# Patent

## Title
Methods and means for removal of a selected DNA sequence

## Abstract

A method is described for the exact removal of a selected subfragment from a DNA molecule by intrachromosomal recombination between two directly repeated DNA sequences using a rare-cleaving double stranded break inducing DNA endonuclease expressed under control of a micro-spore specific promoter. This method can be applied in a method for the exact exchange of a target DNA fragment for a DNA fragment of interest in plant cells and plants.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. National Stage filing of International Application No. PCT/EP2006/003086, filed Mar. 31, 2006, which claims benefit of European Patent Application No. 05075781.4, filed Apr. 4, 2005, and U.S. Provisional Patent Application No. 60/669,243, filed Apr. 7, 2005, the disclosures of each of which are hereby incorporated by reference.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The current invention relates to method and means that allow the efficient removal of a selected part of a DNA sequence of interest previously introduced into said plant, such as e.g. a selectable or screenable marker gene without resorting to in vitro culture during the removal step. The removal method can be used as part of a method for exact exchange in plant cells and plants of a target 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.
2. Background Art
The removal of selected sub-fragments of foreign DNA introduced into plant cells or plants, but which have subsequently become obsolete or even unwanted, for various reasons, after introduction thereof, has been the subject of intensive research. Examples of such sequences are e.g. selectable marker genes which were necessary for the isolation of transgenic plants but which are no longer required in the mature plants. Methods to achieve efficient elimination thereof mostly rely on site-specific recombination or transposition (see e.g. Hohn et al., Plant BioTechnology pp 139-143).
Siebert and Puchta (2002) described that transgenic sequences flanked by sites of a rare cutting restriction enzyme can be excised efficiently from the genome of a higher eukaryote by homologous recombination as well as by non-homologous end-joining.
WO03/004659 relates to recombination systems and to a method for removing nucleic acid sequence from the chromosomal DNA of eukaryotic organisms. The document also relates to transgenic organisms (preferably plants), containing the described systems or produced by the described methods.
However the described methods mostly require the use of an in vitro culture method to identify or select those plant cells in which the deletion of the DNA sequences to be removed has effectively taken place and to regenerate a plant from such cells.
U.S. patent application 2005/0060769 proposes a method to prepare a recombined transgenic Zea mays plant or plant cell from a first transgenic Zea mays plant cell, wherein the transgene in the recombinant plant or plant cell has an altered genetic structure relative to the genetic structure of the transgene in the first transgenic plant cell, due to homologous recombination-mediated transgene deletion.
Hereinafter, including in the claims, different embodiment of methods and means for the efficient removal of selected subsequence of a part of a DNA molecule previously introduced in the cells of a plant without having to resort to in vitro culture methods, are described.
WO97/30166 or U.S. Pat. No. 6,407,314 describe promoter fragments from a microspore-specific gene from tobacco that can be used for expression of genes in microspores.
Another problem that has been solved by the present invention concerns the targeted and exact exchange through homologous recombination of a target DNA sequence in a cell of a plant for a replacement DNA sequence without leaving footprints of the procedure, and without having to resort to in vitro culture methods after the initial step of homology recombination. To this end, the herein described methods for efficient removal of selected subsequence of a part of a DNA molecule previously inserted in the genome, preferably the nuclear genome of cells of a plant, through intrachromosomal homologous recombination can be conveniently used.
The need to control the site of transgene integration in plants has been recognized early on, 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, pp 155-159). These methods mostly rely on homologous recombination-based transgene integration, a strategy which has been successfully applied in prokaryotes and lower eukaryotes (see e.g. EP0317509 or the corresponding publication by Paszkowski et al., 1988, EMBO J., 7, pp 4021-4026). However, for plants, the predominant mechanism for transgene integration is based on illegitimate recombination which involves little homology between the recombining DNA strands. A major challenge in this area is therefore the detection of the rare homologous recombination events, which are masked by the far more efficient integration of the introduced foreign DNA via illegitimate recombination.
One way of solving this problem is by selecting against the integration events that have occurred by illegitimate recombination, such as exemplified in WO94/17176.
Another way of solving the problem is by activation of the target locus through the induction of double stranded DNA breaks via rare-cutting endonucleases, such as I-SceI. This technique has been shown to increase the frequency of homologous recombination by at least two orders of magnitude using Agrobacteria to deliver the repair DNA to the plant cells (Puchta et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93, pp 5055-5060).
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.
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.
Chilton and Que (2003, Plant Physiol. 133: pp 956-965) and Tzifira et al. (2003, Plant Physiol. 133: pp 1011-1023) report that T-DNA preferentially integrates in double stranded DNA breaks, artificially induced by the rare-cleaving enzymes I-SceI or I-CeuI. The reports also included donor T-DNA vectors which comprised a recognition site for the respective rare-cleaving enzyme.
However, the methods in the prior art frequently rely on the reformation or generation through homology recombination of an intact selectable or screenable marker gene.
Therefore, there remains a need for methods which would allow targeted exchange of virtually any target DNA sequence by a replacement DNA. 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
In one embodiment of the invention a method is described for introduction of a DNA molecule of interest into the genome of a plant cell or plant followed by removal of a subsequence of the DNA molecule of interest, preferably comprising a selectable or screenable marker, comprising the steps of

    
    
        a. Introducing the DNA molecule of interest into the genome of the plant cell, the DNA molecule of interest comprising the subsequence of the DNA molecule flanked by two DNA sequences arranged in direct repeat and further comprising at least one recognition site for a rare cleaving double stranded DNA break inducing (DSBI) enzyme located in the vicinity of, preferably between, the two DNA sequences arranged in direct repeat;
        b. Selecting a plant cell wherein the DNA molecule of interest is integrated in the genome and regenerating a plant from the plant cell;
        c. Crossing the plant with a second plant comprising a DSBI enzyme encoding chimeric gene, the chimeric gene comprising the following operably linked DNA segments:
        
            i. a microspore specific promoter fragment, such as a promoter fragment selected from the nucleotide sequence of SEQ ID No. 3;
            ii. a DNA region encoding a rare cleaving double stranded DNA break inducing enzyme recognizing the recognition site, such as an endonuclease selected from the group of I-Sce I, I-Chu I, I-Dmo I, I-Cre I, I-Csm I, PI-Fli I, Pt-Mtu I, I-Ceu I, I-Sce II, I-Sce III, HO, PI-Civ I, PI-Ctr I, PI-Aae I, PI-BSU I, PI-DhaI, PI-Dra I, PI-Mav I, PI-Mch I, PI-Mfu I, PI-Mfl I, PI-Mga I, PI-Mgo I, PI-Min I, PI-Mka I, PI-Mle I, PI-Mma I, PI-Msh I, PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I, PI-Pfu I, PI-Rma I, PI-Spb I, PI-Ssp I, PI-Fac I, PI-Mja I, PI-Pho I, PI-Tag I, PI-Thy I, PI-Tko I or PI-Tsp I or a chimeric endonuclease comprising a Zn finger DNA binding domain and a DNA cleavage domain;
            iii. a transcription termination and polyadenylation region;
        
        
        d. Selecting a progeny plant (F1-plant) comprising the DNA molecule of interest and the DSBI enzyme encoding chimeric gene;
        e. Crossing the progeny plant with another plant whereby the progeny plant is used as pollen donor;
        f. Selecting a population of progeny plants (F2-population) which comprises the DSBI enzyme encoding chimeric gene; and
        g. Selecting a progeny plant wherein subsequence of the DNA molecule has been deleted by homologous recombination between the two DNA sequences arranged in direct repeat and optionally
        h. Crossing the progeny plant wherein the subsequence of the DNA molecule has been deleted, with another plant; and
        i. Obtaining a population of progeny plants (F3-plants) and selecting plants which do not contain the rare cleaving DSBI enzyme encoding chimeric gene.
    
