BRPI0716427B1 - METHOD FOR INTRODUCING AN EXOGENOUS SEQUENCE IN THE GENOME OF A PLANT CELL - Google Patents

Description

Report of the Invention Patent for "METHOD FOR INTRODUCING AN EXOGENOUS SEQUENCE IN THE GENOME OF A PLANT CELL".

TECHNICAL FIELD

[001] This description refers to the fields of genome design, gene targeting, chromosomal integration, and protein expression directed at plants.

BACKGROUND

A major area of interest in agriculture, especially in light of determining the complete nucleotide sequences of a number of plant genomes, is the directed alteration of genome sequences. In particular, the ability to convert endogenous plant sequences would facilitate numerous applications, such as, for example, optimizing crop traits that affect nutritional value, yield, stress tolerance, pathogen resistance, and resistance to agrochemicals and / or adaptation of plants for use as biological factors for the production of pharmaceutical or industrial chemical compounds.

In eukaryotes, attempts have been made to alter genomic sequences in cultured cells taking advantage of the natural phenomenon of homologous recombination. See, for example, Capecchi (1989) Science 244: 1288-1292; United States Patents Nos. 6,528,313 and 6,528,314. If a polynucleotide has sufficient homology to the genomic region containing the sequence to be altered, it is possible for part or all of the polynucleotide sequence to replace the genomic sequence with homologous recombination. However, the frequency of homologous recombination under these circumstances is extremely low. In addition, the frequency of insertion of the exogenous polynucleotide at genomic locations lacking sequence homology exceeds the homologous recombination frequency by several orders of magnitude.

[004] The introduction of a double stranded break in genomic DNA in the region of the genome that supports homology in an exogenous polynucleotide has been shown to stimulate homologous recombination at this site thousands of times in cultured cells. Rouet et al. (1994) Mol. Cell. Biol. 14: 8096-8106; Choulika et al. (1995) Mol. Cell. Biol. 15: 1968-1973; Donoho et al. (1998) Mol. Cell. Biol. 18: 4070-4078. See also Johnson et al. (2001) Biochem. Soc. Trans. 29: 196-201; and Yanez et al. (1998) Gene Therapy 5: 149-159. In these methods, DNA splitting in the desired genomic region was accompanied by the insertion of a recognition site for a meganuclease (ie, an endonuclease whose recognition sequence is so large that it does not occur, or only rarely, in the genome of interest). in the desired genomic region.

However, homologous recombination stimulated by meganuclease divaving occurs either in the fortuitous presence of, or at the directed insertion of an appropriate meganuclease recognition site in the vicinity of the genomic region to be altered. Since meganuclease recognition sites are rare (or non-existent) in a typical plant genome, an insertion of a suitable meganuclease recognition site is contaminated with the same difficulties as associated with other genomic changes, these methods do not being widely applicable.

Thus, there remains a need for compositions and methods for directed sequence alteration in any plant genome, and for compositions and methods for directed introduction of exogenous sequences into a genome.

SUMMARY

[007] The present disclosure provides compositions and methods for cell chromatin di-divonation in a region of interest, and / or homologous recombination in a predetermined region of interest in plant cells. Plant cells can be from monocots or dicot plants, and also include cultured cells, cells in a plant at any stage of development, and plant cells that have been removed from a total plant, and whose cells (or their descendants) will be returned to the plant.

A region of interest in cellular chromatin may be, for example, a genomic sequence, or portion thereof. Compositions include fusion polypeptides comprising a designed zinc finger binding domain (e.g., a zinc finger binding domain having a new specificity), and a splitting domain, and fusion polypeptides comprising a zinc finger binding domain. projected zinc finger, and a half-domain of divagem. Splitting domains and splitting half-domains may be obtained, for example, from various restriction endonucleases and / or homing endonucleases.

In one aspect, there is described herein a vector comprising first and second DNA sequences, in which (i) the first sequence is homologous to a third sequence, and the second sequence is homologous to a fourth sequence; (ii) the third and fourth sequences are chromosomal DNA sequences; and (iii) the edges near the third and fourth sequences are separated by at least 1 pair of nucleotides. In certain embodiments, the third and fourth sequences are endogenous sequences.

In any of the vectors described herein, at least one of the first or second sequences has a length of 100 nucleotides. In addition, any of the vectors described herein may further comprise a fifth sequence, in which the fifth sequence: (a) is interposed between the first and second sequences; and (b) is an exogenous sequence. In certain embodiments, the fifth sequence has a size of at least 1 base pair, but may be larger than 22 kilobase pairs.

Vectors (e.g., the fifth sequence) may also comprise sequences encoding a protein or portions of a protein. In certain embodiments, the coding protein sequence encodes a selectable marker (e.g. green fluorescent protein (GFP), β-glucuronidase (GUS), phosphinothricin N-acetyl transferase (PAT, BAR), neomycin phosphotransferase, β-lactamase, catechol dioxigenase, α-amylase, tyrosinase, β-galactosidase, luciferase, aequorin, EPSP synthase, nitrilase, acetolactate synthase (ALS), dihydrofolate reductase (DHFR), dalapon dehalogenase and anthranilate synthase). In other embodiments, the coding protein sequence (e.g., the fifth sequence) encodes a protein portion or protein portion, for example, a sequence that is homologous to chromosomal sequences.

In still other embodiments, the vectors (e.g. the fifth sequence) comprise one or more transcriptional regulatory sequences. In still other embodiments, vectors (e.g., fifth sequence) may comprise a wild-type equivalent of a mutant chromosomal sequence or, alternatively, a mutant equivalent of a wild-type chromosomal sequence.

In any of the vectors described herein, the first sequence may have at least 35% homology to the third sequence. Similarly, in any of the vectors described herein, the second sequence may have at least 35% homology to the fourth sequence. In some embodiments, the first sequence is at least 35% to 50%, at least 50% to 70%, at least 70% to 80%, at least 80% to 85%, at least 85% to 90%, at least 90% to 95%, at least 95%, 96%, 97%, 98%, 99% or 100% homology to the third sequence. In some embodiments, the second sequence is at least 35% to 50%, at least 50% to 70%, at least 70% to 80%, at least 80% to 85%, at least 85% to 90%, at least 90% to 95%, at least 95%, 96%, 97%, 98%, 99% or 100% homology to the fourth sequence.

In yet another aspect, a method for introducing an exogenous sequence into the genome of a plant cell is described herein, the method comprising the steps of: (a) contacting the cell with any of the vectors described above; and (b) expressing one or more nucleases in the cell, wherein one or more nucleases cleave chromosomal DNA within 0.4 to 3 kilobase pairs of or of the third or fourth sequence; such that chromosomal DNA splitting in step (b) stimulates the incorporation of the targeting vector into the genome by homologous recombination. In certain embodiments, one or more nucleases are fusions between the dividing domain of an IIS Type restriction endonuclease and a projected zinc finger binding domain.

In yet another aspect, there is provided a method for expressing a protein in a plant cell, the method comprising the steps of: (a) contacting the cell with a vector as described herein and (b) expressing one or more nucleases in the cell, in which one or more nucleases cleave chromosomal DNA within 0.1 to 3 kilobases of pairs of or third or fourth sequences; such that chromosomal DNA splitting in step (b) stimulates the incorporation of the vector into the genome by homologous recombination. In certain embodiments, one or more nucleases are fusions between the dividing domain of an IIS Type restriction endonuclease and a projected zinc finger binding domain.

In another aspect, a targeting vector comprising: (a) first and second coding sequences is described herein; and (b) first, second and third target sequences; wherein the sequences are arranged in the order: first target sequence, first coding sequence, second target sequence, second coding sequence, third target sequence; additionally in which the first target sequence is homologous to a first chromosome sequence, and the third target sequence is homologous to a second chromosome sequence. The first and / or second coding sequence may encode a selectable marker, or alternatively, the first and / or second coding sequence may encode a protein that is not a selectable marker. The first and second chromosomal sequences may be endogenous chromosomal sequences. Additionally, the vectors may comprise one or more repeats of the first, second and / or third target sequences.

In another aspect, a method for introducing an exogenous sequence into the genome of a plant cell is provided, the method comprising the steps of: (a) contacting the cell with a targeting vector as described in the preceding paragraph; and (b) expressing one or more nucleases in the cell, wherein one or more nucleases cleave chromosomal DNA within 0.1 to 3 kilobase pairs of any of the first or second chromosomal sequences; such that chromosomal DNA splitting in step (b) stimulates incorporation of the target vector into the genome by homologous recombination. In certain embodiments, one or more nucleases comprise a split half domain; for example, nuclease is a fusion between the diving domain of an IIS Type restriction endonuclease and a designed zinc finger binding domain.

In another aspect, a method for expressing a protein in a plant cell is provided, the method comprising the steps of: (a) contacting the cell with a target vector as described in the two paragraphs above; and (b) expressing one or more nucleases in the cell, wherein one or more nucleases cleave chromosomal DNA within 0.1 to 3 kilobase pairs of any of the first or second chromosomal sequences; such that chromosomal DNA splitting in step (b) stimulates the incorporation of the target vector into the genome by homologous recombination. In certain embodiments, one or more nucleases are fusions between the dividing domain of an IIS Type restriction endonuclease and a projected zinc finger binding domain.

In a still further aspect, a transgenic plant cell obtained according to any of the methods described herein is also provided.

In another aspect, there is provided herein a plant comprising a transgenic plant cell obtained as described herein.

Also provided is a method for removing sequences from the genome of a transgenic plant cell comprising first and second coding sequences; and first, second and third target sequences; wherein the sequences are arranged in the order: first target sequence, first coding sequence, second target sequence, second coding sequence, third target sequence, wherein the method comprises: (a) providing a transgenic plant cell as described herein; and (b) expressing the first and second nucleases in the cell, in which the first nuclease cleaves in the first target sequence and the second nuclease cleaves in the second target sequence.

In a further aspect, disclosed herein is a method for sequencing from the genome of a transgenic plant cell comprising first and second coding sequences; and first, second and third target sequences; wherein the sequences are arranged in the order: first target sequence, first coding sequence, second target sequence, second coding sequence, third target sequences, wherein the method comprises: (a) providing a transgenic plant cell as described herein; and (b) expressing the first and second nucleases in the cell, wherein the first nuclease cleaves in the second target sequence and the second nuclease cleaves in the third target sequence.

In yet another aspect, there is provided herein a method for sequencing from the genome of a transgenic plant cell comprising first and second coding sequences; and first, second and third target sequences; wherein the sequences are arranged in the order: first target sequence, first coding sequence, second target sequence, second coding sequence, third target sequence, wherein the method comprises: (a) providing a transgenic plant cell as described herein; and (b) expressing the first and second nucleases in the cell, wherein the first nuclease cleaves at the first target sequence and the second nuclease cleaves at the third target sequence.

In another aspect, a method for homologous intramolecular recombination in the genome of a cell (e.g., plant cell) is provided, the method comprising the steps of: (a) providing a DNA segment comprising a first sequence that is homologous to a second sequence; and (b) contacting said DNA segment with a nuclease, wherein the nuclease cleaves the DNA segment to a third sequence. In certain embodiments, the DNA segment is endogenous to the cell. In certain embodiments, homologous recombination occurs on a chromosome, for example, when DNA between the first and second sequences is taken from the chromosome. Sequences taken from the chromosome may encode, for example, all or part of a selectable marker. The removed DNA may be replaced by an exogenous sequence, for example, wherein the method further comprises: introducing a polynucleotide into the cell, wherein the polynucleotide comprises: (a) fourth and fifth sequences, wherein the fourth sequence is homologous to sequences not withdrawn in proximity to the first sequence, and the fifth sequence is homologous to sequences not withdrawn in proximity to the second sequence; and (b) the exogenous sequence (e.g., a selectable marker, a protein or portion of a protein other than a selectable marker, an RNA such as siRNA, etc.). In any of the methods provided herein, the selectable marker may be, for example, green fluorescent protein (GFP), β-glucuronidase (GUS), fosfinotricin N-acetyl transferase (PAT, BAR), neomycin phosphotransferase, β-lactamase, catechol. dioxigenase, α-amylase, tyrosinase, β-galactosidase, luciferase, aequorin, EPSP synthase, nitrilase, acetolactate synthase (ALS), dihydrofolate reductase (DHFR), dalapon dehalogenase and anthranilate synthase.

In either method, the third sequence (i.e., the nuclease cleaved sequence) may be unique in the genome and / or the nuclease may be a pair of fusion proteins, in which each fusion protein is a fusion. between a dial half domain (for example, the IIS Type restriction endonuclease dial domain) and a projected zinc finger binding domain. Additionally, the third sequence may be between the first and second sequences (i.e., homologous sequences) and / or at least 1 base pair from the first and / or second sequences.

In either method described herein, one or both of the first and second sequences may be exogenous to the organism.

