WO2006074956A1 - Procedes de transformation de plantes ameliores - Google Patents

Procedes de transformation de plantes ameliores Download PDF

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Publication number
WO2006074956A1
WO2006074956A1 PCT/EP2006/000311 EP2006000311W WO2006074956A1 WO 2006074956 A1 WO2006074956 A1 WO 2006074956A1 EP 2006000311 W EP2006000311 W EP 2006000311W WO 2006074956 A1 WO2006074956 A1 WO 2006074956A1
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WIPO (PCT)
Prior art keywords
dna
plant cell
break inducing
rare
cleaving enzyme
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PCT/EP2006/000311
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English (en)
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Kathleen D'halluin
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Bayer Bioscience N.V.
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Publication of WO2006074956A1 publication Critical patent/WO2006074956A1/fr

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    • 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
    • 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
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • 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
    • 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
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination

Definitions

  • This invention relates to methods for the stable introduction of exogenous DNA in the genome of plants, particularly the nuclear genome, using rare-cleaving enzymes capable of provoking double stranded breaks at the sites flanking the DNA of interest to be inserted in the genome. Such transformation methods lead to higher transformation efficiencies.
  • the current invention provides such improved transformation methods whereby the DNA of interest which is to be introduced in the genome of a plant or plant cell, is flanked by recognition sites for rare cleaving meganucleases which induce a double stranded DNA break.
  • the exogenous DNA is then introduced into the plant cell using conventional methods of DNA introduction, and by temporary action of the rare-cleaving meganucleases, the DNA of interest is released and is inserted into the genomic DNA of the plant cell in an more efficient manner than with the conventional techniques.
  • WO 00/46386 describe gene repair involving the induction of double-stranded DNA cleavage at a chromosomal target site, induced by e.g. the homing endonuclease I-Scel.
  • the described methods involve induction of a double stranded cleavage in the genome of the cell at a site of interest, followed by the introduction of targeting DNA which comprises DNA homologous to the region surrounding the site of interest.
  • PCT application 2004/03122 published as WO2005/049842 describes modified DNA sequences encoding meganucleases, such as I-Scel, adapted for expression in plant cells, and the use thereof to insert target DNA at a specific location in the genome of plants.
  • Thermes et al. (2002, Mech. Dev. 118 : pp91-98) report that I-Scel meganuclease mediates highly efficient transgenesis in fish when the transgenes of interest were flanked by two I- Scel meganuclease recognition sites and co-injected together with the I-Scel meganuclease enzyme into medaka embryos at the one-cell stage.
  • An advertisment on the web-site of Cellectis (www.cellectis.com) refers to this tool for improving the efficiency of classical transgenesis as InVoLin®.
  • the document remains silent as to the applicability of this improved transformation technique to species other than fish.
  • WO03/025183 is a international patent application corresponding to the above mentioned publication. It concerns a method for in vivo generation of a linear polynucleotide with 5' and 3' free ends from a vector having no free end, said linear polynucleotide being integrated into the host cell genome.
  • the vector having no free end comprises the polynucleotide to be linearized or excised flanked by a cleavage site, said cleavage site being preferably not found in the host cell genome.
  • the teaching of this specification is directed towards increasing efficiency of transgenesis in animals, particularly fish, although it is suggested without any experimental basis that the described methods could also be used for plants.
  • a method for transforming a plant cell particularly for increasing transformation efficiency for a plant cell, comprising the steps of