    


In another embodiment of the invention, a method is provided for exchanging a target DNA sequence in the genome, particularly the nuclear genome, of a plant for a DNA sequence of interest comprising the following steps:

    
    
        a. Inducing a first double stranded DNA break at a preselected site in the genome of a cell of a plant, the preselected site being located within the target DNA sequence or in the vicinity of the target DNA sequence;
        b. Introducing a DNA molecule of interest into the plant cell, the DNA molecule comprising
        
            i. The DNA sequence of interest located between two flanking DNA regions having at least 80% sequence homology, preferably 100% sequence homology to a DNA region flanking the target DNA sequence, and preferably flanking the preselected site in the genome of the plant cell;
            ii. A selectable or screenable marker gene located between the flanking DNA regions, the selectable or screenable marker gene further being located between one of the flanking DNA regions and a partial flanking DNA region, comprising part of the one of the flanking DNA regions, located in direct repeat;
            iii. A recognition site for a DSBI enzyme located between the one of the flanking DNA regions and the partial flanking DNA region located in direct repeat;
        
        
        c. Selecting a population of plant cells comprising the selectable or screenable marker;
        d. Selecting a plant cell wherein the DNA sequence of interest (and the selectable or screenable marker) has been introduced by homologous recombination through the flanking DNA regions, and regenerating a plant from the plant cell;
        e. Crossing the regenerated plant or a progeny plant thereof comprising the selectable marker gene with a plant comprising a rare cleaving double stranded break inducing (“DSBI”) enzyme encoding chimeric gene, the chimeric gene comprising the following operably linked DNA segments:
        
            i. a microspore specific promoter;
            ii. a DNA region encoding a double stranded DNA break inducing enzyme recognizing the recognition site located in the DNA of interest;
            iii. a transcription termination and polyadenylation region;
        
        
        f. Selecting a progeny plant (F1-plant) comprising the selectable or screenable marker gene and the DSBI enzyme encoding chimeric gene;
        g. Crossing the progeny plant with another plant whereby the progeny plant is used as pollen donor;
        h. Selecting a population of progeny plants (F2-population) which comprises the DSBI enzyme encoding chimeric gene; and
        i. Selecting a progeny plant wherein the selectable or screenable marker gene is deleted by homologous recombination between the one of the flanking DNA regions and a partial flanking DNA region comprising part of the one of the flanking DNA regions.
    
    


The invention relates to the plants obtainable by the above described methods
In yet another embodiment, the invention relates to a plant comprising a rare cleaving DSBI enzyme encoding chimeric gene, such as the chimeric gene of SEQ ID NO 6 from nucleotide 1941 to nucleotide 3913, the chimeric gene comprising the following operably linked DNA segments:

    
    
        i. a microspore specific promoter such as a promoter fragment selected from the nucleotide sequence of SEQ ID No 3 or a functional fragment thereof;
        ii. a DNA region encoding a double stranded DNA break inducing enzyme recognizing the recognition site located in the DNA of interest, such as an endonuclease selected from the group of I-Sce I, I-Chu I, I-Dmo I, I-Cre I, I-Csm I, PI-Fli I, Pt-Mtu I, I-Ceu I, I-Sce II, I-Sce III, HO, PI-Civ I, PI-Ctr I, PI-Aae I, PI-BSU I, PI-DhaI, PI-Dra I, PI-Mav I, PI-Mch I, PI-Mfu I, PI-Mfl I, PI-Mga I, PI-Mgo I, PI-Min I, PI-Mka I, PI-Mle I, PI-Mma I, PI-Msh I, PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I, PI-Pfu I, PI-Rma I, PI-Spb I, PI-Ssp I, PI-Fac I, PI-Mja I, PI-Pho I, PI-Tag I, PI-Thy I, PI-Tko I or PI-Tsp I or a chimeric endonuclease comprising a Zn finger DNA binding domain and a DNA cleavage, particularly the DNA region comprising the nucleotide sequence of SEQ ID No 1 or SEQ ID No 2; and
        iii. a transcription termination and polyadenylation region.
    
    


The invention also relates to the chimeric gene described above.
In another embodiment of the invention, a DNA vector is provided for exchanging a target DNA sequence in the genome of a plant cell for a DNA sequence of interest through the induction of a double stranded break at a preselected site within the target sequence or in the vicinity thereof, the DNA vector comprising

    
    
        a. the DNA sequence of interest located between two flanking DNA regions having at least 80% sequence homology, preferably a 100% sequence homology to a DNA region flanking the target DNA sequence and flanking the preselected site;
        b. a selectable or screenable marker gene located between the flanking DNA regions, the selectable or screenable marker gene further being located between one of the flanking DNA regions and a partial flanking DNA region comprising part of the one of the flanking DNA regions located in direct repeat; and
        c. a recognition site for a DSBI enzyme located between the one of the flanking DNA regions and the partial flanking DNA region located in direct repeat.
    
    





BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1 to 3 represent different embodiments of the method to remove a selected subpart of a DNA of interest which is or has been introduced into a cell of a plant. They are for illustration purposes only and should not be used to construe the claims in a limiting manner.
FIG. 1 is schematic representation of a method for introducing a DNA of interest having a selected subpart comprising a selectable or screenable marker gene into a cell of a plant and subsequently removing the selected subpart of the DNA of interest. Trait: represents any DNA sequence of interest; DSB: recognition site for a double stranded break inducing enzyme (“DSBIE”); SMG1: selectable marker gene or screenable marker gene; drs: direct repeat sequence; SMG2: selectable or screenable marker gene associated with the DSBIE encoding chimeric gene; MSP: microspore specific promoter; 3′: transcription termination and polyadenylation signal;
FIG. 2 is a schematic representation of a method allowing exact replacement of a target DNA sequence with a replacement DNA sequence. DSB1: recognition site for a first double stranded break inducing enzyme; FS1: flanking sequence 1; FS2: flanking sequence 2; DSB2: recognition site for a second double stranded break inducing enzyme; SMG1: selectable marker gene 1 or screenable marker gene 1; SMG2: selectable marker gene 2 or screenable marker gene 2; DSBIE: double stranded break inducing enzyme; dr1: direct repeat sequence 1 (which is similar or identical to the direct repeat sequence 2 that is part of flanking sequence 2; also indicated herein as “partial flanking DNA region”); MSP: microspore specific promoter; 3′: transcription termination and polyadenylation signal.
FIG. 3 is a schematic representation of a method allowing exact replacement of a target DNA sequence with a replacement DNA sequence similar to the method illustrated in FIG. 2. dr1 in this case is a direct repeat sequence which is part from flanking sequence 1 and which is similar or identical to the direct repeat sequence 2 (dr2).



