g., a tobacco rattle virus (TRV) vector, a potato virus X vector, a pea early browning virus vector, or a barley stripe mosaic virus vector) that encodes the endonuclease.
  • TRV is a bipartite RNA plant virus that can be used to transiently deliver protein coding sequences to plant cells.
  • the TRV genome can be modified to encode a ZFN or TALE nuclease by replacing TRV nucleotide sequence with a subgenomic promoter and the ORF for the endonuclease.
  • TRV vector can be useful because TRV infects dividing cells and therefore can modify germ line cells specifically. In such cases, expression of the endonuclease encoded by the TRV can occur in germ line cells, such that HR at the target site is heritable.
  • a geminivirus vector contains both a repair template and an endonuclease encoding sequence
  • the geminivirus can be deconstructed such that it encodes only the proteins needed for viral replication. Since a deconstructed geminivirus vector has a much larger capacity for carrying sequences that are heterologous to the virus, it is noted that the repair template may be longer than 800 nt.
  • An exemplary system using a deconstructed vector is described in the Example below.
  • the construct(s) containing the repair template and, in some cases, the endonuclease encoding sequence can be delivered to a plant cell using, for example, biolistic bombardment.
  • the repair template and endonuclease sequences can be delivered using Agrobacterium -mediated transformation, insect vectors, grafting, or DNA abrasion, according to methods that are standard in the art, including those described herein.
  • any suitable method can be used to determine whether GT has occurred at the target site.
  • a phenotypic change can indicate that a repair template sequence has been integrated into the target site.
  • PCR-based methods also can be used to ascertain whether a genomic target site contains a repair template sequence, and/or whether precise recombination has occurred at the 5′ and 3′ ends of the repair template. A schematic depicting an example of such a technique is provided in FIG.
  • the cabbage leaf curl virus (CaLCuV) is a bipartite, circular single-stranded DNA virus that can infect Arabidopsis plants when delivered by microprojectile bombardment. Initiating viral infection requires the delivery of two plasmids containing sequence for both genomes (A and B components; FIG. 1 ). The viral sequences are partially duplicated, containing two direct repeats of the origin of replication flanking the viral genome. Consequently, delivery of these plasmids to plant cell nuclei results in replicational release of full-length, circular geminivirus genomes.
  • CaLCuV A components encoding repair template sequence, the coat protein (AR-1) coding sequence was replaced with desired sequence. AR-1 is required for insect-transmission of the virus, but it is not required for viral amplification and systemic spreading. Because of this, approximately 800 nucleotides can be added to the A component genome without preventing its ability to infect.
  • Viral vectors encoding repair templates targeting the ADH1 and gus::nptII loci use the pCPCbLCVA.007 backbone.
  • pCPCbLCVA.007 is a plasmid initially designed for viral induced gene silencing (VIGS). It encodes a partially duplicated A component with the AR-1 nucleotide sequence replaced with a multicloning site (MCS).
  • VIGS viral induced gene silencing
  • An ADH1-targeting repair template was constructed for ligation into pCPCbLCVA.007.
  • the template for amplifying the ADH1 repair template was genomic DNA from Arabidopsis thaliana (ecotype Columbia). To isolate genomic DNA, about 100 mg of leaf tissue was frozen in liquid nitrogen and ground to a fine powder.
  • CTAB buffer 2.0 g hexadecyl trimethyl-ammonium bromide (CTAB)
  • EDTA ethylenediaminetetraacetic acid di-sodium salt
  • pH adjusted to 5.0 per 100 mL of solution was added and the samples were incubated at 65° C. for 20 min. Samples were centrifuged for 5 minutes at 12,000 RPM and the supernatant was transferred to a clean microfuge tube. 500 ⁇ A of chloroform was added and the samples were inverted for 5 minutes at room temperature.
  • Repair templates targeting ADH1 were designed to encode a unique 18 bp modification sequence (5′-GAGCTCAGTACTGC ATGC-3′; SEQ ID NO:1) flanked by arms of homology to the ADH1-ZFN target site.
  • Several repair templates were constructed with varying lengths of homology for each arm. In total, four repair templates were made with 491, 391, 291, or 191 nucleotides of homology in each arm.
  • the modification was designed to remove the native ZFN binding site, which prevents cleavage of the repair template before and after GT.
  • left and right homology sequences were amplified from Arabidopsis genomic DNA using primers NB177+NB128 and NB178+NB129 for 491 bp homology arms, NB104+NB128 and NB112+NB129 for 391 bp homology arms, NB105+NB207 and NB113+NB208 for 291 bp homology arms, and NB106+NB207 and NB114+NB208 for 191 bp homology arms, respectively.
  • Primer sequences are provided in Table 1.
  • the reverse primers for the left homology arm and the forward primers for the right homology arm contained complementary 18 bp linkers encoding the modification sequence.
