Rationally designed meganucleases with altered sequence specificity and dna-binding affinity

Abstract

To provide gene therapy for treatment of pathogenic infections, and for in vitro applications in diagnostics and research.SOLUTION: The invention provides a method for treating a human disease by gene therapy in a eukaryote, by using a recombinant meganuclease having altered specificity for at least one recognition sequence half-site relative to a wild-type I-CREI meganuclease. The recombinant meganuclease comprises a polypeptide having at least 85% sequence similarity to residues 2-153 of the I-CREI meganuclease of a specific sequence, has specificity for a recognition sequence half-site which differs by at least one base pair from a half-site within an I-CREI meganuclease recognition sequence selected from the group consisting of specific sequences, and comprises at least one modification which is not an excluded modification.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the fields of molecular biology and recombinant nucleic acid engineering. In particular, the present invention relates to the synthesis of DNA fragments. A. Rationally designed, non-naturally occurring meganucleotides with altered recognition sequence specificity and / or affinity. Furthermore, the present invention relates to a method for producing such meganucleases, and , relates to methods for producing recombinant nucleic acids and organisms with such meganucleases. [Background technology]

[0002] Related Applications: This application is a joint venture of U.S. Provisional Patent Application No. 60 / 727, filed October 18, 2005. This application claims the benefit of priority to US Pat. No. 5,512, the entire disclosure of which is incorporated herein by reference. The present application is hereby incorporated by reference.

[0003] GOVERNMENT FUNDING This invention was made with funding from the National Institutes of Health, National Institute of Medical Sciences. Grants 2R01-GM-0498712, 5F32-GM072322, and 5 DP1 This invention was supported in part by grant number 0000122. Accordingly, the U.S. Government also grants a license to use the invention. Some rights may be reserved.

[0004] Genome engineering involves inserting, deleting, or substituting specific gene sequences within a genome. and other ways of manipulating it, but many therapeutic and The development of effective tools for genome modification has been a longstanding challenge. , a major target in gene therapy, agricultural engineering, and synthetic biology (PORTEU S ET AL.(2005),NAT.BIOTECHNOL.23;967-73; TZFIRA ET AL.(2005),TRENDS BIOTECHNOL.23 :567-9;MCDANIEL ET AL.(2005),CURR.OPIN.B IOTECHNOL. 16:476-83). General methods for inserting or modifying DNA sequences. The method involves the generation of a transgenic DNA fragment flanked by sequences homologous to a genomic target. A sequence and selecting or screening for the desired homologous recombination event. Recombination with transgenic DNA occurs rarely, but It can be stimulated by a double-strand break in the genomic DNA at the site. There are a number of methods for creating DNA double-strand breaks, including, for example, irradiation and chemical treatment. These methods are not able to stimulate recombination efficiently. However, double-strand breaks are inserted randomly into the genome and they cause mutations. Currently, there are no known genes that are specifically targeted to specific regions in the chromosomal background. However, it is not possible to target gene modification, so there is a large gap in the ability to manipulate the genome as desired. This is a major obstacle.

[0005] One way to achieve this goal is to identify genes that are sufficiently abundant to be present at only a single site in the genome. Nucleases with specificity for relatively large sequences are used to create double-strand breaks in the target conformation. The goal is to stimulate homologous recombination at that site (e.g., PORTEUS E (See T AL. (2005), NAT. BIOTECHNOL. 23:967-73) The effectiveness of this strategy is demonstrated by the engineered zinc finger DNA-binding of the FOKI restriction enzyme. Chimeric fusions between the nuclease domain and a non-specific nuclease domain can be used to identify nucleases from various organisms. This has been revealed in PORTEUS (2006), MOL THER 13: 438-46;WRIGHT ET AL.(2005), PLANT J.44:69 3-705;URNOV ET AL.(2005),NATURE 435:646- 51) These artificial zinc finger nucleases stimulate site-specific recombination. However, these nucleases have non-specific functions resulting from the downregulation of the nuclease domain. They retain specific, residual cleavage activity, often cleaving unwanted sites (S MITH ET AL.(2000),NUCLEIC ACIDS RES.28:3 361-9). These unwanted truncations can lead to mutations and toxicity in the treated organism. (PORTEUS ET AL. (2005), NAT. BIOTE CHNOL.23:967-73).