DETAILED EMBODIMENTS OF THE INVENTION
The current invention is based on the finding that selected sequences of a DNA molecule which are flanked by two direct repeats, and which are located in the neighborhood of a recognition site for a rare-cleaving double stranded DNA break inducing enzyme can be efficiently removed when the plant comprising such DNA is first crossed with a plant comprising a chimeric gene encoding the double stranded DNA break inducing rare-cleaving enzyme under control of a microspore-specific promoter, and pollen of a resulting plant is used to pollinate a receptor plant.
Thus, the invention is in one embodiment directed towards the use of plant comprising a chimeric gene encoding a double stranded DNA break inducing rare-cleaving endonuclease under control of a microspore specific promoter, to remove, by crossing, a DNA fragment located in the vicinity of a recognition site for the double stranded DNA break inducing rare-cleaving endonuclease and further located between two sequences located in direct repeat orientation (see FIG. 1). The expression of the rare cleaving DSBI endonuclease in the microspore during the pollen formation is sufficient to induce double stranded DNA breaks and thereby significantly stimulates the intrachromosomal homologous recombination between the directly repeated sequences, resulting in a removal of the sequences located between these directly repeated sequences.
In other words, in one embodiment of the invention, a method for introduction of a DNA molecule of interest into the genome of a plant cell or plant followed by removal of a subsequence of that DNA molecule is provided comprising the steps of

    
    
        a. Introducing that DNA molecule of interest into the genome of the plant cell comprising the subsequence of that DNA molecule flanked by two DNA sequences arranged in direct repeat and further comprising at least one recognition site for a double stranded DNA break inducing (DSBI) rare cleaving endonuclease located between the two DNA sequences arranged in direct repeat;
        b. Selecting a plant cell wherein the DNA molecule of interest is integrated in the genome and regenerating a plant from the plant cell;
        c. Crossing the plant with a second plant comprising a DSBI enzyme encoding chimeric gene, the chimeric gene comprising the following operably linked DNA segments:
        
            i. a microspore specific promoter;
            ii. a DNA region encoding a rare cleaving double stranded DNA break inducing enzyme recognizing the recognition site;
            iii. a transcription termination and polyadenylation region;
        
        
        d. Selecting a progeny plant (F1-plant) comprising the DNA molecule of interest and the DSBI enzyme encoding chimeric gene;
        e. Crossing the progeny plant with another plant whereby the progeny plant is used as pollen donor;
        f. Selecting a population of progeny plants (F2-population) which comprises the DSBI enzyme encoding chimeric gene; and
        g. Selecting a progeny plant wherein the subsequence of the DNA molecule of interest has been deleted by homologous recombination between the two DNA sequences arranged in direct repeat.
    
    


As used herein, a “double stranded DNA break inducing rare-cleaving endonuclease” is an enzyme capable of inducing a double stranded DNA break at a particular nucleotide sequence, called the “recognition site”. Rare-cleaving endonucleases, also sometimes called mega-nucleases have a recognition site of 14 to 40 consecutive nucleotides. Therefore, rare-cleaving endonucleases have a very low frequency of cleaving, even in the larger plant genomes. Homing endonucleases constitute a family of such rare-cleaving endonucleases. They may be encoded by introns, independent genes or intervening sequences, and present striking structural and functional properties that distinguish them from the more classical restriction enzymes, usually from bacterial restriction-modification Type II systems. Their recognition sites have a general asymmetry which contrast to the characteristic dyad symmetry of most restriction enzyme recognition sites. Several homing endonucleases encoded by introns or inteins have been shown to promote the homing of their respective genetic elements into allelic intronless or inteinless sites. By making a site-specific double strand break in the intronless or inteinless alleles, these nucleases create recombinogenic ends, which engage in a gene conversion process that duplicates the coding sequence and leads to the insertion of an intron or an intervening sequence at the DNA level.
A well characterized homing endonuclease is I-SceI. I-SceI is a site-specific endonuclease, responsible for intron mobility in mitochondria in Saccharomyces cerevisea. The enzyme is encoded by the optional intron Sc LSU.1 of the 21S rRNA gene and initiates a double stranded DNA break at the intron insertion site generating a 4 bp staggered cut with 3′OH overhangs. The recognition site of I-SceI endonuclease extends over an 18 bp non-symmetrical sequence (Colleaux et al. 1988 Proc. Natl. Acad. Sci. USA 85: 6022-6026). The amino acid sequence for I-SceI and a universal code equivalent of the mitochondrial I-SceI gene have been provided by e.g. WO 96/14408. WO 96/14408 further discloses a number of variants of I-SceI protein which are still functional.
PCT application PCT/EP04/013122 (incorporated herein by reference) provides synthetic nucleotide sequence variants of I-SceI which have been optimized for expression in plants. The nucleotide sequence of such synthetic I-Sce I coding regions is set forth in SEQ ID No 1 in UIPAC code. The symbols of the UIPAC code have their usual meaning i.e. N=A or C or G or T; R=A or G; Y=C or T; B=C or G or T (not A); V=A or C or G (not T); D=A or G or T (not C); H=A or C or T (not G); K=G or T; M=A or C; S=G or C; W=A or T.
A list of other rare cleaving DSB inducing enzymes and their respective recognition sites is provided in Table I of WO 03/004659 (pages 17 to 20) (incorporated herein by reference). These include I-Sce I, I-Chu I, I-Dmo I, I-Cre I, I-Csm I, PI-Fli I, Pt-Mtu I, I-Ceu I, I-Sce II, I-Sce III, HO, PI-Civ I, PI-Ctr I, PI-Aae I, PI-BSU I, PI-DhaI, PI-Dra I, PI-Mav I, PI-Mch I, PI-Mfu I, PI-Mfl I, PI-Mga I, PI-Mgo I, PI-Min I, PI-Mka I, PI-Mle I, PI-Mma I, PI-Msh I, PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I, PI-Pfu I, PI-Rma I, PI-Spb I, PI-Ssp I, PI-Fac I, PI-Mja I, PI-Pho I, PI-Tag I, PI-Thy I, PI-Tko I or PI-Tsp I.
Furthermore, methods are available to design custom-tailored rare-cleaving endonucleases that recognize basically any target nucleotide sequence of choice. 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.
As used herein “flanked by two DNA sequences arranged in direct repeat” indicates that the sequence to be removed from the introduced DNA molecule is immediately preceded and followed by two DNA regions, one at each end, wherein said two DNA regions are essentially similar in nucleotide sequence. The directly repeated sequences need not be identical, but may vary between about 75% to about 100% sequence identity. The shorter the repeated sequence, the more stringent the requirement for sequence similarity preferably is. However, in order to restore the DNA sequence without leaving a footprint, as described hereinafter, the DNA sequences arranged in direct repeat should preferably be identical. For avoidance of doubt, if the two DNA regions essentially similar in nucleotide sequence are contained within a double stranded DNA molecule, these DNA sequences are to be located on the same DNA strand, in the same 5′->3′ direction.
The repeated DNA sequence may be at least 10, 50 or 100 nucleotides in length, but the sequence may of course be larger. It has however been found that repeats longer than 300 nucleotides do not any longer significantly enhance the intrachromosomal homology recombination resulting in the removal of the DNA sequence located between the direct repeat sequences.
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 (×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) Computer-assisted sequence alignment, 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.
Although the DSBI recognition site is preferably located between the directly repeated DNA sequences, this is not essential nor required. Indeed, the DSBI recognition site could also be part of one of the repeated DNA sequences.
As used herein “located in the vicinity” refers to the DSBI being located at a distance of between 500 bp, 1 kbp to 10 kbp from the directly repeated DNA sequences.
The methods herein described require the use of a chimeric gene encoding a rare-cleaving double stranded break inducing enzyme, whereby the coding region for the endonuclease is under control of a microspore specific promoter fragment.
As used herein “a microspore specific promoter region” or “a microspore specific promoter” or a “a microspore specific promoter fragment” is a promoter region or promoter or promoter fragment which can promote transcription selectively, preferably specifically, in the unicellular microspore of a plant. In angiosperm plants, sexual reproduction requires the production of viable male and female gametophytes. Pollen, as the male gametophyte if formed within the anther and is initiated from sporogenous cells, which develop into meiocytes. The meiocyte undergoes meiosis to form a tetrad of haploid microspores, which are subsequently released into the anther locule. Following expansion and vacuolation, an asymmetrical mitosis of the microspore results in bicellular pollen, containing a vegetative and a generative cell. In the majority of species, pollen is shed in bicellular condition. A suitable microspore specific promoter region is described in WO 97/30166 (incorporated herein by reference; see also SEQ ID No 3) as the promoter region from NTM19 gene in tobacco. A functional fragment thereof has been incorporated in the chimeric gene of the Examples (SEQ ID No 6). A microspore specific promoter fragment could include the nucleotide sequence of SEQ ID No 3 from position 1 to position 954 or from position 1 to position 993 or the nucleotide sequence of SEQ ID No 6 from position 1941 to 2926.
As used herein “coding region for a rare cleaving double stranded break inducing endonuclease” or “coding region for a rare cleaving double stranded break inducing enzyme” is a nucleotide sequence which encodes a polypeptide that is characterized as a rare cleaving DSBI enzyme such as the homing endonucleases or the chimeric endonucleases described elsewhere in this application. The coding region may thus comprise any nucleotide sequence that encodes any of the amino acid sequences of the homing endonucleases listed in the following table, which can be found in public databases under the mentioned accession numbers (all herein incorporated by reference):