Accordingly, the present disclosure involves, but is not limited to, the following numbered embodiments: 1. A donor vector comprising first and second DNA sequences; wherein the first sequence is homologous to a third sequence, and the second sequence is homologous to a fourth sequence; wherein the third and fourth sequences are chromosomal DNA sequences; and wherein the edges near the third and fourth sequences are separated by at least 1 pair of nucleotides. 2. The vector of 1, in which the third and fourth sequences are endogenous sequences. 3. The vector of 1 or 2, wherein at least one of the first or second sequences is 100 nucleotides in length. The vector of any 1 to 3, further comprising a fifth sequence, in which the fifth sequence: (a) is interposed between the first and second sequences; and (b) is an exogenous sequence. 5. The vector of 4, in which the fifth sequence has a size of at least 1 base pair. 6. The vector of 4, wherein the fifth sequence comprises sequences encoding a selectable marker. 7. The vector of 6, in which the selectable marker is selected from the group consisting of green fluorescent protein (GFP), β-glucuronidase (GUS), fosfinotricin N-acetyl transferase (PAT, BAR), neomycin phosphotransferase, β- lactamase, catechol dioxigenase, α-amylase, tyrosinase, β-galactosidase, luciferase, aequorin, EPSP synthase, nitrilase, acetolactate synthase (ALS), dihydrofolate reductase (DHFR), dalapon dehalogenase and anthranilate synthase. 8. The vector of 4, wherein the fifth sequence comprises sequences encoding a protein other than a selectable marker. 9. The vector of 4, wherein the fifth sequence comprises one or more transcriptional regulatory sequences. 10. The vector of 4, wherein the fifth sequence comprises sequences encoding a portion of a protein. 11 is a vector of 10, wherein the sequences encoding the protein portion comprise sequences homologous to chromosomal sequences. 12. The vector of 4, wherein the fifth sequence comprises a wild-type equivalent of a mutant chromosomal sequence. 13. The vector of 4, wherein the fifth sequence comprises a mutant equivalent of a wild-type chromosomal sequence. 14. The vector of any one from 1 to 13, wherein the first sequence is at least 35% homologous to the third sequence. 15. The vector of any one from 1 to 14, wherein the second sequence is at least 35% homologous to the fourth sequence. 16. The vector of 14, in which the second sequence is at least 35% homologous to the fourth sequence. A method for introducing an exogenous sequence into the genome of a plant cell, the method comprising the steps of: (a) contacting the cell with a target vector of any one from 1 to 16; and (b) expressing one or more nucleases in the cell, wherein one or more nucleases cleave chromosomal DNA within 3 kilobase pairs of any of the third or fourth sequences; such that chromosomal DNA splitting in step (b) stimulates incorporation of the target vector into the genome by homologous recombination. 18. A method of 17, wherein one or more nucleases are fusions between the dividing domain of an IIS Type restriction endonuclease and a projected zinc finger binding domain. 19. A method for expressing a protein in a plant cell, the method comprising the steps of: (a) contacting the cell with a target vector of 8; and (b) expressing one or more nucleases in the cell, wherein one or more nucleases cleave chromosomal DNA within 3 kilobase pairs of any of the third or fourth sequences; such that chromosomal DNA splitting in step (b) stimulates incorporation of the target vector into the genome by homologous recombination. 20. The method of 19, wherein one or more nucleases are fusions between the dividing domain of an IIS Type restriction endonuclease and a projected zinc finger binding domain. 21. A transgenic plant cell obtained according to the method of 17, 18, 19 or 20. 22. A plant comprising a transgenic plant cell according to 21. 23. A method for homologous intramolecular homologous recombination in the genome of a cell the method comprising the steps of: (a) providing a DNA segment comprising a first sequence that is homologous to a second sequence; and (b) contacting said DNA segment with a nuclease, wherein the nuclease cleaves the DNA segment into a third sequence. 24. A method of 23, in which the DNA segment is endogenous to the cell. 25. The 23 or 24 method, in which homologous recombination occurs on a chromosome. 26. The method of 25, in which DNA between the first and second sequences is taken from the chromosome. 27. The method of any of 23, 24, 25 or 26, wherein the third sequence is unique in the genome. 28. The method of any of 23 to 26, wherein the cell is a plant cell. 29. The method of any of 23 to 28, wherein the nuclease is a pair of fusion proteins, wherein each fusion protein is a fusion between the dividing domain of an IIS Type restriction endonuclease and a binding domain of Zinc finger designed. 30. The method of any of 23 to 29, wherein the third sequence is at least 100 base pairs from the first sequence. 31. The method of any of 23 to 30, wherein the third sequence is at least 100 base pairs from the second sequence. 32. The method of any of 23 to 31, wherein the third sequence occurs between the first and second sequences. 33. The method of either 23 to 32, wherein one of the first or second sequences is exogenous to the organism. 34. The method of any of 23 to 32, wherein both of the first and second sequences are exogenous to the organism. 35. The method of 26, wherein sequences taken from the chromosome encode all or part of a selectable marker. 36. The 35 method, in which the selectable marker is selected from the group consisting of green fluorescent protein (GFP), β-glucuronidase (GUS), fosfinotricin N-acetyl transferase (PAT, BAR), neomycin phosphotransferase, β- lactamase, catechol dioxigenase, α-amylase, tyrosinase, β-galactosidase, luciferase, aequorin, EPSP synthase, nitrilase, acetolactate synthase (ALS), dihydrofolate reductase (DHFR), dalapon dehalogenase and anthranilate synthase. 37. The method of 26, wherein the removed DNA is replaced by an exogenous sequence, the method further comprising: introducing a polynucleotide into the cell, wherein the polynucleotide comprises: (a) fourth and fifth sequences, wherein the fourth sequence is homologous to non-withdrawn sequences in proximity to the first sequence, and the fifth sequence is homologous to non-withdrawn sequences in proximity to the second sequence; and (b) the exogenous sequence. 38. The method of 37, wherein the exogenous sequence is a selectable marker. 39. The 38 method, in which the selectable marker is selected from the group consisting of green fluorescent protein (GFP), β-glucuronidase (GUS), fosfinotricin N-acetyl transferase (PAT, BAR), neomycin phosphotransferase, β- lactamase, catechol dioxigenase, α-amylase, tyrosinase, β-galactosidase, luciferase, aequorin, EPSP synthase, nitrilase, acetolactate synthase (ALS), dihydrofolate reductase (DHFR), dalapon dehalogenase and anthranilate synthase. 40. The method of any one from 23 to 39, wherein the exogenous sequence encodes a protein other than a selectable marker. 41. The method of anyone from 23 to 39, wherein the exogenous sequence encodes an RNA. 42. The method of 41, wherein RNA is an siRNA. 43. A target vector comprising: (a) first and second coding sequences; and (b) first, second and third target sequences; wherein the sequences are arranged in the order: first target sequence, first coding sequence, second target sequence, second coding sequence, third target sequence; additionally in which the first target sequence is homologous to a first chromosome sequence, and the third target sequence is homologous to a second chromosome sequence. 44. The vector 43, in which the first coding sequence encodes a selectable marker. 45. The vector 43 or 44, in which the second coding sequence encodes a selectable marker. 46. The 43 or 45 vector, in which the first coding sequence encodes a protein that is not a selectable marker. 47. The vector of either 43, 44 or 46, wherein the second coding sequence encodes a protein that is not a selectable marker. 48. The vector of any one from 43 to 47, wherein the first and second chromosomal sequences are endogenous chromosomal sequences. 49. The vector of any one of 43 to 48, further comprising one or more repeats of the first target sequence. 50. The vector of any one of 43 to 49, further comprising one or more repeats of the second target sequence. 51. The vector of any one from 43 to 50, further comprising one or more repeats of the third target sequence. 52. A method for introducing an exogenous sequence into the genome of a plant cell, the method comprising the steps of: (a) contacting the cell with a target vector according to any of 43 to 51; and (b) expressing one or more nucleases in the cell, wherein one or more nucleases cleave chromosomal DNA within between 0.1 and 3 kilobase pairs of any of the first or second chromosome sequences; such that chromosomal DNA splitting in step (b) stimulates incorporation of the target vector into the genome by homologous recombination. 53. The method of 52, wherein one or more nucleases are fusions between the dividing domain of an IIS Type restriction endonuclease and a projected zinc finger binding domain. 54. A method for expressing a protein in a plant cell, the method comprising the steps of: (a) contacting the cell with a target vector according to any of 43 to 51; and (b) expressing one or more nucleases in the cell, wherein one or more nucleases cleaves chromosomal DNA within between 0.1 and 3 kilobase pairs of any of the first or second chromosomal sequences; such that chromosomal DNA splitting in step (b) stimulates incorporation of the target vector into the genome by homologous recombination. 55. The method of 54, wherein one or more nucleases are fusions between the dividing domain of an IIS Type restriction endonuclease and a projected zinc finger binding domain. 56. A transgenic plant cell obtained according to the method of 52 or 53. 57. A plant comprising a transgenic plant cell according to 56. 58. A method for sequencing from the genome of a transgenic plant cell wherein the method comprises: (a) providing a transgenic plant cell according to 56; and (b) expressing the first and second nucleases in the cell, in which the first nuclease cleaves in the first target sequence and the second nuclease cleaves in the second target sequence. 59. A method for sequencing from the genome of a transgenic plant cell in which the method comprises: (a) providing a transgenic plant cell according to 56; and (b) expressing the first and second nucleases in the cell, wherein the first nuclease cleaves in the second target sequence and the second nuclease cleaves in the third target sequence. 60. A method for sequencing from the genome of a transgenic plant cell in which the method comprises: (a) providing a transgenic plant cell according to 56; and (b) expressing the first and second nucleases in the cell, wherein the first nuclease cleaves at the first target sequence and the second nuclease cleaves at the third target sequence.

BRIEF DESCRIPTION OF DRAWINGS

Figures 1A and 1B are schematic representations of the target building designated pDAB1585. Figure 1A is a linear representation of various elements of the construction. Figure 1B is a representation of the circular construction. Figures 2A and 2B represent exemplary ZFNs and their target sites. Figure 2A represents the ZFNs and binding regions. Figure 2B represents the target sites.

Figure 3 is a schematic representation of plasmid PDAB1400, Figure 4 is a schematic representation of plasmid pDAB782.

Figure 5 is a schematic representation of plasmid pDAB1582.

Figure 6 is a schematic representation of plasmid PDAB354.

Figure 7 is a schematic representation of plasmid PDAB1583.

Figure 8 is a schematic representation of plasmid pDAB2407.

Figure 9 is a schematic representation of plasmid pDAB1584.

Figure 10 is a schematic representation of plasmid pDAB2418.

Figure 11 is a schematic representation of plasmid pDAB4045.

Figure 12 is a schematic representation of plasmid pDAB1575.

Figure 13 is a schematic representation of plasmid pDAB1577.

Figure 14 is a schematic representation of plasmid pDAB1579.

Figure 15 is a schematic representation of plasmid pDAB1580.

Figure 16 is a schematic representation of plasmid pDAB3401.

Figure 17 is a schematic representation of plasmid pDAB1570.

Figure 18 is a schematic representation of plasmid pDAB1572.

Figure 19 is a schematic representation of plasmid pDAB4003.

Figure 20 is a schematic representation of plasmid pDAB1571.

Figure 21 is a schematic representation of plasmid pDAB7204.

Figure 22 is a schematic representation of plasmid pDAB1573.

Figure 23 is a schematic representation of plasmid pDAB1574.

Figure 24 is a schematic representation of plasmid pDAB1581.

Figure 25 is a schematic representation of plasmid pDAB1576.

Figures 26A and 26B are schematic representations of the target plasmid vector designated pDAB1600. Figure 26A is a linear representation of various elements of the plasmid. Figure 26B is a representation of the circular plasmid.

Figure 27 is a schematic representation of plasmid pDAB7002.

Figure 28 is a schematic representation of plasmid pDAB7025.

Figure 29 is a schematic representation of plasmid pDAB1591.

Figure 30 is a schematic representation of plasmid pcDNA3.1-IL1 -LO-Fokl.

Figure 31 is a schematic representation of plasmid pcDNA3.1 -CD27-L0-Fokl.

Figure 32 is a schematic representation of plasmid pDAB1592.

Figure 33 is a schematic representation of plasmid pDAB1594.

Figures 34 A, B and C are schematic representations of plasmids pDAB1596 and pDAB1598. Figure 34A is a schematic of linearized plasmids. Figure 34B shows pDAB1596. Figure 34C shows pDAB1598.

Figure 35 is a schematic representation of plasmid pDAB1577.

Figure 36 is a schematic representation of plasmid pDAB1578.

Figures 37A and B are schematic representations of plasmid pDAB1601. Figure 37A shows various elements in linear form. Figure 37B shows the circular plasmid.

Figure 38 is a schematic depicting predicted interchromosomal homologous recombination stimulated by IL-1 ZFN-Fokl fusion.

Figure 39 is a scheme depicting a predicted interchromosomal homologous recombination stimulated by the Scd27 ZFN-Fokl fusion.

Figure 40 shows PCR analysis of recombinants. Lanes 1-20 show homologous recombination events from the transformation of NT1-240 with SCD27-Fokl fusion protein construct. The designated lanes 21 and 22 show homologous recombination events from the transformation of NT1-240 with SCD27-Fokl fusion protein construct. Control lanes are shown in the left 3 lanes.

Figure 41 shows Southern blot analysis of recombinants. The designated lanes 1-20 show homologous recombination events from the transformation of NT1-240 with SCD27-Fokl fusion protein construct. The designated lanes 21 and 22 show homologous recombination events from the transformation of NT1-240 with SCD27-Fokl fusion protein construct. The control lanes are shown on the leftmost 2 lines.

Figure 42 is a scheme depicting predicted interchromosomal homologous recombination stimulated by IL-1 ZFN-Fokl fusion.

Figure 43 is PCR analysis confirming that GFP is reconstituted in fluorescent tissues expressing in the IL-1-Fokl fusion protein.

Fig. 44 is a schematic representation of plasmid pSB11.

Figure 45 is a schematic representation of plasmid pSB1. Figure 46 is a schematic representation of plasmid pDAB3872.

Figure 47 is a schematic representation of plasmid PDAB4365, DETAILED DESCRIPTION

Disclosed herein are compositions and methods useful for target cleavage of plant cell chromatin cell and for target alteration of a plant cell nucleotide sequence cell, for example, by target cleavage followed by intrachromosomal homologous recombination, or by target cleavage followed by homologous recombination between an exogenous polynucleotide (comprising one or more cell nucleotide sequence homology regions), and a genomic sequence.