[0006] Recognizes 15-40 base pair cleavage sites commonly found in plant and fungal genomes A group of naturally occurring nucleases may offer a relatively non-toxic alternative for genome engineering. Such "meganucleases" or "homing endonucleases" are often caused by parasitic DNA elements, e.g., group 1 self-splicing introns These nucleases naturally interact with the host genome. This promotes homologous recombination or gene insertion at a specific location in the genome. This occurs by generating double-strand breaks in the chromosome, which recruits the NA repair machinery (S TODDARD (2006), REV. BIOPHYS. 38:49-95). In general, Meganucleases are divided into four families: the LAGLIDADG families family, GIY-YIG family, HIS-CYS box family, and HN These families affect catalytic activity and recognition sequences. They are characterized by structural motifs. For example, members of the LAGLIDADG family Bars are characterized by having one or two copies of the conserved LAGLIDADG motif. (CHEVALIER ET AL. (2001), NUCLEIC ACI See DS RES.29(18):3757-3774. LAGLIDADG meganucleases with a single copy of α form homodimers On the other hand, members with two copies of the LAGLIDADG motif are monomers. Similarly, GIY-YIG family members were found to be 70-100 residue long G It contains IY-YIG modules, two of which contain the four invariant residues required for activity. and contains four to five conserved sequence motifs (VAN ROEY ET AL. (2002 ), NATURE STRUCT.BIOL.9:806-811). HIS-C YS box meganucleases are highly fragmented, distributed over a region containing several hundred amino acid residues. Characterized by a conserved stretch of histidine and cysteine ​​(CHEVA LIER ET AL.(2001),NUCLEIC ACIDS RES.29(1 8):3757-3774). In the case of the NHN family, its members are divided into two pairs. is defined by a motif containing a conserved histidine surrounded by asparagine residues. (CHEVALIER ET AL.(2001), NUCLEIC ACIDS R ES.29(18):3757-3774). These four families of meganucleases The specificity of DNA recognition sequences and catalytic activity of these enzymes differ significantly from each other with respect to structurally conserved regions. are.

[0007] Natural meganucleases, mainly from the LAGLIDADG family, have been site-specific genome sequencing in plants, yeast, Drosophila, mammalian cells, and mice This method preserves the meganuclease recognition sequence, although it has been used effectively to modify Is it a homologous sequence (MONNAT ET AL. (1999), BIOCHEM.BIOPHY S.RES.COMMUN.255:88-93) or a pre-added enzyme containing a recognition sequence Engineered genome (ROUET ET AL. (1994), MOL. CELL BIOL .14:8096-106;CHILTON ET AL.(2003),PLANT PHYSIOL.133:956-65;PUCHTA ET AL.(1996),P ROC.NATL.ACAD.SCI.USA 93:5055-60;RONG ET AL.(2002),GENES DEV.16:1568-81;GOUBLE E T AL.(2006),J.GENE MED.8(5):616-622) Limited.

[0008] Systematic implementation of nuclease-stimulated gene modification allows targeted delivery to existing sites in the genome. It is therefore necessary to use a processing enzyme with specificity targeted towards the NA interruption. Meganucleases can be used to facilitate gene modification at medically or biotechnologically relevant sites. There is great interest in adapting it (PORTEUS ET AL. (2005),NAT.BIOTECHNOL.23:967-73;SUSSMAN ET AL.(2004),J.MOL.BIOL.342:31-41;EPINAT ET AL.(2003),NUCLEIC ACIDS RES.31:2952- 62).

[0009] Meganuclease I obtained from Chlamydomonas reinhardtii -CREI is a member of the LAGLIDADG family and is a 22-synthetic protein in the chloroplast chromosome. It recognizes and cleaves base pair recognition sequences and is a good target for meganuclease redesign. In this wild-type enzyme, each monomer directly contacts nine base pairs in the overall recognition sequence. It is a homodimer that recognizes a single site in this recognition sequence (SUSSMAN et al., L.(2004),J.MOL.BIOL.342:31-41;CHAMES ET AL.(2005),NUCLEIC ACIDS RES.33:E178;SELI GMAN ET AL.(2002),NUCLEIC ACIDS RES.30:3 870-9), or more recently, at position 3 in the recognition sequence (ARRNO ULD ET AL. (2006), J.MOL.BIOL.355:443-58), Genetic selection techniques were used to identify mutations in I-CREI that alter base selectivity. The I-CREI protein-DNA interface directly binds to the DNA bases. Nine contacting amino acids and their potential to form contacts in the modified interface The size of this interface is determined by the size of the cleavage site. The sequence library constructed to select enzymes with significantly altered positions was also sufficient. It presents such complex combinations that it is unlikely that they will ever be extracted. DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0010] There remains a need for nucleases that facilitate precise modification of the genome. a pre-designated, Techniques for generating nucleases with rationally designed recognition sequences and the ability to identify precise sequences Techniques that utilize such nucleases to genetically engineer organisms with modifications There is still demand for. [Means for solving the problem]

