METHODS AND MEANS TO MODIFY A PLANT GENOME AT A NUCLEOTIDE SEQUENCE COMMONLY USED IN PLANT GENOME ENGINEERING

Abstract

Methods and means are provided to modify in a targeted manner the plant genome of transgenic plants comprising chimeric genes wherein the chimeric genes have a DNA element commonly used in plant molecular biology. Re-designed meganucleases to cleave such an element commonly used in plant molecular biology are provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser. No. 13/700,773, filed Jan. 16, 2013, which claims the benefit of a .sctn.371 U.S. National Stage of International Application No. PCT/EP2011/002895, filed Jun. 7, 2011, which claims the benefit of European Patent Application Serial No. 10005941.9, filed Jun. 9, 2010 and U.S. Patent Application Ser. No. 61/355,849, filed Jun. 17, 2010, the contents of which are herein incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named "BCS10-2010-WO1.ST25.txt", created on May 8, 2013, and having a size of 35,000 bytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] The invention relates to the field of agronomy. More particularly, the invention provides methods and means to introduce a targeted modification, including insertion, deletion or substitution, at a precisely localized nucleotide sequence in the genome of a transgenic plant, wherein the nucleotide sequence is comprised within an element or DNA fragment frequently used in plant transgenesis, such as a commonly used selectable marker gene. The modifications are triggered in a first step by induction of a double stranded break at the recognition nucleotide sequence using meganucleases derived from naturally occurring meganucleases which have been re-designed to recognize the recognition site and cleave it.

BACKGROUND ART

[0004] The need to introduce targeted modification of plant genomes, including the control over the location of integration of foreign DNA in plants has become increasingly important, and several methods have been developed in an effort to meet this need (for a review see Kumar and Fladung, 2001, Trends in Plant Science, 6, pp155-159). These methods mostly rely on the initial introduction of a double stranded DNA break at the targeted location.

[0005] Activation of the target locus and/or repair or donor DNA through the induction of double stranded DNA breaks via rare-cutting endonucleases, such as I-SceI. has been shown to increase the frequency of homologous recombination by several orders of magnitude. (Puchta et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93, pp5055-5060; Chilton and Que, Plant Physiol., 2003; D'Halluin et al. 2008 Plant Biotechnol. J. 6, 93-102).

[0006] WO96/14408 describes an isolated DNA encoding the enzyme I-SceI. This DNA sequence can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.

[0007] WO00/46386 describes methods of modifying, repairing, attenuating and inactivating a gene or other chromosomal DNA in a cell through I-SceI double strand break. Also disclosed are methods of treating or prophylaxis of a genetic disease in an individual in need thereof. Further disclosed are chimeric restriction endonucleases.

[0008] In addition, methods have been described which allow the design of rare cleaving endonucleases to alter substrate or sequence-specificity of the enzymes, thus allowing to induce a double stranded break at a locus of interest without being dependent on the presence of a recognition site for any of the natural rare-cleaving endonucleases. Briefly, 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 FokI. 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 WO2004/067736. Custom made meganucleases or redesigned meganucleases with altered sequence specificity and DNA-binding affinity may also be obtained through rational design as described in WO2007/047859.

[0009] WO2007/049095 describes "LADGLIDADG" homing endonuclease variants having mutations in two separate subdomains, each binding a distinct part of a modified DNA target half site, such that the endonuclease variant is able to cleave a chimeric DNA target sequence comprising the nucleotides bound by each subdomain.

[0010] WO2007/049156 and WO 2007/093836 describe I-CreI homing endonuclease variants having novel cleavage specificity and uses thereof.

[0011] WO2007/047859 describes rationally designed meganucleases with altered sequence specificity and DNA binding affinity.

[0012] WO2006/105946 described a method for the exact exchange in plant cells and plants of a target

[0013] DNA sequence for a DNA sequence of interest through homologous recombination, whereby the selectable or screenable marker used during the homologous recombination phase for temporal selection of the gene replacement events can subsequently be removed without leaving a foot-print and without resorting to in vitro culture during the removal step, employing the therein described method for the removal of a selected DNA by microspore specific expression of a double stranded break inducing rare cleaving endonuclease.

[0014] U.S. provisional patent application 60/828,042 and European patent application 06020370.0, and WO2008/037436 describe variants of the methods and means of WO2006/105946 wherein the removal step of a selected DNA fragment induced by a double stranded break inducing rare cleaving endonuclease is under control of a germline-specific promoter. Other embodiments of the method relied on non-homologous endjoining at one end of the repair DNA and homologous recombination at the other end.

[0015] Gao et al. 2009, The Plant Journal, pp 1-11 describe heritable targeted mutagenesis in maize using a designe endonuclease.

[0016] Since the re-designed meganucleases are derived from naturally occurring endonucleases, the available potential recognition sites are not entirely random but appear to have some degree of resemblance to the nucleotide sequence originally recognized by the naturally occurring endonuclease upon which the re-designed meganuclease is based. As stated by Gao et al, 2009 (supra) the structure-based protein design method to modify the DNA-binding characteristics of I-CreI are based on visual inspection of the I-CreI-DNA co-crystal structure leading to a prediction of a a large number of amino acid substitutions that change I-CreI base preference at particular positions in its recognition site. Individual amino acid substitutions were evaluated experimentally, and those that conferred the desired change in base preference were added to a database of mutations that can be "mixed and matched" to generate derivatives of I-CreI that recognize highly divergent DNA sites. In theory, the combinatorial diversity available using the current mutation database is sufficient to target an engineered endonuclease approximately every 1000 bp in a random DNA sequence.

[0017] Accordingly, there still remains a need for functional re-designed meganucleases which can recognize a recognition site in an DNA element or region previously introduced into a transgenic plant as a commonly used part of a transgene, and induce a double branded DNA break in that region with sufficient efficiency, thereby triggering the events required for e.g. insertion of foreign DNA, deletion or substitution by homologous recombination or non-homologous endjoining at the double stranded break site. Identification of such a pair of recognition site and re-designed meganuclease, enhances the available tools to modify a plant genome in a targeted manner, by allowing insertion, deletion or substitution of the DNA in the vicinity of the induced double stranded DNA break at the location of a previously introduced transgene, without having to resort to presence of historically introduced recognition sites for rare-cleaving endonucleases such as e.g. I-SceI (which does not occur naturally in plant cells).

[0018] These and other problems are solved as described hereinafter in the different detailed embodiments of the invention, as well as in the claims.

SUMMARY OF THE INVENTION

[0019] In one embodiment of the invention, a method is provided for introducing a foreign DNA molecule at a predefined site in a genome of a transgenic plant cell comprising the steps of

[0020] a. inducing a double stranded DNA break at the predefined site;

[0021] b. introducing the foreign DNA molecule in the plant cell;

[0022] c. selecting a plant cell wherein the foreign DNA is introduced at the predefined site; and

[0023] d. optionally regenerating the plant cell into a plant

[0024] characterized in that the predefined site is a nucleotide sequence different from a recognition site for a natural occurring meganuclease and that the predefined site is a nucleotide sequence commonly introduced as part of a transgene in a transgenic plant and wherein double stranded DNA break is induced by introduction of a non-naturally occurring single chain meganuclease or a pair of non-naturally occurring meganucleases which recognizes or recognize in concert the predefined site and induces or induce the double stranded break.

[0025] In another embodiment the invention provides a method for introducing a foreign DNA molecule at a predefined site in a genome of a plant cell comprising the steps of

[0026] a. inducing a double stranded DNA break at the predefined site;

[0027] b. introducing the foreign DNA molecule in the plant cell;

[0028] c. selecting a plant cell wherein the foreign DNA is introduced at the predefined site; and

[0029] d. optionally regenerating the plant cell into a plant

[0030] characterized in that the predefined site is comprised within a phosphinotricin acetyl transferase coding region from S. hygroscopicus (bar coding region), which may have the nucleotide sequence of SEQ ID No 3 and that the double stranded DNA break is induced by introduction of a single chain meganuclease or a pair of meganucleases which recognizes or recognize in concert the predefined site and induces or induce the double stranded break. The predefined site may comprise the nucleotide sequence of SEQ ID No 1 or SEQ ID No 2.

[0031] In yet another embodiment, a method is provided for introducing a foreign DNA molecule at a predefined site in a genome of a plant cell comprising the steps of

[0032] a. inducing a double stranded DNA break at the predefined site;

[0033] b. introducing the foreign DNA molecule in the plant cell;

[0034] c. selecting a plant cell wherein the foreign DNA is introduced at the predefined site;

[0035] d. optionally regenerating the plant cell into a plant

[0036] characterized in that the predefined site comprises the nucleotide sequence of SEQ ID No 1 or SEQ ID No. 2 and that the double stranded DNA break is induced by introduction of a single chain meganuclease or a pair of meganucleases which recognizes or recognize in concert the predefined site and induces or induce the double stranded break such as a meganuclease or the pair of meganucleases is/are derived from I-CreI and wherein the following amino acids are present in one of the subunits : S at position 32; Y at position 33; E at position 38; R at position 40; K at position 66; Q at position 80; T at position 42; R at position 77; R at position 68; R at position 70; Q at position 44; I at position 24; S at position 26; S at position 28 and R at position 30 or R at position 70; T at position 44; I at position 24; S at position 26; S at position 28; N at position 30; S at position 32; R at position 33; Q at position 38; Q at position 80; R at position 40; K at position 66; T at position 42; R at position 77 and R at position. Examples of such meganuclease are protein comprising the amino acid sequence of SEQ ID No. 4 and SEQ ID 5, respectively, encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID No. 6 from nucleotide position 2004 to nucleotide position 2525 or the nucleotide sequence of SEQ ID No. 6 from nucleotide position 4885 to nucleotide position 5405.

[0037] In any of the embodiments, the foreign DNA may be comprised within a repair DNA, the repair DNA comprising at least one flanking nucleotide sequence homologous to the upstream or downstream sequence of the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 2. The foreign DNA may comprises a selectable marker gene and/or a plant expressible gene of interest such as of a herbicide tolerance gene, an insect resistance gene, a disease resistance gene, an abiotic stress resistance gene, a enzyme involved in oil biosynthesis, carbohydrate biosynthesis, a enzyme involved in fiber strength or fiber lenght, an enzyme involved in biosynthesis of secondary metabolites.

[0038] The meganuclease or the pair of meganucleases may be expressed from a chimeric gene or a pair of chimeric genes, each comprising a plant expressible promoter operably linked to a coding region encoding the meganuclease or one of the pair of meganucleases, and further operationally linked to a DNA region involved in transcription termination and polyadenylation functional in a plant cell.

[0039] The invention further provides, plant cells and plants and seeds or propagating parts wherein the foreign DNA has been introduced into the predefined site, which have been obtained by the methods herein provided.

[0040] The invention also provides a method of growing a plant wherein the foreign DNA has been introduced into the predefined site, which has been obtained by the methods herein provided comprising the step of applying a chemical to the plant or substrate wherein the plant is grown.

[0041] Yet another embodiment of the invention concerns a process for producing a plant comprising foreign DNA integrated at the bar coding region comprising the step of crossing a plant consisting essentially of the plant cells obtained by the methods of the invention with another plant or with itself and optionally harvesting seeds.

[0042] The invention also concerns a process comprising the step of applying a chemical compound on a plant or a seed of a plant wherein the foreign DNA has been introduced into the predefined site, which has been obtained by the methods herein provided.

[0043] Another embodiment of the invention relates to the ysse of a meganuclease or a pair of meganucleases as herein described to introduce a foreign DNA into the bar coding region in a plant cell.

[0044] Yet another embodiment of the invention relates to the use of a custom made meganuclease to introduce a foreign DNA of interest at a predefined site in a plant cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1: Schematic representation of the recognition site and interactions with amino acids of the different meganuclease monomeric units BAY 39 and BAY40. The recognition site sequence is set forth in SEQ ID NO:1. The complement of the recognition site sequence is set forth in SEQ ID NO:2.

[0046] FIG. 2: Amino acid sequence of BAY 39/40 monomeric unit 2 ("40") (SEQ ID NO:4) (note that the amino acid sequence comprises a SV40 nuclear localization signal (amino acids 1 to 10)).