  • the forward primers for the left homology arm and the reverse primer for the right homology arm contained linkers encoding XbaI and BglII restriction enzyme sites, respectively.
  • PCR reactions were performed in a 25 ⁇ l PCR mix composed of 2.5 ⁇ l of 10 ⁇ NEB Standard Taq buffer, 0.5 ⁇ l of 10 mM dNTPs, 0.5 ⁇ l of 10 ⁇ M primer 1, 0.5 ⁇ l of 10 ⁇ M primer 2, 18.8 ⁇ l of dH 2 O, 0.2 ⁇ l of Taq polymerase, and 2 ⁇ l of genomic DNA ( ⁇ 200 ng).
  • the PCR conditions were 5 minutes at 94° C.
  • Fusion reactions were performed in a 24 ⁇ l PCR mix composed of 2.5 ⁇ l of 10 ⁇ cloned Pfu buffer, 0.5 ⁇ l of 10 mM dNTPs, 14.5 ⁇ l of dH 2 O, 0.5 ⁇ l of Pfu enzyme, and 3 ⁇ l each of the purified amplicons. Fusion conditions were 5 minutes at 94° C. followed by 10 cycles of 30 seconds at 94° C., 30 seconds at 50° C., and 1 minute at 72° C. Following the fusion PCR, 0.5 ⁇ l of 10 ⁇ M primer 1 and 0.5 ⁇ l of 10 ⁇ M primer 2 were added and the samples were run in another PCR. The PCR conditions were 5 minutes at 94° C.
  • the chromosomal target for the repair template is a GUS transgene with ⁇ 300 bp of nucleotide sequence removed from the 3′ end and replaced with a Zif268 target site.
  • GUS-FIX repair templates were designed to contain flanking arms of homology to the target locus (200 bp each) and a 300 bp modification sequence. As a consequence of GT, the coding sequence of GUS is restored. Cells actively expressing GUS can be phenotypically detected by an enzymatic assay.
  • the left homology arm (also containing the 300 bp of GUS-FIX sequence) and the right homology arm were amplified from pDW1269 plasmid DNA using primers NB274+NB271 and NB272+NB275, respectively.
  • the left and right homology arms contained complementary sequences to enable their fusion in OE-PCR.
  • PCR reactions to generate the fragments were performed in a 25 ⁇ l mix composed of 2.5 ⁇ l of 10 ⁇ NEB Standard Taq buffer, 0.5 ⁇ l of 10 mM dNTPs, 0.5 ⁇ l of 10 ⁇ M primer 1, 0.5 ⁇ l of 10 ⁇ M primer 2, 18.8 ⁇ l of dH 2 O, 0.2 ⁇ l of Taq polymerase, and 2 ⁇ l of genomic DNA ( ⁇ 200 ng).
  • the PCR conditions were 5 minutes at 94° C. followed by 30 cycles of 30 seconds at 94° C., 30 seconds at 55° C., and 1 minute at 72° C.
  • the resulting amplicons were resolved by agarose electrophoresis using a 1% gel.
  • DNA bands of expected sizes were purified and ligated together in an OE-PCR. Fusion reactions were performed in a 24 ⁇ l mix composed of 2.5 ⁇ l of 10 ⁇ cloned Pfu buffer, 0.5 ⁇ l of 10 mM dNTPs, 14.5 ⁇ l of dH 2 O, 0.5 ⁇ l of Pfu enzyme, and 3 ⁇ l each of the purified amplicons. Fusion conditions were 5 minutes at 94° C. followed by 10 cycles of 30 seconds at 94° C., 30 seconds at 50° C., and 1 minute at 72° C.
  • DNA bands of expected sizes were purified and ligated in a 10 ⁇ l reaction using T4 DNA ligase.
  • DH5 ⁇ E. coli were transformed with 2 ⁇ l of the ligation mix following standard procedures, and plated onto LB media containing 50 ⁇ g/ml of carbenicillin.
  • the DNA sequence of a resulting clone was confirmed to encode the GUS-FIX repair template sequence.
  • This vector is referred to as CaLCuVA.GUS-FIX.
  • Biolistic bombardment was carried out closely following the protocol described by Muangsan et al., Meth. Mol. Biol., 265:101-115, 2004. Briefly, to prepare microprojectile particles for five bombardments, 5 ⁇ g of each plasmid (CaLCuVA and CaLCuVB) was added to a tube containing 50 ⁇ l of 60 mg/mL gold beads and briefly vortexed. 50 ⁇ l of 2.5 M CaCl 2 was directly added to the samples and immediately pipetted in and out of a tip to break up conglomerates. 20 ⁇ l of 0.1 M spermidine was added and the samples were immediately vortexed for 5 min. The samples were centrifuged at 10,000 RPM for 10 seconds and the supernatant was removed.