[0011] The present invention is based on the identification and characterization of the LAGLIDADG family of meganucleases. When meganuclease contacts a double-stranded DNA recognition sequence, the DNA bases and NA backbone and thereby affect the specificity and activity of the enzyme. This finding is based on the recognition sequence specificity of meganucleases, as explained in detail below. and / or to identify amino acid substitutions that can alter DNA binding affinity and natural Meganuclease capable of recognizing a desired DNA sequence that is not recognized by other meganucleases The present invention has been used to rationally design and develop enzymes. for in vitro applications in research and treatment of bacterial infections and in diagnosis and treatment of minor loci within the genome of an organism. In order to achieve the desired recombination of gene sequences at the Provides a method for using

[0012] Thus, in one embodiment, the present invention provides a method for the production of I-CREI meganucleases, which are capable of producing I-CREI meganucleases with a reduced number of nucleotides compared to wild-type I-CREI meganucleases. and recombinant meganuclei with altered specificity for at least one recognition sequence half-site. In this embodiment, the meganuclease comprises the wild-type I-CR of SEQ ID NO: 1. Has at least 85% sequence similarity to residues 2-153 of EI meganuclease The recombinant meganuclease comprises a polypeptide having the sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: A half portion of the I-CREI meganuclease recognition sequence selected from SEQ ID NO: 4 and SEQ ID NO: 5 and specific for a recognition sequence half-site that differs from the base pair by at least one base pair. The recombinant meganucleases are not exclusive modifications found in the prior art, as listed in Table 5. It contains at least one modification.

[0013] In another embodiment, the present invention provides a method for producing at least one I-MSOI meganuclease having ... Providing recombinant meganucleases with altered specificity for another recognition sequence half-site In this embodiment, the meganuclease is the I-MSOI meganuclease of SEQ ID NO: 6. polypeptides having at least 85% sequence similarity to residues 6-160 of ATP wherein the recombinant meganuclease is selected from SEQ ID NO: 7 and SEQ ID NO: 8. A recognition sequence that differs by at least one base pair from a half-site within the SOI meganuclease recognition sequence The recombinant meganuclease has specificity for the half-sites and is not known in the art. The amino acid sequence includes at least one modification listed in Table 7, but is not an exclusive modification.

[0014] In another embodiment, the present invention provides a method for identifying a target I-SCEI meganuclease that has a recognition sequence different from that of a wild-type I-SCEI meganuclease. In this embodiment, a recombinant meganuclease with altered specificity for The nuclease is a nucleotide sequence corresponding to residues 3-186 of the I-SCEI meganuclease of SEQ ID NO: 9. The recombinant meganucleic acid comprises a polypeptide having at least 85% sequence similarity to the The enzyme has the I-SCEI meganuclease recognition sequences of SEQ ID NO: 10 and SEQ ID NO: 11. and the recombinant megagene has specificity for a recognition sequence that differs by at least one base pair from the The cleavage is not an exclusive modification recognized in the prior art, but is at least one of the modifications listed in Table 9. Contains one modification.

[0015] In another embodiment, the present invention provides at least one I-CEUI meganuclease that is at least as potent as the wild-type I-CEUI meganuclease. Providing recombinant meganucleases with altered specificity for another recognition sequence half-site In this embodiment, the meganuclease is the I-CEUI meganuclease of SEQ ID NO: 12. and a polypeptide having at least 85% sequence similarity to residues 5-211 of the enzyme. However, the recombinant meganuclease is selected from SEQ ID NO: 13 and SEQ ID NO: 14. - A recognition sequence that differs by at least one base pair from a half-site within the CEUI meganuclease recognition sequence The recombinant meganuclease has specificity for the half-sites and is not known in the art. The compound contains at least one modification listed in Table 11, which is not an exclusive modification.

[0016] The meganucleases of the present invention may be modified at one, two, or more positions within the recognition sequence. To affect the sequence specificity of the meganuclease, one of the modifications disclosed in this application may be The meganuclease may comprise one, two, or more than two of the enzymes disclosed in the present application. The invention may include only novel modifications that are recognized in the prior art or may incorporate the novel modifications disclosed in this application. However, what is specifically excluded is what is known in the art. It is a recombinant meganuclease containing only the engineered modifications.