Genomic sequences include those present on chromosomes, eptsomes, organelle genomes (e.g., mitochondria, chloroplasts), artificial chromosomes, and any other type of nucleic acid present in a cell, such as, for example, amplified sequences, chromosomes of double minutes, and the genomes of endogenous bacteria or infection, and viruses. Genomic sequences may be normal (ie, wild type) or mutant; Mutant sequences may comprise, for example, insertions, withdrawals, translocations, rearrangements, and / or point mutations. A genomic sequence may also comprise one of a number of different alleles.

[6677] Compositions useful for target cleavage and recombination include fusion proteins comprising a dividing domain (or a dividing half-domain) and a zinc finger binding domain, polynucleotides encoding these proteins, and combinations of polypeptides and polynucleotides encoding polypeptide. A zinc finger binding domain may comprise one or more zinc appendages (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc appendages), and may be designed to bind to any genomic sequence. Thus, by identifying a target genomic region of interest in which dividing or recombination is desired, one may, according to the methods disclosed herein, construct one or more fusion proteins comprising a dividing domain (or half-domain). diving) and a zinc finger domain designed to recognize a target sequence in said genomic region. The presence of such fusion protein (or proteins) in a cell will result in the fusion protein (s) binding to its binding and dividing site (s) within or near said genomic region. In addition, if an exogenous polynucleotide homologous to the genomic region is also present in such a cell, homologous recombination occurs at a high rate between the genomic region and the exogenous polynucleotide.

General Practice of the methods, as well as preparation and use of the compositions disclosed herein, employ, unless otherwise indicated, conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, Recombinant DNA and related fields as are within the skill in the art. These techniques are fully explained in the literature. See, for example, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press, 1989, and Third Edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987, and periodic updates; the METHODS IN ENZYMOLOGY series, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third Edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (P.M. Wassarman and A, P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol 119, "Chromatin Protocols" {P.B. Becker, ed.) Humana Press, Totowa, 1999, Definitions The terms "nucleic acid", "polynucleotide", and "oligonucleotide" are used interchangeably, and refer to a linearly conforming deoxyribonucleotide or ribonucleotide polymer or circular, and in the form of single or double filament. For the purpose of the present disclosure, these terms are not construed as limiting with respect to the length of a polymer. The terms may involve known analogs of natural nucleotides as well as nucleotides that are modified on the base, sugar and / or phosphate moieties (e.g., phosphorothioate supports). In general, an analog of a particular nucleotide has the same base paired specificity; that is, an analog of A will pair with T.

The terms "polypeptide", "peptide" and "protein11" are interchangeably used to refer to an amino acid residue polymer.The term also applies to amino acid polymers wherein one or more amino acids are chemical analogs or derivatives. corresponding naturally occurring amino acids.

"Binding" refers to a non-covalent sequence-specific interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need to be sequence specific (eg, contact phosphate residues on a DNA support), considering that the interaction as a whole is sequence specific. Such interactions are generally characterized by a dissociation constant (Kd) of 10 "6 M" 1 or less. "Affinity" refers to binding resistance: increased binding affinity being correlated with a lower Kd.

A "binding protein" is a protein that is capable of non-covalently binding to another molecule. A binding protein may bind to, for example, a DNA molecule (a DNA binding protein), an RNA molecule (an RNA binding protein), and / or a protein molecule (a binding protein). of protein). In the case of a protein binding protein, it may bind itself (to form homodimers, homotrimers, etc.), and / or it may bind to one or more molecules of a different protein or proteins. A binding protein may have more than one type of binding activity. For example, zinc finger proteins have DNA binding activity, RNA binding activity, and protein binding activity.

A "zinc finger DNA binding protein" (or binding domain) is a protein, or domain within a larger protein, that binds to DNA in a specific sequence manner through one or more zinc appendages, which are amino acid sequence regions within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.

The zinc appendage binding domain may be "designed" to bind to a predetermined nucleotide sequence. Non-limiting examples of methods for zinc finger protein design are design and selection. A drawn zinc finger protein is the non-naturally occurring protein whose design / composition results primarily from rational criteria. Rational design criteria include application of substitution rules and computerized algorithms for processing information in a database that stores information from existing ZFP drawings and binding data. See, for example, United States Patents 6,140,081; 6,453,242; 6,534,261; and 6,785,613; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496; and United States Patents 6,746,838; 6,866,997; and 7,030,215.

A "selected" zinc finger protein is a protein not found in nature whose production results primarily from an empirical process, such as phage description, interaction trap, or hybrid selection. See, for example, US 5,789,538; US 5,925,523; US 6,007,988; US 6,013,453; US 6,200,759; US 6,733,970; US RE39,229; and WO95 / 19431; WO96 / 06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197 and WO 02/099084.

The term "sequence" refers to a nucleotide sequence of any length, which may be DNA or RNA; it may be linear, circular or branched, and may be either single or double ribbon. The term "donor sequence" refers to a nucleotide sequence that is inserted into a genome. A donor sequence may be of any length, for example, between 2 and 25,000 nucleotides in length (or any integer between or above them), preferably between about 100 and 5,000 nucleotides in length (or any integer in between), more. preferably between about 200 and 2,500 nucleotides in length.

A "homologous sequence" refers to a first sequence that shares a degree of sequence identity with a second sequence, and whose sequence may be identical to that of the second sequence. A "non-identical homologous sequence" refers to a first sequence that shares a degree of sequence identity with a second sequence but whose sequence is not identical to that of the second sequence. For example, a polynucleotide comprising the wild type sequence of a mutant gene is homologous and not identical to the mutant gene sequence. In certain embodiments, the degree of homology between the two sequences is sufficient to permit homologous recombination between them using normal cellular mechanisms. Two non-identical homologous sequences may be of any length, and their degree of nonhomology may be as small as a single nucleotide (for example, for correction of a genomic point mutation by target homologous recombination), or greater than 10. or more kilobases (for example, for insertion of a gene at a predetermined location on a chromosome). Two polynucleotides comprising non-identical homologous sequences need not be of the same length. For example, an exogenous polynucleotide (i.e. donor polynucleotide) of between 20 and 10,000 nucleotides, or nucleotide pair, may be used.

Techniques for nucleic acid determination and amino acid sequence identity are known in the art. Typically, such techniques include determining the mRNA nucleotide sequence for a gene, and / or determining the amino acid sequence thereby encoded, and comparing these sequences to a second nucleotide, or amino acid sequence. Genomic sequences can also be determined and compared in this way. In general, identity refers to an exact nucleotide to nucleotide or amino acid to amino acid match of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) may be compared by determining their percent identity. The percent identity of two sequences, whether nucleic acid or amino acid sequence, is the number of exact matches between two aligned sequences divided by the length of the shortest sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the algorithm. of local homology by Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981). This algorithm can be applied to the amino acid sequence by the use of a classification matrix developed by Dayhoff, Atlas of Protein Sequence and Structure. M.O. Dayhoff ed., 5 suppl. 3: 353-358, National Biomedical Research Foundation, Washington, D.C., USA, and standardized by Gribskov, Nucl. Acids Res. 14 (6): 6745-6763 (1986). An exemplary implementation of this algorithm for determining percent identity of a sequence is provided by the Genetics Computer Group (Madison, WI) in the "BestFit" utility application. The default parameters for this method are described in the Wisconsín Sequence Analysts Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wl). A preferred method of establishing percent identity in the context of the present disclosure is to use copyright protected MPSRCH packaging by the University of Edinburgh developed by John F. Collins and Shane S. Sturrok and distributed by IntelliGenetics, Inc. ( Mountaín View, CA), From this set of packages, the Smith-Waterman algorithm can be employed where foul parameters are used for the classification table (eg open gap penalty of 12, open gap penalty of one , and a clearance of six). From the generated data, the value of "Match" reflects the string identity. Other suitable programs for calculating percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following penalty parameters: genetic code = standard; filter = none; tape = both; cut = 60; wait = 10; Array = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, Gene Bank + EMBL + DDBJ + PDB + Gene Bank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs can be found on the internet. With respect to the sequences described herein, the desired degree range of sequence identity is approximately 35% to 100%, and any integer between them. Typically percent identities between sequences are at least 35% -40%; 40% -45%; 45% -50%; 50% -60%; 60% -70%; 70-75%, preferably 80-82%, more preferably 85-90%, even more preferably 92%, even more preferably 95%, and most preferably 98% sequence identity.

Alternatively, the degree of sequence similarity between polynucleotides can be determined by polynucleotide hybridization under conditions that allow stable duplex formation between homologous regions, followed by single stranded specific nuclease (s) digestion, and determination. in size of the digested fragments. Two nucleic acids, or two polypeptide sequences, are substantially homologous to each other when the sequences exhibit at least about 70% -75%, preferably 80% -82%, more preferably 85% -90%, even more preferably 92%. even more preferably 95%, and most preferably 98% of sequence activity over a defined length of molecules as determined using the above methods. As used herein, substantially homologous also refers to sequences showing complete identity to a specified DNA, or polypeptide sequence. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. The definition of the appropriate hybridization conditions is within the skill of the art. (See, for example, Sambrook et al., Supra; Acid Nucleic Hybridization: A Practical Approach, editors B.D. Hames and S.J. Higgins, (1985) Oxford; Washington, DC; IRL Press).

Selective hybridization of two nucleic acid fragments can be determined as follows. The degree of sequence identity between two nucleic acid molecules affects the efficiency and resistance of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit hybridization of a sequence completely identical to a target molecule. Inhibition of completely identical sequence hybridization can be evaluated using hybridization assays that are well known in the art (e.g. Southern blotting (DNA), Northern blotting (RNA), solution hybridization, or the like, see Sambrook, et. al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, NY). Such assays may be conducted using varying degrees of selectivity, for example using conditions ranging from low to high stringency. If low stringency conditions are employed, the absence of nonspecific binding can be assessed using a secondary probe that still lacks a partial degree of sequence identity (e.g., a probe having less than about 30% of sequence identity with the target molecule) such that, in the absence of nonspecific binding events, the secondary probe will not hybridize to the target. When using a hybridization-based detection system, an acid probe The nucleic acid is chosen, which is complementary to a reference nucleic acid sequence, and then by selection of appropriate probe conditions, and the reference sequence hybridizes to or binds one another to form a nucleic acid sequence. Duplex molecule. A nucleic acid molecule that is capable of selectively hybridizing to a reference sequence under moderately stringent hybridization conditions typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length, having at least approximately 70% sequence identity with the selected nucleic acid probe sequence. Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length, having a sequence identity of more than about 90-95% with the acid probe sequence selected nucleic acid. Hybridization conditions useful for probe / reference sequence hybridization, where the probe and reference sequence have a specific degree of sequence identity, can be determined as known in the art (see, for example, Acid nucleic hybridization: A Practical Approach, editors B, D, Hames and SJ, Higgins, (1985) Oxford; Washington, DC; IRL Press), [0092] Conditions for hybridization are well known to those skilled in the art. Hybridization stringency refers to the degree to which hybridization conditions disadvantage the formation of unequal nucleotide-containing hybrids, with higher stringency correlated with a lower tolerance for unequal hybrids. Factors affecting hybridization stringency are well known to those skilled in the art, and include, but are not limited to, temperature, pH, ionic strength, and concentration of organic solvents such as formamide and dimethyl sulfoxide. Known to those skilled in the art, hybridization stringency is increased by higher temperatures, lower ionic resistance, and lower solvent concentrations.

With respect to stringency conditions for hybridization, it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of the sequences, the base composition of the various sequences, salt concentrations and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., extra d sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as variation of washing conditions. Selection of a particular set of hybridization conditions is selected by following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloninq: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, ΝΎ. ).

"Recombination" refers to a process of exchange of genetic information between two polynucleotides. For the purpose of this disclosure, "homologous recombination (HR)" refers to the specialized form of such exchange that occurs, for example, during repair of double strand breaks in cells. This process requires nucleotide homology sequence, use of a "donor" molecule for template repair of a "target" molecule (ie, the molecule experiencing double stranded breaking), and is variably known as "conversion". non-intersecting gene "or" short stretch gene conversion "because this leads to the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such a transfer may involve correction of heteroduplex DNA mismatch that forms between the broken target and the donor, and / or "synthesis dependent braided annealing", wherein the donor is used to rebuild. - synthesize genetic information that becomes part of the target, and / or related processes. Such specialized HR often results in a change in the sequence of the target molecule such that part or all of the donor polynucleotide sequence is incorporated into the polynucleotide.

"Cleavage" refers to the breakdown of the covalent support of a DNA molecule. Cleavage may be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-filament cleavage and double-filament cleavage are possible, double-filament cleavage can occur as a result of two distinct double-filament cleavage events. DNA cleavage can result in the production of either moderate ends or staggered ends. In certain embodiments, fusion polypeptides are used for double-stranded DNA cleavage.

A "cleavage domain" comprises one or more polypeptide sequences that have catalytic activity for DNA cleavage. A cleavage domain may be contained within a single polypeptide chain, or cleavage activity may result from the association of two (or more) polypeptides.

A "cleavage half-domain" is a polypeptide sequence that together a second (or identical or different) polypeptide forms a complex having splitting activity (preferably double stranded splitting activity).

"Chromatin" is the nucleoprotein structure comprising the cellular genome. Cellular chromatin comprises nucleic acid, primarily DNA, and protein, including histone and nonhistone chromosomal proteins. Most eukaryotic cell chromatin exists in the form of nucleosomes, wherein a nucleosome nucleus comprises approximately 150 base pairs of DNA associated with an octamer comprising two each of histones H2A, H2B, H3 and H4; and articulating DNA (of varying length depending on the organism) extends between nucleosome nuclei. A histone H1 molecule is usually associated with the articulating DNA. For purposes of the present disclosure, the term "chromatin" is meaningful to encompass all types of prokaryotic and eukaryotic cellular nucleoproteins. Cellular chromatin includes both chromosome and episomic chromatin.