  • the gold-bead pellet was resuspended in 250 ⁇ l of 100% ethanol and then centrifuged at 10,000 rpm for 10 sec. Supernatants were removed and the samples were resuspended in 65 ⁇ l of 100% ethanol. The particles were then stored on ice until bombardment. To prepare the assembly for the microprojectile particles, macrocarrier holders and macrocarriers were soaked in 95% ethanol, air-dried, and assembled. 10 ⁇ l of resuspended particles were then spotted onto the center of the macrocarrier and allowed to air-dry.
  • Biolistic bombardment was carried out in a horizontal laminar flow hood using a PDS-1000 He system (Bio-Rad).
  • a non-sterile rupture disk (1100 psi) was dipped in 100% isopropanol and placed into the upper assembly.
  • the macrocarrier launch assembly (MCLA) was then prepared by dipping a metal stopping screen in 95% ethanol, and then placing the dried screen onto the opening of the lower assembly.
  • the macrocarrier and macrocarrier holder were inverted and placed above the stopping screen.
  • the retaining ring was screwed in, and the MCLA was placed into the top rack of the chamber. A single pot containing four plants was then placed in the chamber directly beneath the MCLA.
  • a vacuum of 28 in was created, and helium was added to the upper chamber until the rupture disk burst. Bombarded plants were then removed from the chamber and returned to a covered flat. Between bombardments of different constructs, the chamber was cleaned with 70% ethanol. This procedure was repeated for additional infections. By following these methods, infection was successfully initiated in majority of the bombarded plants (75-100%).
  • infected Arabidopsis plants were placed in a flat with approximately 1 L of fertilizer solution and moved back to the growth chamber. A clear plastic dome was used to cover the plants for seven days post infection. Infection was noticeable 8-10 dpi by curling of rosette leaves.
  • plants containing an XVE ADH1-ZFN transgene were induced by exposure to ⁇ -estradiol (Sigma E2758) by spraying and watering.
  • the spray contained 0.01% Silwet L-77 (Vac-In-Stuff) and 20 ⁇ M ⁇ -estradiol, while the water contained only 20 ⁇ M ⁇ -estradiol. Induction was carried out by continuously spraying (approximately once a day) and watering (approximately twice a week) for 10-14 days.
  • genomic DNA was extracted from somatic plant tissue. A single rosette leaf and cauline leaf were collected from each infected plant. Care was taken when choosing leaves in order to minimize the likelihood of detecting recombination between plasmid molecules and genomic DNA. Criteria for choosing rosette leaves were 1) healthy leaf tissue with no obvious necrotic lesions, and 2) leaves growing on the periphery of the pot—away from damage caused by biolistic bombardment. Plant genomic DNA was extracted following the CTAB procedure as described above.
  • enrichment PCR was performed on purified genomic DNA.
  • Enrichment PCR is designed to detect ZFN-induced NHEJ mutations at the ADH1 target locus—an indirect assay for verifying nuclease activity. This procedure relies on a restriction enzyme site positioned in or near the target site spacer sequence. In essence, if the nuclease is not active, then target site amplicons will be completely digested by the restriction enzyme. On the other hand, if the nuclease is active there will be a population of target site amplicons with destroyed restriction enzymes sites that will not be digested by the restriction enzyme. Thus, detection of a digestion-resistant band suggests that the nuclease was actively creating DSBs.
  • genomic DNA from induced and non-induced plants was digested with BstXI (NEB) in a 10 ⁇ l solution following standard procedures. Immediately following digestion, 2 ⁇ l of the solution was used as a template for PCR in a reaction containing of 2.5 ⁇ l of 10 ⁇ NEB Standard Taq buffer, 0.5 ⁇ l of 10 mM dNTPs, 0.5 ⁇ l of 10 ⁇ M primer NB161, 0.5 ⁇ l of 10 ⁇ M primer NB154, 18.8 ⁇ l of dH 2 O, 0.2 ⁇ l of Taq polymerase, and 2 ⁇ l of the digested solution ( ⁇ 200 ng genomic DNA). The PCR conditions were 5 minutes at 94° C.
  • DNA isolated from infected plants is a mixture of plant genomic DNA and virus genomic DNA.
  • Primers were designed to recognize viral sequence (non-repair template sequence) in the CaLCuV A plasmid ( FIG. 6 , left panel), and to amplify across the entire repair template sequence.
  • PCR reactions contained 2.5 ⁇ l of 10 ⁇ NEB Standard Taq buffer, 0.5 ⁇ l of 10 mM dNTPs, 0.5 ⁇ l of 10 ⁇ M primer NB153, 0.5 ⁇ l of 10 ⁇ M primer NB158, 18.8 ⁇ l of dH 2 O, 0.2 ⁇ l of Taq polymerase, and 2 ⁇ l of purified genomic DNA ( ⁇ 200 ng).