DSBI enzyme
Accession number







I-AniI
P03880


I-CvuI
P56347


I-CreI
P05725


I-ChuI
Q32001


I-CpaI - I-CpaIII - I-CpaIV - I-CpaV
Q39562/Q8WKZ5/Q8WKZ6/



Q8WKZ8


I-CpaII
Q39559


I-CeuI
P32761


I-DmoI
P21505


I-SceI
P03882


I-SceII
P03878


I-SceIII
Q9ZZX3


PI-SceI
P17255


I-NanI
Q25535


I-NitI
Q25567


I-NjaI
Q25568


I-PpoI
Q94702


PI-PfuI
O73954


PI-PkoI
P77933


PI-PkoII
P77933


PI-PspI
Q51334


PI-TfuI
P74918


PI-TfuII
P74918


PI-ThyI
Q9HH05


PI-ThyII
Q9HH05


PI-TliI
P30317


PI-TliII
P30317


I-TevI
P13299


I-TevII
P07072


I-TevIII
Q38419









It will be clear that for expression of the endonucleases under the control of a microspore specific promoter fragment, the coding region should be adapted so that the universal codon language is used to encode the above mentioned polypeptides. The coding region may further be optimized for expression in plants and the synthetic coding region have a nucleotide sequence which has been designed to fulfill the following criteria:

    
    
        a) the nucleotide sequence encodes a functional rare cleaving double stranded break inducing endonuclease,
        b) the nucleotide sequence has a GC content of about 50% to about 60%
        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;
        d) the nucleotide does not comprise a nucleotide sequence selected from the group consisting of CCAAT, ATTGG, GCAAT and ATTGC;
        e) the nucleotide sequence does not comprise a sequence selected from the group consisting of ATTTA, AAGGT, AGGTA, GGTA or GCAGG;
        f) the nucleotide sequence does not comprise a GC stretch consisting of 7 consecutive nucleotides selected from the group of G or C;
        g) the nucleotide sequence does not comprise a GC stretch consisting of 5 consecutive nucleotides selected from the group of A or T; and
        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).
    
    


The double stranded break inducing enzyme may comprise, but need not comprise, a nuclear localization signal (NLS) [Raikhel, Plant Physiol. 100: 1627-1632 (1992) and references therein], such as the NLS of SV40 large T-antigen [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.
Although the methods for removal are herein described as involving an active step of introduction of a DNA molecule of interest, followed by removal of selected subfragment thereof, it will be clear that the removal method of the invention can be used to remove any sequence located between direct DNA repeats, provided that a DSBI enzyme can be found or engineered that recognizes a DSBI recognition site in the vicinity of the repeated DNA sequences.
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.
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.
The DNA may be integrated by homologous recombination or non-homologous end-joining methods involving a double stranded break induction at a preselected site as described e.g. in PCT/EP04/013122.
In one particular embodiment of the invention, the method of removal may be used in combination with DNA insertion, deletion or replacement by targeted homologous recombination, and wherein the targeted DNA insertion is achieved using a selectable or screenable marker, followed by verification in the population of plant cells or plants comprising the selectable or screenable marker of those plant cells or plants wherein the targeted DNA insertion occurred by homologous recombination. When the flanking sequences and direct repeats are appropriately chosen, this method results in exact replacement of the target DNA for a DNA of interest, without any remainder (“footprint”) of the DNA molecule of interest used to achieve the replacement. The method of removal further does not need any additional in vitro culture, thereby avoiding that somaclonal variations are generated. An schematical outline of the method can be found in FIGS. 2 and 3.
Interestingly, it has been observed that using the methods as described in PCT/EP04/013122 for targeted insertion of foreign DNA of interest through homologous recombination, those transformation events wherein the foreign DNA is indeed inserted through homologous recombination represent a relatively high proportion (in the order of 1 to 5%) of the total population of events wherein the DNA is incorporated in the plant chromosome by any means. Accordingly, there is no need to rely on the generation or recreation through the homologous recombination of a DNA sequence resulting in a recognizable phenotype (such as the creation of an intact selectable marker gene after homologous recombination) to identify those events whereby the DNA is inserted by homologous recombination. Rather, a selectable or screenable marker gene can be included in the DNA region between the flanking DNA sequences followed by analysis of a relatively small number of transformed plant cells or plants, for identification of those transformation events wherein targeted DNA insertion occurred through homologous recombination.
Thus, in this embodiment of the invention, a method is provided for exchanging a target DNA sequence in cells of a plant for a DNA sequence of interest (or a foreign DNA) comprising the following steps:

    
    
        Inducing a first double stranded DNA break at a preselected site in the genome of the cell, the preselected site being located within the target DNA sequence or in the vicinity of said target DNA sequence;
        Introducing a DNA molecule of interest (of foreign DNA) into the plant cell, whereby the DNA molecule comprises the following operably linked DNA fragments:
        
            i. a DNA molecule of interest located between two flanking DNA regions having at least 80% sequence homology, preferably 100% sequence homology to a DNA region flanking the target DNA sequence and flanking the preselected site in the genome of the plant cell;
            ii. A selectable or screenable marker gene located between the flanking DNA regions, whereby the selectable or screenable marker gene is further located between one of the flanking DNA regions and another copy of at least part of the mentioned one of the flanking DNA regions located in direct repeat (also indicated as partial flanking DNA sequence);
            iii. A recognition site for a DSBI enzyme located between the one of the flanking DNA regions and the partial flanking DNA region located in direct repeat;
        
        
        Selecting a population of plant cells comprising the selectable or screenable marker;
        Selecting a plant cell wherein the selectable or screenable marker has been introduced by homologous recombination through the flanking DNA regions and regenerating a plant from the plant cell;
        Crossing the regenerated plant or a progeny plant thereof comprising the selectable marker gene with a plant comprising a DSBI enzyme encoding chimeric gene, the chimeric gene comprising the following operably linked DNA segments:
        
            a microspore specific promoter;
            a DNA region encoding a double stranded DNA break inducing enzyme recognizing the recognition site located in the DNA of interest;
            a transcription termination and polyadenylation region;
        
        
        Selecting a progeny plant (F1-plant) comprising the selectable or screenable marker gene and the DSBI enzyme encoding chimeric gene;
        Crossing the progeny plant with another plant whereby the progeny plant is used as pollen donor;
        Selecting a population of progeny plants (F2-population) which comprises the DSBI enzyme encoding chimeric gene; and
        Selecting a progeny plant within said F2 population wherein the selectable or screenable marker gene is deleted by homologous recombination between the one of the flanking DNA regions and a partial flanking DNA region comprising part of the one of the flanking DNA regions.
    