A "chromosome" is a chromatin complex comprising all or a portion of the genome of a cell. The genome of a cell is often characterized by its karyotype, which is the collection of all chromosomes that comprise the cell genome. The genome of a cell may comprise one or more chromosomes.

An "episome" is a replicating nucleic acid, nucleoprotein complex or other structure comprising a nucleic acid that is not part of the chromosomal karyotype of a cell. Examples of episomes include plasmids and certain viral genomes.

An "accessible region" is a site in cellular chromatin in which a target site present in nucleic acid may be linked by an exogenous molecule that recognizes the target site. Without wishing to be bound by any particular theory, it is believed that an accessible region is one that is not wrapped in a nucleosome structure. The distinct structure of an accessible region can often be detected by its sensitivity to chemical and enzymatic probes, for example nucleases.

A "target site" or "target sequence" is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided that sufficient conditions for binding exist. For example, the 5'-GAATTC-3 'sequence is a target site for Eco RI restriction endonuclease.

An "exogenous" molecule is a molecule that is not normally present in a cell but can be introduced into a cell by one or more genetic, biochemical, or other methods. "Normal cell presence" is determined with respect to the particular stage of development and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic muscle development is an exogenous molecule relative to an adult muscle cell. Similarly, a heat shock induced molecule is an exogenous molecule with respect to a cell without heat shock. An exogenous molecule may comprise, for example, a working version of a malfunctioning endogenous molecule, or a malfunctioning version of a normally functioning endogenous molecule.

An exogenous molecule may be, among other things, a small molecule, as generated by a combinatorial chemical process, or a macromolecule, such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules. Nucleic acids include DNA and RNA, may be single stranded or double stranded; may be linear, branched or circular; and can be of any length. Nucleic acids include those capable of duplexing as well as triplex-forming nucleic acids. See, for example, United States Patent Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases and helicases.

An exogenous molecule may be the same type of molecule as an endogenous molecule, for example, an exogenous protein, or nucleic acid. For example, an exogenous nucleic acid may comprise a viral genome of infection, an Agrogacterium tumefacians T-braid, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell. Methods for introducing exogenous molecules into cells are known to those skilled in the art, and include, but are not limited to, lipid-mediated transfer (ie, liposomes including neutral and cationic lipids), electroporation, direct injection, cell fusion. , particle bombardment, calcium phosphate coprecipitation, DEAE-dextran mediated transfer, and viral vector mediated transfer.

By contrast, an "endogenous" molecule is one that is normally present in a particular cell at a particular stage of development under particular environmental conditions. For example, an endogenous nucleic acid may comprise a chromosome, the genome of a mitochondria, chloroplast or other organelle, or a naturally occurring episomal nucleic acid. Additional endogenous molecules may include proteins, for example transcription factors and enzymes.

A "fusion" molecule is a molecule to which two or more subunit molecules are preferably and covalently linked. Subunit molecules may be of the same chemical type of molecule, or may be of different chemical types of molecules. Examples of the first type of fusion molecule include, but are not limited to, fusion proteins (e.g., a fusion between a ZFP DNA binding domain and a splice domain), and fusion nucleic acids (e.g., a nucleic acid encoding the fusion protein described above). Examples of the second type of fusion molecule include, but are not limited to, a fusion between a triplex-forming nucleic acid and a polypeptide, and a fusion between a minor slot ligand and a nucleic acid.

Expression of a fusion protein in a cell may result from the distribution of the fusion protein to the cell, or the distribution of a polynucleotide encoding the fusion protein to a cell into which the polynucleotide is transcribed, and transcription. is translated to generate the fusion protein. Transunion, polypeptide splitting, and polypeptide binding may also be involved in the expression of a protein in a cell. Methods for delivering polynucleotide and polypeptide to cells are presented elsewhere in this description.

A "gene" for the purpose of this disclosure includes a DNA region encoding a gene product (see below) as well as all DNA regions that regulate gene product production, whether or not such regulatory sequences are adjacent to the coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, isolators, boundary elements, origins of replication. , matrix fixation sites, and local control regions.

"Gene expression" refers to the conversion of information, contained in a gene, into a gene product. A gene product may be the direct transcriptional product of a gene (for example, mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA), or a protein produced by translation of an mRNA. The gene product also includes RNAs that are modified by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

"Modulation" of gene expression refers to a change in the activity of a gene. Expression modulation may include, but is not limited to, gene activation and gene repression.

"Plant" cells include, but are not limited to, monocot or dicot cells. Non-limiting examples of monocots include cereal plants such as corn, rice, barley, oats, sorghum, rye, sugar cane, pineapple, onion, banana, and coconut. Non-limiting examples of dicots include tobacco, tomato, sunflower, cotton, sugar beet, potato, lettuce, melon, soybean, canola (rapeseed), and alfalfa. Plant cells can be from any part of the plant, and / or from any stage of plant development.

A "region of interest" is any region of cellular chromatin, such as, for example, a gene or non-coding sequence within or adjacent to a gene, where it is desirable to bind to a molecule. exogenous. Binding may be for the purpose of target DNA splitting, and / or target recombination. A region of interest may be present in a chromosome, an episome, an organelle genome (eg, mitochondrial, chloroplast), or a viral infection genome, for example. A region of interest may be within the coding region of a gene, within transcribed non-coding regions, such as, for example, leader sequences, trailing or intron sequences, or within non-transcribed or upstream regions. or downstream of the coding region. A region of interest may be as small as a single nucleotide pair or up to 25,000 nucleotide pairs in length, or any integral value of nucleotide pairs.

The terms "operably linked" and "operably linked" (or "operably linked") are used interchangeably with reference to a juxtaposition of two or more components (such as sequence elements), wherein the components are arranged, such as whereas both components function normally and allow the possibility that at least one of the components may mediate a function that is performed under at least one of the other components. By way of illustration, a transcriptional regulatory sequence, such as a promoter, is operably linked to a coding sequence if the transcriptional regulatory sequence controls the transcriptional level of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptional regulatory sequence is generally operably linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence that is operably linked to a coding sequence even though they are not contiguous.

With respect to fusion polypeptides, the term "operably linked" may refer to the fact that each of the components performs the same function in binding to the other component as it would be if it were not thereby bound. For example, with respect to a fusion polypeptide in which a ZFP DNA binding domain is fused to a dividing domain, the ZFP DNA binding domain and the dividing domain are operatively linked if, in the fusion polypeptide, the binding domain portion of ZFP DNA is capable of binding to its target site and / or its binding site, while the divider domain is capable of cleaving DNA in the vicinity of the target site.

A "functional fragment" of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full length protein, polypeptide or nucleic acid, yet retains the same function as the length protein. polypeptide or nucleic acid. A functional fragment may have more, less, or the same number of residues as the corresponding native molecule, and / or may contain one or more amino acid or nucleotide substitutions. Methods for determining the function of one nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well known in the art. Similarly, methods for determining protein function are well known. For example, the DNA binding function of a polypeptide may be determined, for example, by filter binding, electrophoretic mobility alteration, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. See, Ausubel et al., Supra. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays or complementation, both genetic and biochemical. See, for example, Fields et al. (1989) Nature 340: 245-246; United States Patent No. 5,585,245 and PCT WO 98/44350.

Target Sites The disclosed methods and compositions include fusion proteins comprising a dividing domain (or a half-dividing domain) and a zinc finger domain, wherein the zinc finger domain, by binding to a Cell chromatin sequence (for example, a target site, or a binding site) directs the activity of the diving domain (or half-domain domain) to the vicinity of the sequence and therefore induces divagem in the vicinity of the sequence. -target. As placed somewhere in this description, a zinc finger domain can be designed to bind virtually any desired sequence. Accordingly, upon identification of a region of interest containing a sequence in which splitting or recombination is desired, one or more zinc appendage binding domains may be designed to bind to one or more sequences in the region of interest. Expression of a fusion protein comprising a zinc finger binding domain and a dividing domain (or two fusion proteins each comprising a zinc finger binding domain and a half dividing domain), in a cell, divides the region of interest.

Selection of a cell chromatin sequence for binding by a zinc finger domain (e.g., a target site) may be accompanied, for example, according to the methods disclosed in the same applicant's United States Patent No. 6,453,242 (September 17, 2002), which also discloses methods for assigning ZFPs to bind to a selected sequence. It will be clear to one skilled in the art that simple visual inspection of a nucleotide sequence can also be used to select a target site. Accordingly, any means for target location selection may be used in the claimed methods.

Target sites are generally composed of a plurality of adjacent sub-targets. A sub-target refers to the sequence (usually either a nucleotide trio, or a nucleotide quartet, which may overlap with a nucleotide with an adjacent quartet) linked by an individual zinc finger. See, for example, WO 02/077227. If the braid that a zinc finger protein makes the most contact with is called the "primary recognition braid" or "primary contact braid" target filament, some zinc finger proteins bind to three base trios in the filament. target, and to a fourth base in the non-target braid. A target site generally has a length of at least 9 nucleotides and is consequently linked by a zinc finger binding domain comprising at least three zinc appendages. However, binding of, for example, 4 appendix binding domains to 12 nucleotide target sites, 5 appendix binding domains to 15 nucleotide binding sites, or 6 appendix binding domains to 18 nucleotide target sites. nucleotide is also possible. As will be apparent, binding of larger binding domains (e.g., 7-, 8-, 9-appendix, and more) to larger binding sites is also possible.

Target site need not be a multiple of three nucleotides. For example, in cases where cross-braided interactions occur (see, for example, U.S. Patent 6,453,242 and WO 02/077227), one or more of the individual zinc appendages of a multi-appendix binding domain may bind. to overlay quartet subplots. As a result, a three appendage protein may bind to 10 nucleotide sequences, in which the tenth nucleotide is part of a quartet linked by a terminal appendage, a four appendage protein may bind to 13 nucleotide sequences, in which the thirteenth nucleotide is part of a quartet linked by a terminal appendix, etc.

The length and nature of amino acid linker sequences between individual zinc appendages in a multi-appendix binding domain also affect binding to a target sequence. For example, the presence of a so-called "noncanonical articulator", "long articulator", or "structured articulator" between adjacent zinc appendages in a multi-appendage binding domain may allow those appendages to bind to subplaces that they are not immediately adjacent. Non-limiting examples of such articulators are described, for example, in United States Patent No. 6,479,626 and WO 01/53480. Consequently, one or more subplots, at a target site for a zinc finger binding domain, may be separated from one another by 1, 2, 3, 4, 5 or more nucleotides. To provide one example, however, a four-appendage binding domain may bind to a 13-nucleotide target site comprising, in sequence, two contiguous 3 nucleotide sublocals, one intervention nucleotide, and two contiguous triage sublocals.

Sequence distance (e.g., target sites) refers to the number of nucleotides or nucleotide pair intervening between two sequences, as measured from the edges of the sequences closest to one another.

In certain embodiments where splitting relies on the binding of two zinc finger domains / splitting half-domain fusion molecules to separate target sites, the two target loci may be in opposite DNA strands. In other embodiments, both target sites are on the same strand of DNA. Zinc Finger Binding Domains A zinc finger binding domain comprises one or more zinc appendages. Miller etal, (1985) EMBO J, 4: 1609-1614; Rhodes (1993) Scientific American Feb.:56-65; United States Patent No. 6,453,242. Typically, a single zinc finger domain is about 30 amino acids in length. Structural studies have shown that each zinc finger domain (motif) contains two beta sheets (held on a beta loop that contains the two invariant cysteine residues), and an alpha spiral (containing the two invariant histidine residues), which are maintained in a particular conformation by the coordination of a zinc atom by the two cysteines and the two histidines.

Zinc appendices include both canonical C2H2 zinc appendices (ie those in which the zinc ion is coordinated by two cysteine and histidine residues), and non-canonical zinc appendages such as, for example, appendices. zinc C3H (those where the zinc ion is coordinated by three cysteine residues and one histidine residue), and C4 zinc appendices (those where the zinc ion is coordinated by four cysteine residues). See also WO 02/057293.

Zinc finger binding domains can be designed to bind to a sequence of choice. See, for example, Beerli et al. (2002) Nature BiotechnoL 20: 135-141; Rabo et al. (2001) Am. Rev. Biochem. 70: 313-340; Isalan et al. (2001) Nature Biotechnol 19: 656-660; Segai et af. (2001) Curr. Op. Biotechnoi. 12: 632-637; Choo etal. (2000) Curr. Opin. Struct. Biol. 10: 411-416. A designed zinc finger binding domain can have a binding specificity compared to a naturally occurring zinc finger protein. Design methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, use of databases comprising nucleotide sequence trio (or quartet) and individual amino acid zinc finger sequence, wherein each trio or quartet of nucleotide sequences is associated with one or more sequences. amino acids from zinc appendages that bind to the particular trio or quartet of sequence. See, for example, U.S. Patent Nos. 6,453,242 and 6,534,261. Additional design methods are disclosed, for example, in United States Patents 6,746,838; 6,785,613; 6,866,997; and 7,030,215.

Exemplary selection methods, including phage description and two-hybrid systems, are disclosed in United States Patent 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB 2,338,237.

The enhancement of binding specificity for zinc appendage binding domain has been described, for example, in U.S. Patent No. 6,794,136.