[0017] In another aspect, the present invention provides a method for detecting a non-sequence-specific binding affinity for double-stranded DNA. The present invention provides a recombinant meganuclease with altered degree of double-stranded DNA recognition sequence. This is achieved by modifying the meganuclease residues that contact the backbone. The modifications increase or decrease the binding affinity, and therefore the overall activity of the enzyme. Furthermore, the increase / decrease in binding and activity can be correlated with a decrease / increase in sequence specificity. Therefore, the present invention provides a method for modifying DNA-binding affinity. This provides a means to globally alter sequence specificity by

[0018] Thus, in one embodiment, the present invention provides a method for producing a nuclease having a high affinity for I-CREI compared to wild-type I-CREI meganuclease. and to provide a recombinant meganuclease with altered binding affinity to double-stranded DNA. The meganuclease of the present invention is the I-CREI meganuclease of SEQ ID NO: 1. polypeptides having at least 85% sequence similarity to residues 2-153, DNA binding affinity is (1) (A) E80 by H, N, Q, S, T, K or R, D 137, I81, L112, P29, V64 or Y66 substitution, or (B) K or corresponds to one substitution selected from the substitution of T46, T140 or T143 by R (2) (A) H, N, Q, S, or T, D or E for K34, K48, R51, K82, K116 or K139 Substitution, or (B) I81, L112, P29, V64, Y66, T4 by D or E 6. At least one corresponding to one substitution selected from the substitutions of T140 or T143 It is reduced by modification of

[0019] In another embodiment, the present invention provides a method for producing a double-stranded D-type I-MSOI meganuclease compared to wild-type I-MSOI meganuclease. The present invention provides a recombinant meganuclease with altered binding affinity to NA. The meganuclease in SEQ ID NO: 6 is composed of residues 6-16 of the I-MSOI meganuclease. 0, but contains a polypeptide having at least 85% sequence similarity to the DNA-binding parent The degree of harmony is (1) (A) E147, I85, G8 by H, N, Q, S, T, K or R 6 or Y118, or (B) substitution of Q41, N70, S87, T by K or R one selected from the substitutions 88, H89, Q122, Q139, S150 or N152 or conversely, (2)(A) K36, R51, K123, K143 or by H, N, Q, S, T, D or E (B) Substitution of R144, or I85, G86, Y118, Q41, N by D or E Substitution of 70, S87, T88, H89, Q122, Q139, S150 or N152 is reduced by at least one modification corresponding to one substitution selected from:

[0020] In another embodiment, the present invention provides a method for producing a double-stranded D-type I-SCEI meganuclease compared to a wild-type I-SCEI meganuclease. The present invention provides a recombinant meganuclease with altered binding affinity to NA. The meganuclease in SEQ ID NO: 9 is a nucleotide sequence consisting of residues 3-18 of the I-SCEI meganuclease. 6, but contains a polypeptide with at least 85% sequence similarity to the DNA-binding parent. The degree of harmony is (1) (A) D201, L19, L8 by H, N, Q, S, T, K or R Substitution of 0, L92, Y151, Y188, I191, Y199 or Y222, or ( B) N15, N17, S81, H84, N94, N120, T156 by K or R , N157, S159, N163, Q165, S166, N194 or S202 or by at least one modification corresponding to one substitution selected from Conversely, (2)(A) K20, K23, K63 by H, N, Q, S, T, D or E, Substitution of K122, K148, K153, K190, K193, K195 or K223 , or (B) L19, L80, L92, Y151, Y188, I19 by D or E 1, Y199, Y222, N15, N17, S81, H84, N94, N120, T15 6. N157, S159, N163, Q165, S166, N194 or S202 The amino acid sequence is reduced by at least one modification corresponding to one of the substitutions selected from the group consisting of:

[0021] In another embodiment, the present invention provides a method for producing a double-stranded D-type I-CEUI meganuclease compared to wild-type I-CEUI meganuclease. The present invention provides a recombinant meganuclease with altered binding affinity to NA. The meganuclease in accordance with the present invention is a nuclease comprising residues 5-2 of the I-CEUI meganuclease of SEQ ID NO: 12. 11, but contains polypeptides with at least 85% sequence similarity to Affinity is (1) (A) D25 or D12 by H, N, Q, S, T, K or R 8, or (B) S68, N70, H94, S117, N120 by K or R , N129 or H172 (2) (A) H, N, Q, S, T, D, or E, or (B ) S68, N70, H94, S117, N120, N129 or by D or E at least one modification corresponding to one of the substitutions selected from H172 do.