    


Thus, as used herein “a preselected site” indicates a particular nucleotide sequence in the plant nuclear genome, located in or near the target DNA sequence at which location it is desired to insert the foreign DNA or to exchange the target DNA sequence. A person skilled in the art would be able to either choose a double stranded DNA break inducing (“DSBI”) enzyme recognizing the selected target nucleotide sequence or engineer such a DSBI endonuclease. Alternatively, a DSBI endonuclease recognition site may be introduced into the plant genome using any conventional transformation method or by conventional breeding using a plant line having a DSBI endonuclease recognition site in its genome, and any desired foreign DNA may afterwards be introduced into that previously introduced preselected target site.
The double stranded DNA breaks in the transforming DNA molecule may be induced conveniently by transient introduction of a plant-expressible chimeric gene comprising a plant-expressible promoter region operably linked to a DNA region encoding a double stranded break inducing enzyme. The DNA region encoding a double stranded break inducing enzyme may be a synthetic DNA region, such as but not limited to, a synthetic DNA region whereby the codons are chosen according to the design scheme as described elsewhere in this application for I-SceI encoding regions. The endonuclease itself, as a protein, could also be introduced into the plant cells, e.g. by electroporation. However, the endonuclease can also be provided in a transient manner by introducing into the genome of a plant cell or plant, a chimeric gene comprising the endonuclease coding region operably linked to an inducible plant-expressible promoter, and providing the appropriate inducible compound for a limited time prior to, during or immediately after introduction of the transforming DNA molecule. The endonuclease could also be provided as an RNA precursor encoding the endonuclease.
The double stranded break inducing enzyme may comprise, but need not comprise, a nuclear localization signal (NLS) [Raikhel, Plant Physiol. 100: 1627-1632 (1992) and references therein], such as the NLS of SV40 large T-antigen [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.
As used herein, the “target DNA sequence” is the DNA sequence located in the genome of the plant cell which is modified, by addition, deletion or substitution.
As used herein “flanking DNA regions” are DNA sequences having homology to the DNA regions respectively upstream or downstream of the target DNA sequence. 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.
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 bp 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.
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.
Preferably, the preselected site and the further mentioned recognition sequence are recognized by different rare cleaving double stranded break inducing endonucleases.
The mentioned “partial flanking DNA region” indicates that the DNA region comprises at least a portion of the flanking DNA region adjacent to DNA region to be deleted and which usually will comprise the selectable or screenable marker. It is clear that the partial flanking DNA sequence may also be equal in length to the flanking DNA sequence or even comprise a longer flanking DNA sequence.
“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, β-glucoronidase (GUS), R-locus genes, green fluorescent protein and the likes.
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).
In another embodiment of the invention, the DNA removal method described herein may be combined with a method for DNA insertion at a preselected site in the genome of a cell, based on non-homologous end-joining.
Accordingly, the invention provides a method for inserting a selected DNA molecule at a predetermined location in the genome, preferably the nuclear genome of a plant cell, comprising the following steps:

    
    
        Inducing a first double stranded DNA break at a preselected site in the genome of the cell, the preselected site preferably being located within a target DNA sequence;
        Introducing a foreign DNA molecule into the plant cell, whereby the DNA molecule comprises the following operably linked DNA fragments:
        
            the selected DNA molecule of interest;
            A selectable or screenable marker gene preceded or followed by a repeat DNA region having at least 80% sequence identity to one of the genomic DNA regions located adjacent to the preselected site whereby the DNA region is located in direct repeat with the genomic copy thereof upon insertion of the foreign DNA molecule in the preselected site by non-homologous end joining;
            A recognition site for a rare cleaving DSBI enzyme located in the region of the foreign DNA comprising said repeat DNA region and said selectable marker gene;
        
        
        Selecting a population of plant cells comprising the selectable or screenable marker;
        Selecting a plant cell wherein the selectable or screenable marker has been introduced by non homologous end-joining at the preselected site and regenerating a plant from the plant cell;
        Crossing the regenerated plant or a progeny plant thereof comprising the selectable marker gene with a plant comprising a DSBI enzyme encoding chimeric gene, the chimeric gene comprising the following operably linked DNA segments:
        
            a microspore specific promoter;
            a DNA region encoding a double stranded DNA break inducing enzyme recognizing the recognition site located in the DNA of interest;
            a transcription termination and polyadenylation region;
        
        
        Selecting a progeny plant (F1-plant) comprising the selectable or screenable marker gene and the DSBI enzyme encoding chimeric gene;
        Crossing the progeny plant with another plant whereby the progeny plant is used as pollen donor;
        Selecting a population of progeny plants (F2-population) which comprises the DSBI enzyme encoding chimeric gene; and
        Selecting a progeny plant within said F2 population wherein the selectable or screenable marker gene is deleted by homologous recombination between the repeat DNA region and the genomic DNA regions located adjacent to the preselected site.
    
    


The above mentioned method can be conveniently used to interrupt any DNA sequence of choice, such as e.g. a polypeptide coding region, a biologically active RNA encoding DNA sequence, a promoter region, a regulatory region, a recognition site for protein or RNA binding etc.
In this embodiment, events wherein the DNA molecule has been inserted by non-homologous end-joining can be conveniently identified by e.g. a PCR reaction using a primer sequence recognizing a genomic sequence located in the vicinity of the preselected site, and which further preferably does not recognize the foreign DNA, and a primer within the foreign DNA molecule. Upon insertion of the foreign DNA by non-homologous end-joining at the preselected a DNA fragment will be amplified. Such DNA fragment would not be amplified when a the foreign DNA is randomly integrated.
It will be appreciated that the means and methods of the invention may be used in any plant capable of reproduction through pollen, 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, Lenma spp., Nicotiana spp., Arabidopsis, alfalfa, barley, bean, corn, cotton, flax, pea, rape, rice, rye, safflower, sorghum, soybean, sunflower, tobacco, wheat, asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery, cucumber, eggplant, lettuce, onion, oilseed rape, pepper, potato, pumpkin, radish, spinach, squash, tomato, zucchini, almond, apple, apricot, banana, blackberry, blueberry, cacao, cherry, coconut, cranberry, date, grape, grapefruit, guava, kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passion fruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry, strawberry, tangerine, walnut and watermelon.
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 DNA sequence 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.
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.
The following non-limiting Examples describe the removal of a selected subfragment from an introduced DNA molecule using a double strand DNA break inducing enzyme, such as I-SceI, expressed under control of a microspore specific promoter encoding chimeric gene.
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.
Throughout the description and Examples, reference is made to the following sequences:

    SEQ ID No 1: nucleotide sequence of synthetic I-SceI coding region (UIPAC code).
    SEQ ID No 2: nucleotide sequence of synthetic I-SceI coding region.
    SEQ ID No 3: nucleotide sequence of microspore selective NTM19 gene including promoter region
    SEQ ID No 4: nucleotide sequence of the T-DNA of pTCV63
    SEQ ID No 5: nucleotide sequence of the T-DNA of pTCV64
    SEQ ID No 6: nucleotide sequence of the T-DNA of pTCV72


EXAMPLES
Removal of a Selectable Marker Gene by Intrachromosomal Homologous Recombination (IHR)
A recombination assay to detect removal of a selected DNA fragment has been developed based on the restoration of an egfp-bar fusion gene after removal of a selectable marker gene (hyg) ( ̃2000 bp) by intrachromosomal homologous recombination (IHR) between directly repeated sequences (part of egfp sequences; either about 300 bp or about 600 bp). One of the repeat sequences is flanked by an I-SceI (and Zinc finger Zif268) recognition site giving the possibility to create a DSB between the repeats. In order to allow the IHR during transition from one generation to another, the I-SceI endonuclease was placed under control of a microspore specific promoter (pNTM19).
Using standard recombinant DNA techniques, the following DNA molecules were constructed for use in the following experiments:

    
    
        1. pTCV63: with short direct repeat sequences ( ̃300 bp) containing the following operably linked DNA constructs:
        
            p35S3: a CaMV35S promoter fragment
            egf(short): a first part the eGFP coding sequence comprising a 300 bp overlap with the subsequently named GFP sequence
            a recognition site for I-SceI endonuclease
            a recognition site for Zif268 Zn finger containing DNA binding protein
            pCsVMV: a cassava vein mosaic virus promoter fragment
            hyg: coding region for hygromycin resistance
            3′35S: 3′ transcription termination and polyadenylation signal
            gfp(short): the 3′ part of the eGFP coding sequence, comprising a direct repeat of 300 bp sequences of the previous egf portion of this plasmid, and wherein the coding region is translationally linked to a bar gene coding region
            3′nos: a 3′ transcription termination and polyadenylation signal from the nopaline synthase gene.
        
        
    
    


This plasmid was introduced into Agrobacterium tumefaciens and the resulting strain (A4330) was used to generate transgenic tobacco plants (G7NT001).

    
    
        2. pTCV64: with long direct repeat sequences ( ̃600 bp) containing the following operably linked DNA constructs:
        
            p35S3: a CaMV35S promoter
            egf(long): a first part the eGFP coding sequence comprising a 600 bp overlap with the subsequently named gfp sequence
            a recognition site for I-SceI endonuclease
            a recognition site for Zif268 Zn finger containing DNA binding protein
            pCsVMV: a cassava vein mosaic virus promoter
            hyg: coding region for hygromycin resistance
            3′35S: 3′ transcription termination and polyadenylation signal
            gfp(long): the 3′ part of the efgp coding sequence, comprising a direct repeat of 600 bp sequences of the previous egf construct, and wherein the coding region is translationally linked to a bar gene coding region
            3′nos: a 3′ transcription termination and polyadenylation signal from the nopaline synthase gene
        
        
    
    


This plasmid was introduced into Agrobacterium tumefaciens and the resulting strain (A4364) was used to generate transgenic tobacco plants (G7NT004)

    
    
        3. pTCV72:
        
            pnos: a nopaline synthase promoter
            neo: neomycine phosphotransferase II coding region
            3′ocs: a 3′ transcription termination and polyadenylation signal from the octopine synthase gene;
            pNTM19: a microspore specific promoter fragment
            I-SceI: coding region for the endonuclease I-SceI
            3′nos: a 3′ transcription termination and polyadenylation signal from the CaMV 35S transcript
        
        
    
    


This plasmid was introduced into Agrobacterium tumefaciens and the resulting strain (A4331) was used to generate transgenic tobacco plants (G7NT005)
From three independent single copy transformed tobacco lines of each G7NT001 and G7NT004 crosses have been made with two independent single copy transformed lines comprising the chimeric gene encoding I-SceI under control of a microspore specific promoter (G7NT005) using G7NT005 as male plant whereby the progeny lines were indicated as follows:

    G7NT001-0001×G7NT005-0001>04TDNT000001
    G7NT001-0002×G7NT005-0001>04TDNT000002
    G7NT001-0003×G7NT005-0001>04TDNT000003
    G7NT001-0001×G7NT005-0002>04TDNT000004
    G7NT001-0002×G7NT005-0002>04TDNT000005
    G7NT001-0003×G7NT005-0002>04TDNT000006
    G7NT004-0001×G7NT005-0001>04TDNT000007
    G7NT004-0002×G7NT005-0001>(no progeny)
    G7NT004-0003×G7NT005-0001>04TDNT000012
    G7NT004-0001×G7NT005-0002>04TDNT000008
    G7NT004-0002×G7NT005-0002>04TDNT000010
    G7NT004-0003×G7NT005-0002>04TDNT000011


From each crossing 200 seeds have been sown on Km (200 mg/L), 200 seeds on Hyg (50 mg/L) and 200 seeds on Km(200 mg/L)+Hyg(50 mg/L) to check normal transmission of transgenes. There was a quite normal transmission of the different transgenes for most of the crossings (note that for some crossings contamination problems and seed quality problems were encounter (see following table):










No of seedlings resistant to the respective selective agent















no KmR +




no KmR
no HygR
HygR



no seedlings/
seedlings/
seedlings/
seedlings/


Line
50 seeds
200 seeds
200 seeds
200 seeds





G7NT001-0001x
32
47/150
55
28/150


G7NT005-0001


G7NT001-0001x
32
29
51
15


G7NT005-0002


G7NT001-0002x
32
89
64
59


G7NT005-0001


G7NT001-0002x
46
69
94
42


G7NT005-0002


G7NT001-0003x
47
92
93
53


G7NT005-0001


G7NT001-0003x
48
88
85
47


G7NT005-0002


G7NT004-0001x
49
92
65/150
44


G7NT005-0001


G7NT004-0002x
47
73/150
89
34/150


G7NT005-0001


G7NT004-0002x
49
58/150
98
60


G7NT005-0002


G7NT004-0003x
39
63
69
50


G7NT005-0001


G7NT004-0003x
45
60
91
22


G7NT005-0002









From each of these 12 crossings, a few KmR+HygR progeny plants have been transferred to the greenhouse for being used as pollinator of WT SR1 plants. From these 12 crossings each time three KmR+HygR plants have been used as pollinator of WT SR1 plants according to the following scheme:



  
    S
    ⁢
    
      
    
    ⁢
    R
    ⁢
    
      
    
    ⁢
    1
    ×
    04
    ⁢
    TDNT
    ⁢
    
      
    
    ⁢
    000001
    ⁢
    
      
    
    ⁢
    
      -
    
    ⁢
    001
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      002
    
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      003
    
  




  
    S
    ⁢
    
      
    
    ⁢
    R
    ⁢
    
      
    
    ⁢
    1
    ×
    04
    ⁢
    TDNT
    ⁢
    
      
    
    ⁢
    000002
    ⁢
    
      
    
    ⁢
    
      -
    
    ⁢
    001
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      002
    
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      003
    
  




  
    S
    ⁢
    
      
    
    ⁢
    R
    ⁢
    
      
    
    ⁢
    1
    ×
    04
    ⁢
    TDNT
    ⁢
    
      
    