Provided that an individual zinc finger binds to a three nucleotide sequence (i.e. trio) (or a four nucleotide sequence that overlaps with a nucleotide with the fourth nucleotide binding site of a finger) adjacent zinc), the length of a sequence to which a zinc finger binding domain is designed to bind (for example, a target sequence) will determine the number of zinc appendages in a zinc finger binding domain. designed. For example, for ZFPs where appendix motifs do not bind to overlapping sublocals, a six nucleotide target sequence is bound by a three appendage binding domain; a nine nucleotide target sequence is linked by a three appendage binding domain, etc. As noted herein, binding sites for individual (i.e. subclocal) zinc appendages at a target site need not be contiguous, but may be separated by one or more nucleotides, depending on the length and nature of amino acid sequences between the appendages. of zinc (ie inter-appendage articulators) in an appendix binding domain.

In a multi-appendix zinc finger-binding domain, adjacent zinc appendages may be separated by approximately 5 amino acid linker amino acid sequences (so-called "canonical" inter-appendage linkers) or, alternatively, by one or more non-linkers. -canonicals. See, for example, U.S. Patent Nos. 6,453,242 and 6,534,261. For designed zinc appendage binding domain comprising more than three appendages, insertion of longer ("non-canonical") interappendicular aticulators between certain of the zinc appendages may be preferred as it may increase affinity and / or specificity. binding by the binding domain. See, for example, United States Patent No. 6,479,626 and WO 01/53480. Consequently, the multiappendix zinc appendage binding domain can also be characterized with respect to the presence and location of non-canonical interappendix articulators. For example, a six-appended zinc finger binding domain comprising three appendages (joined by two canonical inter-appendage articulators), one long articulator, and three additional appendages (joined by two canonical interappex articulators) is denoted a 2x3 configuration. Similarly, a binding domain comprising two appendages (with a canonical articulator between them), a long articulator, and two additional appendages (joined by a canonical articulator), is denoted a 2x2 protein. A protein comprising three units of two appendages (each of which the two appendages are joined by a canonical articulator), and in which each unit of two appendages is joined to two adjacent appendix units by a long articulator, is referred to as one. 3x2 protein.

[00131] The presence of a nonconical inter-appendage articulator between two adjacent zinc appendages in a multi-appendix binding domain often allows the two appendages to bind to subclasses that are not immediately contiguous in the target sequence. Consequently, there may be gaps in one or more nucleotides between sub-sites at a target site; that is, a target site may contain one or more nucleotides that are not contacted by a zinc finger. For example, a 2x2 zinc finger binding domain can bind to two six nucleotide sequences separated by one nucleotide, that is, it binds to a 13 nucleotide target site. See also Moore et al. (2001a) Proc. Natl. Acad. Know. USA 98: 1432-1436; Moore et al. (2001b) Proc. Natl. Acad. Know. USA 98: 1437-1441 and WO 01/53480.

As mentioned earlier, a sub-target is a three- or four-nucleotide sequence that is linked by a single zinc finger. For certain proposals, a two-appended unit is denoted a connector module. A binding module can be obtained by, for example, selecting two adjacent appendages in the context of a multi-appendix protein (usually three appendages) that bind to six particular target nucleotide sequences. Alternatively, modules may be constructed by mounting individual zinc appendages. See also WO 98/53057 and WO 01/53480, Cleavage Domains The domain-splice portion of the fusion proteins disclosed herein can be obtained from any endonudease or exonuclease. Exemplary endonudeases from which a dividing domain may be derived include, but are not limited to, self-directional restriction endonudeases. See, for example, Catalog 2002-2003, New England Biolabs, Beverly, MA; and Belfort et al. (1997} Acid nudeics Res. 25: 3379-3388, Additional DNA cleaving enzymes are known (e.g., S1 Nuclease; Mung bean nuclease; Pancreatic DNase I; micrococcal nuclease; HO endonudease yeast; see also Lingun et al. ( eds.) Nudeases, Cold Spring Harbor Labyrous Press, 1993), One or more of these enzymes (or functional fragments thereof) may be used as a source of cleavage domains and dividing half-domains.

Similarly, a dividing half-domain (e.g., fusion proteins comprising a zinc finger binding domain and a dividing half-domain) may be derived from any nuclease or portion thereof as set forth above which requires dimming for diving activity. In general, two fusion proteins are required for splitting if the fusion proteins comprise cleavage half domains. Alternatively, a simple protein comprising two cleavage half domains may be used. The two splitting half-domains may be derived from the same endonudease (or functional fragments thereof), or each cleavage half-domain may be derived from a different endonudease (or functional fragments thereof). In addition, the target sites for the two fusion proteins are preferably disposed relative to one another such that the binding of the two fusion proteins to their respective sites occurs at the half-spacing domains in a spatial orientation to each other. , which allows the splitting half-domains to form a functional splitting domain, for example by dimerization. Thus, in certain embodiments, the edges near the target sites are separated by 5-8 nucleotides, or by 15-18 nucleotides. However, any integral number of nucleotides or nucleotide pairs can intervene between two target sites (e.g., 2 to 50 nucleotides or more). In general, the dividing point occurs between the target locations.

Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequence-specific DNA binding (at a recognition site), and DNA dividing at or near the binding site. Certain restriction enzymes (eg, Type IIS) cleave DNA at sites removed from the recognition site, and have detachable binding and splitting domains. For example, the IIS Fok I Type enzyme catalyzes double stranded DNA splitting into 9 nucleotides from its recognition site in one braid and 13 nucleotides from its recognition site in the other. See, for example, United States Patent 5,356,802; 5,436,150 and 5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Know. USA 89: 4275-4279; Li et al. (1993) Proc. Natl. Acad. Know. USA 90: 2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Know. USA 91: 883-887; Kim et al. (1994b) J. Biol. Chem. 269: 31.978-31.982. Thus, in one embodiment, fusion proteins comprise the dividing domain (or dividing half-domain) of at least one IIS Type restriction enzyme and one or more zinc appendage binding domains, which may or may not be designed. .

An exemplary IIS Type restriction enzyme, whose dividing domain is separable from the binding domain, is Fok I. This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc.

Natl. Acad. Know. USA 95: 10,570-10,575. Accordingly, for the purpose of the present disclosure, the portion of the Fok I enzyme used in the disclosed fusion proteins is considered a dividing half-domain. Thus, for double-stranded targeting and / or targeting of cell sequences using zinc-Fo / c I finger fusions, two fusion proteins, each comprising a Fok1-dividing half-domain, can be used to reconstitute a catalytically active dialing domain. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two Fok1 splitting half-domains may also be used. Parameters for target cleavage and target sequence alteration using Fok I zinc finger fusions are provided anywhere in this description.

A split domain or split half domain can be any portion of a protein that retains dividing activity, or that retains the ability to multimerize (e.g., dimerize) to form a functional divide domain.

Exemplary IIS Type restriction enzymes are listed in Table 1. Additional restriction enzymes also contain linkage and separable splitting domains, and these are contemplated by the present disclosure. See, for example, Roberts et al. (2003) Acid nucleics Res. 31: 418-420.

Table 1: Some Restriction Enzymes Type IIS Aar I BsrB I SspD51 Ace III BsrDI Sth1321 Aci I BstF51 Sts I

Hello I Btr I TspDT I

Bae I Bts I TspGW I

Bbr71 Cdi I Tth11111 Bbv I CjeP I UbaP I

Bbv II Drd II Bsa I

BbvC I Eci I BsmB I

Bcc I Eco31 I

Bce83 I Eco57 I

BceA I Eco57M I

Bcef I Esp3 I

Bcg I Fau I

BciV I Fin I

Bfi I Fok I

Bin I Gdi II

Bmg I Gsu I

Bpu10 I Hga I

BsaX I Hin4 II

Bsb I HphI

BscA I Ksp632 I

BscG I Mbo II

BseR I Mly I

BseY I Mme I

Bsi I

Bsml Pfl 1108 I

BsmA I Ple I

BsmF I Ppi I

Bsp24 I Psr I

BspG I RleA I

BspM I Sap I

BspNC I SfaN I

Bsr I Yes I

Diving Domain Zinc Finger-Mergers Domain Methods for delineating and constructing fusion proteins (and polynucleotides encoding them) are known to those skilled in the art. For example, methods for fusion protein delineation and construction comprising zinc finger proteins (and polynucleotides encoding them) are described in the same United States Patents 6,453,242 and 6,534,261. In certain embodiments, polynucleotides encoding such fusion proteins are constructed. These polynucleotides may be inserted into a vector and the vector may be introduced into a cell (see below for further description regarding vectors and methods for introducing polynucleotides into cells).

In certain embodiments of the methods described herein, a fusion protein comprises a zinc finger binding domain and a dividing half-domain from the restriction enzyme Fok I, and two such fusion proteins are expressed in one fusion protein. cell. Expression of two fusion proteins in a cell may result from the distribution of two of the proteins to the cell; delivery of a protein and nucleic acid encoding one of the proteins to the cell; distribution of two nucleic acids, each encoding one of the proteins to the cell; or by delivery of a single nucleic acid, which encodes both proteins to the cell. In further embodiments, a fusion protein comprises a single polypeptide chain comprising two splitting half-domains and a zinc finger binding domain. In this case, a single fusion protein is expressed in a cell and, without wishing to be bound by theory, is believed to cleave DNA as a result of formation of an intramolecular dimer of the dividing half domains.

In certain embodiments, the fusion protein components (e.g., ZFP-Fok I fusions) are arranged such that the zinc finger domain is closer to the amino terminus of the fusion protein, and the half domain of divagem is closer to the carboxy terminal. This mirrors the relative orientation of the splitting domain in naturally occurring dimerization splitting domains, such as those derived from the Fok I enzyme, where the DNA binding domain is closer to the amino terminus, and the dividing half-domain. is closer to the carboxy terminal. In these embodiments, the dimerization of the dividing half-domains to form a functional nuclease is provided by the binding of fusion proteins to sites on opposite DNA strands, with the 5 'ends of the binding sites being proximal to one another.

In further embodiments, the fusion protein components (e.g., ZFP-Fok I fusions) are arranged such that the dividing half domain is closer to the amino terminus of the fusion protein, and the finger domain zinc is closer to the carboxy terminal. In these embodiments, the dimerization of the dividing half-domains to form a functional nuclease is provided by the binding of fusion proteins to sites on opposite DNA strands, with the 3 'ends of the binding sites being proximal to one another.

In still further embodiments, a first fusion protein contains the divider half-domain closest to the amino terminus of the fusion protein, and the zinc finger domain closest to the carboxy terminus, and a second fusion protein is disposed such that the zinc finger domain is closer to the amino terminus of the fusion protein, and the dividing half-domain is closer to the carboxy terminus. In these embodiments, both fusion proteins bind to the same DNA strand, with the first fusion protein binding site containing the zinc finger domain closest to the carboxy terminal located on the 5 'side of the second fusion protein binding site. fusion containing the zinc finger domain closest to the amino terminus.

In certain embodiments, the fusion proteins disclosed, the amino acid sequence between the zinc finger domain and the dividing domain (or half-dividing domain) is referred to as the "ZC linker". The ZC articulator is to be distinguished from the interappendix articulators discussed above. See, for example, United States Patent Publications 20050064474A1 and 20030232410, and International Patent Publication W005 / 084190, for details on obtaining divan optimizing ZC articulators. Methods for Target Cleavage The disclosed methods and compositions may be used to cleave DNA in a region of interest in cellular chromatin (for example, at a desired or predetermined location in a genome, for example, in a gene, or mutant). , or wild type). For such target DNA splitting, a zinc finger binding domain is designed to bind to a target site at or near the predetermined dividing site, and a fusion protein comprising the designed zinc finger binding domain, and a divide domain is expressed in a cell. Upon binding of the zinc finger portion of the fusion protein to the target site, DNA is cleaved near the target site by the dividing domain. The exact dividing location may depend on the length of the ZC handler.

Alternatively, two fusion proteins, each comprising a zinc finger binding domain and a dividing half domain, are expressed in a cell, and bind to target sites that are juxtaposed in such a way that a functional dividing domain is reconstituted, and DNA is cleaved in the vicinity of the target sites. In one embodiment, splitting occurs between the target sites of the two zinc finger binding domains. One or both of the zinc finger binding domains may be designed.

For target cleavage using a zinc-finger binding domain-domain-splice fusion polypeptide, the binding site may involve the divider site, or the proximal edge of the binding site may be 1, 2, 3, 4, 5, 6, 10, 25, 50 or more nucleotides (or any integer between 1 and 50 nucleotides) from the divider site. The exact location of the binding site with respect to the divider location will depend on the particular divider domain, and the length of the ZC pivot. For methods in which two fusion polypeptides, each comprising a zinc finger binding domain and a split half domain, are used, the binding sites generally extend at the split site. Thus, the border near the first binding site may be 1, 2, 3, 4, 5, 6, 10, 25 or more nucleotides (or any integral value between 1 and 50 nucleotides), one on one side of the binding site. and the border near the second binding site may be 1, 2, 3, 4, 5, 6, 10, 25 or more nucleotides (or any integer between 1 and 50 nucleotides) on the other side of the dividing site. . Methods for mapping in vitro and in vivo dividing sites are known to those skilled in the art.