  • a first DNA molecule e.g. by direct DNA transfer methods such as electroporation, micro-projectile bombardment, PEG mediated DNA introduction or silicon whisker mediated DNA introduction, into a plant cell, such as a protoplast or tissue or explant, whereby the first DNA molecule comprises a DNA sequence of interest, flanked at least at one end, or on both ends , by a recognition site for a double-stranded DNA break inducing rare-cleaving enzyme such as I-Sce I, I-Chu I, I-Dmo I, I-Cre I, I-Csm I, PI-FIi I, Pt-Mtu I, I-Ceu I, I-Sce II, I-Sce III, HO, Pi-Civ I, PI-Ctr I, PI-Aae I, PI-BSU I, PI-Dhal, PI-Dra I, PI-Mav I, PI-Mch I, PI-M
  • the double-stranded break inducing rare- cleaving enzyme e.g. by introduction of a second DNA molecule comprising a plant- expressible promoter operably linked to a DNA region encoding said double stranded DNA break inducing rare-cleaving enzyme, optionally optimized for expression in plant cells; and a transcription termination and polyadenylation DNA region; and
  • the invention relates to the use of a double stranded DNA break inducing rare cleaving endonuclease, such as I-Sce I, I-Chu I, I-Dmo I, I-Cre I, I-Csm I, PI-FIi I, Pt-Mtu I, I-Ceu I, I-Sce II, I-Sce III, HO, Pi-Civ I, PI-Ctr I, PI-Aae I, PI-BSU I, PI-Dhal, PI-Dra I, PI-Mav I, PI-Mch I, PI-Mfu I, PI-MfI I, PI-Mga I, PI-Mgo I, PI-Min I, PI-Mka I, PI-MI
  • rare cleaving endonuclease such as I-Sce I, I-Chu I, I-Dmo I, I-Cre I, I-C
  • the current invention is based on the finding that inclusion of one or two recognition sites for a double-stranded DNA break inducing rare-cleaving enzyme, in a tranforming DNA, preferably flanking the DNA sequence of interest to be introduced, can enhance the transformation frequency when the transforming DNA is introduced in plant cells in combination with the double-stranded DNA break inducing rare-cleaving enzyme.
  • the invention is directed towards the use of a double stranded DNA break inducing rare cleaving endonuclease in combination with a transforming DNA molecule comprising at least one recognition site for said double stranded DNA break inducing rare cleaving endonuclease flanking the DNA sequence of interest, to improve transformation frequency of plant cells.
  • a "double stranded DNA break inducing rare-cleaving endonuclease” is an enzyme capable of inducing a double stranded DNA break at a particular nucleotide sequence, called the "recognition site”.
  • Rare-cleaving endonucleases also sometimes called mega-nucleases have a recognition site of 14 to 40 consecutive nucleotides. Therefore, rare- cleaving endonuclease have a very low frequency of cleaving, even in the larger plant genomes.
  • Homing endonucleases constitute a family of such rare-cleaving endonucleases.
  • these nucleases By making a site-specific double strand break in the intronless or inteinless alleles, these nucleases create recombinogenic ends, which engage in a gene conversion process that duplicates the coding sequence and leads to the insertion of an intron or an intervening sequence at the DNA level.
  • I-Scel A well characterized homing endonuclease is I-Scel.
  • I-Scel is a site-specific endonuclease, responsible for intron mobility in mitochondria in Saccharomyces cerevisea.
  • the enzyme is encoded by the optional intron Sc LSU.1 of the 21 S rRNA gene and initiates a double stranded DNA break at the intron insertion site generating a 4 bp staggered cut with 3 'OH overhangs.
  • the recognition site of I-Scel endonuclease extends over an 18 bp nonsymmetrical sequence (Colleaux et al. 1988 Proc. Natl. Acad. ScL USA 85: 6022-6026).
  • WO 96/14408 The amino acid sequence for I-Scel and a universal code equivalent of the mitochondrial I-Scel gene have been provided by e.g. WO 96/14408. WO 96/14408 further discloses that a number of variants of I-Scel protein which are still functional.
  • PCT application 2004/03122 provides synthetic nucleotide sequence variants of I-Scel which have been optimized for expression in plants.
  • the nucleotide sequence of such synthetic I-Sce I coding regions is set forth in SEQ ID No 1 in UIPAC code.
  • chimeric restriction enzymes can be prepared using hybrids between a zinc-finger domain designed to recognize a specific nucleotide sequence and the non-specific DNA-cleavage domain from a natural restriction enzyme, such as Fokl.
  • a natural restriction enzyme such as Fokl.
  • Such methods have been described e.g. in WO 03/080809, WO94/18313 or WO95/09233 and in Isalan et al, 2001, Nature Biotechnology 19, 656- 660; Liu et al. 1997, Proc. Natl. Acad. ScI USA 94, 5525-5530).
  • Another way of producing custom-made meganucleases, by selection from a library of variants, is described in WO 2004/067736.
  • the "efficiency of transformation” or "frequency of transformation” as used herein can be, measured by the number of transformed cells (or transgenic calli grown from individual transformed cells) that are recovered under standard experimental conditions (i.e. standardized or normalized with respect to amount of cells contacted with foreign DNA, amount of delivered DNA, type and conditions of DNA delivery, general culture conditions etc).