[0047] FIG. 3: Amino acid sequence of BAY 39/40 monomeric unit 1 ("39") (SEQ ID NO:5) (note that the amino acid sequence comprises an SV40 nuclear localization signal (amino acids 1 to 10)).

[0048] FIG. 4: Amino acid sequence of the single chain BAY 39/40 meganuclease (SEQ ID NO:18) (note that the amino acid sequence comprises an SV40 nuclear localization signal (amino acids 1-12) and a linker region (amino acids 168 to 205)).

[0049] FIG. 5: Alignment of the nucleotide sequence of PCR amplicons around the recognition site of the bar coding regions, in phosphinotricin sensitive lines derived from a transgenic plant comprising a plant expressible bar gene and plant expressible genes for the monomeric units of BAY39/40 . 1. nucleotide sequence of a control sample (SEQ ID NO:7); 2. nucleotide sequence of a phosphinotricin tolerant line (SEQ ID NO:8); 3-9: nucleotide sequences of phosphinotricin sensitive lines (SEQ ID NO:9-15, respectively).

DESCRIPTION OF DIFFERENT EMBODIMENTS OF THE INVENTION

[0050] The current invention is based on the observation that functional re-designed meganucleases can be obtained which specifically recognize and cleave a nucleotide sequence (SEQ ID No. 1 and SEQ ID No. 2--FIG. 1), which can be found in the nucleotide sequence of the coding region of the phosphinotricin acetyltransferase gene from Streptomyces hygroscopicus (bar gene) (Thompson, C., Movva, R., Tizard, R., Crameri, R., Davies, J., Lauwereys, M. ans Botterman, J. (1987) Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus. The EMBO Journal 6: 2519-2523 (Accession X05822)), which nucleotide sequence is present in a commonly used selectable marker gene in plant transgenesis.

[0051] SEQ ID No. 3 represents the nucleotide sequence of the bar gene. The complement of the recognition site of SEQ ID No. 1 (SEQ ID No. 2) corresponds to the nucleotide sequence of SEQ ID No. 3 from nucleotide 132 to 153. The herein described meganucleases are thus capable of recognizing and cleaving a nucleotide sequence in transgenic plants comprising a plant-expressible gene which has a plant expressible promoter operable linked to a DNA region encoding the phosphinotricin acetyltransferase gene from Streptomyces hygroscopicus (bar) and followed by a 3' transcription termination and polyadenylation region functional in plants, the bar coding region comprising the nucleotide sequence which is the complement of the nucleotide sequence of SEQ ID No. 1, such as SEQ ID No. 3.

[0052] The bar coding region has been incorporated in a number of transgenic plants which have been, are or will be commercialized including plants comprising the following events:

[0053] Chicory (Cichorium intybus):

[0054] Events RM3-3, RM3-4, RM3-6 as described in regulatory file 97-148-01p

[0055] Oilseeed rape (Brassica napus)

[0056] Event MS1 as described in regulatory files DD95-04 (CA) or 98-278-01p (US)

[0057] Event MS8 as described in regulatory files DD96-17 (CA) or 98-278-01p (US) or WO 2001/041558

[0058] Event RF1 as described in regulatory files DD95-04 (CA) or 98-278-01p (US)

[0059] Event RF2 as described in regulatory files DD95-04 (CA) or 98-278-01p (US)

[0060] Event RF3 as described in regulatory files DD96-17 (CA) or 98-278-01p (US) or WO 2001/041558

[0061] Events PHY14, PHY35, PHY36 as described in Japanese deregulatory files

[0062] Cotton (Gossypium hirsutum)

[0063] Event LLcotton 25 as described in regulatory files 02-042-01p (US) or WO 2003/013224

[0064] Event T303-40 as described in WO2008/122406

[0065] Event GHB119 as described in regulatory file 08-340-01p (US) or WO2008/151780

[0066] Corn (Zea mays)

[0067] Event TC-6275 (=DAS-06275-8) as described in regulatory file 03-181-01p (US)

[0068] Event Bt176 as described in regulatory file 94-319-01p (US)

[0069] Event B16 (=DLL25) as described in US deregulation dossier 95-145-01p or WO9506128

[0070] Event DBT418 as described in US deregulation dossier 96-291-01p (US)

[0071] Event ZMA101

[0072] Event CBH351 as described in US deregulation dossier 9'7-265-01p (US)

[0073] Event MS3 as described in US deregulation file 95-228-01p (US)

[0074] Rice (Oryza sativa)

[0075] Event LLRice62 as described in US deregulation dossier 98-329-01p or WO 2001/083818

[0076] Event LLRICE601 as described in US deregulation dossier 06-234-01p or U.S. patent application 2008289060

[0077] Soybean (Glycine max)

[0078] Events W62 and W98 described in regulatory file 96-068-01p(US)

[0079] Transgenic plants containing these events therefore contain a recognition sequence for the meganucleases herein described and are suitable subjects for the methods of the invention.

[0080] Furthermore, the plant expressible bar gene is used generally as a selectable marker and numerous transgenic plants have been generated which are also suitable subjects for the methods of the invention.

[0081] Accordingly, in one embodiment, the invention relates to a method for introducing a foreign

[0082] DNA molecule at a predefined or preselected site in a genome of a transgenic plant cell comprising the steps of

[0083] a. inducing a double stranded DNA break at the predefined site;

[0084] b. introducing the foreign DNA molecule in said plant cell; and

[0085] c. selecting a plant cell wherein the foreign DNA is introduced at the predefined site;

[0086] wherein the predefined site is a nucleotide sequence different from a recognition site for a natural occurring meganuclease and is a nucleotide sequence commonly introduced as part of a transgene in a transgenic plant and wherein double stranded DNA break is induced by introduction of a non-naturally occurring single chain meganuclease or a pair of non-naturally occurring meganuclease monomeric units which recognizes or recognize togethter the predefined site and induces or induce the double stranded break.

[0087] As used herein, "a nucleotide sequence commonly introduced as a part of a transgene in plants" refers to a nucleotide sequence of a DNA region that has been used previously as an element of a chimeric gene introduced in plants, whereby transgenic plants are readily available, particularly whereby the transgenic plants have been, are or will be commercialized and regulatory approvals have been applied for and are publicly available. Several databases are available which summarize and provide information on applications for regulatory approvals including the GM crop database of the Center of Environmental risk assessment which can be consulted online (http://www.cera-gmc.org/?action=gm_crop_database&) or the summary list of the Petitions of

[0088] Nonregulated Status Granted or Pending by APHIS, available online at http://www.aphis.usda.gov/brs/not_reg.html.

[0089] DNA regions commonly introduced as part of a transgene in plants include promoter regions such as the 35S promoter of the CaMV 35S transcript (Odell et al. (1985), Nature 313 : 810-812); the FMV 35S promoter (Richins R. D., Scholthof H. B., Shepherd R. J. (1987) Sequence of the figwort mosaic virus (caulimovirus group). Nucleic Acids Research 15: 8451-8466); the promoter of the small subunit of Arabidopsis thaliana Rubisco gene (Krebbers E., Seurinck J., Herdies L., Cashmore A. R., Timko M. P. (1988). Four genes in two diverged subfamilies encode the ribulose-1,5-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana. Plant Molecular Biology, 11, 745-759); the Casava Vein Mosaic Virus promoter

[0090] (Verdaguer et al (1996) Plant Mol. Biol. 31: 1129 or Verdaguer et al (1998) Plant Mol. Biol. 37: 1055); the Actin2 promoter from Arabidopsis (An Y. Q., McDowell J. M., Huang S., McKinney E. C., Chambliss S., Meagher R. B. (1996) Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. The Plant Journal 10: 107-121) or rice (McElroy D., Zhang W., Cao J., Wu R. (1990) Isolation of an efficient actin promoter for use in rice transfomation. The Plant Cell 2: 163-171); the Histone H3 promoter or histone H4 promoter (Chaboute M, Chaubet N, Philipps G, Ehling M and Gigot C (1987) Genomic organization and nucleotide sequences of two histone H3 and two histone H4 genes of Arabidopsis thaliana. Plant Mol. Biol. 8: 179-191); the promoter of the maize (Zea mays) ubiquitin-1 gene (Christensen et al (1992) Plant Mol. Biol. 18: 675); 5' UTR leader sequences such as the cab22L leader (Harpster

[0091] M, Townsend J, Jones J, Bedbrook J and Dunsmuir P.(1988) Relative strengths of the 35S cauliflower mosaic virus, 1', 2' and nopaline synthase promoters in transformed tobacco, sugarbeet and oilseed rape callus tissue. Mol Gen Genet. 212:182-190); or 5' tev (Carrington J and Freed D (1990) Cap-independent enhancement of translation by a plant potyvirus 5' nontranslated region. J Virol 64(4): 1590-1597); a 3' end of the nopaline synthase gene (Depicker A., Stachel S., Dhaese P., Zambryski P., Goodman H. M. (1982). Nopaline synthase: transcript mapping and DNA sequence. Journal of Molecular and Applied Genetics 1, 561-573); a 3' end of the octopine synthase gene (De Greve H., Dhaese P., Seurinck J., Lemmers M., Van Montagu M., Schell J. (1982). Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene. Journal of Molecular and Applied Genetics, 1, 499-511); the CaMV35S terminator (Sanfacon et al (1991) Genes Dev. 5: 141) transcription termination and polyadenylation region of gene 7 of the octopine type T-DNA vector (D'Haese et al, 1983, The EMBO Journal, 2, 419-426) and selectable markers such as bar (Thompson, C., Movva, R., Tizard, R., Crameri, R., Davies, J., Lauwereys, M. ans Botterman, J. (1987) Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus. The EMBO Journal 6: 2519-2523 (Accession X05822)); pat (Wohlleben, W., Arnold, W., Broer, I., Hillemann, D., Strauch, E. and Puhler, A. Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tu494 and its expression in Nicotiana tabacum. Gene 70 (1), 25-37 (1988)); 2mepsps (sequence 4 from U.S. Pat. No. 6566587 or EMBL number AR337832); CP4 (Padgette S. R., Re D., Barry G., Eichholtz D., Delannay X., Fuchs R. L., Kishore G. M., Fraley R. T. (1996).

[0092] New weed control opportunities: development of soybeans with a Roundup Ready gene. In Herbicide-Resistant Crops: Agricultural, Environmental, Econ . . . , neo Accession V00618; Beck et al (1982) Gene 19(3) p327-336); or hpt (Kaster et al., (1983), NAR 11, 6895-6911).

[0093] A preferred DNA region in the context of this invention is the nucleotide sequence of the coding region of the bar gene as mentioned above.

[0094] The redesigned meganucleases described herein are based on the naturally occurring meganuclease I-CreI for use as a scaffold. I-CreI is a homing endonuclease found in the chloroplasts of Chlamydomonas rheinhardti (Thompson et al. 1992, Gene 119, 247-251). This endonuclease is a homodimer that recognizes a pseudo-palindromic 22 by DNA site in the 23SrRNA gene and creates a double stranded DNA break that is used from the introduction of an intron. I-CreI is a member of a group endonucleases carrying a single LAGLIDADG motif. LAGLIDADG enzymes contain one or two copies of the consensus motif. Single-motif enzymes, such as I-CreI function as homodimers, whereas double-motif enzymes are monomers with two separate domains. Accordingly, when re-designing meganucleases derived from an I-CreI scaffold to recognize a 22 by nucleotide sequence of interest, two monomeric units are designed, each recognizing a part of the 22 by recognition site, which are needed in concert to induce a double stranded break at the 22 by recognition site. Concerted action may be achieved by linking the two monomeric units into one single chain meganuclease, or may also be achieved by promoting the formation of heterodimers, as described e.g. in WO2007/047859.

[0095] The amino acid sequence of a naturally occurring I-CreI is provided as SEQ ID No. 16. To re-design I-CreI monomeric units such that the heterodimers thereof recognize the nucleotide sequence of SEQ ID No. 1 and/or 2 the following amino acids were introduced at the mentioned positions:

[0096] 1. in meganuclease unit 1:

[0097] a. S at position 32;

[0098] b. Y at position 33;

[0099] c. Eat position 38;

[0100] d. R at position 40;

[0101] e. K at position 66;

[0102] f. Q at position 80;

[0103] g. T at position 42;

[0104] h. R at position 77;

[0105] i. Rat position 68;

[0106] j. R at position 70;

[0107] k. Q at position 44;

[0108] l. I at position 24;

[0109] m. S at position 26;

[0110] n. Sat position 28;

[0111] o. R at position 30.