Accordingly, the methods described herein may employ a designed zinc finger binding domain fused to a divider domain. In certain cases, the binding domain is designed to bind to a target sequence, at or near, whose splitting is desired. The fusion protein, or a polynucleotide encoding it, is introduced into a plant cell. Once introduced into or expressed in the cell, the fusion protein binds to the target sequence, and cleaves at or near the target sequence. The exact location of the divide depends on the nature of the divide domain and / or the presence and / or nature of the articulating sequences between the link and the divide domains. In cases where two fusion proteins, each comprising a dividing half-domain, are used, the distance between the edges near the binding sites may be 1, 2, 3, 4, 5, 6, 7, 8, 9. , 10, 25 or more nucleotides (or any integer between 1 and 50 nucleotides). Optimal levels of dividing may also depend on both the distance between the binding sites of the two fusion proteins (see, for example, Smith et al. (2000) Acid nucleics Res. 28: 3361-3369; Bibikova et al. (2001 ) Mol. Cell Biol. 21: 289-297), and the length of the ZC pivot on each fusion protein. See also United States Patent Publication 20050064474A1 and International Patent Publications W005 / 084190, W005 / 014791 and W003 / 080809.

In certain embodiments, the divide domain comprises two split half domains, both of which are part of a single polypeptide comprising a binding domain, a first split half domain and a second split half domain. The dividing half-domains may have the same or different amino acid sequences as they function to cleave the DNA.

Dividing half-domains may also be provided in separate molecules. For example, two fusion polypeptides may be introduced into a cell in which each polypeptide comprises a binding domain and a half-domain of domain. The dividing half-domains may have the same amino acid sequence or different amino acid sequences, provided they function to cleave the DNA. Additionally, the binding domains bind to the target sequences that are typically arranged such that, upon binding of the fusion polypeptides, the two splitting half-domains are presented in a spatial orientation relative to each other, allowing for reconstitution. of a dividing domain (e.g., by dimerizing the half domains), thereby positioning the half domains relative to each other to form a functional dividing domain, resulting in cell chromatin splitting into a region of interest . Typically, splitting by the reconstituted splitting domain occurs at a location located between the two target sequences. One or both of the proteins may be designed to bind to their target site.

The two fusion proteins may bind in the region of interest at the same opposite polarity, and their binding sites (ie, target sites) may be separated by any number of nucleotides, for example from 0 to 200 nucleotides. , or any full value between them. In certain embodiments, the binding sites for two fusion proteins, each comprising a zinc finger binding domain and a dividing half domain, may be located between 5 and 18 separate nucleotides, for example, 5-8 nucleotides. separated, or 15-18 separate nucleotides, or 6 separate nucleotides, or 16 separate nucleotides, as measured from the edge of each binding site closest to the other binding site, and splitting occurs between the binding sites.

The site at which DNA is cleaved is generally between the binding sites for the two fusion proteins. Double stranded DNA breakdown often results from two single stranded breaks, or "incisions" spaced 1, 2, 3, 4, 5, 6, or more apart, (for example, native Fok I double stranded DNA splitting results from single strand breaks 4 nucleotides apart). As such, dividing does not occur at exactly opposite locations in each strand of DNA. In addition, the structure of the fusion proteins and the distance between target sites may influence whether splitting occurs adjacent to a single nucleotide pair, or if splitting occurs at multiple sites. However, for many applications, including target recombination and target mutagenesis (see below), dividing within a range of nudeotides is generally suffident, and particular base pairing is not required.

As noted above, the fusion protein (s) may be introduced as polypeptides and / or polynucleotides. For example, two polynucleotides, each comprising sequences encoding one of the aforementioned polypeptides, may be introduced into a cell, and when the polypeptides are expressed, and each binds to its target sequence, splitting occurs at or near the sequence. -target. Alternatively, a single polynucleotide comprising sequences encoding both fusion polypeptides is introduced into a cell. Polynucleotides may be DNA, RNA or any modified forms, or analogs, or DNA and / or RNA.

To enhance divinity specificity, additional compositions may also be employed in the methods described herein. For example, single-dial media domains may exhibit limited double-filament dialing activity. In methods in which two fusion proteins, each containing three appendage zinc finger domain, a half-domain of division, are introduced into the cell, or protein specifies a target site of approximately 9 nudeotides. Although the aggregated 18 nudeotide target sequence is similarly unique in a mammalian genome, any given 9 nudeotide targeting site occurs on average approximately 23,000 times in the human genome. Thus, nonspecific splitting due to local specific binding of a simple half-domain can occur. Accordingly, the methods described herein contemplate the use of a dominant negative mutant of a dividing half-domain such as Fok (or a nucleic acid encoding the same) that is expressed in a cell together with the two fusion proteins. The dominant negative mutant is able to dimerize but is unable to cleave, as well as blocking the dividing activity of a half domain to which it is dimerized. By providing the dominant molar excess negative mutant for fusion proteins, only regions in which both fusion proteins are linked will have a fairly high local concentration of functional divider half-domains for dimerization and divider to occur. two fusion proteins are bound, their dividing half-domain forms a dimer with the dominant negative mutant half-domain, and undesirable, non-specific dividing does not occur, [00155] Three catalytic amino acid residues in the half-domain of Fok I data were identified: Asp 450, Asp 467 and Lys 469. Bitinaite ef al. (1998) Proc. Nati. Acad, I know. USA 95: 10,570-10,575. Thus, one or more mutations in one of these residues can be used to generate a dominant negative mutation. Additionally, many of the catalytic amino acid residues of other Type IIS endonucleases are known and / or may be determined, for example, by alignment with Fok I sequences, and / or by generation and testing of mutants for catalytic activity. Dimerization of Domain Mutations in Clivaqem's Mid Domain [00156] Methods for target cleavage involving the use of fusions between a ZFP and a split half domain (such as, for example, a ZFPiFokl fusion) requires the use of two such fusion molecules, each generally directed to a distinct target sequence. Target sequences for the two fusion proteins may be chosen such that target cleavage is directed to a single site in a genome, as discussed above. A potential source of reduced dividing specificity may result from homodimerization of one of the dividing half-domain ZFPs / fusions. This may be due, for example, to the presence in one genome of inverted repeats of the target sequences for one of the two ZFP / split-domain fusions located so as to allow two copies of the same fusion protein to bond with an orientation and spacing that allow formation of a functional dimer.

One approach to reducing the likelihood of this type of aberrant divagem in sequences other than the intended target site involves generation of divider half-domain variants that minimize or prevent homodimerization. Preferably, one or more amino acids in the mid-domain region involved in their dimerization are altered. In the crystal structure of the Fok? Protein dimer, the structure of the dividing half-domains is reported to be similar to the arrangement of the dividing half-domains during Fok? Wah et al (1998) Proc. Natl. Acad. Know. USA 95: 10564-10569. This structure indicates that amino acid residues at positions 483 and 487 play a key role in the dimerization of Fok? The structure also indicates that amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 are all closed tightly to the interface. of dimerization to influence dimerization. Accordingly, amino acid sequence changes at one or more of the aforementioned positions will likewise alter the dimerization properties of the dividing half domain. Such changes may be introduced, for example, by constructing a library containing (or encoding) different amino acid residues at these positions and selecting variants with the desired properties, or by rationally delineated individual mutants. In addition to homodimerization prevention, it is also possible that some of these mutations may increase the dividing efficiency above that obtained with two wild-type dividing half domains.

Accordingly, alteration of a Fok \ dividing half-domain into any amino acid residue that affects dimerization can be used to prevent one of a pair of ZFP / Fok \ fusions from supporting homodimerization that can lead to divodation. unwanted sequences. Thus, for target cleavage using a pair of ZFP / Fok \ fusions, one or both of the fusion proteins may comprise one or more amino acid changes that inhibit autodimerization, but allow heterodimerization of the two fusion proteins to occur such that splitting occurs. occurs at the desired target location. In certain embodiments, alterations are present in both fusion proteins, and the alterations have additive effects; that is, homodimerization of any fusion leading to aberrant splitting is minimized or abolished, while heterodimerization of the two fusion proteins is facilitated compared to that obtained with wild-type splitting half-domains. Methods for Target Alteration of Genomic Sequences and Target Recombination Also described herein are methods of replacing a genomic sequence (e.g., a region of interest in cell chromatin) with a non-identical homologous sequence (i.e. recombination). target). Previous attempts to replace particular sequences have involved contacting a cell with a polynucleotide comprising sequences that support homology to a chromosomal region (i.e., a donor DNA), followed by selection of cells in which the donor DNA molecule had supported homologous recombination in the genome. . The success rate of these methods is low due to poor homologous recombination efficiency and a high frequency of non-specific donor DNA insertion into regions of the genome other than the target site.

The present description provides methods of target sequence alteration characterized by a higher target recombination efficiency and a lower frequency of non-specific insertion events. The methods involve the production and use of engineered zinc appendage binding domain fused to divanding domains (or divaging half-domains) to produce one or more double stranded target breaks in cellular DNA. Because double stranded breaks in cellular DNA stimulate cellular repair mechanisms several thousand times in the vicinity of the dividing site, such target cleavage allows sequence alteration or substitution (via homology driven repair) at virtually any site in the genome.

In addition to the fusion molecules described herein, target substitution of a selected genomic sequence also requires the introduction of the substitution (or donor) sequence. The donor sequence may be introduced into the cell prior to, concurrently with or subsequent expression of the fusion protein (s). The donor polynucleotide contains sufficient homology for a genomic sequence to support homologous recombination (or homology driven repair) between it and the genomic sequence to which it supports homology. Approximately 25, 50 100, 200, 500, 750, 1,000, 1,500, 2,000 nucleotides or more of sequence homology between a donor sequence and a genomic sequence (or any integral value between 10 and 2,000 nucleotides or more) will support homologous recombination between these. Donor sequences may range in length from 10 to 5,000 nucleotides (or any integral nucleotide value in between), or greater. It will be readily apparent that the donor sequence is not typically not identical with the genomic sequence it replaces. For example, the donor polynucleotide sequence may contain one or more single base changes, insertions, withdrawals, inversions or rearrangements with respect to the genomic sequence, provided that sufficient homology to chromosomal sequences is present. Alternatively, a donor sequence may contain a non-homologous sequence flanked by two regions of homology. Additionally, donor sequences may comprise a vector molecule containing sequences that are not homologous to the region of interest in the cellular chromatin. Generally, the homologous region (s) of a donor sequence will have at least 50% sequence identity to a genomic sequence with which recombination is desired. In certain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity may be present, depending on the length of the donor polynucleotide.

A donor molecule may contain several discontinuous regions of cell chromatin homology. For example, for targeting sequences not normally present in a region of interest, said sequences may be present in a donor nucleic acid molecule and flanked by regions of sequence homology in the region of interest.

To simplify assays (e.g., hybridization, PCR, restriction enzyme digestion) for successful determination of the donor sequence, certain sequence differences may be present in the donor sequence as compared to the genomic sequence. Preferably, if located in a coding region, such nucleotide sequence differences will not change the amino acid sequence, or will produce dumb changes in the amino acid (ie, changes that do not affect protein structure or function). The donor polynucleotide may optionally contain sequence changes corresponding to the zinc finger domain binding sites in the region of interest, to prevent splitting of donor sequences that have been introduced into cell chromatin by homologous recombination.

The donor polynucleotide may be single stranded or double stranded DNA or RN A, and may be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence may be protected (e.g., from exonucleolytic degradation) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues are added to the 3 'terminus of a linear molecule and / or autocomplementary oligonucleotides are attached at one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Know. USA 84: 4959-4963; Nehls et al. (1996) Science 272: 886-889. Additional methods for protection of exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group (s) and the use of modified internucleotide bonds such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose, or residues. of deoxyribose.

A polynucleotide may be introduced into a cell as part of a vector molecule having additional sequences such as, for example, origins of replication, promoters, and genes encoding antibiotic resistance. In addition, donor polynucleotides may be introduced as exposed nucleic acid, as nucleic acid complexed with an agent, such as a liposome or poloxamer, or may be distributed by bacteria or viruses (e.g. Agrobacterium, Rhizobium sp. NGR234, Sinorbizoboium meiiiotl, Mesorhizoblum loti, tobacco mosaic virus, potato X virus, cauliflower mosaic virus, and cassava vein mosaic virus See, for example, Ghung et al. (2006) Trends Plant Sci 11 (1) : 1-4.

Without being bound by theory, it appears that the presence of a double stranded break in a cell sequence, coupled with the presence of an exogenous DNA molecule having homology to a region adjacent to, or surrounding the break, activates mechanisms. cells that repair breakdown by transferring sequence information from the donor molecule to the cell sequence (e.g., genomic or chromosomal); that is, by a homology driven repair process, also known as "gene conversion". Applicant's methods advantageously combine the powerful targeting capabilities of ZFPs designed with a split domain (or split half domain) to specifically target a double stranded break to the genome region in the insertion of exogenous sequences is desired.

For alteration of a chromosome sequence, it is not necessary for the total donor sequence to be copied to the chromosome, since much of the donor sequence is copied to effect the desired sequence change.

The efficiency of insertion of donor sequences by homologous recombination is inversely related to the distance in cellular DNA between the standard braided break and the location at which recombination is desired. In other words, higher homologous recombination efficiencies are observed when the double strand break is closest to the place where recombination is desired. In cases where a precise recombination site is not predetermined (for example, the desired recombination event may occur over a genomic sequence interval), the donor nucleic acid length and sequence, together with the site (s) Divation values are selected to obtain the desired recombination event. In cases where the desired event is designed to change the sequence of a single nucleotide pair into a genomic sequence, cellular chromatin is cleaved within 10,000 nucleotides on either side of that nucleotide pair. In certain embodiments, splitting occurs within 1,000, 500, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, or 2 nucleotides, or any integer between 2 and 1,000 nucleotides, on either side of the nucleotide pair whose sequence is to be changed.