  • the PCR conditions were 5 minutes at 94° C. followed by 35 cycles of 30 seconds at 94° C., 30 seconds at 55° C., and 1 minute at 72° C. 10 ⁇ l of the PCR sample was loaded onto a 1.0% agarose gel.
  • repair templates equal to or less than 715 bp were stably replicated in plant cells. For this reason, only viruses carrying repair templates equal or less than 715 bp were assessed in the subsequent experiments. Based on these experiments, it was concluded that first generation geminiviral vectors effectively amplified and disseminated repair templates in Arabidopsis plants.
  • Nested PCR was performed to detect modified ADH1 loci. Primers were designed to amplify the ADH1 locus approximately 700 bp upstream and downstream of the ZFN target sequence. The resulting amplicons were then used as a template for a nested PCR, with primers that specifically recognize the unique 18 bp modification sequence and ADH1 sequence outside the homology arms carried by the virus.
  • the ADH1 locus was amplified in a PCR reaction containing 2.5 ⁇ l of 10 ⁇ NEB Standard Taq buffer, 0.5 ⁇ l of 10 mM dNTPs, 0.5 ⁇ l of 10 ⁇ M primer NB257, 0.5 ⁇ l of 10 ⁇ M primer NB258, 18.8 ⁇ l of dH 2 O, 0.2 ⁇ l of Taq polymerase, and 2 ⁇ l of purified genomic DNA ( ⁇ 200 ng).
  • the PCR conditions were 5 minutes at 94° C. followed by 15 cycles of 30 seconds at 94° C., 30 seconds at 55° C., and 1 minute at 72° C. Amplicons were column purified using the QIAquick Gel Extraction Kit.
  • Purified amplicons were then used as templates for three nested PCRs.
  • the first PCR checked for the 5′ modification junction using primers NB154 and NB264.
  • the second PCR checked for the 3′ modification junction using primers NB263 and NB155.
  • the third PCR was a control for template amplification and used primers NB155 and NB154.
  • PCR was performed using Expand Long Template PCR system (Roche) in a reaction containing 2.5 ⁇ l buffer 1, 0.5 ⁇ l 10 mM dNTPs, 0.5 ⁇ l of 10 ⁇ M primer 1, 0.5 ⁇ l of 10 ⁇ M primer 2, 0.2 ⁇ l of the Taq/Tgo polymerase mix, 17.8 ⁇ l dH 2 O, and 3 ⁇ l of purified amplicons.
  • the PCR conditions were 5 minutes at 94° C. followed by 30 cycles of 30 seconds at 94° C., 30 seconds at 55° C., and 1 minute at 72° C. Amplicons were run on a 1% agarose gel.
  • GT was stimulated at the gus::nptII transgene.
  • plants containing a stably integrated gus::nptII transgene were infected with CaLCuVA.GUS-FIX and CaLCuVB following the procedures described above.
  • Zif268::FokI was transiently delivered to plants 8 dpi by TRV.
  • TRV is a bipartite RNA plant virus that can be used to transiently deliver protein coding sequences to plant cells.
  • TRV was modified to express Zif268::FokI by replacing the 2b and 2c nucleotide sequences with a subgenomic promoter and the ORF for the Zif268::FokI.
  • Infection was carried out by syringe infiltration of Agrobacterium carrying T-DNA coding for both TRV genomes. Briefly, GV3101 Agrobacterium carrying T-DNA encoding for TRV1 and TRV2-Zif268 were grown overnight at 28° C. in 3 mL of LB medium containing 50 ⁇ g/mL kanamycin and 50 ⁇ g/mL gentamycin.
  • One mL of the culture was transferred to 100 mL LB medium containing 50 ⁇ g/mL kanamycin and 50 ⁇ g/mL gentamycin and grown overnight at 28° C. until they reached an OD of approximately 1.0. Solutions were then centrifuged at 7000 RPM for 10 minutes and resuspended in 50 mL of MMAi solution (0.5 g MS salts, 0.195 g MES, 2 g sucrose, 100 ⁇ l of 200 mM acetosyringone per 100 mL at pH 5.6) followed by shaking at 50 rpm for 2 hours.
  • MMAi solution 0.5 g MS salts, 0.195 g MES, 2 g sucrose, 100 ⁇ l of 200 mM acetosyringone per 100 mL at pH 5.6
  • TRV1 and TRV2-Zif268 Solutions of Agrobacterium containing TRV1 and TRV2-Zif268 were mixed in a 1:1 ratio and syringe infiltrated into three rosette leaves per plant. TRV and geminivirus infected plants were moved to a growth chamber under 12 h light/12 h dark conditions at 22-24° C. for 15 days.