  • the frequency of transformation can be expressed as the number of transgenic calli obtained per 100 callus pieces transformed.
  • frequency of transformation can be expressed as the number of transgenic calli per 10 6 intitial protoplasts used.
  • the frequency of transformation can be expressed as the number of transgenic calli per plate or Petri-dish.
  • protoplasts When protoplasts are used to generate transformed calli, the latter usually originate from one unit of cultured cells during the regeneration process and may thus correspond to one transformation event, comprising the same transgenes integrated at the same genomic positions.
  • transgenic calli can also originate from independent transformation events, particularly when using transformation of cultured tissues such as (suspension) calli or explants.
  • transformation frequencies are expressed by the number of transgenic plant calli/ 100 initial callus pieces or per petridish, it may be that the actual transformation frequencies are even higher as a transgenic callus scored as one event may actually consist of several independent transformation events.
  • transformation efficiency indicates that the transformation frequencies resulting from the methods according to the invention are higher than when similar conventional transformation methods are used without the presence of recognition sites for DSB inducing rare cleaving enzyme in the transforming DNA or without the transient provision of the DSB inducing rare-cleaving endonuclease.
  • the invention relates to a method for transforming a plant cell, particularly for increasing transformation efficiency for a plant cell.
  • a DNA molecule comprising a DNA sequence of interest is introduced into plant cells using any suitable DNA transfer method, whereby the DNA sequence of interest is flanked at least at one end by a recognition site for a double-stranded DNA break inducing rare-cleaving enzyme. Further a suitable double-stranded break inducing rare-cleaving enzyme, which can recognize the recognition sites is transiently provided to the plant cells.
  • Transgenic plant cell wherein said DNA sequence of interest is integrated into the genome of said plant cell can be isolated, as known in the art, and may be further regenerated into intact transgenic plants.
  • the recognition sites can be oriented in such a way that upon binding and cleaving of the endonuclease, the endonuclease remains associated with the DNA to be inserted (DNA sequence of interest) or alternatively with the remainder of the DNA molecule or vector.
  • the double stranded DNA breaks in the transforming DNA molecule may be induced conveniently by transient introduction of a plant-expressible chimeric gene comprising a plant-expressible promoter region operably linked to a DNA region encoding a double stranded break inducing enzyme.
  • the endonuclease itself, as a protein, could also be introduced into the plant cells, e.g. by electroporation.
  • the endonuclease can also be provided in a transient manner by introducing into the genome of a plant cell or plant, a chimeric gene comprising the endonuclease coding region operably linked to an inducible plant-expressible promoter, and providing the appropriate inducible compound for a limited time prior to, during or immediately after introduction of the transforming DNA molecule.
  • the endonuclease could also be provided as an RNA precursor encoding the endonuclease.
  • the transforming DNA molecule can be transferred into plant cells using any conventional method, including but not limited to direct DNA transfer method.
  • direct DNA transfer is any method of DNA introduction into plant cells which does not involve the use of natural Agrobacterium spp. and which is capable of introducing DNA into plant cells. This includes methods well known in the art such as introduction of DNA by electroporation into protoplasts, introduction of DNA by electroporation into intact plant cells or partially degraded tissues or plant cells, introduction of DNA through the action of agents such as PEG and the like, into protoplasts, use of silicon whiskers, and bombardment with DNA coated microprojectiles.
  • the means and methods of the invention are particularly useful for corn or tobacco, but may also be used in other plants with similar effects, particularly in cereal plants including wheat, oat, barley, rye, rice, turfgrass, sorghum, millet or sugarcane plants.