[0022] The meganucleases of the present invention have the ability to affect DNA-binding affinity, and thus are capable of binding to the nucleases disclosed in this application. The backbone may include one, two, or more of the following modifications of contact residues: Furthermore, these modifications that affect DNA-binding affinity are due to specific modifications within the recognition sequence. of the aforementioned base-contacting residues, which alter the sequence specificity of the recombinant meganuclease at a given position. It may be combined with one or more of the novel modifications, or in combination with the prior art modifications described above. The novel modifications may be combined with modifications of the prior art. By combining carboxyl and base contact modifications, It is possible to rationally design enzymes with desired specificity and activity. For example, DNA-binding proteins that reverse the loss of affinity resulting from design changes to base-contacting residues. It is possible to design an increased degree of saturation, reducing sequence specificity and increasing the recognition sequence for the enzyme. It is also possible to design affinity reductions that broaden the set.

[0023] In another aspect, the present invention provides a method for modifying the affinity for homo- or heterodimer formation. The present invention provides a rationally designed meganuclease monomer with a dimer formation affinity of 100%. , either by the same monomer (i.e., homodimer formation) or by different monomers (i.e., , heterodimer formation), can be measured, for example, by a reference wild-type meganuclease These recombinant meganucleases are capable of synthesizing the nuclease dimers. Modification of amino acid residues present at the protein-protein interface between monomers These modifications promote heterodimer formation and have non-palindromic recognition sequences. These can be used to create meganucleases.

[0024] Thus, in another embodiment, the present invention provides a method for the synthesis of a dinucleotide analogue by a reference meganuclease monomer. The present invention provides a recombinant meganuclease with an altered affinity for nucleomer formation. In the same manner, the recombinant monomer comprises residues 2-1 of the I-CREI meganuclease of SEQ ID NO: 1. 53, but in a dimeric form. Affinity for synthesis is determined by (A) substitution of K7, K57, or K96 with D or E, or (B) a substitution selected from the substitution of E8 or E61 by K or R; Based on such recombinant monomers, the present invention provides Furthermore, (1) a small amount of nucleotides corresponding to residues 2-153 of the I-CREI meganuclease of SEQ ID NO: 1 a first polypeptide having at least 85% sequence similarity, said first polypeptide being resistant to dimer formation; Affinity is determined by substitution selected from (A) substitution of K7, K57, or K96 with D or E. (2) a first polypeptide that is altered by at least one modification corresponding to a substitution; At least 85% of residues 2-153 of I-CREI meganuclease in column number 1 a second polypeptide having sequence similarity and an affinity for dimer formation of (B) at least one corresponding to a substitution selected from the substitution of E8 or E61 by K or R; Recombinant meganuclease heterodimer containing a second polypeptide altered by one modification Provide a dimer.

[0025] In another embodiment, the present invention provides a method for detecting dimer formation by a reference meganuclease monomer. In this embodiment, a recombinant meganuclease monomer is provided that has an altered affinity for the The recombinant monomer is a nucleotide sequence of residues 6-160 of the I-MSOI meganuclease of SEQ ID NO: 6. polypeptides having at least 85% sequence similarity to The affinity for R302 was determined by (A) substituting D or E for R302, or (B) substituting K or R for R302. at least one corresponding to a substitution selected from the substitutions of D20, E11 or Q64 by Based on such recombinant monomers, the present invention further provides: ) at least 85% of residues 6-160 of the I-MSOI meganuclease of SEQ ID NO: 6 % sequence similarity, and the affinity for dimer formation is A) at least one modification corresponding to a substitution selected from the substitution of R302 by D or E (1) a first polypeptide modified by the addition of a nucleotide sequence; and (2) a I-MSOI meganucleotide sequence of SEQ ID NO: 6. A second polypeptide with at least 85% sequence similarity to residues 6-160 of lyase (B) D20, E1 by K or R and modified by at least one modification corresponding to a substitution selected from the group consisting of substitutions Q1 and Q64. The present invention provides a recombinant meganuclease heterodimer comprising a second polypeptide that is capable of binding to the nuclease.

[0026] In another embodiment, the present invention provides a method for detecting dimer formation by a reference meganuclease monomer. In this embodiment, a recombinant meganuclease monomer is provided that has an altered affinity for the The recombinant monomer is a nucleotide sequence of residues 5-21 of the I-CEUI meganuclease of SEQ ID NO: 12. 1, but does not form dimers. The affinity for It is modified by at least one modification corresponding to a substitution selected from the substitutions of E152. Based on such recombinant monomers, the present invention further provides: (1) I-CEU of SEQ ID NO: 12; I. A nucleotide sequence with at least 85% sequence similarity to residues 5-211 of meganuclease I. 1 polypeptide, wherein the affinity for dimer formation is R9 by (A) D or E a first polynucleotide modified by at least one modification corresponding to a substitution selected from the group consisting of and (2) a peptide corresponding to residues 5-211 of the I-CEUI meganuclease of SEQ ID NO: 12. a second polypeptide having at least 85% sequence similarity to the (B) the affinity for E152 corresponds to a substitution selected from the group consisting of K and R Recombinant Meganuclea Comprising a Second Polypeptide Altered by at Least One Modification The present invention provides a heterodimer of phosphodiesterase.