  • plants were analyzed for cells expressing functional GUS protein. Fifteen days after TRV infection and 23 days after geminivirus infection, plants were stained overnight at 37° C. in an X-Gluc solution (0.052 g X-Gluc (GoldBio), 5 mL 1M sodium phosphate, 0.1 mL Triton X per 100 mL). Plants were removed from the stain and incubated in 75% ethanol for 2-3 days to remove chlorophyll (which helped with visualizing the blue staining) Plants were visualized using a stereoscope. If GT occurred, spots of blue were observed where one or multiple cells had reconstituted GUS expression.
  • X-Gluc solution 0.052 g X-Gluc (GoldBio), 5 mL 1M sodium phosphate, 0.1 mL Triton X per 100 mL. Plants were removed from the stain and incubated in 75% ethanol for 2-3 days to remove chlorophyll (which helped with visualizing the blue staining) Plants were visualized using a
  • FIG. 8 shows images of plants co-infected with CaLCuVA.GUS-FIX and CaLCuVB (or with either plasmid alone) that were stained in X-gluc.
  • the spotty patches of blue staining in the rosette leaves and in the newly developed tissue suggested that GT had occurred.
  • An exemplary method for generating bean yellow dwarf virus (BeYDV) replicons in plant cells involves delivery of one or two plasmids or T-DNA molecules that encode the trans-acting replication-associated proteins, Rep/RepA, and direct duplications of the large intergenic region (LIR) flanking sequence encoding the small intergenic region (SIR; FIGS. 9-10 ).
  • LIR large intergenic region
  • SIR small intergenic region
  • virus replication is initiated by Rep protein binding to LIR sequence on a circular dsDNA genome.
  • the geminivirus genome is linearized and contains flanking LIR sequences (also referred to as an LSL vector)
  • Rep proteins bind to the LIR sequences and release circularized, single-stranded geminiviral replicons (GVRs).
  • Replicons can then be used as a template for replicase-mediated genome amplification. Consequently, any sequence present inside the flanking LIRs will be present in the replicon. Eliminating coat protein and movement protein sequence abolishes cell-cell movement, but significantly lessens genome-size restraints imposed by plasmodesmata. To compensate for loss of cell-cell movement, Agrobacterium was used to direct GVR production in specific cells. To facilitate cloning of endonuclease and repair template sequence into an LSL destination vector, MultiSite Gateway cloning technology (Invitrogen) was implemented.
  • the first block was designed to contain LIR::DEM2 splice acceptor (last 62 nt of the DEM2 intron)::tobacco etch virus (TEV) 5′ UTR (last 93 nt of the TEV 5′ UTR)::attR1::chloramphenicol resistance gene (CmR).
  • the second block contained ccdB::attR2::SIR.
  • the third block contained 2 ⁇ 35S::TEV 5′ UTR (first 38 nt of the TEV 5′ UTR)::DEM2 splice donor (first 32 nt of the DEM2 intron)::LIR.
  • LIR and SIR sequences were obtained from the mild BeYDV isolate (GenBank accession number DQ458791.1).
  • pFZ19 was used as a template for PCR amplification using primers NB326 and NB327.
  • PCR solutions contained 2.5 ⁇ l of 10 ⁇ cloned Pfu buffer, 0.5 ⁇ l of 10 mM dNTPs, 0.5 ⁇ l of 10 ⁇ M primer NB326, 0.5 ⁇ l of 10 ⁇ M primer NB327, 18.5 ⁇ l of dH 2 O, 0.5 ⁇ l of Pfu enzyme, and 2 ⁇ l of plasmid DNA ( ⁇ 20 ng).
  • PCR cycling included 5 minutes at 94° C., followed by 30 cycles of 30 seconds at 94° C., 30 seconds at 55° C., and 2 minutes at 72° C.
  • PCR amplicons were column purified using the QIAquick gel extraction kit.
  • OE-PCR solutions contained 2.5 ⁇ l of 10 ⁇ cloned Pfu buffer, 0.5 ⁇ l of 10 mM dNTPs, 0.5 ⁇ l of 10 ⁇ M primer NB327, 0.5 ⁇ l of 10 ⁇ M primer NB325, 14.5 ⁇ l of dH 2 O, 0.5 ⁇ l of Pfu enzyme, and 2 ⁇ l of purified amplicons, NB330 (2 ng) and NB331 (2 ng).
  • PCR cycling consisted of 5 minutes at 94° C., followed by 30 cycles of 30 seconds at 94° C., 30 seconds at 55° C., and 4 minutes at 72° C.
  • Amplicons and 1 ⁇ g of pBluescript KS+ vector were digested with Kpnl and XbaI. Digested fragments were purified and ligated following standard procedures. The resulting ligation was transformed into DH5 ⁇ E. coli cells following standard procedures.
  • pBlock1 sequence verified plasmid containing block 1
  • Blocks 2 and 3 were constructed using similar methods. Construction of Block 2 first required amplification and purification of ccdB::attR2 from pFZ19 using primers NB328 and NB332.