As detailed above, the binding sites for two fusion proteins, each comprising a zinc finger binding domain and a half-dividing domain, may be located 5-8 or 15-18 separate nucleotides, as shown below. measured from the edge of each binding site closest to the other binding site, and dividing occurs between the binding sites. Whether splitting occurs at a single site or at multiple sites between the binding sites is immaterial, since cleaved genomic sequences are replaced by donor sequences. Thus, for efficient alteration of the sequence of a single nucleotide pair by target recombination, the midpoint of the region between the binding sites is within 10,000 nucleotides of that nucleotide pair, preferably within 1,000 nucleotides, or 500 nucleotides, or 200 nucleotides, or 100 nucleotides, or 50 nucleotides, or 20 nucleotides, or 10 nucleotides, or 5 nucleotides, or 2 nucleotides, or one nucleotide, or in the nucleotide pair of interest.

In certain embodiments, a homologous chromosome may serve as the donor polynucleotide. Thus, for example, correction of a mutation in a heterozygote can be achieved by engineered fusion proteins that bind to and cleave the mutant sequence on a chromosome, but do not cleave the wild-type sequence on the homologous chromosome. Double stranded breakage on the mutation-bearing chromosome stimulates a homology-based "gene conversion" process in which the wild-type sequence from the homologous chromosome is copied to the cleaved chromosome, thereby restoring two copies of the wild-type sequence. .

Methods and compositions are also provided that may increase target recombination levels including, but not limited to, the use of additional ZFP-functional domain fusions to activate expression of genes involved in homologous recombination, such as, for example. , members of the RAD52 epistasis group (e.g. Rad50, Rad51, Rad51B, Rad51C, Rad51D, Rad52, Rad54, Rad54B, Mre11, XRCC2, XRCC3), genes whose products interact with the aforementioned gene products (e.g., BRCA1, BRCA2) and / or genes in the NBS1 complex. See, for example, Boyko et al. (2006) Plant Physiology 141: 488-497 and LaFarge et al. (2003) Acid nucleics Res 31 (4): 1148-1155. Similarly functional domain ZFP-fusions may be used, in combination with the methods and compositions disclosed herein, to suppress expression of genes involved in non-homologous terminal coupling (e.g., Ku70 / 80, XRCC4, poly (ADP ribose) polymerase, DNA ligase 4). See, for example, Riha et al. (2002) EMBO 21: 2819-2826; Freisner et al. (2003) Plant J. 34: 427-440; Chen et al. (1994) European Journal of Biochemistry 224: 135-142. Methods for activating and repressing gene expression using fusions between a zinc finger binding domain and a functional domain are disclosed, for example, in U.S. Patent Nos. 6,534,261; 6,824,978 and б. 933,113. Additional repression methods include the use of target antisense oligonucleotides and / or small interference RNA (siRNA or RNAi) targeting the gene sequence to be repressed.

As an alternative to, or in addition to activation of expression of gene products involved in homologous recombination, fusions of these proteins (or functional fragments thereof) with a target zinc finger binding domain to the region of interest may be used to recruit these proteins (recombination proteins) to the region of interest, thereby increasing their local concentration and further stimulating homologous recombination processes. Alternatively, a polypeptide involved in homologous recombination as described above (or a functional fragment thereof) may be part of a triple fusion protein comprising a zinc finger binding domain, a dividing domain (or dividing half-domain) and the recombination protein (or functional fragment thereof). Additional proteins involved in recombination-related gene conversion and chromatin remodeling, which may be used in the aforementioned methods and compositions, include histone acetyltransferases (e.g., Esalp, Tip60), histone methyltransferases (e.g., Dotlp), histone kinases and histone phosphatases. See also Bhat et al. (1999) PlantJ. 33: 455-469.

Further increases in target recombination efficiency in cells comprising a zinc finger / nuclease fusion molecule and a donor DNA molecule are achieved by blocking the cells in the G2 phase of the cell cycle when repair processes are performed. drive units are maximally active. Such apprehensions can be achieved in a number of ways. For example, cells may be treated with, for example, drugs, compounds and / or small molecules that influence cell cycle progression to seize cells in the G2 phase. Exemplary molecules of this type include, but are not limited to, compounds that affect microtubule polymerization (e.g., vinblastine, nocodazole, Taxol), DNA-interacting compounds (e.g. dichloride diamine with s-platinum (ll), cisplatin). , doxorubicin) and / or compounds that affect DNA synthesis (e.g., thymidine, hydroxyurea, L-mimosin, etoposide, 5-fluorouracil). Further increases in recombination efficiency are achieved by the use of histone deacetylase inhibitors (HDAC) (eg sodium butyrate, trichostatin A) that alter the chromatin structure to produce genomic DNA more accessible to the cell recombination machine.

Additional methods for cell cycle arrest include overexpression of proteins that inhibit CDK cell cycle kinases activity, for example by introducing a protein-encoding cDNA into the cell, or by introducing a designed ZFP into the cell. which activates expression of the gene encoding the protein. Cell cycle seizure is also achieved by inhibiting the activity of cyclins and CDKs, for example by using RNAi methods (eg, United States Patent No. 6,506,559), or by introducing a designed ZFP into the cell. which represses expression of one or more genes involved in cell cycle progression such as, for example, cyclin and / or CDK genes. See, for example, U.S. Patent No. 6,534,261 for methods for the synthesis of zinc finger proteins designed for gene expression regulation.

Alternatively, in certain cases, target cleavage is conducted in the absence of a donor polynucleotide (preferably at the S or G2 phase), and recombination occurs between homologous chromosomes.

Expression Vectors A nucleic acid encoding one or more ZFPs or ZFP fusion proteins may be cloned into a vector for transformation into prokaryotic or eukaryotic cells for replication and / or expression. Vectors may be prokaryotic vectors, for example, plasmids, or transport vectors, insect vectors, or eukaryotic vectors. A nucleic acid encoding a ZFP can also be cloned into an expression vector for administration to a plant cell.

To express ZFPs or ZFP fusion proteins, sequences encoding ZFPs or ZFP fusions are typically subcloned into an expression vector containing a promoter for directing transcription. Suitable bacterial and eukaryotic promoters are well known in the art and described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989; 3rd ed., 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., supra. Bacterial expression systems for expressing ZFP are available from, for example, E. coli, Bacillus sp., and Salmonella (Paiva et al., Gene 22: 229- 235 (1983)) Kits for such expression systems are commercially available Eukaryotic expression systems for mammalian, yeast, and insect cells are well known to those skilled in the art, and are also commercially available.

The promoter used to direct expression of a nucleic acid-encoding ZFP depends on the particular application. For example, a suitable strong constitutive host cell promoter is typically used for expression and purification of ZFPs.

In contrast, when a ZFP is administered in vivo for plant gene regulation (see, "Acid nucleic delivery to plant cells" section below), either a constitutive promoter or an inducible promoter is used, depending on the particular use of the plant. ZFP. Non-limiting examples of plant promoters include promoter sequences derived from A. thaliana ubiquitin-3 (ubi-3) (Callis, et al., 1990, J. Biol. Chem. 265-12486-12493); A. tumifaciens mannopine synthase (Amas) (Petolino et al., United States Patent No. 6,730,824); and / or Cassava Vein Mosaic Virus (CsVMV) (Verdaguer et al., 1996, Plant Molecular Biology 31: 1129-1139). See also Examples.

In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for expression of nucleic acid in either prokaryotic or eukaryotic host cells. A typical expression cassette thus contains a promoter operably linked, for example, to a ZFP-encoding nucleic acid sequence, and signals required, for example, for efficient transcript polyadenylation, transcriptional termination, ribosome binding sites , or translation termination. Additional elements of the cassette may include, for example, enhancers, and heterologous coupling signals.

The particular expression vector used to carry genetic information in the cell is selected with respect to the intended use of ZFP, for example, expression in plants, animals, bacteria, fungi, protozoa, etc. (see expression vectors described below). Standard bacterial and animal expression vectors are known in the art, and are described in detail, for example, in United States Patent Publication 20050064474A1, and International Patent Publications 005/084190, W005 / 014791 and W003 / 080809.

Standard transfection methods can be used to produce bacterial, mammalian, yeast or insect cell lines expressing large amounts of protein, which can then be purified using standard techniques (see, for example). , Colley et al., J. Biol. Chem. 264: 17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Eukaryotic epocherotic cell transformations are performed according to standard techniques (see, for example, Morrison, J. Bact. 132: 349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymoiogy 101: 347- 362 (Wu et al., Eds., 1983).

Any of the well known procedures for introducing nucleotide foreign sequence into such host cells can be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, ultrasonic methods (eg, sonoporation), liposomes, microinjection, exposed DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of these. of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell (see, for example, Sambrook et al., supra). It is only necessary that the particular genetic design procedure used be able to successfully introduce at least one gene into the host cell capable of expressing the protein of choice.

Nucleic Acid Distribution to Plant Cells As noted above, DNA constructs can be introduced into (e.g., into the genome of) a desired plant host by a variety of conventional techniques. For reviews of such techniques see, for example, Weissbach & Weissbach Methods for Plant Molecular Biology (1988, Academic Press, N.Y.) Section VIII, pp. 421-463; and Grierson & Corey, Plant Molecular Biology (1988, 2d Ed), Blackie, London, Ch. 7-9.

For example, DNA construct may be introduced into plant cell genomic DNA using techniques such as electroporation and microinjection of plant cell protoplasts, or DNA constructs may be introduced directly into plant tissue. using biological methods such as DNA particle bombardment (see, for example, Klein et al (1987) Nature 327: 70-73). Alternatively, the DNA constructs may be combined with suitable T-DNA flanking regions, and introduced into a conventional Agrobacterium tumefaciens host vector. Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example, Horsch et al (1984) Science 233: 496-498, and Fraley et al (1983) Proc. Nat'l. Acad. Know. USA 80: 4803.

In addition, gene transfer can be achieved using non-Agrobacterium viruses or bacteria, such as Rhizobium sp. NGR234, Sinorhizoboium meliloti, Mesorhizobium loti, potato X virus, cauliflower mosaic virus, and cassava vein mosaic virus, and / or tobacco mosaic virus, See, for example, Chung et al. (2006) Trends Plant I know. 11 (1): 1-4.

The virulence functions of the host Agrobacterium tumefaciens will direct the insertion of the adjacent marker construct and DNA into the plant cell DNA when the cell is infected by the bacterium using binary T DNA vector (Bevan (1984) Nuc. Acid Res. 12: 8711 -8721), or a co-cultivation procedure (Horsch et al (1985) Science 227: 1229-1231). Generally, the Agrobacterium transformation system is used to design dicot plants (Bevan et al (1982) Ann. Rev. Genet 16: 357-384; Rogers et al (1986) Methods Enzymol. 118: 627-641). The Agrobacterium transformation system can also be used to transform as well as transfer DNA to monocotyledonous plants and plant cells. See United States Patent No. 5,591,616; Hernalsteen et al (1984) EMBO J 3: 3039-3041; Hooykass-Van Slogteren et al (1984) Nature 311: 763-764; Grimsley et al (1987) Nature 325: 1677-179; Boulton et al (1989) Plant Mol. Biol. 12: 31-40; and Gould et al (1991) Plant Physiol. 95: 426-434.

Alternative gene transfer and transformation methods include, but are not limited to, protoplast transformation via calcium-, polyethylene glycol (PEG) - or electroporation-mediated understanding of exposed DNA (see Paszkowski et al. (1984)). ) EMBO J 3: 2717-2722, Potrykus et al. (1985) Molec Gen. Genet 199: 169-177, Fromm et al. (1985) Proc. Nat. Acad. Sci. USA 82: 5824-5828; and Shimamoto (1989) Nature 338: 274-276), and plant tissue electroporation (D'Halluin et al. (1992) Cell of plant 4: 1495-1505). Additional methods for plant cell transformation include microinjection, understanding of silicon carbide mediated DNA (Kaeppler et al. (1990) Cell of plant Reporter 9: 415-418), and microprojectile bombardment (see Klein et al. (1988 ) Proc. Nat. Acad. Sci. USA 85: 4305-4309; and Gordon-Kamm et al. (1990) Cell of plant 2: 603-618).

The disclosed methods and compositions may be used to insert exogenous sequences at a predetermined location in a plant cell genome. This is useful since expression of a transgene introduced into a plant genome critically depends on its site of integration. Accordingly, genes encoding, for example, nutrients, antibiotics, or therapeutic molecules can be inserted by target recombination into regions of a plant genome favorable for their expression.

Transformed plant cells that are produced by any of the above transformation techniques may be cultured to regenerate the total plant having the transformed genotype and thus the desired phenotype. Such regeneration techniques occur in the manipulation of certain phytohormones in a tissue culture growth medium, typically occurring in a biocide and / or herbicide marker that has been introduced along with the desired nucleotide sequences. Plant regeneration of cultured protoplasts is described in Evans, et al., "Protoplasts Isolation and Culture" in Handbook of Cell of Plant Culture, pp. 124-176, Macmillian Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration may also be obtained from plant callus, explants, organs, pollens, embryos or parts thereof. Such regeneration techniques are described generally in Klee et al (1987) Ann. Rev. of Plant Phys. 38: 467-486.

Nucleic acids introduced into a plant cell can be used to confer desired traits on essentially any plant. A wide variety of plant systems can be designed for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present disclosure, and the various transformation methods mentioned above. In preferred embodiments, target plant and plant cells for design include, but are not limited to, monocotyledonous and dicotyledonous plants, such as crops including grain crops (e.g., wheat, rice, millet, barley), fruit crops ( tomatoes, apples, pears, strawberries, oranges), fodder crops (eg alfalfa), root vegetable crops (eg carrots, potatoes, sugar beans, yams), leafy vegetable crops (eg lettuce, spinach; flowering plants (eg petunia, rose, chrysanthemum), conifers and pine trees (eg pine, spruce); plants used for phytoremediation (eg heavy metal accumulation plants) oil crops (eg sunflower, rapeseed), and plants used for experimental purposes (eg Arabidopsis) Thus, the disclosed methods and compositions have use over a wide range of plants, including, but not s not limited to, species of the genus Asparagus, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucurbita, Daucus, Glycine, Gossypium, Hordeum, Lactuca, Lycopersicon, Malus, Manihot, Nicotiana, Oryza, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale, Solanum, Sorghum, Triticum, Vitis, Vigna, and Zea.