    ⁢
    000003
    ⁢
    
      
    
    ⁢
    
      -
    
    ⁢
    001
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      002
    
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      003
    
  




  
    S
    ⁢
    
      
    
    ⁢
    R
    ⁢
    
      
    
    ⁢
    1
    ×
    04
    ⁢
    TDNT
    ⁢
    
      
    
    ⁢
    000004
    ⁢
    
      
    
    ⁢
    
      -
    
    ⁢
    001
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      002
    
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      003
    
  




  
    S
    ⁢
    
      
    
    ⁢
    R
    ⁢
    
      
    
    ⁢
    1
    ×
    04
    ⁢
    TDNT
    ⁢
    
      
    
    ⁢
    000005
    ⁢
    
      
    
    ⁢
    
      -
    
    ⁢
    001
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      002
    
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      003
    
  




  
    S
    ⁢
    
      
    
    ⁢
    R
    ⁢
    
      
    
    ⁢
    1
    ×
    04
    ⁢
    TDNT
    ⁢
    
      
    
    ⁢
    000006
    ⁢
    
      
    
    ⁢
    
      -
    
    ⁢
    001
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      002
    
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      003
    
  




  
    S
    ⁢
    
      
    
    ⁢
    R
    ⁢
    
      
    
    ⁢
    1
    ×
    04
    ⁢
    TDNT
    ⁢
    
      
    
    ⁢
    000007
    ⁢
    
      
    
    ⁢
    
      -
    
    ⁢
    001
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      002
    
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      003
    
  




  
    S
    ⁢
    
      
    
    ⁢
    R
    ⁢
    
      
    
    ⁢
    1
    ×
    04
    ⁢
    TDNT
    ⁢
    
      
    
    ⁢
    0000012
    ⁢
    
      
    
    ⁢
    
      -
    
    ⁢
    001
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      002
    
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      003
    
  




  
    S
    ⁢
    
      
    
    ⁢
    R
    ⁢
    
      
    
    ⁢
    1
    ×
    04
    ⁢
    TDNT
    ⁢
    
      
    
    ⁢
    000008
    ⁢
    
      
    
    ⁢
    
      -
    
    ⁢
    001
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      002
    
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      003
    
  




  
    S
    ⁢
    
      
    
    ⁢
    R
    ⁢
    
      
    
    ⁢
    1
    ×
    04
    ⁢
    TDNT
    ⁢
    
      
    
    ⁢
    0000010
    ⁢
    
      
    
    ⁢
    
      -
    
    ⁢
    001
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      002
    
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      003
    
  




  
    S
    ⁢
    
      
    
    ⁢
    R
    ⁢
    
      
    
    ⁢
    1
    ×
    04
    ⁢
    TDNT
    ⁢
    
      
    
    ⁢
    0000011
    ⁢
    
      
    
    ⁢
    
      -
    
    ⁢
    001
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      002
    
  




  
    
      
    
    ⁢
    
      
        -
      
      ⁢
      003
    
  



From each progeny of these crosses (see following tables) 50 seeds have been sown on non-selective substrate to determine the germination frequency, 50 seeds on kanamycin to determine the transmission rate of the NTM19-I-SceI gene and about 4000 seeds on PPT for determining the frequency of IHR during transition from one generation to the other. The number of PPTR seedlings which are also KmR determines whether or not there is an effect of DSB induction by NTM19-ISceI endonuclease on the frequency of IHR during transition from one generation to the other.
The results of the progeny analysis of 22 progenies are summarized in tables A, B and C.
There is a very strong effect of NTM19-I-SceI on the frequency of IHR during transition from one generation to another as all PPTR seedlings are also KmR!
It has to be remarked that a large part of the PPTR and GFPF seedlings did not develop further into plants and died off due to the toxic effect of GFP.











TABLE A






Germination

No PPTR and
No KmR seedlings/



frequency (no
No KmR
GFPF
No of PPTR and



seedlings/50
seedlings/50
seedlings/no
GFPF seedlings


Cross
seeds)
seeds
of seeds
screened for kmR







SR1x04TDNT000001-001
43
24
77/4348
5/5


short repeat


(1.77%)


SR1x04TDNT000001-002
49
20
79/4835
23/23


short repeat


(1.63%)


SR1x04TDNT000001-003
47
22
98/4827
27/27


short repeat


(2.03%)


SR1x04TDNT000002-001
47
23
33/4762
4/4


short repeat


(0.69%)


SR1x04TDNT000004-001
49
30
123/4798 
36/36


short repeat


(2.6%)


SR1x04TDNT000004-002
48
23
100/4745
32/32


short repeat


(2.1%)


SR1x04TDNT000004-003
48
15
118/4665 
6/6


short repeat


(2.5%)


SR1x04TDNT000005-001
49
25
94/4665
16/16


short repeat


(2.01%)


SR1x04TDNT000005-002
48
20
47/4690
7/7


short repeat


(1%)


SR1x04TDNT000005-003
48
22
120/4658 
16/18 (2 S or R?)


short repeat


(2.6%)


SR1x04TDNT000006-001
47
28
136/4665 
24/24


short repeat


(2.9%)


SR1x04TDNT000006-003
49
20
77/4650
12/12


short repeat


(1.66%)






















TABLE B











N° KmR








seedlings/








N° of PPTR



Germination



N° PPTR and
and GFPF



frequency
N° KmR
N° HygR
N° of
GFPF
seedlings



(n° seedlings/
seedlings/
seedlings/
KmR + HygR/
seedlings/N°
screened


Cross
50 seeds)*
50 seeds
50 seeds
100 seeds
of seeds**
for KmR





















SR1x04TDNT00000
23
14
12
13
44/4973
33/33


3-001 short repeat




(0.89%)**


SR1x04TDNT00000
20
16
11
16
46/4857
46/46


3-003 short repeat




(0.95%)**


SR1x04TDNT00000
19
7
7
7
16/4915
16/16


7-001 long repeat




(0.33%)**


SR1x04TDNT00000
28
17
12
12
33/4890
33/33


8-001 long repeat




(0.7%)** 


SR1x04TDNT00000
20
7
8
8
33/4840
33/33


8-003 long repeat




(0.69%)**


SR1x04TDNT00001
16
10
9
9
14/4312
14/14


2-003 long repeat




(0.32%)**





*the progenies mentioned in this table were sown at the same moment. Due to a too drastic sterilization with bleach, there was a bad and irregular germination (for most lines <50%).


**This means that the N° of PPTR and GFPF seedlings/N° of seeds is an underestimation with at least a factor 2 as the germination frequency is for most lines less than 50%



















TABLE C











N° KmR








seedlings/








N° of PPTR



Germination



N° PPTR and
and GFPF



frequency
N° KmR
N° HygR
N° of
GFPF
seedlings



(n° seedlings/
seedlings/
seedlings/
KmR + HygR/
seedlings/n°
screened


Cross
50 seeds)*
50 seeds
50 seeds
100 seeds
of seeds**
for KmR*





















SR1x04TDNT00000
50
20
26
9
7/1330
NT*


2-002 short repeat




(0.5%)


SR1x04TDNT00000
50
30
18
25
9/1355
NT*


2-003 short repeat




 (0.66%)


SR1x04TDNT00000
50
20
21
25
24/1389 
NT*


3-002 short repeat




(1.7%)


SR1x04TDNT00000
50
25
25
17
3/1346
NT*


7-003 long repeat




(0.2%)





*NT: not tested yet






Moreover all PPTR and GFPF seedlings are indeed hygromycin sensitive, demonstrating the hyg gene has indeed been removed by intrachromosomal recombination in the IHR locus.