[0112] 2. in meganuclease unit 2:

[0113] p. R at position 70;

[0114] q. T at position 44;

[0115] r. I at position 24;

[0116] s. S at position 26;

[0117] t. S at position 28;

[0118] u. N at position 30;

[0119] v. S at position 32;

[0120] w. Rat position 33;

[0121] x. Q at position 38;

[0122] y. Q at position 80;

[0123] z. R at position 40;

[0124] aa. K at position 66;

[0125] bb. T at position 42;

[0126] cc. R at position 77;

[0127] dd. R at position 68.

[0128] A schematic representation thereof is provided in Figures &. The re-designed double stranded break inducing enzyme may comprise, but need not comprise, a nuclear localization signal (NLS), such as the NLS of SV40 large T-antigen [Raikhel, Plant Physiol. 100: 1627-1632 (1992) and references therein] [Kalderon et al. Cell 39: 499-509 (1984)]. The nuclear localization signal may be located anywhere in the protein, but is conveniently located at the N-terminal end of the protein. The nuclear localization signal may replace one or more of the amino acids of the double stranded break inducing enzyme. It should be noted that if the re-designed meganuclease has been provided with a NLS at the N-terminus of the protein, such as a 10 amino acid NLS of SV40, the amino acid positions would be shifted (increased) accordingly. Likewise, in the event two monomeric units are linked into a single chain meganuclease, the position of the second unit will also be shifted.

[0129] Re-designed meganucleases suitable for the invention may comprise an amino acid sequence as represented in SEQ ID No. 4 and 5 (monomeric units which can cleave the recognition site as a heterodimer).

[0130] Conveniently, the redesigned meganuclease(s) can be provided by expression of a plant expressible recombinant gene(s) encoding such meganuclease(s). To this end, a DNA region comprising a nucleotide sequence encoding a re-designed meganuclease or meganuclease monomeric unit can be operably linked to a plant-expressible promoter and a DNA region involved in transcription termination and polyadenylation and introduced into a plant or plant cells. The recombinant gene(s) encoding redesigned meganuclease(s) may be introduced transiently or stably.

[0131] For the purpose of the invention, the term "plant-operative promoter" and "plant-expressible promoter" mean a promoter which is capable of driving transcription in a plant, plant tissue, plant organ, plant part, or plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell.

[0132] Promoters that may be used in this respect are constitutive promoters, such as the promoter of the cauliflower mosaic virus (CaMV) 35S transcript (Hapster et al.,1988, Mol. Gen. Genet. 212: 182-190), the CaMV 19S promoter (U.S. Pat. No. 5,352,605; WO 84/02913; Benfey et al., 1989, EMBO J. 8:2195-2202), the subterranean clover virus promoter No 4 or No 7 (WO 96/06932), the Rubisco small subunit promoter (U.S. Pat. No. 4,962,028), the ubiquitin promoter (Holtorf et al., 1995, Plant Mol. Biol. 29:637-649), T-DNA gene promoters such as the octopine synthase (OCS) and nopaline synthase (NOS) promoters from Agrobacterium, and further promoters of genes whose constitutive expression in plants is known to the person skilled in the art.

[0133] Further promoters that may be used in this respect are tissue-specific or organ-specific promoters, preferably seed-specific promoters, such as the 2S albumin promoter (Joseffson et al., 1987, J. Biol. Chem. 262:12196-12201), the phaseolin promoter (U.S. Pat. No. 5,504,200; Bustos et al., 1989, Plant Cell 1.(9):839-53), the legumine promoter (Shirsat et al., 1989, Mol. Gen. Genet. 215(2):326-331), the "unknown seed protein" (USP) promoter (Baumlein et al., 1991, Mol. Gen. Genet. 225(3):459-67), the napin promoter (U.S. Pat. No. 5,608,152; Stalberg et al., 1996, Planta 199:515-519), the Arabidopsis oleosin promoter (WO 98/45461), the Brassica Bce4 promoter (WO 91/13980), and further promoters of genes whose seed-specific expression in plants is known to the person skilled in the art.

[0134] Other promoters that can be used are tissue-specific or organ-specific promoters like organ primordia-specific promoters (An et al., 1996, Plant Cell 8: 15-30), stem-specific promoters (Keller et al., 1988, EMBO J. 7(12): 3625-3633), leaf-specific promoters (Hudspeth et al., 1989, Plant Mol. Biol. 12: 579-589), mesophyl-specific promoters (such as the light-inducible Rubisco promoters), root-specific promoters (Keller et al., 1989, Genes Dev. 3: 1639-1646), tuber-specific promoters (Keil et al., 1989, EMBO J. 8(5): 1323-1330), vascular tissue-specific promoters (Peleman et al., 1989, Gene 84: 359-369), stamen-selective promoters (WO 89/10396, WO 92/13956), dehiscence zone-specific promoters (WO 97/13865), and the like.

[0135] Nucleotide sequences encoding re-designed meganucleases suitable for the invention may comprise the nucleotide sequence of SEQ ID No. 6 from nucleotide position 2004 to nucleotide position 2525 or the nucleotide sequence of SEQ ID No. 6 from nucleotide position 4885 to nucleotide position 5405. To facilitate cloning and other recombinant DNA techniques, it may be advantageous to include an intron functional in plants into the region encoding a meganuclease, particularly a single chain meganuclease.

[0136] The DNA region encoding the re-designed meganuclease may be optimized for expression in plants by adapting GC content, codon usage, elimination of unwanted nucleotide sequences. The coding region may further be optimized for expression in plants and the synthetic coding region may have a nucleotide sequence which has been designed to fulfill the following criteria:

[0137] a) the nucleotide sequence encodes a functional redesigned homing endonuclease as herein described;

[0138] b) the nucleotide sequence has a GC content of about 50% to about 60%;

[0139] c) the nucleotide sequence does not comprise a nucleotide sequence selected from the group consisting of GATAAT, TATAAA, AATATA, AATATT, GATAAA, AATGAA, AATAAG, AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA and CATAAA;

[0140] d) the nucleotide does not comprise a nucleotide sequence selected from the group consisting of CCAAT, ATTGG, GCAAT and ATTGC;

[0141] e) the nucleotide sequence does not comprise a sequence selected from the group consisting of ATTTA, AAGGT, AGGTA, GGTA or GCAGG;

[0142] f) the nucleotide sequence does not comprise a GC stretch consisting of 7 consecutive nucleotides selected from the group of G or C;

[0143] g) the nucleotide sequence does not comprise a AT stretch consisting of 5 consecutive nucleotides selected from the group of A or T; and

[0144] h) the nucleotide sequence does not comprise codons coding for Leu, Ile, Val, Ser, Pro, Thr, Ala that comprise TA or CG duplets in positions 2 and 3 (i.e. the nucleotide sequence does not comprise the codons TTA, CTA, ATA, GTA, TCG, CCG, ACG and GCG).

[0145] It will also be clear that the terms used to describe the method such as "introduction of a DNA fragment" as well as "regeneration of a plant from the cell" do not imply that such DNA fragment necessarily needs to be introduced by transformation techniques. Indeed, it will be immediately clear to the person skilled in the art that the DNA molecule of interest may also be introduced by breeding or crossing techniques from one plant to another.

[0146] However, it will be clear that the DNA molecule of interest may be introduced into the plant cells by any method known in the art, including Agrobacterium mediated transformation but also by direct DNA transfer methods. The transforming DNA molecule can be transferred into plant cells using any conventional method, including but not limited to direct DNA transfer method. As used herein "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.

[0147] The capability of inducing a double stranded break at a preselected site opens up several potential applications. Foreign DNA of interest may be introduced into the preselected site either by homologous recombination, or in the process of non-homologous endjoining. The double stranded break may also be used to induce the formation of small deletions or insertions at the preselected site, thereby potentially inactivating the chimeric gene comprising the nucleotide sequence of the preselected site. The double stranded break at the preselected site will also facilitate replacement of a DNA region in the vicinity of that site for a DNA region of interest e.g. as described in WO 06/105946, WO08/037436 or WO08/148559.

[0148] To insert foreign DNA by homologous recombination at the preselected site, the foreign DNA may be comprised within a repair DNA, wherein the foreign DNA is flanked by at least one flanking DNA region having a nucleotide sequence which is similar to the nucleotide sequence of the DNA region upstream or downstream of the preselected site. The repair DNA may comprise the foreign DNA to be inserted flanked by two flanking DNA regions, upstream and downstream of the foreign DNA and which are similar to nucleotide sequence of the DNA region upstream or downstream of the preselected sites.

[0149] As used herein "a flanking DNA region" is a DNA with a nucleotide sequences having homology to the DNA regions respectively upstream or downstream of the target DNA sequencen or preselected site. This allows to better control the insertion of the foreign DNA or the DNA molecule of interest. Indeed, integration by homologous recombination will allow precise joining of the foreign DNA fragment to the plant nuclear genome up to the nucleotide level.

[0150] The flanking DNA regions may vary in length, and should be at least about 10 nucleotides in length. However, the flanking region may be as long as is practically possible (e.g. up to about 100-150 kb such as complete bacterial artificial chromosomes (BACs)). Preferably, the flanking region will be about 50 by to about 2000 bp. Moreover, the regions flanking the foreign DNA of interest need not be identical to the DNA regions flanking the preselected site and may have between about 80% to about 100% sequence identity, preferably about 95% to about 100% sequence identity with the DNA regions flanking the preselected site. The longer the flanking region, the less stringent the requirement for homology. Furthermore, it is preferred that the sequence identity is as high as practically possible in the vicinity of the location of exact insertion of the foreign DNA. Furthermore, to achieve exchange of the target DNA sequence without changing the DNA sequence of the adjacent DNA sequences, the flanking DNA sequences should preferably be identical to the DNA regions flanking the preselected site.

[0151] Moreover, the regions flanking the foreign DNA of interest need not have homology to the regions immediately flanking the preselected site, but may have homology to a DNA region of the nuclear genome further remote from that preselected site. Insertion of the foreign DNA will then result in a removal of the target DNA between the preselected insertion site and the DNA region of homology. In other words, the target DNA located between the homology regions will be substituted for the foreign DNA of interest.

[0152] The foreign DNA to be inserted may also comprise a selectable or screenable marker, which may or may not be removed after insertion.

[0153] "Selectable or screenable markers" as used herein have there usual meaning in the art and include, but are not limited to plant expressible phosphinotricin acetyltransferase, neomycine phosphotransferase, glyphosate oxidase, glyphosate tolerant EPSP enzyme, nitrilase gene, mutant acetolactate synthase or acetohydroxyacid synthase gene, .beta.-glucoronidase (GUS), R-locus genes, green fluorescent protein and the likes.

[0154] The selection of the plant cell or plant wherein the selectable or screenable marker and the rest of the foreign DNA molecule has been introduced by homologous recombination through the flanking DNA regions can e.g. be achieved by screening for the absence of sequences present in the transforming DNA but located outside of the flanking DNA regions. Indeed, presence of sequences from the transforming DNA outside the flanking DNA regions would indicate that the transformed plant cells origination by random DNA insertion. To this end, selectable or screenable markers may be included in the transforming DNA molecule outside of the flanking DNA regions, which can then be used to identify those plant cells which do not have the selectable or screenable markers located outside of the transforming DNA and which may have arisen by homologous recombination through the flanking DNA regions. Alternatively, the transforming DNA molecule may contain selectable markers outside the flanking DNA regions that allow selection for the absence of such genes (negative selectable marker genes).

[0155] It will be clear that the methods according to the invention allow insertion of any DNA of interest including DNA comprising a nucleotide sequence with a particular nucleotide sequence signature e.g. for subsequent identification. The DNA of interest may also be one or more plant expressible gene(s) including but not limited to a herbicide tolerance gene, an insect resistance gene, a disease resistance gene, an abiotic stress resistance gene, an enzyme involved in oil biosynthesis or carbohydrate biosynthesis, an enzyme involved in fiber strength and/or length, an enzyme involved in the biosynthesis of secondary metabolites.