  • Purified amplicons were added to an OE-PCR with NB344 and primers NB328+NB329 to generate the complete nucleotide sequence for block 2.
  • Purified amplicons were ligated into pBluescript KS+ with XbaI and SacI and transformed into ccdB-resistant XL-1 Blue cells to generate pBlock2.
  • Construction of block 3 first required PCR amplification of 2 ⁇ 35S sequence from pMDC32 using primers NB333+NB334.
  • purified amplicons were used in an OE-PCR with NB335 and NB336 using primers NB333 and NB337.
  • pBlock1 and pBlock3 were digested with BsaI and gel purified. Primers NB338 and NB339 were dephosphorylated, annealed, ligated into pBlock1 and pBlock3 vector backbones, and transformed into DH5 ⁇ to generate pBlock1 HP and pBlock3HP.
  • pBlock1HP, pBlock2, pBlock3HP, pCAMBIA1300 were digested with SbfI+XbaI, XbaI+XhoI, XhoI+SbfI, and SbfI, respectively. Fragments of the expected sizes were gel purified, ligated, and transformed into ccdB-resistant XL-1 Blue cells following standard protocols for 4-way ligations. The resulting plasmid (pLSL, FIG. 10C ) was sequence verified and used as a destination vector for MultiSite Gateway cloning.
  • a nuclease-entry vector was constructed for MultiSite Gateway cloning into pLSL (pNJB091; FIG. 10B ).
  • pNJB091 Four unique restriction enzyme sites immediately upstream of two FokI coding sequences allows for sequential cloning of custom-designed DNA binding domains.
  • pZHY013 a modified pCR8 entry vector encoding FokI heterodimer sequences; FIG. 10A
  • NB318 were digested with BsmI and EcoRV. Digested fragments were gel purified, ligated and transformed into DH5 ⁇ cells following standard protocols.
  • a donor-entry vector was constructed for MultiSite Gateway cloning into pLSL (pNJB080; FIG. 10B ). Two unique pairs of restriction enzyme sites flanking ccdB and CmR selection markers permit efficient cloning of repair templates.
  • sequence encoding the CmR and ccdB genes was amplified by PCR from pFZ19 using NB316+NB317 primers. Amplicons were purified and used in an OE-PCR with NB314 and primers NB315 and NB317.
  • PCR solutions contained 2.5 ⁇ l of 10 ⁇ cloned Pfu buffer, 0.5 ⁇ l of 10 mM dNTPs, 0.5 ⁇ l of 10 ⁇ M NB315, 0.5 ⁇ l of 10 ⁇ M NB317, 16.5 ⁇ l of dH 2 O, 0.5 ⁇ l of Pfu enzyme, 2 ⁇ l of purified amplicons, and 2 ⁇ l of 10 ⁇ M NB314.
  • PCR cycling included 5 minutes at 94° C., followed by 30 cycles of 30 seconds at 94° C., 30 seconds at 55° C., and 3 minutes at 72° C. Resulting amplicons were gel purified following standard procedures.
  • MultiSite Gateway recombination with pNEL1R5 into pLSL positions repair template sequence between two transcriptional-termination sequences (upstream Nos-T sequence and downstream SIR sequence).
  • the studies herein may benefit from flanking termination sequences. For example, transcriptional gene silencing is facilitated through production of RNA molecules with homology to an endogenous gene. Reducing read-through transcription of repair template sequence may decrease unintentional silencing of targeted genes.
  • the first plasmid encodes the Rep/RepA coding sequence downstream of an estradiol-inducible XVE promoter (pXVEREP), such that when integrated into the plant genome, Rep/RepA expression can be induced by exposing plant tissue to ⁇ -estradiol.
  • the second plasmid encodes Rep/RepA downstream of a 2 ⁇ 35S promoter (p35SREP).
  • RepA For each plasmid, WT RepA and mutant RepA (RepA LxCxQ; Liu et al., Virology 256:270-279, 1999) versions are created (pXVEREPLxCxQ and p35SREPLxCxQ).
  • RepA interacts with the host cell's retinoblastoma (RB) protein, sequestering its repressive activity on E2F. This promotes entry into S phase, and, in turn, provides the invading geminivirus with replication machinery needed to amplify its genome.
  • RB retinoblastoma
  • the studies described herein may benefit from a RepA protein that does not interact with RB.
  • WT and mutant Rep/RepA coding sequences were amplified by OE-PCR using NB319, NB320, and NB322, and primers NB323 and NB324 (WT Rep/RepA), or using NB319, NB321, and NB322, and primers NB323 and NB324 (mutant Rep/RepA).