  • the methods of the invention can also be applied to any plant including but not limited to cotton, canola, oilseed rape, soybean, vegetables, potatoes, Lemna spp., Nicotiana spp., Arabidopsis, alfalfa, barley, bean, corn, cotton, flax, pea, rape, rice, rye, safflower, sorghum, soybean, sunflower, tobacco, wheat, asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery, cucumber, eggplant, lettuce, onion, oilseed rape, pepper, potato, pumpkin, radish, spinach, squash, tomato, zucchini, almond, apple, apricot, banana, blackberry, blueberry, cacao, cherry, coconut, cranberry, date, grape, grapefruit, guava, kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passion fruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry, strawberry, tangerine, walnut
  • the plants obtained by the methods described herein may be further crossed by traditional breeding techniques with other plants to obtain progeny plants comprising the targeted DNA insertion events obtained according to the present invention.
  • SEQ ID No 1 nucleotide sequence of synthetic I-Scel coding region (UIPAC code).
  • SEQ ID No 2 nucleotide sequence of synthetic I-Scel coding region.
  • SEQ ID No 3 nucleotide sequence of an I-Scel recognition site.
  • SEQ ID No 4 nucleotide sequence of pTRR26
  • SEQ ID No 5 nucleotide sequence of pIB82
  • SEQ ID No 6 nucleotide sequence of pIB83
  • SEQ ID No 7 nucleotide sequence of pIB84
  • SEQ ID No 8 nucleotide sequence of pCV93
  • SEQ IDNo 9 nucleotide sequence of pCV78
  • Example 1 DNA molecules for transformation.
  • pTRR26 (SEQ ID No 4) is a DNA vector containing a CaMV 35S promoter, a DNA region coding for I-Sce I meganuclease and a 3 '35S termination and polyadenylation region.
  • B. pIB82 is a DNA vector designed as repair DNA for experiments on homologous recombination. It contains a defective phosphinotricin acetyl transferase gene under control of a CaMV 35S promoter. It also contains a neomycin phosphotranferase gene under control of a nopaline synthase promoter and followed by a 3' termination and polyadenylation signal from octopine synthase. Sequence of this vector is provided in SEQ ID No 5.
  • C. pIB83 is a DNA vector almost identical to pIB82 but which contains a I-Scel recognition site, flanking the 3' end of the bar gene (SEQ ID No 6).
  • D. pIB84 is also a DNA vector almost identical to IB82 but which contains two I-Scel sites, one flanking the 3 ' end of the bar gene and the other one flanking the chimeric neomycin phosphotransferase gene.
  • the orientation of the I-Sce I sites is such that the I-Sce I endonuclease remains attached to the excised transforming DNA and not to the vector part (SEQ ID No 7).
  • E. pCV93 is a DNA vector comprising a phosphinotricin acetyltransferase coding region under control of a CaMV35S promoter and a 3 'end of a nopaline synthase gene involved in transcription termination and polyadenylation. This chimeric gene is flanked at both sites by a I-Sce I recognition site (SEQ ID No 8).
  • F. pCV78 A synthetic DNA fragment encoding I-Sce I, having the nucleotide sequence of SEQ ID No 2 (optimized for expression in plants) was synthesized and operably linked to a CaMV35S promoter and a CaMV35S 3' termination and polyadenylation signal (yielding plasmid pCV78; SEQ ID No 9).
  • Example 2 Increase of the efficiency of transformation in tobacco.
  • Protoplasts of wild type SRl tobacco plants were electroporated, using a BioRad GenePulser® II RF module (600V/cm, 50 kHz, 1 pulse of 10 ms, 25% modulation) with either 1,5 pmol pIB82, PIB83 or pIB84 DNA, respectively in the presence or absence of 1.5 pmol I-Scel encoding DNA pTRR26.
  • 3 to 5 electrocuvettes, containing about 1 x 10 6 protoplasts were electroporated.
  • Two weeks after electroporation protoplast derived colonies were plated on plant growth media containing 200 mg/L kanamycin. The results (mean transformation efficiency) of the transformation experiments are summarized in Table I. Table I. Mean transformation frequencies in tobacco.
  • Example 3 Increase of the efficiency of transformation in corn.
  • Suspension culture cells of maize line derived from HE89 were subjected to microparticle bombardment, using a BioRAD PPS_1000/He Biolistic Particle delivering systems essentially as described by Sanford et al., 1992 whereby the particles were coated with either pCV93 DNA (transforming DNA) alone, or with a mix of pCV93 DNA and pCV78 DNA (encoding a synthetic I-Scel site).
  • pCV93 DNA transforming DNA
  • pCV78 DNA encoding a synthetic I-Scel site
  • the transformation frequency can be increased at least by a factor 1.5 using transforming DNA flanked by I-Sce I recognition sites, in the presence of I-Scel encoding DNA. It should be noted that this number may be an underestimate, as the independent PPT-resistant calli may be the result of more than one transformation event.