[0027] Recombinant meganuclease monomers or heteronucleases with altered affinity for dimer formation The telodimers may further comprise one, two, or more of the aforementioned modifications of base contact residues; One, two, or three or more of the above backbone contact residue modifications, or a combination of both Thus, for example, the base contacts of the monomers can be sequence specific. The backbone contacts of the monomers can be modified to change the DNA binding site. It can be modified to change binding affinity, and the protein-protein interface can be modified to affect dimer formation. The monomers can be combined with similarly modified monomers to achieve the desired sequence specificity and activity. It is now possible to produce rationally designed meganuclease heterodimers with

[0028] In another aspect, the present invention provides a rationally designed mega-computer system as described and enabled herein. Various methods are provided for the use of nucleases. These methods involve the genetic modification of producing engineered cells and organisms; treating diseases through gene therapy; Recombinant meganucleotides for treating systemic infections and for in vitro applications in diagnostics and research This involves using a enzyme.

[0029] Thus, in this aspect, the invention provides a gene comprising an exogenous sequence of interest inserted into a chromosome. A method for producing a genetically modified eukaryotic cell, comprising: subjecting the cell to (I) (II) a nucleic acid sequence encoding the meganuclease of the present invention; and (II) a nucleic acid sequence comprising the sequence of interest. The method provides a method for achieving this by transfecting the cell with a second nucleic acid sequence containing the Upon completion, the meganuclease produces a cut site in the chromosome, and the sequence of interest is inserted by homologous recombination. The fragment is inserted into the break site in the chromosome by end-joining or non-homologous end-joining.

[0030] Alternatively, in another aspect, the invention provides a gene comprising a foreign sequence of interest inserted into a chromosome. A method for producing a genetically modified eukaryotic cell, comprising injecting the cell with a megagene of the present invention. and transfecting the cells with a nucleic acid containing the sequence of interest. In this case, the meganuclease is A cleavage site is generated within the chromosome, and the sequence of interest is inserted by homologous recombination or non-homologous end joining. and inserted into the chromosome at the break site.

[0031] In another aspect, the present invention provides a method for genetically modifying a target sequence in a chromosome by disrupting the target sequence. A method for producing a modified eukaryotic cell, comprising injecting the cell with a meganuclease gene of the present invention. The present invention provides a method for achieving this by transfecting a cell with a nucleic acid encoding a protein. In this process, the meganuclease creates a cleavage site in the chromosome, and the target sequence is located at the cleavage site. It is destroyed by non-homologous end joining at this position.

[0032] In another aspect, the invention provides a method for producing a genetically modified organism, the method comprising: producing a genetically modified eukaryotic cell according to the aforementioned method, and This is achieved by cultivating modified eukaryotic cells to produce genetically modified organisms. In these embodiments, the eukaryotic cell is a gamete, a zygote, a blastocyst cell, It is possible to choose from embryonic stem cells and protoplast cells.

[0033] In another aspect, the present invention provides a method for treating disease by gene therapy in eukaryotes. The method comprises: (I) infecting at least one cell of the eukaryotic organism with a meganuclear vector of the invention; (II) a first nucleic acid sequence encoding an enzyme, and (II) a second nucleic acid sequence containing a sequence of interest. In this case, the meganuclease is A break is created in the chromosome and the sequence of interest is inserted by homologous recombination or non-homologous end joining. The insertion of a sequence of interest into a chromosome provides gene therapy for disease.

[0034] Alternatively, in another aspect, the present invention provides a method for treating disease by gene therapy in eukaryotic organisms. a method for treating a eukaryotic organism, the method comprising: injecting a megakaryotic cell of the present invention into at least one cell of the eukaryotic organism; nuclease and transfecting the cells with a nucleic acid containing the sequence of interest. In this case, the meganuclease is inserted into the chromosome. The sequence of interest is then inserted into the target DNA by homologous recombination or non-homologous end joining. The insertion of the sequence of interest into the chromosome at the breakpoint is then performed to identify the gene for the disease. Bringing treatment.

[0035] In another aspect, the invention provides a method for detecting a target sequence in a eukaryotic chromosome. a method for treating a disease by gene therapy that disrupts at least one of the eukaryotic cells; Both cells are transfected with a nucleic acid encoding a meganuclease of the invention. In this case, the meganuclease is The target sequence is disrupted by homologous recombination or non-homologous end joining. Disruption of the target sequence provides a gene therapy for the disease.