  • PCR solutions consisted of 2.5 ⁇ l of 10 ⁇ cloned Pfu buffer, 0.5 ⁇ l of 10 mM dNTPs, 0.5 ⁇ l of 10 ⁇ M NB323, 0.5 ⁇ l of 10 ⁇ M NB324, 14.5 ⁇ l of dH 2 O, 0.5 ⁇ l of Pfu enzyme, and 2 ⁇ l of each DNA component.
  • PCR cycling included 5 minutes at 94° C., followed by 30 cycles of 30 seconds at 94° C., 30 seconds at 55° C., and 3 minutes at 72° C. Resulting amplicons were purified using the QIAquick gel extraction kit.
  • pLSL was modified to encode NLS-tagged green fluorescent protein (pLSLGFP) or beta-glucuronidase (pLSLGUS).
  • GFP and GUS nucleotide sequence were amplified from, respectively, pTC23 and pNB67 using primers NB362 and NB363, and primers NB448 and NB449.
  • Forward and reverse primers contained XbaI and AatII restriction enzyme sites, respectively for cloning into pNB091.
  • the resulting vectors were used in a MultiSite Gateway recombination reaction with pLSL and pNB098 (a modified version of pNB080 with a repair template to correct a non-functional gus::nptII transgene) to generate pLSLGFP and pLSLGUS.
  • These vectors were sequence verified and transformed into Agrobacterium tumefaciens GV3101 by the freeze-thaw method. Single colonies of transformed Agrobacterium were grown overnight in a shaker at 28° C. in 5 mL of LB starter culture with 50 ⁇ g/ml kanamycin and 50 ⁇ g/ml gentamicin.
  • Circular replicons were detected by PCR using primers NB415 and NB416. Template switching was minimized by using the Expand Long Template PCR mix (Roche) following manufacturer's protocols. Strong amplification of LIR sequence only from samples co-transformed with p35SREP suggests that GVRs were present in the transformed cells ( FIG. 15 , bottom). Taken together, these data illustrate that GVRs can facilitate transient delivery of reporter proteins.
  • pLSL was modified to encode a Zif268::FokI ZFN.
  • Zif268::FokI sequence was amplified from pDW1345 using primers NB379 and NB380.
  • Forward and reverse primers contained XbaI and AatII restriction enzyme sites for cloning into pNJB091.
  • the resulting vector was used in a MultiSite Gateway recombination reaction with pLSL and pNB098 to generate pLSLZ.D.
  • the resulting vectors were sequence verified and transformed into Agrobacterium tumefaciens GV3101 by the freeze-thaw method.
  • Target sequence for Zif268 is present within a gus::nptII reporter gene that is stably integrated in the genome of N. tabacum plants ( FIG. 16 ).
  • Leaf tissue was syringe infiltrated with Agrobacterium containing pLSLZ.D, or coinfiltrated with Agrobacterium containing pLSLZ.D and p35SREP.
  • Plant DNA was extracted seven dpi, replicational release was verified ( FIG. 17 ), and Zif268 target sequence was analyzed for ZFN-induced non-homologous end joining (NHEJ) mutations.
  • NHEJ non-homologous end joining
  • a 484 bp DNA sequence, encoding the Zif268 target sequence was amplified by PCR using primers NB422 and NB424.
  • the resulting amplicons were purified and used as a template in a second PCR with primers NB396 and NB307 ( FIG. 18 ).
  • the PCR product was digested overnight with MseI and separated on an agarose gel. Cleavage-resistant products, present only in the pLSLZD and p35SREP lane, were cloned and sequenced ( FIG. 18 ). Six out of eight sequenced clones contained mutations at the Zif268 target sequence.
  • Replicon-mediated expression of a ZFN monomer is predicted to be efficient due to its relatively small coding sequence (the Zif268::FokI gene is 897 nt) and minimal sequence repeats.
  • pLSL was modified to encode two TALE nuclease sequences separated by a T2A translational-skipping sequence (pLSLT).
  • Target sequence for the TALE nuclease pair is present within two endogenous ALS genes, SuRA and SuRB (Zhang et al., Plant Physiol. 161:20-27, 2012, FIG. 16 ). WT N.
  • the CRISPR/Cas system functions to protect bacteria and archaea against invading foreign nucleic acid. It was previously demonstrated that targeted DNA double-strand breaks (DSBs) could be created in mammalian cells by expression of the Cas9 endonuclease and a programmable guide RNA (gRNA). We tested whether the CRISPR/Cas system is functional in plant cells using GVRs to deliver the components necessary for targeted DNA cleavage.
  • the LSL T-DNA was modified to encode a plant codon-optimized Cas9 followed by gRNA driven by an AtU6 RNA polymerase III promoter.
  • the gRNA was designed to recognize a site in SuRA and SuRB approximately 100 bp downstream of the T30 TALEN target ( FIG.
  • the data demonstrate that the CRISPR/Cas system can be used to make targeted modifications to plant genomes and that GVRs can simultaneously deliver gRNA and the Cas9 endonuclease.