[0156] Herbicide-tolerance genes include a gene encoding the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Examples of such EPSPS genes are the AroA gene (mutant CT7) of the bacterium Salmonella typhimurium (Comai et al., 1983, Science 221, 370-371), the CP4 gene of the bacterium Agrobacterium sp. (Barry et al., 1992, Curr. Topics Plant Physiol. 7, 139-145), the genes encoding a Petunia EPSPS (Shah et al., 1986, Science 233, 478-481), a Tomato EPSPS (Gasser et al., 1988, J. Biol. Chem. 263, 4280-4289), or an Eleusine EPSPS (WO 01/66704). It can also be a mutated EPSPS as described in for example EP 0837944, WO 00/66746, WO 00/66747 or WO02/26995. Glyphosate-tolerant plants can also be obtained by expressing a gene that encodes a glyphosate oxido-reductase enzyme as described in U.S. Pat. Nos. 5,776,760 and 5,463,175. Glyphosate-tolerant plants can also be obtained by expressing a gene that encodes a glyphosate acetyl transferase enzyme as described in for example WO 02/36782, WO 03/092360, WO 05/012515 and WO 07/024782. Glyphosate-tolerant plants can also be obtained by selecting plants containing naturally-occurring mutations of the above-mentioned genes, as described in for example WO 01/024615 or WO 03/013226. EPSPS genes that confer glyphosate tolerance are described in e.g. U.S. patent application Ser. Nos. 11/517,991, 10/739,610, 12/139,408, 12/352,532, 11/312,866, 11/315,678, 12/421,292, 11/400,598, 11/651,752, 11/681,285, 11/605,824, 12/468,205, 11/760,570, 11/762,526, 11/769,327, 11/769,255, 11/943801 or 12/362,774. Other genes that confer glyphosate tolerance, such as decarboxylase genes, are described in e.g. U.S. patent applications Ser. Nos. 11/588,811, 11/185,342, 12/364,724, 11/185,560 or 12/423,926.

[0157] Other herbicide tolereance genes may encode an enzyme detoxifying the herbicide or a mutant glutamine synthase enzyme that is resistant to inhibition, e.g. described in U.S. patent application Ser. No. 11/760,602. One such efficient detoxifying enzyme is an enzyme encoding a phosphinothricin acetyltransferase (such as the bar or pat protein from Streptomyces species). phosphinothricin acetyltransferases are for example described in U.S. Pat. Nos. 5,561,236; 5,648,477; 5,646,024; 5,273,894; 5,637,489; 5,276,268; 5,739,082; 5,908,810 and 7,112,665.

[0158] Herbicide-tolerance genes may also confer tolerance to the herbicides inhibiting the enzyme hydroxyphenylpyruvatedioxygenase (HPPD). Hydroxyphenylpyruvatedioxygenases are enzymes that catalyze the reaction in which para-hydroxyphenylpyruvate (HPP) is transformed into homogentisate. Plants tolerant to HPPD-inhibitors can be transformed with a gene encoding a naturally-occurring resistant HPPD enzyme, or a gene encoding a mutated or chimeric HPPD enzyme as described in WO 96/38567, WO 99/24585, and WO 99/24586, WO 2009/144079, WO 2002/046387, or U.S. Pat. No. 6,768,044. Tolerance to HPPD-inhibitors can also be obtained by transforming plants with genes encoding certain enzymes enabling the formation of homogentisate despite the inhibition of the native HPPD enzyme by the HPPD-inhibitor. Such plants and genes are described in WO 99/34008 and WO 02/36787. Tolerance of plants to HPPD inhibitors can also be improved by transforming plants with a gene encoding an enzyme having prephenate deshydrogenase (PDH) activity in addition to a gene encoding an HPPD-tolerant enzyme, as described in WO 2004/024928. Further, plants can be made more tolerant to HPPD-inhibitor herbicides by adding into their genome a gene encoding an enzyme capable of metabolizing or degrading HPPD inhibitors, such as the CYP450 enzymes shown in WO 2007/103567 and WO 2008/150473.

[0159] Still further herbicide tolerance genes encode variant ALS enzymes (also known as acetohydroxyacid synthase, AHAS) as described for example in Tranel and Wright (2002, Weed Science 50:700-712), but also, in U.S. Pat. Nos. 5,605,011, 5,378,824, 5,141,870, and 5,013,659. The production of sulfonylurea-tolerant plants and imidazolinone-tolerant plants is described in U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937; and 5,378,824; and international publication WO 96/33270. Other imidazolinone-tolerance genes are also described in for example WO 2004/040012, WO 2004/106529, WO 2005/020673, WO 2005/093093, WO 2006/007373, WO 2006/015376, WO 2006/024351, and WO 2006/060634. Further sulfonylurea- and imidazolinone-tolerance gebnes are described in for example WO 07/024782 and U.S. Patent Application No. 61/288958.

[0160] Insect resistance gene may comprising a coding sequence encoding:

[0161] 1) an insecticidal crystal protein from Bacillus thuringiensis or an insecticidal portion thereof, such as the insecticidal crystal proteins listed by Crickmore et al. (1998, Microbiology and Molecular Biology Reviews, 62: 807-813), updated by Crickmore et al. (2005) at the Bacillus thuringiensis toxin nomenclature, online at: http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/), or insecticidal portions thereof, e.g., proteins of the Cry protein classes Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1F, Cry2Ab, Cry3Aa, or Cry3Bb or insecticidal portions thereof (e.g. EP 1999141 and WO 2007/107302), or such proteins encoded by synthetic genes as e.g. described in and U.S. patent application Ser. No. 12/249,016 ; or

[0162] 2) a crystal protein from Bacillus thuringiensis or a portion thereof which is insecticidal in the presence of a second other crystal protein from Bacillus thuringiensis or a portion thereof, such as the binary toxin made up of the Cry34 and Cry35 crystal proteins (Moellenbeck et al. 2001, Nat. Biotechnol. 19: 668-72; Schnepf et al. 2006, Applied Environm. Microbiol. 71, 1765-1774) or the binary toxin made up of the Cry1A or Cry1F proteins and the Cry2Aa or Cry2Ab or Cry2Ae proteins (U.S. patent application Ser. No. 12/214,022 and EP 08010791.5); or

[0163] 3) a hybrid insecticidal protein comprising parts of different insecticidal crystal proteins from Bacillus thuringiensis, such as a hybrid of the proteins of 1) above or a hybrid of the proteins of 2) above, e.g., the Cry1A.105 protein produced by corn event MON89034 (WO 2007/027777); or

[0164] 4) a protein of any one of 1) to 3) above wherein some, particularly 1 to 10, amino acids have been replaced by another amino acid to obtain a higher insecticidal activity to a target insect species, and/or to expand the range of target insect species affected, and/or because of changes introduced into the encoding DNA during cloning or transformation, such as the Cry3Bb1 protein in corn events MON863 or MON88017, or the Cry3A protein in corn event MIR604; or

[0165] 5) an insecticidal secreted protein from Bacillus thuringiensis or Bacillus cereus, or an insecticidal portion thereof, such as the vegetative insecticidal (VIP) proteins listed at: http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html, e.g., proteins from the VIP3Aa protein class; or

[0166] 6) a secreted protein from Bacillus thuringiensis or Bacillus cereus which is insecticidal in the presence of a second secreted protein from Bacillus thuringiensis or B. cereus, such as the binary toxin made up of the VIP1A and VIP2A proteins (WO 94/21795); or

[0167] 7) a hybrid insecticidal protein comprising parts from different secreted proteins from Bacillus thuringiensis or Bacillus cereus, such as a hybrid of the proteins in 1) above or a hybrid of the proteins in 2) above; or

[0168] 8) a protein of any one of 5) to 7) above wherein some, particularly 1 to 10, amino acids have been replaced by another amino acid to obtain a higher insecticidal activity to a target insect species, and/or to expand the range of target insect species affected, and/or because of changes introduced into the encoding DNA during cloning or transformation (while still encoding an insecticidal protein), such as the VIP3Aa protein in cotton event COT102; or

[0169] 9) a secreted protein from Bacillus thuringiensis or Bacillus cereus which is insecticidal in the presence of a crystal protein from Bacillus thuringiensis, such as the binary toxin made up of VIP3 and Cry1A or Cry1F (U.S. Patent Appl. Nos. 61/126083 and 61/195019), or the binary toxin made up of the VIP3 protein and the Cry2Aa or Cry2Ab or Cry2Ae proteins (U.S. patent application Ser. No. 12/214,022 and EP 08010791.5).

[0170] 10) a protein of 9) above wherein some, particularly 1 to 10, amino acids have been replaced by another amino acid to obtain a higher insecticidal activity to a target insect species, and/or to expand the range of target insect species affected, and/or because of changes introduced into the encoding DNA during cloning or transformation (while still encoding an insecticidal protein)

[0171] An "insect-resistant gene as used herein, further includes transgenes comprising a sequence producing upon expression a double-stranded RNA which upon ingestion by a plant insect pest inhibits the growth of this insect pest, as described e.g. in WO 2007/080126, WO 2006/129204, WO 2007/074405, WO 2007/080127 and WO 2007/035650.

[0172] Abiotic stress tolerance genes include

[0173] 1) a transgene capable of reducing the expression and/or the activity of poly(ADP-ribose) polymerase (PARP) gene in the plant cells or plants as described in WO 00/04173, WO/2006/045633, EP 04077984.5, or EP 06009836.5.

[0174] 2) a transgene capable of reducing the expression and/or the activity of the PARG encoding genes of the plants or plants cells, as described e.g. in WO 2004/090140.

[0175] 3) a transgene coding for a plant-functional enzyme of the nicotineamide adenine dinucleotide salvage synthesis pathway including nicotinamidase, nicotinate phosphoribosyltransferase, nicotinic acid mononucleotide adenyl transferase, nicotinamide adenine dinucleotide synthetase or nicotine amide phosphorybosyltransferase as described e.g. in EP 04077624.7, WO 2006/133827, PCT/EP07/002433, EP 1999263, or WO 2007/107326.

[0176] Enzymes involved in carbohydrate biosynthesis include those described in e.g. EP 0571427, WO 95/04826, EP 0719338, WO 96/15248, WO 96/19581, WO 96/27674, WO 97/11188, WO 97/26362, WO 97/32985, WO 97/42328, WO 97/44472, WO 97/45545, WO 98/27212, WO 98/40503, WO99/58688, WO 99/58690, WO 99/58654, WO 00/08184, WO 00/08185, WO 00/08175, WO 00/28052, WO 00/77229, WO 01/12782, WO 01/12826, WO 02/101059, WO 03/071860, WO 2004/056999, WO 2005/030942, WO 2005/030941, WO 2005/095632, WO 2005/095617, WO 2005/095619, WO 2005/095618, WO 2005/123927, WO 2006/018319, WO 2006/103107, WO 2006/108702, WO 2007/009823, WO 00/22140, WO 2006/063862, WO 2006/072603, WO 02/034923, EP 06090134.5, EP 06090228.5, EP 06090227.7, EP 07090007.1, EP 07090009.7, WO 01/14569, WO 02/79410, WO 03/33540, WO 2004/078983, WO 01/19975, WO 95/26407, WO 96/34968, WO 98/20145, WO 99/12950, WO 99/66050, WO 99/53072, U.S. Pat. No. 6,734,341, WO 00/11192, WO 98/22604, WO 98/32326, WO 01/98509, WO 01/98509, WO 2005/002359, U.S. Pat. No. 5,824,790, U.S. Pat. No. 6,013,861, WO 94/04693, WO 94/09144, WO 94/11520, WO 95/35026 or WO 97/20936 or enzymes involved in the production of polyfructose, especially of the inulin and levan-type, as disclosed in EP 0663956, WO 96/01904, WO 96/21023, WO 98/39460, and WO 99/24593, the production of alpha-1,4-glucans as disclosed in WO 95/31553, U.S. 2002031826, U.S. Pat. No. 6,284,479, U.S. Pat. No. 5,712,107, WO 97/47806, WO 97/47807, WO 97/47808 and WO 00/14249, the production of alpha-1,6 branched alpha-1,4-glucans, as disclosed in WO 00/73422, the production of alternan, as disclosed in e.g. WO 00/47727, WO 00/73422, EP 06077301.7, U.S. Pat. No. 5,908,975 and EP 0728213, the production of hyaluronan, as for example disclosed in WO 2006/032538, WO 2007/039314, WO 2007/039315, WO 2007/039316, JP 2006304779, and WO 2005/012529.