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Abstract

L'invention concerne des procédés permettant d'introduire de manière stable de l'ADN exogène dans le génome de plantes, notamment dans le génome nucléaire, au moyen d'enzymes à sites de clivages rares qui peuvent provoquer des cassures bicaténaires au niveau d'un site adjacent à l'ADN d'intérêt à insérer dans le génome. Ces procédés de transformation permettent d'obtenir une efficacité de transformation plus élevée.
PCT/EP2006/000311 2005-01-14 2006-01-11 Procedes de transformation de plantes ameliores WO2006074956A1 (fr)

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Application Number Priority Date Filing Date Title
EP05075072.8 2005-01-14
EP05075072 2005-01-14
US64517505P 2005-01-19 2005-01-19
US60/645,175 2005-01-19

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012168124A1 (fr) 2011-06-06 2012-12-13 Bayer Cropscience Nv Méthodes et moyens pour modifier le génome d'une plante en un site présélectionné
WO2012168910A1 (fr) 2011-06-10 2012-12-13 Basf Plant Science Company Gmbh Protéine de fusion de nucléase et ses utilisations
US8338157B2 (en) 2008-03-11 2012-12-25 Precision Biosciences, Inc. Rationally-designed meganuclease variants of lig-34 and I-crei for maize genome engineering
US8912392B2 (en) 2007-06-29 2014-12-16 Pioneer Hi-Bred International, Inc. Methods for altering the genome of a monocot plant cell

Citations (5)

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WO1996014408A2 (fr) * 1994-11-07 1996-05-17 Institut Pasteur Sequence nucleotidique codant l'enzyme i-scei et ses utilisations
WO2000046386A2 (fr) * 1999-02-03 2000-08-10 The Children's Medical Center Corporation Reparation genique a induction de clivage d'adn bicatenaire en un site cible chromosomique
WO2003004659A2 (fr) * 2001-07-04 2003-01-16 Sungene Gmbh & Co. Kgaa Systemes de recombinaison et procedes pour retirer des sequences d'acide nucleique du genome d'organismes eucaryotes
WO2003025183A2 (fr) * 2001-09-14 2003-03-27 Cellectis Integration aleatoire d'un polynucleotide par linearisation in vivo
WO2004106496A2 (fr) * 2003-05-27 2004-12-09 Tosk, Inc. Methodes et compositions a utiliser dans la recombinaison homologue dans des cellules de plantes

Patent Citations (5)

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Publication number Priority date Publication date Assignee Title
WO1996014408A2 (fr) * 1994-11-07 1996-05-17 Institut Pasteur Sequence nucleotidique codant l'enzyme i-scei et ses utilisations
WO2000046386A2 (fr) * 1999-02-03 2000-08-10 The Children's Medical Center Corporation Reparation genique a induction de clivage d'adn bicatenaire en un site cible chromosomique
WO2003004659A2 (fr) * 2001-07-04 2003-01-16 Sungene Gmbh & Co. Kgaa Systemes de recombinaison et procedes pour retirer des sequences d'acide nucleique du genome d'organismes eucaryotes
WO2003025183A2 (fr) * 2001-09-14 2003-03-27 Cellectis Integration aleatoire d'un polynucleotide par linearisation in vivo
WO2004106496A2 (fr) * 2003-05-27 2004-12-09 Tosk, Inc. Methodes et compositions a utiliser dans la recombinaison homologue dans des cellules de plantes

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Title
CHILTON M-D M ET AL: "Targeted integration of T-DNA into the tobacco genome at double-stranded breaks: New insights on the mechanism of T-DNA integration", PLANT PHYSIOLOGY, AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD, US, vol. 133, no. 3, November 2003 (2003-11-01), pages 956 - 965, XP002319963, ISSN: 0032-0889 *
PUCHTA H ET AL: "HOMOLOGOUS RECOMBINATION IN PLANT CELLS IS ENHANCED BY IN VIVO INDUCTION OF DOUBLE STRAND BREAKS INTO DNA BY A SITE-SPECIFIC ENDONUCLEASE", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 21, no. 22, 11 November 1993 (1993-11-11), pages 5034 - 5040, XP000571587, ISSN: 0305-1048 *
TZFIRA T ET AL: "Site-specific integration of Agrobacterium tumefaciens T-DNA via double-stranded intermediates", PLANT PHYSIOLOGY, AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD, US, vol. 133, no. 3, November 2003 (2003-11-01), pages 1011 - 1023, XP002319962, ISSN: 0032-0889 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8912392B2 (en) 2007-06-29 2014-12-16 Pioneer Hi-Bred International, Inc. Methods for altering the genome of a monocot plant cell
US8338157B2 (en) 2008-03-11 2012-12-25 Precision Biosciences, Inc. Rationally-designed meganuclease variants of lig-34 and I-crei for maize genome engineering
WO2012168124A1 (fr) 2011-06-06 2012-12-13 Bayer Cropscience Nv Méthodes et moyens pour modifier le génome d'une plante en un site présélectionné
WO2012168910A1 (fr) 2011-06-10 2012-12-13 Basf Plant Science Company Gmbh Protéine de fusion de nucléase et ses utilisations
US9758796B2 (en) 2011-06-10 2017-09-12 Basf Plant Science Company Gmbh Nuclease fusion protein and uses thereof

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