No of HygR seedlings/No of PPTR and GFPF


Cross
seedlings screened for HygR







SR1x04TDNT000012-003
 0/11


SR1x04TDNT000008-001
 0/12


SR1x04TDNT000008-003
 0/11


SR1x04TDNT000001-002
0/8


SR1x04TDNT000005-003
0/7


SR1x04TDNT000006-003
0/7









From the segregation analysis of 18 progeny populations, it can be concluded that there is a very strong effect of NTM19-I-SceI on the frequency of IHR during transition from one generation to another as all PPTR seedlings are also KmR.
The progeny of a crossing between SR1 (female) and 04TDNT00000X-00Y will normally segregate into:

    
    
        25% with only NTM19-ISceI endonuclease
        25% with only the HR construct
        25% with both NTM19-I-SceI endonuclease+IHR construct
        25% neither NTM19-I-SceI endonuclease nor IHR construct
    
    


The fact that all PPTR seedlings are also KmR shows that all HR recombinants occur only in the fraction which contains both the I-SceI endonuclease under control of a NTM19 microspore specific promoter as well as the HR construct. Our results show that in the best case up to 11% of the microspores which contain both NTM19-ISceI endonuclease+IHR construct has undergone intrachromosomal homologous recombination resulting in the restoration of a defective egfp-bar fusion gene (SR1×04TDNT000006-001). As no IHR recombinants resulting in a functional egfp-bar gene were obtained in the fraction which contain only the IHR construct, we may conclude that either spontaneous IHR (in absence of targeted DSB induction in the microspores) does not occur or if spontaneous IHR does occur, it does not result in the restoration of a defective egfp-bar fusion gene. In contrast, DSB-induced IHR in the microspores allows more precise intrachromosomal homologous recombination resulting in the restoration of a defective egfp-bar fusion gene.
Sequence analysis showed that no footprints are left after removal of the selectable marker mediated by DSB-induced IHR in the microspores.

## Claims

1. A method for exchanging a target DNA sequence in the genome of a plant for a DNA sequence of interest comprising:
a. inducing a first double stranded DNA break at a preselected site in the genome of a cell of a plant, said preselected site being located within said target DNA sequence or in the vicinity of said target DNA sequence;
b. introducing a repair DNA molecule into said plant cell, said DNA molecule comprising
i. said DNA sequence of interest located between two flanking DNA regions having at least 80% sequence homology to a DNA region flanking said target DNA sequence, and flanking said preselected site in the genome of said plant cell;
ii. a selectable or screenable marker gene located between said flanking DNA regions, said selectable or screenable marker gene further being located between one of the flanking DNA regions and another copy of at least part of said one of the flanking DNA regions immediately adjacent to the target DNA sequence located in direct repeat; and
iii. a recognition site for a DSBI enzyme located between said one of the flanking DNA regions and said partial flanking DNA region located in direct repeat;

c. selecting a population of plant cells comprising said selectable or screenable marker;
d. selecting a plant cell wherein said selectable or screenable marker has been introduced by homologous recombination through said flanking DNA regions and regenerating a plant from said plant cell;
e. crossing said regenerated plant or a progeny plant thereof comprising said selectable marker gene with a plant comprising a DSBI enzyme encoding chimeric gene, said chimeric gene comprising the following operably linked DNA segments:
i. a microspore specific promoter;
ii. a DNA region encoding a double stranded DNA break inducing (DSBI) enzyme recognizing said recognition site located in said DNA of interest;
iii. a transcription termination and polyadenylation region;

f. selecting a progeny plant (F1-plant) comprising said selectable or screenable marker gene and said DSBI enzyme encoding chimeric gene;
g. crossing said progeny plant with another plant whereby said progeny plant is used as pollen donor;
h. selecting a population of progeny plants (F2-population) which comprises said DSBI enzyme encoding chimeric gene; and
i. selecting a progeny plant wherein said selectable or screenable marker gene is deleted by homologous recombination between said one of the flanking DNA regions and said another copy of at least part of said one of the flanking DNA regions immediately adjacent to the target DNA sequence, located in direct repeat.

2. The method of claim 1 wherein said first double stranded break at said preselected site is induced by introduction of a first DSBI enzyme, said first DSBI enzyme not recognizing said recognition site for a DSBI enzyme located in said DNA of interest.

3. The method of claim 2, wherein said first DSBI enzyme and said DSBI enzyme recognizing said recognition site located in said DNA of interest are two different DSBI enzymes, wherein said enzymes are I-Sce I, I-Chu I, I-Dmo I, I-Cre I, I-Csm I, PI-Fli I, Pt-Mtu I, I-Ceu I, I-Sce II, I-Sce III, HO, PI-Civ I, PI-Ctr I, PI-Aae I, PI-BSU I, PI-DhaI, PI-Dra I, PI-Mav I, PI-Mch I, PI-Mfu I, PI-Mfl I, PI-Mga I, PI-Mgo I, PI-Min I, PI-Mka I, PI-Mle I, PI-Mma I, PI-Msh I, PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I, PI-Pfu I, PI-Rma I, PI-Spb I, PI-Ssp I, PI-Fac I, PI-Mja I, PI-Pho I, PI-Tag I, PI-Thy I, PI-Tko I, PI-Tsp I, or a chimeric endonuclease comprising a Zn finger DNA binding domain and a DNA cleavage domain.

4. The method of claim 1, wherein said DSBI enzyme recognizing said recognition site for a DSBI enzyme located in said DNA of interest is I-Sce I.

5. The method of claim 4, wherein said DNA region encoding said double stranded DNA break inducing enzyme comprises the nucleotide sequence of SEQ ID No 1 or SEQ ID No 2.

6. The method of claim 1, wherein said microspore specific promoter comprises a promoter comprising the nucleotide sequence of SEQ ID No 3 or a functional fragment thereof.

7. The method of claim 1, wherein said DSBI encoding chimeric gene comprises the nucleotide sequence of SEQ ID No. 6 from nucleotide 1941 to 3913.

8. A DNA vector for exchanging a target DNA sequence in the genome of a plant cell for a DNA sequence of interest through the induction of a double stranded break at a preselected site within said target sequence or in the vicinity thereof, said DNA vector comprising:
a. said DNA sequence of interest located between two flanking DNA regions having at least 80% sequence homology to a DNA region flanking said target DNA sequence and flanking said preselected site;
b. a selectable or screenable marker gene located between said flanking DNA regions, said selectable or screenable marker gene further being located between one of the flanking DNA regions and a partial flanking DNA region comprising part of said one of the flanking DNA regions immediately adjacent to the target DNA sequence located in direct repeat; and
c. a recognition site for a double stranded DNA break inducing (DSBI) enzyme located between said one of the flanking DNA regions and said partial flanking DNA region located in direct repeat.

9. The method of claim 1, wherein the preselected site is located within said target DNA.

10. The method of claim 1, wherein the preselected site is located within 500 bp from the directly repeated DNA sequences.

11. The method of claim 1, wherein direct repeats immediately precede and follow the selectable or screenable marker gene.

12. The method of claim 11, wherein the direct repeats are identical.

13. The method of claim 10, wherein direct repeats immediately precede and follow the selectable or screenable marker gene.

14. The method of claim 13, wherein the direct repeats are identical.