[0177] It is also an embodiment of the invention to provide chimeric genes encoding re-designed meganucleases as herein described, wherein the chimeric gene comprise a plant expressible promoter operably linked to a DNA region encoding a protein comprising an amino acid sequence corresponding to the amino acid sequence of I-CreI as a scaffold comprising a S at position 32; Y at position 33; E at position 38; R at position 40; K at position 66; Q at position 80; T at position 42; R at position 77; R at position 68; R at position 70; Q at position 44; I at position 24; S at position 26; S at position 28 and R at position 30 or R at position 70; T at position 44; I at position 24; S at position 26; S at position 28; N at position 30; S at position 32; R at position 33; Q at position 38; Q at position 80; R at position 40; K at position 66; T at position 42; R at position 77 and R at position 68 such as the protein comprising the amino acid sequence of SEQ ID 4 or SEQ ID 5.

[0178] It will be appreciated that the means and methods of the invention may be used in any plant including corn, tobacco, 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 (Angiospermae or Gymnospermae) 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 and sugar 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 and watermelon.

[0179] It is also an object of the invention to provide plant cells and plants generated according to the methods of the invention. Gametes, seeds, embryos, either zygotic or somatic, progeny or hybrids of plants comprising the DNA insertion events, which are produced by traditional breeding methods, are also included within the scope of the present invention. Such plants may contain a heterologous or foreign DNA sequence inserted at or instead of a target sequence, and will only be different from their progenitor plants by the presence of this heterologous DNA or DNA sequence post exchange.

[0180] 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.

[0181] The plants and seeds according to the invention may be further treated with a chemical compound, such as a chemical compound selected from the following lists:

[0182] Fruits/Vegetables Herbicides: Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine, Trifluralin, Fluazifop, Glufosinate, Halosulfuron Gowan, Paraquat, Propyzamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam

[0183] Fruits/Vegetables Insecticides: Aldicarb, Bacillus thuriengiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin, Abamectin, Cyfluthrin/beta-cyfluthrin, Esfenvalerate, Lambda-cyhalothrin, Acequinocyl, Bifenazate, Methoxyfenozide, Novaluron, Chromafenozide, Thiacloprid, Dinotefuran, Fluacrypyrim, Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr, Cyazypyr, Triflumuron, Spirotetramat, Imidacloprid, Flubendiamide, Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen, Cyanopyrafen, Clothianidin, Thiamethoxam, Spinotoram, Thiodicarb, Flonicamid, Methiocarb, Emamectin-benzoate, Indoxacarb, Fenamiphos, Pyriproxifen, Fenbutatin-oxid

[0184] Fruits/Vegetables Fungicides: Ametoctradin, Azoxystrobin, Benthiavalicarb, Boscalid,

[0185] Captan, Carbendazim, Chlorothalonil, Copper, Cyazofamid, Cyflufenamid, Cymoxanil, Cyproconazole, Cyprodinil, Difenoconazole, Dimetomorph, Dithianon, Fenamidone, Fenhexamid, Fluazinam, Fludioxonil, Fluopicolide, Fluopyram, Fluoxastrobin, Fluxapyroxad, Folpet, Fosetyl, Iprodione, Iprovalicarb, Isopyrazam, Kresoxim-methyl, Mancozeb, Mandipropamid, Metalaxyl/mefenoxam, Metiram, Metrafenone, Myclobutanil, Penconazole, Penthiopyrad, Picoxystrobin, Propamocarb, Propiconazole, Propineb, Proquinazid, Prothioconazole, Pyraclostrobin, Pyrimethanil, Quinoxyfen, Spiroxamine, Sulphur, Tebuconazole, Thiophanate-methyl, Trifloxystrobin

[0186] Cereals herbicides: 2.4-d, amidosulfuron, bromoxynil, carfentrazone-e, chlorotoluron, chlorsulfuron, clodinafop-p, clopyralid, dicamba, diclofop-m, diflufenican, fenoxaprop, florasulam, flucarbazone-na, flufenacet, flupyrsulfuron-m, fluroxypyr, flurtamone, glyphosate, iodosulfuron, ioxynil, isoproturon, mcpa, mesosulfuron, metsulfuron, pendimethalin, pinoxaden, propoxycarbazone, prosulfocarb, pyroxsulam, sulfosulfuron, thifensulfuron, tralkoxydim, triasulfuron, tribenuron, trifluralin, tritosulfuron

[0187] Cereals Fungicides: Azoxystrobin, Bixafen, Boscalid, Carbendazim, Chlorothalonil, Cyflufenamid, Cyproconazole, Cyprodinil, Dimoxystrobin, Epoxiconazole, Fenpropidin, Fenpropimorph, Fluopyram, Fluoxastrobin, Fluquinconazole, Fluxapyroxad, Isopyrazam, Kresoxim-methyl, Metconazole, Metrafenone, Penthiopyrad, Picoxystrobin, Prochloraz, Propiconazole, Proquinazid, Prothioconazole, Pyraclostrobin, Quinoxyfen, Spiroxamine, Tebuconazole, Thiophanate-methyl, Trifloxystrobin

[0188] Cereals Insecticides: Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin, .beta.-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Pirimicarb, Methiocarb, Sulfoxaflor

[0189] Maize Herbicides: Atrazine, Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-) Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor, Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione, Foramsulfuron, Topramezone, Tembotrione, Saflufenacil, Thiencarbazone, Flufenacet, Pyroxasulfon

[0190] Maize Insecticides: Carbofuran, Chlorpyrifos, Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin, Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide, Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb, .beta.-Cyfluthrin, Cypermethrin, Bifenthrin, Lufenuron, Tebupirimphos, Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid, Dinetofuran, Avermectin

[0191] Maize Fungicides: Azoxystrobin, Bixafen, Boscalid, Cyproconazole, Dimoxystrobin, Epoxiconazole, Fenitropan, Fluopyram, Fluoxastrobin, Fluxapyroxad, Isopyrazam, Metconazole, Penthiopyrad, Picoxystrobin, Propiconazole, Prothioconazole, Pyraclostrobin, Tebuconazole, Trifloxystrobin

[0192] Rice Herbicides: Butachlor, Propanil, Azimsulfuron, Bensulfuron, Cyhalofop, Daimuron, Fentrazamide, Imazosulfuron, Mefenacet, Oxaziclomefone, Pyrazosulfuron, Pyributicarb, Quinclorac, Thiobencarb, Indanofan, Flufenacet, Fentrazamide, Halosulfuron, Oxaziclomefone, Benzobicyclon, Pyriftalid, Penoxsulam, Bispyribac, Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione, Tefuryltrione, Oxadiazone, Fenoxaprop, Pyrimisulfan

[0193] Rice Insecticides: Diazinon, Fenobucarb, Benfuracarb, Buprofezin, Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid, Chromafenozide, Clothianidin, Ethiprole, Flubendiamide, Rynaxypyr, Deltamethrin, Acetamiprid, Thiamethoxam, Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Cypermethrin, Chlorpyriphos, Etofenprox, Carbofuran, Benfuracarb, Sulfoxaflor

[0194] Rice Fungicides: Azoxystrobin, Carbendazim, Carpropamid, Diclocymet, Difenoconazole, Edifenphos, Ferimzone, Gentamycin, Hexaconazole, Hymexazol, Iprobenfos (IBP), Isoprothiolane, Isotianil, Kasugamycin, Mancozeb, Metominostrobin, Orysastrobin, Pencycuron, Probenazole, Propiconazole, Propineb, Pyroquilon, Tebuconazole, Thiophanate-methyl, Tiadinil, Tricyclazole, Trifloxystrobin, Validamycin

[0195] Cotton Herbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn, Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate, Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron, Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron

[0196] Cotton Insecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin, Deltamethrin, Abamectin, Acetamiprid, Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin, Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl, Flonicamid Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin, Spirotetramat

[0197] Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran, Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen, Sulfoxaflor

[0198] Cotton Fungicides: Azoxystrobin, Bixafen, Boscalid, Carbendazim, Chlorothalonil, Copper, Cyproconazole, Difenoconazole, Dimoxystrobin, Epoxiconazole, Fenamidone, Fluazinam, Fluopyram, Fluoxastrobin, Fluxapyroxad, Iprodione, Isopyrazam, Isotianil, Mancozeb, Maneb, Metominostrobin, Penthiopyrad, Picoxystrobin, Propineb, Prothioconazole, Pyraclostrobin, Quintozene, Tebuconazole, Tetraconazole, Thiophanate-methyl, Trifloxystrobin

[0199] Soybean Herbicides: Alachlor, Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl, Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin, Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim, Glufosinate

[0200] Soybean Insecticides: Lambda-cyhalothrin, Methomyl, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr, Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole, Deltamethrin, .beta.-Cyfluthrin, gamma and lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb, beta-Cyfluthrin

[0201] Soybean Fungicides: Azoxystrobin, Bixafen, Boscalid, Carbendazim, Chlorothalonil, Copper, Cyproconazole, Difenoconazole, Dimoxystrobin, Epoxiconazole, Fluazinam, Fluopyram, Fluoxastrobin, Flutriafol, Fluxapyroxad, Isopyrazam, Iprodione, Isotianil, Mancozeb, Maneb, Metconazole, Metominostrobin, Myclobutanil, Penthiopyrad, Picoxystrobin, Propiconazole, Propineb, Prothioconazole, Pyraclostrobin, Tebuconazole, Tetraconazole, Thiophanate-methyl, Trifloxystrobin

[0202] Sugarbeet Herbicides: Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate, Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim, Triflusulfuron, Tepraloxydim, Quizalofop

[0203] Sugarbeet Insecticides: Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Deltamethrin, 13-Cyfluthrin, gamma/lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran

[0204] Canola Herbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate, Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop, Clethodim, Tepraloxydim

[0205] Canola Fungicides: Azoxystrobin, Bixafen, Boscalid, Carbendazim, Cyproconazole, Difenoconazole, Dimoxystrobin, Epoxiconazole, Fluazinam, Fluopyram, Fluoxastrobin, Flusilazole, Fluxapyroxad, Iprodione, Isopyrazam, Mepiquat-chloride, Metconazole, Metominostrobin, Paclobutrazole, Penthiopyrad, Picoxystrobin, Prochloraz, Prothioconazole, Pyraclostrobin, Tebuconazole, Thiophanate-methyl, Trifloxystrobin, Vinclozolin

[0206] Canola Insecticides: Carbofuran, Thiacloprid, Deltamethrin, Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran, .beta.-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole, Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on

[0207] As used herein "comprising" is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein.

[0208] A chimeric gene comprising a DNA region which is functionally or structurally defined may comprise additional DNA regions etc.

[0209] As used herein, "plant part" includes any plant organ or plant tissue, including but not limited to fruits, seeds, embryos, meristematic regions, callus tissue, leaves, roots, shoots, flowers, gametophytes, sporophytes, pollen, and microspores.

[0210] For the purpose of this invention, the "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (.times.100) divided by the number of positions compared. A gap, i.e. a position in an alignment where a residue is present in one sequence but not in the other, is regarded as a position with non-identical residues. The alignment of the two sequences is performed by the Needleman and Wunsch algorithm (Needleman and Wunsch 1970). The computer-assisted sequence alignment above, can be conveniently performed using standard software program such as GAP which is part of the Wisconsin Package Version 10.1 (Genetics Computer Group, Madison, Wis., USA) using the default scoring matrix with a gap creation penalty of 50 and a gap extension penalty of 3.

[0211] It will be clear that whenever nucleotide sequences of RNA molecules are defined by reference to nucleotide sequence of corresponding DNA molecules, the thymine (T) in the nucleotide sequence should be replaced by uracil (U). Whether reference is made to RNA or DNA molecules will be clear from the context of the application.

[0212] The following non-limiting Examples describe the use of a re-designed meganuclease to modify plants at the site of a 3'gene 7 nucleotide sequence already present in the plant genome.