One skilled in the art will recognize that after the expression cassette is stably incorporated into transgenic plants and confirmed to be operable, it may be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques may be used, depending on the species to be crossed.

[00193] A transformed plant cell, callus, tissue or plant can be identified and isolated by selecting or classifying plant material designed for traits encoded by the marker genes present in the transforming DNA. For example, selection may be accomplished by growing the projected plant material into the medium containing an inhibitory amount of the antibiotic or herbicide to which the transformation gene construct confers resistance. In addition, transformed plants and plant cells may also be identified by classifying the activities of any visible marker genes (e.g., β-glucuronidase, luciferase, B or C1 genes) that may be present in recombinant nucleic acid constructs. Such selection and classification methodologies are well known to those skilled in the art.

Physical and biochemical methods may also be used to identify plant transformants or plant cells containing inserted gene constructs. These methods include, but are not limited to: 1) Southern analysis or PCR amplification for detection and determination of recombinant DNA insert structure; 2) Northern blotting, S1 RNase protection, primer extension or PCR-reverse transcriptase amplification for detection, and examination of RNA transcripts of gene constructs; 3) enzyme assays for enzyme detection or ribozyme activity, where such gene products are encoded by the gene construct; 4) protein gel electrophoresis, Western blotting techniques, immunoprecipitation, or enzyme linked immunoassays, where the gene construction products are proteins. Additional techniques, such as in situ hybridization, enzyme blotting, and immunoblotting, may also be used to detect the presence or expression of recombinant construct in specific plant organs and tissues. The methods for performing all of these assays are well known to those skilled in the art.

Effects on gene manipulation using the methods disclosed herein can be observed by, for example, northern blotting of RNA (e.g., mRNA) isolated from the tissues of interest. Typically, if the amount of mRNA has increased, it may be assumed that the corresponding endogenous gene is being expressed at a higher rate than before. Other methods of measuring gene and / or CYP74B activity may be used. Different types of enzyme assays may be used depending on the substrate used and the method of detecting the increase or decrease of a reaction product or byproduct. In addition, expressed levels of and / or CYP74B protein may be immunochemically measured, ie ELISA, RIA, EIA and other antibody based assays well known to those skilled in the art, such as by electrophoretic (or blotting or western blotting). The transgene may be selectively expressed in some plant tissues or at some stages of development, or the transgene may be expressed in substantially all plant tissues substantially throughout their life cycle. However, any combinatorial mode of expression is also applicable.

The present disclosure also involves seeds of the transgenic plants described above in which the seed has a transgene or gene construct. The present disclosure further involves the above described progeny, clones, cell lines or plant cell in which said progeny, clone, cell line or cell has the transgene or gene construct.

ZFPs and expression vectors encoding ZFPs may be administered directly to the plant for target cleavage and / or recombination. [00198] The administration of effective amounts is by any of the commonly used routes for introducing ZFP at last contact with the plant. plant cell to be treated. ZFPs are administered in any suitable manner, preferably with pharmaceutically acceptable carriers. Suitable methods of administering such modulators are available and well known to those skilled in the art, and while more than one route may be used to administer a particular composition, a particular route may often provide a more immediate and more effective reaction than another. via.

Vehicles may also be used and are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions that are available (see, for example, Remington's Pharmaceutical Sciences, 17th ed. 1985)). Applications The methods and compositions discussed for target cleavage can be used to induce mutations in a genomic sequence. Target cleavage can also be used to create gene deletions (e.g., for target validation or functional genomics), and to facilitate target insertion of a sequence into a genome (ie gene deletion). Insertion may be by substitution of chromosomal sequences by homologous recombination or by target integration, wherein a new sequence (i.e. a sequence not present in the region of interest), flanked by sequences homologous to the region of interest on the chromosome , is inserted at a predetermined target location. The same methods can also be used to replace a wild type sequence with a mutant sequence, or to convert one allele to a different allele.

Target cleavage of infection or integrated plant pathogens can be used to treat pathogenic infections in a plant host, for example by dividing the pathogen genome such that its pathogenicity is reduced or eliminated. Additionally, target cleavage of genes encoding plant virus receptors may be used to block expression of such receptors, thereby preventing viral infection and / or viral diffusion in the plant.

[00202] Exemplary plant pathogens include, but are not limited to, plant viruses such as Alfamoviruses, Alphacryptoviruses, Badnaviruses, Betacryptoviruses, Bigeminiviruses, Bromoviruses, Bymoviruses, Capilloviruses, Carlaviruses, Carmoviruses, Caulimoviruses, Cyoviruses, Cyoviruses, Cyoviruses, Cyoviruses, Cyoviruses, Dianthoviruses, Enamoviruses, Fabaviruses, Fijiviruses, Furoviruses, Hordeiviruses, Hybrigeminiviruses, Idaeoviruses, llarviruses, Ipomoviruses, Luteoviruses, Machlomoviruses, Macluraviruses, Marafiviruses, Monogeminiviruses, Nanaviruses, Necroviruses, Nepoviruses, Nucleorhabdoviruses, Oryzaviruses, Ourmiaviruses, Phytoreoviruses, Potexviruses, Potyviruses, Rymoviruses, satellite RNAs, satelliviruses, Sequiviruses, Sobemoviruses, Tenuiviruses, Tobamoviruses, Tobraviruses, Tombusviruses, Tospoviruses, Trichoviruses, Tymoviruses, Umbraviruses, Varicosaviruses and Waikaviruses; fungal pathogens such as rust (e.g. Ustilaginales), rust (Uredinales), rust (Clavicepts pupurea) and downy mildew; molds (Oomycetes) such as Phytophthora infestans (potato rust; bacterial pathogens such as Erwinia (eg E. herbicola), Pseudomonas (eg P. aeruginosa, P. syringae, P. fluorescense and P. putida), Ralstonia (e.g. R. solanacearum), Agrobacterium and Xanthomonas, nematelmints (Nematoda), and Phytomyxea (Polymyxa and Plasmodiophora).

The disclosed methods for target recombination may be used to replace any genomic sequence with a non-identical homologous sequence. For example, a mutant genomic sequence may be substituted for its wild-type equivalent, thereby providing methods for treating plant diseases; provides resistance to plant pathogens; increases crop yields, etc. In the similar manner, an allele of a gene may be replaced by a different allele using the target recombination methods disclosed herein.

In either case, a region of interest comprises a mutation, and the donor polynucleotide comprises the corresponding wild-type sequence. Similarly, a wild-type genomic sequence may be replaced by a mutant sequence if desired. For example, overexpression of an oncogene may be reversed by either mutating the gene or replacing its control sequences with sequences that support a lower non-pathogenic level of expression. Indeed, any pathology dependent on a particular genomic sequence, in any way, can be corrected or alleviated using the methods and compositions disclosed herein.

Target cleavage and target recombination may also be used to alter non-coding sequences (e.g., regulatory sequences such as promoters, enhancers, primers, terminators, binding sites) to alter the expression levels of a gene product. Such methods may be used, for example, for therapeutic purposes, validation studies of functional and / or target genomics.

The target modification of chromatin structure, as disclosed in the same applicant's WO 01/83793, may be used to facilitate binding of fusion proteins to cellular chromatin.

In additional embodiments, one or more fusions between a zinc finger binding domain and a recombinase (or functional fragment thereof) may be used, in addition to or instead of the zinc finger fusions of the domain domain disclosed herein. , to facilitate target recombination. See, for example, U.S. Patent No. 6,534,261 and Akopian et al (2003) Proc. Natl. Acad. Know. USA 100: 8688-8691.

In further embodiments, the disclosed methods and compositions are used to provide fusions of transcriptionally activated ZFP binding domains or repression domains that require dimerization (or homodimerization or heterodimerization) for their activity. In these cases, a fusion polypeptide comprises a zinc finger binding domain and a functional domain monomer (e.g., a dimeric transcriptional activation monomer or repression domain). Binding of two such fusion polypeptides to correctly situated target sites permits dimerization to reconstitute a functional transcriptional activation or repression domain.

Additionally, as disclosed above, the methods and compositions set forth herein may be used for targeting integration of exogenous sequences into a region of interest in the genome of a cell, for example, where divagen increases insertion via homology-dependent mechanisms ( for example, insertion of a donor sequence comprising an exogenous sequence together with one or more sequences that are either identical or homologous but not identical with a predetermined genomic sequence (i.e. a target site)).

The donor sequence typically contains sufficient homology in the flanking regions of the exogenous sequence to support homologically directed repair of a double stranded break in a genomic sequence, thereby inserting the exogenous sequence at the genomic target site. Therefore, the donor nucleic acid may be of any size sufficient to support integration of the exogenous sequence by homology-dependent repair mechanisms (e.g., homologous recombination). Without wishing to be bound by any particular theory, the regions of homology flanking the exogenous sequence are thought to provide breakage of chromosome ends with a template for resynthesis of genetic information at the site of double stranded breakage. In certain embodiments, two of the identical sequences, or two of the homologous but non-identical sequences (or one of each) are present, flanking the exogenous sequence. An exogenous sequence (either exogenous nucleic acid or exogenous polynucleotide) is one that contains a nucleotide sequence that is not normally in the region of interest.

Exemplary exogenous sequences include, but are not limited to, cDNAs, promoter sequences, enhancer sequences, epitope tags, marker genes, dividing enzyme recognition sites, and various types of expression constructs. See, for example, United States Patent No. 6,833,252. Additional exemplary unidirectional endocucleases include l-Cei / l, Pl-Pspl, Pl-Sce, 1-ScelV, l-Csml, l-Panl, l-Scell, l-Ppol, l-Scelll, l-Crel, \ -Tv \, \ -Tv \\ and \ -Tev \\\. Their recognition sequences are known. See also United States Patent No. 5,420,032; Belfort et al. (1997) Acid nucleics Res. 25: 3379-3388; Dujon et al. (1989) Gene 82: 115-118; Perler et al. (1994) Acid nucleics Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12: 224-228; Gimble et al. (1996) J. Mol. Biol. 263: 163-180; Argast et al. (1998) J. Mol. Biol. 280: 345-353, and the New England Biolabs catalog.

Marker genes include, but are not limited to, sequences encoding proteins that mediate antibiotic resistance (e.g. ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding colored or fluorescent proteins or luminescent substances (e.g. green fluorescent protein, increased green fluorescent protein, red fluorescent protein, luciferase), and proteins that mediate increased cell growth and / or gene amplification (e.g. dihydrofolate reductase). Exemplary marker genes thus include, but are not limited to, β-glucuronidase (GUS), phosphinothricin N-acetyl transferase (PAT, BAR), neomycin phosphotransferase, β-lactamase, catechol dioxigenase, Î ± -amylase, tyrosinase, β-galactosidase , luciferase, aequorin, EPSP synthase, nitrilase, acetolactate synthase (ALS), dihydrofolate reductase (DHFR), dalapon dehalogenase and anthranilate synthase. In certain embodiments, target integration is used to insert an RNA expression construct, for example, sequences responsible for the regulated expression of microRNA or siRNA. Additional promoters, enhancers, and regulatory sequences as described above may also be incorporated into an RNA expression construct.

Claims (6)

Method for introducing an exogenous sequence into the genome of a plant cell, characterized in that it comprises the steps of: (a) contacting the cell with a donor DNA vector comprising a first, second and fifth DNA sequence; and the fifth sequence comprises an exogenous DNA vector sequence to be introduced and is interposed between the first and second donor DNA vector sequences; wherein the first sequence has at least 90% to 95% homology to a third sequence in the plant cell genome, the second sequence has at least 90% to 95% homology to a fourth sequence in the plant cell genome. plant, wherein the third and fourth sequences in the plant cell genome are chromosomal DNA sequences and the proximal ends of the third and fourth sequences are separated by at least one pair of nucleotides; and (b) expressing one or more nucleases in the cell, wherein one or more nucleases are each a pair of fusion proteins, wherein each fusion protein is a fusion between the dividing domain of a Fok I restriction endonuclease. and an engineered zinc finger binding domain, and wherein one or more nucleases cleave chromosomal DNA into one or more corresponding target sequences between the third and fourth sequences, wherein one or more nucleases cleave chromosomal DNA at the locating 0.4 to 3 kilo base pairs of the third or fourth sequence in the plant cell genome; such that the chromosomal DNA splitting in step (b) stimulates the incorporation of the fifth DNA sequence into the genome by homologous recombination. Method according to claim 1, characterized in that the exogenous sequence is a selectable marker. Method according to claim 2, characterized in that the selectable marker is selected from the group consisting of green fluorescent protein (GFP), β-glucuronidase (GUS), phosphinothricin N-acetyl transferase (PAT, BAR ), neomycin phosphotransferase, β-lactamase, catechol dioxigenase, Î ± -amylase, tyrosinase, β-galactosidase, luciferase, aequorin, EPSP synthase, nitrilase, acetolactate reductase (ALS), dihydrofolate reductase (DHFR), dalapon desalogenase. Method according to claim 1, characterized in that the fifth sequence comprises one or more transcriptional regulatory sequences. Method according to claim 1, characterized in that the fifth sequence comprises sequences encoding a protein or a portion of a protein. Method according to claim 1, characterized in that the fifth sequence comprises a wild equivalent of a mutant chromosomal sequence, or a mutant equivalent of a wild-type chromosomal sequence.