[0036] In another aspect, the present invention provides a method for the prevention of viral or prokaryotic pathogens in eukaryotic hosts. A method for treating an infection by disrupting a target sequence in the genome of the pathogen. In this case, the meganuclease creates a break in the chromosome. The target sequence is amplified by (1) non-homologous end joining at the cleavage site or (2) ) destroyed by homologous recombination with a second nucleic acid, and the destruction of the target sequence prevents the infection. This provides a treatment that

[0037] More generally, in another aspect, the invention provides a method for detecting a target gene for at least one base position of a recognition sequence. A method for rationally designing a recombinant meganuclease with altered specificity, comprising the steps of: 1) determining at least a portion of the three-dimensional structure of a reference meganuclease-DNA complex; (2) identifying amino acid residues that form a base contact surface at the base position; and (3) the β-carbon of at least the first residue at the interface and at least the first base at the base position; (4) (A) determining the distance between the first base and the second base; For the first residue, group 1 and group 2 are members of G, C, T, or A, as appropriate. and / or group 2; or (B) selecting a group more than 6 angstroms from said first base. The first residue at the more distal position is a member of the appropriate set of G, C, T, and A. Amino acids that promote the desired change by selecting certain group 2 and / or group 3 substitutions The method for achieving this is provided by specifying the substitution. This method is also applicable to other contact residues of the same base. Repeat for other contact residues to other bases at the same position, and then for other positions. That's fine.

[0038] In yet another aspect, the present invention provides recombinant meganucleotides with increased DNA-binding affinity. A method for rationally designing a meganuclease, comprising: (1) determining a reference meganuclease-DNA complex; (2) determining at least a portion of the three-dimensional structure of the backbone; (3) (A) Identifying contact residues with negatively charged or hydrophobic side chains. (B) selecting substituents with uncharged / polar or positively charged side chains for the group; or For contact residues with uncharged / polar side chains, substitutions with positively charged side chains are selected. and identifying amino acid substitutions that increase DNA-binding affinity. Conversely, the present invention provides a method for producing recombinant meganuclei with reduced DNA-binding affinity. A method for rationally designing a meganuclease, comprising: (1) generating a reference meganuclease-DNA; (2) determining at least a portion of the three-dimensional structure of the complex; and (3) (A) Identify contact residues with positively charged side chains. (B) selecting substitutions with uncharged / polar or negatively charged side chains, or (C) hydrophobic and For contact residues with uncharged / polar side chains, substitutions with negatively charged side chains were selected. Therefore, the method to achieve this is to identify amino acid substitutions that decrease DNA-binding affinity. Provide the law.

[0039] These and other aspects and embodiments of the present invention are described in the following detailed description of the invention. It will be apparent to one skilled in the art based on this. BEST MODE FOR CARRYING OUT THE INVENTION

[0040] 1.1 Introduction The present invention relates, in part, to a method for producing a double-stranded DNA recognition sequence by a meganuclease. It forms specific contacts with the NA bases and nonspecific contacts with the DNA backbone, This affects the recognition sequence specificity and DNA-binding affinity of the enzyme. Identification of specific amino acid residues in the LAGLIDADG family of meganucleases This discovery is based on the characterization and characterization of meganucleases, as described in detail below. Amino acid substitutions in meganucleases that can alter specificity and / or affinity and to recognize desired DNA sequences not recognized by natural meganucleases. and / or have a higher specificity and / or affinity than natural meganucleases. The method was used to rationally design and develop meganucleases with increased or decreased Furthermore, DNA-binding affinity affects not only enzyme activity but also sequence specificity, The present invention provides rationally designed meganucleases that have altered activity compared to naturally occurring meganucleases. Furthermore, the present invention provides a method for producing a dimer by contacting monomers. Residues at the interface of the β-glucan complex have been modified to promote heterodimer formation. Finally, the present invention provides for the biotechnology of recombinant cells and organisms. In addition to uses in the production of steroids, as disclosed in this application, gene therapy, anti-pathogen, anti-cancer and the use of rationally designed meganucleases in in vitro applications.

[0041] As a general matter, the present invention provides: (1) a method for detecting individual bases in a double-stranded DNA recognition sequence; sequence-specific binding, or (2) to the phosphodiester backbone of a double-stranded DNA molecule The meganuclease contains modified amino acid residues at multiple sites due to non-specific binding. The present invention provides methods for generating rationally designed LAGLIDADG meganucleases. However, since enzyme activity correlates with DNA-binding affinity, the DNA recognition sequence Varying the amino acids involved in binding to dsDNA can increase the overall binding affinity to dsDNA. The specificity of the meganuclease will be increased or decreased through specific base pair interactions. It may also alter the activity of the meganuclease. Varying the amino acid responsible for binding to the amine alters the overall binding affinity to double-stranded DNA. By increasing or decreasing the degree of binding to the recognition sequence, not only the activity of the enzyme but also The degree of specificity or degeneracy may vary.