[0213] Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR--Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

[0214] Throughout the description and Examples, reference is made to the following sequences:

[0215] SEQ ID No. 1: nucleotide sequence of the recognition site of the re-designed meganucleases BAY 39/BAY40

[0216] SEQ ID No. 2: nucleotide sequence of the complement of the recognition site of the re-designed meganucleases BAY 39/BAY40

[0217] SEQ ID No. 3: nucleotide sequence of the bar gene coding region

[0218] SEQ ID No. 4: amino acid sequence of the meganuclease BAY39/40 monomeric unit 1

[0219] SEQ ID No. 5: amino acid sequence of the meganuclease BAY39/40 monomeric unit 2

[0220] SEQ ID No. 6: nucleotide sequence of the T-DNA vector pCV177 expressing a pair of heterodimer meganucleases BAY 35 and BAY36

[0221] SEQ ID No. 7: nucleotide sequence of the PCR amplicon of the bar coding region around the BAY39/40 recognition site (control)

[0222] SEQ ID No. 8: nucleotide sequence of the PCR amplicon of the bar coding region around the BAY39/40 recognition site (PPT tolerant line)

[0223] SEQ ID No. 9: nucleotide sequence of the PCR amplicon of the bar coding region around the BAY39/40 recognition site (PPT sensitive line 1)

[0224] SEQ ID No. 10: nucleotide sequence of the PCR amplicon of the bar coding region around the BAY39/40 recognition site (PPT sensitive line 2)

[0225] SEQ ID No. 11: nucleotide sequence of the PCR amplicon of the bar coding region around the BAY39/40 recognition site (PPT sensitive line 3)

[0226] SEQ ID No. 12: nucleotide sequence of the PCR amplicon of the bar coding region around the BAY39/40 recognition site (PPT sensitive line 4)

[0227] SEQ ID No. 13: nucleotide sequence of the PCR amplicon of the bar coding region around the BAY39/40 recognition site (PPT sensitive line 5)

[0228] SEQ ID No. 14: nucleotide sequence of the PCR amplicon of the bar coding region around the BAY39/40 recognition site (PPT sensitive line 6)

[0229] SEQ ID No. 15: nucleotide sequence of the PCR amplicon of the bar coding region around the BAY39/40 recognition site (PPT sensitive line 7)

[0230] SEQ ID No. 16: amino acid sequence of I-CreI natural variant

EXAMPLES

[0231] All re-designed meganucleases described herein have been designed by Precision BioSciences Inc., 104 T.W. Alexander Drive, Research Triangle Park, N.C. 27713.

Example 1

Description of the T-DNA Vectors Encoding Re-Designed Meganucleases According to the Invention

[0232] Using conventional recombinant DNA techniques a chimeric gene encoding a pair of re-designed meganuclease monomers which as a heterodimer recognize the nucleotide sequence of SEQ ID No. 1 or 2 (hd BAY39/40) comprising the following operably linked DNA fragments:

[0233] a DNA region encoding the CaMV35S promoter (SEQ ID No 6 from nt position 1516 to nt position 1933)

[0234] a DNA region comprising the BAY 39/40 monomeric unit 1 encoding region, operably linked to a SV40 NLS at the N-terminus (SEQ ID No 6 from nt position 2004 to 2525)

[0235] a DNA region involved in 3' end transcription termination and polyadenylation from nopaline synthase gene (SEQ ID No 6 from nt position 2530 to 2783)

[0236] a DNA region encoding the CaMV35S promoter (SEQ ID No 6 from nt position 4397 to nt position 4814)

[0237] a DNA region comprising the BAY 39/40 monomeric unit 2, operably linked to a SV40 NLS at the N-terminus (SEQ ID No 6 from nt position 4885 to 5405)

[0238] a DNA region involved in 3' end transcription termination and polyadenylation from nopaline synthase gene (SEQ ID No 6 from nt position 5411 to 5664).

[0239] The nucleotide sequence of the resulting plasmid is represented in SEQ ID No. 6.

Example 2

Description of the Target Tobacco Line and Assay

[0240] In order to develop an assay for double stranded DNA break induction, a phosphinotricin tolerant tobacco transgenic plant line was selected that contained a bar coding region under control of plant-expressible promoter.

[0241] This transgenic line was used as starting material in a transformation wherein the chimeric genes encoding hd BAY39/40 meganucleases were stably introduced together with a plant expressible chimeric gene comprising a hygromycinphosphotransferase conferring resistance to hygromycine.

[0242] After double stranded DNA break induction at the recognition site in the bar coding region through expression of the plant expressible chimeric genes encoding the BAY39/40 heterodimer, the break can be repaired by non-homologous end-joining in the absence of repair DNA, resulting in deletion or insertion of one or more base pairs, thereby disrupting the bar coding region, resulting in phosphinotricin sensitivity.

[0243] Several plant lines exhibiting phosphinotricine sensitivity and hygromycin resistance were selected. From these plant lines DNA fragment were amplified by PCR using primers located at either side of the recognition site (SEQ ID No 1 or 2) in the bar coding region, and the nucleotide sequence of the amplicons was determined. An alignment of the different nucleotide sequences is represented in FIG. 4.

[0244] It is clear that in the PPT sensitive plant lines (3 to 9), the recognition site for BAY39/40 has been altered by deletion(3 to 8) or insertion (9) whereas no alteration was found in PPT resistant plant lines (2).

[0245] From these experiments it can thus be concluded that hdBAY39/40 exhibits cleavage activity at the preselected site.

Example 3

Targeted Insertion by Non-Homologous End-Joining

[0246] Co-delivery of pCV177 comprising the chimeric genes encoding hd BAY39/40 meganucleases and repair DNA comprising a selectable marker such as a plant-expressible chimeric gene comprising a 2mepsp coding region (without further homology to the target region) to plant cells comprising a plant expressible chimeric bar gene integrated in their genome and selection of phosphinotricin sensitive plants tolerant to the selection compound such as glyphosate allows identification of the plant cells wherein the repair DNA is integrated in the bar coding region.

Example 4

Targeted Insertion by Homologous End-Joining

[0247] Co-delivery of pCV177 comprising the chimeric genes encoding hd BAY39/40 meganucleases and repair DNA comprising a selectable marker such as a plant-expressible chimeric gene comprising a 2mepsp coding region flanked upstream by flanking sequences comprising a nucleotide sequence with sequence similarity to the bar coding region of SEQ ID No 3 from nucleotide 1 to nucleotide 132 and flanked downstream by flanking sequences comprising a nucleotide sequence with sequence similarity to the bar coding region of SEQ ID No 3 from nucleotide 154 to nucleotide 552,

[0248] to plant cells comprising a plant expressible chimeric bar gene integrated in their genome and selection of phosphinotricin sensitive plants tolerant to the selection compound such as glyphosate allows identification of the plant cells wherein the repair DNA is integrated in the bar coding region.