  • GVRs were assessed for their ability to achieve GT through the coordinated delivery of nucleases and repair templates.
  • the target for modification was the defective gus::nptII gene, which can be repaired by correcting a 600 bp deletion that removes part of the coding sequences of both GUS and NPTII.
  • Zif268::FokI in pLSLZ.D is a us::NPTII repair template ( FIG. 21 ).
  • Cells having undergone GT will stain blue when incubated in a solution with the GUS substrate X-Gluc. Random integration of the repair template or read-through transcription from viral promoters should not produce functional GUS protein due to 703 nt missing from the 5′ coding sequence.
  • pLSLZ.D and p35SREP were engineered to encode Zif268::FokI and a us:NPTII repair template (p35SZ.D).
  • Multisite Gateway recombination was performed using plasmids pMDC32, pNB098 and pNB091.
  • GVRs may promote GT, including high levels of nuclease expression, high levels of repair template production and pleotropic Rep and RepA activity.
  • the coding sequence Zif268::FokI was replaced with GFP. Consistent with the stimulatory effect DSBs have on recombination, we observed a significant decrease in blue sectors when Zif268::FokI was removed ( FIG. 25 , top left).
  • the target gene was the endogenous SuRB gene.
  • a repair template present downstream of the T30 TALEN pair on pLSLT, contained 1 kb of sequence homologous to the SuRB locus flanking NPTII coding sequence.
  • the NPTII coding sequence is placed in-frame with the SuRB coding sequence, resulting in the production of a SuRB::NPTII fusion protein.
  • Agrobacterium containing pLSLT and p35SREP were grown overnight at 28° C.
  • Amplification of a ⁇ 1.2 kb product suggests this plant was produced from a cell that has undergone GT. Amplification of the 5′ junction may suggest that the GT event was ‘one-sided’ (e.g. following invasion of the repair template by a free 3′ end of the chromosomal DNA, the NPTII sequence is copied and then the break is sealed by illegitimate recombination).
  • Genomic DNA from several lines of hygromycin-resistant potato plants was isolated and assessed for the presence of p35SREP T-DNA and circular replicons. Amplification of a 440 bp sequence from Rep/RepA and a 714 bp sequence from replicon nucleotide sequence from plant line 10 suggests GVRs are present in potato cells ( FIG. 32 ). Interestingly, expression of Rep/RepA does not elicit an observable hypersensitive response. This was demonstrated by verifying expression of Rep/RepA in phenotypically-normal hygromycin-resistant plants by RT-PCR using primers that detect Rep/RepA RNA sequence ( FIG. 33 ).
  • pLSLGUS and p35SREP were transformed into Agrobacterium tumefaciens (AGL1) by the freeze-thaw method.
  • Agrobacterium was grown overnight at 28° C. to an OD 600 of 1 and diluted in LB media to an OD 600 of 0.2.
  • Half leaves were fully infiltrated with Agrobacterium encoding pLSLGUS or coinfiltrated with pLSLGUS and p35SREP.
  • leaf tissue was stained eleven dpi in X-Gluc solution. Chlorophyll was removed using 80% ethanol, and leaf images were taken ( FIG. 34 ).
  • the presence of GUS-expressing cells only in tissue transformed with pLSLGUS and p35SREP ( FIG. 35 ) suggested GVRs can drive transient protein expression in tomato leaf tissue.
  • an LSL T-DNA was constructed with cis-acting replication sequences from the Wheat dwarf virus (WDV) ( FIG. 36 ).
  • Rep/RepA coding sequence was positioned inside the flanking LIR sequences, just downstream of the complementary sense LIR promoter.
  • WDV LSL plasmids containing the GFP gene (WDV-GFP) were delivered to wheat ( Triticum aestivum cultivar Bobwhite), Setaria ( Setaria viridis ) and maize ( Zea mays cultivar A188), by particle bombardment. Three days post bombardment, tissue was assessed for GFP expression. Enhanced expression of GFP was observed in wheat calli ( FIG. 37 ), Setaria calli ( FIG. 38 ), and corn embryos ( FIG. 39 ) when delivered WDV-GFP.
  • WDV replicons are replicating and promoting GFP expression.
  • a WDV replicon was engineered to contain the T30 TALEN pair followed by a repair template designed to correct the non-functional gus::nptII gene ( FIG. 40 , top).
  • Leaf tissue also was transformed with conventional T-DNA containing the T30 TALEN pair followed by the us::NPTII repair template. Blue sectors observed in leaf tissue delivered GVR T-DNA and conventional T-DNA suggests that gus::nptII gene function was restored through GT in a subset of leaf cells ( FIG. 40 , bottom).

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US11384360B2 (en) 2022-07-12
WO2013192278A1 (fr) 2013-12-27
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US20210054388A1 (en) 2021-02-25
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