Sequence CWU 1

1

18122DNAArtificialRecognition site for BAY39/40 1gacgaggtcg tccgtccact cc 22222DNAArtificialComplement of the recognition site for BAY39/40 2ggagtggacg gacgacctcg tc 223552DNAArtificialCoding region of the phosphinotricin actelyltransferase gene derived from S. hygroscopicus (bar) 3atgagcccag aacgacgccc ggccgacatc cgccgtgcca ccgaggcgga catgccggcg 60gtctgcacca tcgtcaacca ctacatcgag acaagcacgg tcaacttccg taccgagccg 120caggaaccgc aggagtggac ggacgacctc gtccgtctgc gggagcgcta tccctggctc 180gtcgccgagg tggacggcga ggtcgccggc atcgcctacg cgggcccctg gaaggcacgc 240aacgcctacg actggacggc cgagtcgacc gtgtacgtct ccccccgcca ccagcggacg 300ggactgggct ccacgctcta cacccacctg ctgaagtccc tggaggcaca gggcttcaag 360agcgtggtcg ctgtcatcgg gctgcccaac gacccgagcg tgcgcatgca cgaggcgctc 420ggatatgccc cccgcggcat gctgcgggcg gccggcttca agcacgggaa ctggcatgac 480gtgggtttct ggcagctgga cttcagcctg ccggtaccgc cccgtccggt cctgcccgtc 540accgagatct ga 55246234DNAArtificialPlasmid pCV177 4gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140tcctttttgc tagcgagagg cggtttgcgt attggctaga gcagcttgcc aacatggtgg 1200agcacgacac tctcgtctac tccaagaata tcaaagatac agtctcagaa gaccaaaggg 1260ctattgagac ttttcaacaa agggtaatat cgggaaacct cctcggattc cattgcccag 1320ctatctgtca cttcatcaaa aggacagtag aaaaggaagg tggcacctac aaatgccatc 1380attgcgataa aggaaaggct atcgttcaag atgcctctgc cgacagtggt cccaaagatg 1440gacccccacc cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc 1500aagtggattg atgtgaacat ggtggagcac gacactctcg tctactccaa gaatatcaaa 1560gatacagtct cagaagacca aagggctatt gagacttttc aacaaagggt aatatcggga 1620aacctcctcg gattccattg cccagctatc tgtcacttca tcaaaaggac agtagaaaag 1680gaaggtggca cctacaaatg ccatcattgc gataaaggaa aggctatcgt tcaagatgcc 1740tctgccgaca gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa 1800gacgttccaa ccacgtcttc aaagcaagtg gattgatgtg atatctccac tgacgtaagg 1860gatgacgcac aatcccacta tccttcgcaa gacccttcct ctatataagg aagttcattt 1920catttggaga ggacacgctg aaatcaccag tctctctcta caaatctatc tctctcgagc 1980tttcgcagat ctgtcgaacc acc atg gca ccg aag aag aag cgc aag gtg cat 2033 Met Ala Pro Lys Lys Lys Arg Lys Val His 1 5 10 atg aac acc aag tac aac aag aag ttc ctg ctc tac ctg gcg ggc ttc 2081Met Asn Thr Lys Tyr Asn Lys Lys Phe Leu Leu Tyr Leu Ala Gly Phe 15 20 25 gtg gac ggg gac ggc tcc atc atc gcc tcc atc tcc ccg aac cag tcc 2129Val Asp Gly Asp Gly Ser Ile Ile Ala Ser Ile Ser Pro Asn Gln Ser 30 35 40 cgc aag ttc aag cat cag ctg cgc ctc acc ttc acc gtc acc cag aag 2177Arg Lys Phe Lys His Gln Leu Arg Leu Thr Phe Thr Val Thr Gln Lys 45 50 55 aca cag cgc cgt tgg ttc ctc gac aag ctg gtg gac aag atc ggg gtg 2225Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Lys Ile Gly Val 60 65 70 ggc aag gtg cgc gac cgc ggc agc gtc tcc gac tac cgc ctg tcc cag 2273Gly Lys Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Arg Leu Ser Gln 75 80 85 90 atc aag cct ctg cac aac ttc ctg acc cag ctc cag ccc ttc ctg aag 2321Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys 95 100 105 ctc aag cag aag cag gcc aac ctc gtg ctg aag atc atc gag cag ctg 2369Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu 110 115 120 ccc tcc gcc aag gaa tcc ccg gac aag ttc ctg gag gtg tgc acc tgg 2417Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp 125 130 135 gtg gac cag atc gcc gct ctg aac gac tcc aag acc cgc aag acc act 2465Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr 140 145 150 tcc gag acc gtc cgc gcc gtt cta gac agt ctc tcc gag aag aag aag 2513Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys 155 160 165 170 tcg tcc ccc tagcatgccg ttcaaacatt tggcaataaa gtttcttaag 2562Ser Ser Pro attgaatcct gttgccggtc ttgcgatgat tatcatataa tttctgttga attacgttaa 2622gcatgtaata attaacatgt aatgcatgac gttatttatg agatgggttt ttatgattag 2682agtcccgcaa ttatacattt aatacgcgat agaaaacaaa atatagcgcg caaactagga 2742taaattatcg cgcgcggtgt catctatgtt actagatcgg gcccgggaat aaaatatctt 2802tattttcatt acatctgtgt gttggttttt tgtgtgaatc gatagtacta acatacgctc 2862tccatcaaaa caaaacgaaa caaaacaaac tagcaaaata ggctgtcccc agtgcaagtg 2922caggtgccag aacatttctc tgctagcctc atgaccaaaa tcccttaacg tgagttttcg 2982ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 3042ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 3102ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 3162ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 3222ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 3282tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 3342tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 3402tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 3462tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 3522gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 3582tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 3642ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 3702gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 3762gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc 3822cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 3882ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta 3942cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 4002ggaaacagct atgaccatga ttacgccaag cttgagaggc ggtttgcgta ttggctagag 4062cagcttgcca acatggtgga gcacgacact ctcgtctact ccaagaatat caaagataca 4122gtctcagaag accaaagggc tattgagact tttcaacaaa gggtaatatc gggaaacctc 4182ctcggattcc attgcccagc tatctgtcac ttcatcaaaa ggacagtaga aaaggaaggt 4242ggcacctaca aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga tgcctctgcc 4302gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt 4362ccaaccacgt cttcaaagca agtggattga tgtgaacatg gtggagcacg acactctcgt 4422ctactccaag aatatcaaag atacagtctc agaagaccaa agggctattg agacttttca 4482acaaagggta atatcgggaa acctcctcgg attccattgc ccagctatct gtcacttcat 4542caaaaggaca gtagaaaagg aaggtggcac ctacaaatgc catcattgcg ataaaggaaa 4602ggctatcgtt caagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag 4662gagcatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga 4722tatctccact gacgtaaggg atgacgcaca atcccactat ccttcgcaag acccttcctc 4782tatataagga agttcatttc atttggagag gacacgctga aatcaccagt ctctctctac 4842aaatctatct ctctcgagct ttcgcagatc tgtcgaacca cc atg gca ccg aag 4896 Met Ala Pro Lys 175 aag aag cgc aag gtg cat atg aac acc aag tac aac gag gag ttc ctg 4944Lys Lys Arg Lys Val His Met Asn Thr Lys Tyr Asn Glu Glu Phe Leu 180 185 190 ctc tac ctg gcg ggc ttc gtg gac ggg gac ggc tcc atc atc gcc tcc 4992Leu Tyr Leu Ala Gly Phe Val Asp Gly Asp Gly Ser Ile Ile Ala Ser 195 200 205 atc tcc ccg cgc cag tcc tac aag ttc aag cat gag ctg cgc ctc acc 5040Ile Ser Pro Arg Gln Ser Tyr Lys Phe Lys His Glu Leu Arg Leu Thr 210 215 220 225 ttc cag gtc acg cag aag aca cag cgc cgt tgg ttc ctc gac gag ctg 5088Phe Gln Val Thr Gln Lys Thr Gln Arg Arg Trp Phe Leu Asp Glu Leu 230 235 240 gtg gac gag atc ggg gtg ggc aag gtg cgc gac cgc ggc agc gtc tcc 5136Val Asp Glu Ile Gly Val Gly Lys Val Arg Asp Arg Gly Ser Val Ser 245 250 255 gac tac cgc ctg tcc cag atc aag cct ctg cac aac ttc ctg acc cag 5184Asp Tyr Arg Leu Ser Gln Ile Lys Pro Leu His Asn Phe Leu Thr Gln 260 265 270 ctc cag ccc ttc ctg gag ctc aag cag aag cag gcc aac ctc gtg ctg 5232Leu Gln Pro Phe Leu Glu Leu Lys Gln Lys Gln Ala Asn Leu Val Leu 275 280 285 aag atc atc gag cag ctg ccc tcc gcc aag gaa tcc ccg gac aag ttc 5280Lys Ile Ile Glu Gln Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe 290 295 300 305 ctg gag gtg tgc acc tgg gtg gac cag atc gcc gct ctg aac gac tcc 5328Leu Glu Val Cys Thr Trp Val Asp Gln Ile Ala Ala Leu Asn Asp Ser 310 315 320 aag acc cgc aag acc act tcc gag acc gtc cgc gcc gtt cta gac agt 5376Lys Thr Arg Lys Thr Thr Ser Glu Thr Val Arg Ala Val Leu Asp Ser 325 330 335 ctc tcc gag aag aag aag tcg tcc ccc tagcatgccg ttcaaacatt 5423Leu Ser Glu Lys Lys Lys Ser Ser Pro 340 345 tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa 5483tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg 5543agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa 5603atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg 5663gcccgggaat aaaatatctt tattttcatt acatctgtgt gttggttttt tgtgtgaatc 5723gatagtacta acatacgctc tccatcaaaa caaaacgaaa caaaacaaac tagcaaaata 5783ggctgtcccc agtgcaagtg caggtgccag aacatttcgg taccgagctc gaattcactg 5843gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 5903gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 5963tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg 6023catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc 6083gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 6143ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 6203aggttttcac cgtcatcacc gaaacgcgcg a 62345173PRTArtificialSynthetic Construct 5Met Ala Pro Lys Lys Lys Arg Lys Val His Met Asn Thr Lys Tyr Asn 1 5 10 15 Lys Lys Phe Leu Leu Tyr Leu Ala Gly Phe Val Asp Gly Asp Gly Ser 20 25 30 Ile Ile Ala Ser Ile Ser Pro Asn Gln Ser Arg Lys Phe Lys His Gln 35 40 45 Leu Arg Leu Thr Phe Thr Val Thr Gln Lys Thr Gln Arg Arg Trp Phe 50 55 60 Leu Asp Lys Leu Val Asp Lys Ile Gly Val Gly Lys Val Arg Asp Arg 65 70 75 80 Gly Ser Val Ser Asp Tyr Arg Leu Ser Gln Ile Lys Pro Leu His Asn 85 90 95 Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys Leu Lys Gln Lys Gln Ala 100 105 110 Asn Leu Val Leu Lys Ile Ile Glu Gln Leu Pro Ser Ala Lys Glu Ser 115 120 125 Pro Asp Lys Phe Leu Glu Val Cys Thr Trp Val Asp Gln Ile Ala Ala 130 135 140 Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr Ser Glu Thr Val Arg Ala 145 150 155 160 Val Leu Asp Ser Leu Ser Glu Lys Lys Lys Ser Ser Pro 165 170 6173PRTArtificialSynthetic Construct 6Met Ala Pro Lys Lys Lys Arg Lys Val His Met Asn Thr Lys Tyr Asn 1 5 10 15 Glu Glu Phe Leu Leu Tyr Leu Ala Gly Phe Val Asp Gly Asp Gly Ser 20 25 30 Ile Ile Ala Ser Ile Ser Pro Arg Gln Ser Tyr Lys Phe Lys His Glu 35 40 45 Leu Arg Leu Thr Phe Gln Val Thr Gln Lys Thr Gln Arg Arg Trp Phe 50 55 60 Leu Asp Glu Leu Val Asp Glu Ile Gly Val Gly Lys Val Arg Asp Arg 65 70 75 80 Gly Ser Val Ser Asp Tyr Arg Leu Ser Gln Ile Lys Pro Leu His Asn 85 90 95 Phe Leu Thr Gln Leu Gln Pro Phe Leu Glu Leu Lys Gln Lys Gln Ala 100 105 110 Asn Leu Val Leu Lys Ile Ile Glu Gln Leu Pro Ser Ala Lys Glu Ser 115 120 125 Pro Asp Lys Phe Leu Glu Val Cys Thr Trp Val Asp Gln Ile Ala Ala 130 135 140 Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr Ser Glu Thr Val Arg Ala 145 150 155 160 Val Leu Asp Ser Leu Ser Glu Lys Lys Lys Ser Ser Pro 165 170 768DNAArtificialPCR amplicon of bar coding region around the BAY 39/40 recognition site (control) 7cggtcaactt ccgtaccgag ccgcaggaac cgcaggagtg gacggacgac ctcgtccgtc 60tgcgggac 68868DNAArtificialPCR amplicon of bar coding region around the BAY 39/40 recognition site (PPT tolerant plant linel) 8cggtcaactt ccgtaccgag ccgcaggaac cgcaggagtg gacggacgac ctcgtccgtc 60tgcgggac 68967DNAArtificialPCR amplicon of bar coding region around the BAY 39/40 recognition site (PPT sensitive plant linel 1) 9cggtcaactt ccgtaccgag ccgcaggaac cgcaggagtg gacgacgacc tcgtccgtct 60gcgggac 671027DNAArtificialPCR amplicon of bar coding region around the BAY 39/40 recognition site (PPT sensitive plant linel 2) 10cggtcaactt ccgtccgtct gcgggac 271161DNAArtificialPCR amplicon of bar coding region around the BAY 39/40 recognition site (PPT sensitive plant linel 3) 11cggtcaactt ccgtaccgag ccgcaggaac cgcaggagtg gacctcgtcc gtctgcggga 60c 611249DNAArtificialPCR amplicon of bar coding region around the BAY 39/40 recognition site (PPT sensitive plant linel 4) 12cggtcaactt ccgtaccgag ccgcaggaac cctcgtccgt ctgcgggac 491348DNAArtificialPCR amplicon of bar coding region around the BAY 39/40 recognition site (PPT sensitive plant linel 5) 13cggtcaactt ccgtaccgag ccgcaggaac ctcgtccgtc tgcgggac 481456DNAArtificialPCR amplicon of bar coding region around the BAY 39/40 recognition site (PPT sensitive plant linel 6) 14cggtcaactt ccgtaccgag ccgcaggaac cgcacgacct cgtccgtctg cgggac 561568DNAArtificialPCR amplicon of bar coding region around the BAY 39/40 recognition site (PPT sensitive plant linel 7) 15cggtcaactt ccgtaccgag ccgcaggaac cgcaggagtg gacggacgac ctcgtccgtc 60tgcgggac 6816163PRTArtificialI-CreI 16Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe 1 5 10 15 Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser 20 25 30 Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Glu Lys 35 40 45 Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val 50 55 60 Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu 65 70 75 80 Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys

85 90 95 Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu 100 105 110 Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp 115 120 125 Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr 130 135 140 Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys 145 150 155 160 Ser Ser Pro 174925DNAArtificial Sequencevector 17ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtacggc 660cgtcaaggcc aagcttcccg tgggggatcc accatacatg gagtcaaaaa ttcagatcga 720ggatctaaca gaactcgccg tgaagactgg cgaacagttc atacagagtc ttttacgact 780caatgacaag aagaaaatct tcgtcaacat ggtggagcac gacactctcg tctactccaa 840gaatatcaaa gatacagtct cagaagacca aagggctatt gagacttttc aacaaagggt 900aatatcggga aacctcctcg gattccattg cccagctatc tgtcacttca tcaaaaggac 960agtagaaaag gaaggtggca cctacaaatg ccatcattgc gataaaggaa aggctatcgt 1020tcaagatgcc tctgccgaca gtggtcccaa agatggaccc ccacccacga ggagcatcgt 1080ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg gattgatgtg atatctccac 1140tgacgtaagg gatgacgcac aatcccacta tccttcgcaa gacccttcct ctatataagg 1200aagttcattt catttggaga ggactcgaga attaagcaaa agaagaagaa gaagaagtcc 1260aaaacc atg gct aaa ccg cct aag aaa aag cgg aag gtt cat atg aat 1308 Met Ala Lys Pro Pro Lys Lys Lys Arg Lys Val His Met Asn 1 5 10 acc aaa tac aac aaa gaa ttc ctt ctc tac cta gct ggt ttc gta gac 1356Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe Val Asp 15 20 25 30 gga gat gga tct att atc gca tca att agc cct cgg caa tcg tac aaa 1404Gly Asp Gly Ser Ile Ile Ala Ser Ile Ser Pro Arg Gln Ser Tyr Lys 35 40 45 ttc aag cat gaa ctg cga ctt act ttc caa gtg aca cag aaa acc caa 1452Phe Lys His Glu Leu Arg Leu Thr Phe Gln Val Thr Gln Lys Thr Gln 50 55 60 agg aga tgg ttc ctt gat aaa ctc gtg gac gaa atc ggc gtt gga aag 1500Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val Gly Lys 65 70 75 gtc aga gat aga ggg tcg gtg tcc gac tat agg ctc agt cag att aaa 1548Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Arg Leu Ser Gln Ile Lys 80 85 90 cct ttg cat aac ttc cta act caa ctt caa cca ttt ctg aaa ttg aag 1596Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys Leu Lys 95 100 105 110 cag aag cag gtaagtttct gcttctacct ttgatatata tataataatt 1645Gln Lys Gln atcattaatt agtagtaata taatatttca aatatttttt tcaaaataaa agaatgtagt 1705atatagcaat tgcttttctg tagtttataa gtgtgtatat tttaatttat aacttttcta 1765atatatgacc aaaacatggt gatgtgcag gca aat ctg gtt ctc aag ata ata 1818 Ala Asn Leu Val Leu Lys Ile Ile 115 120 gag caa cta cca agc gca aag gaa tct cca gac aag ttt ttg gaa gtg 1866Glu Gln Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val 125 130 135 tgt acc tgg gtt gac caa atc gca gct ttg aat gat tcc aag aca cga 1914Cys Thr Trp Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg 140 145 150 aag aca act tct gag act gtg aga gca gtc ctt gat tca tta ccc ggt 1962Lys Thr Thr Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Pro Gly 155 160 165 tcg gtt ggt ggc tta agc cct agt cag gct agt tct gcc gct agt tct 2010Ser Val Gly Gly Leu Ser Pro Ser Gln Ala Ser Ser Ala Ala Ser Ser 170 175 180 185 gcc tca agc tct cca ggt tct ggg ata tcc gaa gcc ctt aga gct ggt 2058Ala Ser Ser Ser Pro Gly Ser Gly Ile Ser Glu Ala Leu Arg Ala Gly 190 195 200 gct act aag agc aag gag ttt ctc ctg tat tta gcc gga ttt gtt gat 2106Ala Thr Lys Ser Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe Val Asp 205 210 215 ggg gat ggt tca atc att gcc tct atc tca cca aat cag agc cgt aag 2154Gly Asp Gly Ser Ile Ile Ala Ser Ile Ser Pro Asn Gln Ser Arg Lys 220 225 230 ttt aag cac caa ctg agg ttg aca ttc acc gtg aca cag aag act caa 2202Phe Lys His Gln Leu Arg Leu Thr Phe Thr Val Thr Gln Lys Thr Gln 235 240 245 aga aga tgg ttt ctg gat aag ctt gtc gat gaa att ggc gtg gga aag 2250Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val Gly Lys 250 255 260 265 gtt cgt gat aga gga tct gtt agt gac tat cgc cta tcc cag att aaa 2298Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Arg Leu Ser Gln Ile Lys 270 275 280 cct ctt cac aac ttc ctg acc cag ctt caa cct ttc ttg aaa tta aag 2346Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys Leu Lys 285 290 295 cag aag cag gct aac ctg gtt ctc aaa atc att gag caa ctc cca tca 2394Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu Pro Ser 300 305 310 gca aaa gaa tca ccg gat aaa ttt ctg gag gta tgc act tgg gta gac 2442Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp Val Asp 315 320 325 caa att gct gct ctg aac gat tca aag act cga aaa acc act agt gag 2490Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr Ser Glu 330 335 340 345 aca gtt cgt gct gtc tta gat tcc ttg tcc gag aaa aag aaa agc tct 2538Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys Ser Ser 350 355 360 ccc tgattcccag ataagggaat tagggttcct atagggtttc gctcatgtgt 2591Pro tgagcatata agaaaccctt agtatgtatt tgtatttgta aaatacttct atcaataaaa 2651tttctaattc ctaaaaccaa aatccagcct gcaggtctag ataagtggga tatcacgtga 2711agcttgcaag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat 2771catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 2831gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 2891ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 2951gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 3011tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 3071cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 3131gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 3191gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 3251gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 3311ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 3371atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 3431tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 3491ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 3551gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 3611ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 3671ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 3731agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 3791ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 3851aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 3911tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 3971cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 4031tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagat ccacgctcac 4091cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 4151ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 4211gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 4271gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 4331gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 4391gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 4451tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 4511aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 4571cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 4631caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 4691cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 4751ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 4811aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 4871tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccac 492518362PRTArtificial SequenceSynthetic Construct 18Met Ala Lys Pro Pro Lys Lys Lys Arg Lys Val His Met Asn Thr Lys 1 5 10 15 Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe Val Asp Gly Asp 20 25 30 Gly Ser Ile Ile Ala Ser Ile Ser Pro Arg Gln Ser Tyr Lys Phe Lys 35 40 45 His Glu Leu Arg Leu Thr Phe Gln Val Thr Gln Lys Thr Gln Arg Arg 50 55 60 Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val Gly Lys Val Arg 65 70 75 80 Asp Arg Gly Ser Val Ser Asp Tyr Arg Leu Ser Gln Ile Lys Pro Leu 85 90 95 His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys Leu Lys Gln Lys 100 105 110 Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu Pro Ser Ala Lys 115 120 125 Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp Val Asp Gln Ile 130 135 140 Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr Ser Glu Thr Val 145 150 155 160 Arg Ala Val Leu Asp Ser Leu Pro Gly Ser Val Gly Gly Leu Ser Pro 165 170 175 Ser Gln Ala Ser Ser Ala Ala Ser Ser Ala Ser Ser Ser Pro Gly Ser 180 185 190 Gly Ile Ser Glu Ala Leu Arg Ala Gly Ala Thr Lys Ser Lys Glu Phe 195 200 205 Leu Leu Tyr Leu Ala Gly Phe Val Asp Gly Asp Gly Ser Ile Ile Ala 210 215 220 Ser Ile Ser Pro Asn Gln Ser Arg Lys Phe Lys His Gln Leu Arg Leu 225 230 235 240 Thr Phe Thr Val Thr Gln Lys Thr Gln Arg Arg Trp Phe Leu Asp Lys 245 250 255 Leu Val Asp Glu Ile Gly Val Gly Lys Val Arg Asp Arg Gly Ser Val 260 265 270 Ser Asp Tyr Arg Leu Ser Gln Ile Lys Pro Leu His Asn Phe Leu Thr 275 280 285 Gln Leu Gln Pro Phe Leu Lys Leu Lys Gln Lys Gln Ala Asn Leu Val 290 295 300 Leu Lys Ile Ile Glu Gln Leu Pro Ser Ala Lys Glu Ser Pro Asp Lys 305 310 315 320 Phe Leu Glu Val Cys Thr Trp Val Asp Gln Ile Ala Ala Leu Asn Asp 325 330 335 Ser Lys Thr Arg Lys Thr Thr Ser Glu Thr Val Arg Ala Val Leu Asp 340 345 350 Ser Leu Ser Glu Lys Lys Lys Ser Ser Pro 355 360

Claims

1. A method for introducing a foreign DNA molecule at a predefined site in a genome of a plant cell comprising the steps of a. inducing a double stranded DNA break at said predefined site; b. introducing said foreign DNA molecule in said plant cell; and c. selecting a plant cell wherein said foreign DNA is introduced at said predefined site; characterized in that said predefined site is comprised within a DNA region encoding a phosphinotricin acetyltransferase as encoded by Streptomyces hygroscopicus (bar coding region) and that said double stranded DNA break is induced by introduction of a single chain meganuclease or a pair of meganucleases which recognizes or recognize in concert said predefined site and induces or induce said double stranded break.

2. The method according to claim 1, wherein said predefined site comprises the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 2.

3. The method according to claim 1 or claim 2, wherein said bar coding region comprises the nucleotide sequence of SEQ ID No. 3.

4. A method for introducing a foreign DNA molecule at a predefined site in a genome of a plant cell comprising the steps of a. inducing a double stranded DNA break at said predefined site; b. introducing said foreign DNA molecule in said plant cell; and c. selecting a plant cell wherein said foreign DNA is introduced at said predefined site; characterized in that said predefined site comprises the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 2 and that said double stranded DNA break is induced by introduction of a single chain meganuclease or a pair of meganucleases which recognizes or recognize in concert said predefined site and induces or induce said double stranded break.

5. The method according to any one of claim 1, 2 or 4, wherein said meganuclease or said pair of meganucleases is/are derived from I-CreI and wherein the following amino acids are present in meganuclease unit 1: a. S at position 32; b. Y at position 33; c. E at position 38; d. R at position 40; e. K at position 66; f. Q at position 80; g. T at position 42; h. R at position 77; i. R at position 68; j. R at position 70; k. Q at position 44; l. I at position 24; m. S at position 26; n. S at position 28; o. R at position 30. and wherein the following amino acids are present in meganuclease unit 2: p. R at position 70; q. T at position 44; r. I at position 24; s. S at position 26; t. S at position 28; u. N at position 30; v. S at position 32; w. R at position 33; x. Q at position 38; y. Q at position 80; z. R at position 40; aa. K at position 66; bb. T at position 42; cc. R at position 77; dd. R at position 68.

6. The method according to any one of claim 1, 2, or 4, wherein said pair of meganucleases obligatory forms heterodimers or wherein said meganuclease is a single chain meganuclease comprising two domains derived from I-CreI covalently connected by a linker

7. The method according to any one of claim 1, 2, or 4, wherein said pair of meganucleases comprises the amino acid sequence of SEQ ID No. 5 and SEQ ID No. 6, respectively, or wherein said pair of meganucleases is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID No. 4 from nucleotide position 2004 to nucleotide position 2525 or to 2522 and the nucleotide sequence of SEQ ID No. 4 from nucleotide position 4885 to nucleotide position 5406 or to 5403 or wherein said single chain meganuclease comprises the amino acid sequence of SEQ ID No. 18 from position 1 to 167 and from position 206 to 362 or wherein said single chain meganuclease is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID No. 17 from nucleotide position 1267 to 1605 and from 1796 to 1956 and from 2071 to 2544 or 2541, or said single chain meganuclease is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID No. 17 from nucleotide position 1267 to 1605 and from 1796 to 2544 or 2541.

8. The method according to any one of claim 1, 2, or 4, wherein said foreign DNA is comprised within a repair DNA, said repair DNA comprising at least one flanking nucleotide sequence homologous to the upstream or downstream sequence of the nucleotide sequence of SEQ ID No. 1.

9. The method according to any one of claim 1, 2, or 4, wherein said meganuclease or said pair of meganucleases is expressed from a chimeric gene or a pair of chimeric genes, each comprising a plant expressible promoter operably linked to a coding region encoding said meganuclease or one of said pair of meganucleases, and further operationally linked to a DNA region involved in transcription termination and polyadenylation functional in a plant cell.

10. The method according to any one of claim 1, 2, or 4, wherein said foreign DNA comprises a selectable marker gene.

11. The method according to any one of claim 1, 2, or 4, wherein said foreign DNA comprises a plant expressible gene of interest, said gene of interest optionally being selected from a herbicide tolerance gene, an insect resistance gene, a disease resistance gene, an abiotic stress resistance gene, an enzyme involved in oil biosynthesis, carbohydrate biosynthesis, an enzyme involved in fiber strength or fiber length, an enzyme involved in biosynthesis of secondary metabolites.

12. The method according to any one of claim 1, 2, or 4 wherein said plant cell is further regenerated into a plant.

13. A plant cell or plant, or seed or propagating material thereof, wherein said foreign DNA has been introduced into said predefined site, obtained by the method according to any one of claim 1, 2, or 4.

14. A method of growing a plant according to claim 13, comprising the step of applying a chemical to said plant or substrate wherein said plant is grown.

15. A process for producing a plant comprising foreign DNA integrated at the bar coding region, comprising the step of crossing a plant consisting essentially of the plant cells of claim 13 with another plant or with itself and optionally harvesting seeds.

16. A process of producing treated seed comprising the step of applying a chemical compound on a seed of the plant according to claim 13.

17. Use of a meganuclease or a pair of meganucleases as described in any one of claim 1, 2, or 4 to introduce a foreign DNA into the bar coding region.

18. Use of a custom made meganuclease to introduce a foreign DNA of interest at a predefined site in a plant cell.

19. A method for introducing a foreign DNA molecule at a predefined site in a genome of a plant cell comprising the steps of a. inducing a double stranded DNA break at said predefined site; b. introducing said foreign DNA molecule in said plant cell; and c. selecting a plant cell wherein said foreign DNA is introduced at said predefined site; characterized in that said double stranded DNA break is induced by introduction of a non-naturally occurring single chain meganuclease or a pair of non-naturally occurring meganucleases which recognizes or recognize in concert said predefined site and induces or induce said double stranded break.

20. A method for introducing a foreign DNA molecule at a predefined site in a genome of a transgenic plant cell comprising the steps of a. inducing a double stranded DNA break at said predefined site; b. introducing said foreign DNA molecule in said plant cell; and c. selecting a plant cell wherein said foreign DNA is introduced at said predefined site; characterized in that said predefined site is a nucleotide sequence different from a recognition site for a natural occurring meganuclease, said predefined site being a nucleotide sequence commonly introduced as part of a transgene in a transgenic plant and wherein double stranded DNA break is induced by introduction of a non-naturally occurring single chain meganuclease or a pair of non-naturally occurring meganucleases which recognizes or recognize in concert said predefined site and induces or induce said double stranded break.

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