[0042] As detailed below, rational design of meganucleases is based on DNA recognition / binding. Applying a set of rules to identify amino acids and select appropriate amino acid changes This rule includes the following: Meganuclear interactions between amino acid side chains and bases in the sense and antisense strands of DNA The relative positions of the amino acid side chains and DNA bases in the enzyme-DNA complex This includes consideration of molecular positioning and non-covalent chemical interactions.

[0043] Finally, the majority of natural meganucleases that bind to DNA as homodimers are pseudo- Recognizes sequences that are either 1 or 2, or are complete palindromes. Therefore, the probability of encountering a palindrome at a sequence site of interest in the genome is extremely low. Therefore, these enzymes can be reprogrammed to recognize sequences of interest in the genome. If we have to calculate the ratio of the two enzyme monomers that recognize different half-sites, Upon heterodimerization, the non-palindromic hybrid recognition sequence is cleaved. Therefore, in one aspect, it is necessary to design two enzyme monomers that can The invention involves the dimerization of two monomers that differ at at least one amino acid position to form heterodimers. The present invention provides a rationally designed meganuclease obtained by forming a dimer. The dimers are rationally designed to form heterodimers that recognize non-palindromic recognition sequences. A mixture of two different monomers can be designed to produce up to three active meganuclei. In addition, or Alternatively, in some cases, it may increase the probability of homodimer or heterodimer formation, or In order to reduce this, the interaction between monomers to form dimers is The amino acid residue is changed.

[0044] Thus, in one aspect, the present invention provides a LAGLIDADG meganuclease and LAGLIDADG meganucleotides containing amino acid changes that alter the specificity and / or activity of the enzyme. In another aspect, the present invention provides methods for the rational design of cleavage enzymes. In another aspect, the present invention provides a rationally designed meganuclease. The desired DNA sequence or locus within the genome can be modified by inserting, deleting, substituting, or Such methods may be used to produce recombinant nucleic acids and organisms that are modified by other manipulations. In yet another aspect, the present invention provides methods of using such rationally designed meganucleases. Rationally designed megakaryons with pathogen-specific or cancer-specific recognition sequences in pathogens or cancer cells A method for reducing the viability of this pathogen or cancer cells using nucleases is provided. .

[0045] 1.2 References and Definitions The patent and scientific literature referred to in this application establishes knowledge that is available to those skilled in the art. Published U.S. patents, approved applications, published patents, and related patent applications cited in this application are Reference materials, including foreign patents and GENBANK database sequences, are incorporated by reference. This application is incorporated by reference in its entirety to the same extent as if each individual patent application was specifically and individually indicated to be incorporated by reference. Included in.

[0046] As used in this application, the term "meganuclease" refers to a nuclease with a recognition sequence greater than 12 base pairs. refers to an endonuclease that binds to double-stranded DNA. Natural meganucleases are It can be a monomer (e.g., I-SceI) or a dimer (e.g., I-CreI). The term meganuclease as used in this application includes monomeric meganucleases, dimeric meganucleases, and dimeric meganucleases, or contacting to form dimeric meganucleases The term "homing endonuclease" refers to a monomer that It is synonymous with the term "proteinase."

[0047] The term "LAGLIDADG meganuclease" as used in this application refers to a LAGLIDADG meganuclease that is not naturally occurring. Meganucleases containing a single LAGLIDADG motif that is a mer-type, or This refers to a meganuclease that contains two LAGLIDADG motifs in its monomeric form. The term "mono-LAGLIDADG meganuclease" as used in the application refers to a single LAG The term "di-LAGLID" as used in this application refers to meganucleases containing a LIDADG motif. The term "ADG meganuclease" is used when it is necessary to distinguish between the two. This refers to meganucleases that contain the LAGLIDADG motif. The two structural domains of the di-LAGLIDADG meganuclease containing L It can be referred to as the AGLIDADG subunit.

[0048] As used in this application, the term "rationally designed" refers to non-naturally occurring and / or genetically engineered The rationally designed meganucleases of the present invention are wild-type or naturally occurring In the amino acid sequence or primary structure, the meganucleases of In addition, the rationally designed meganucleases of the present invention may differ in their amino acid structure, amino acid structure, or quaternary structure. The specificity of the recognition sequence and / or activity of the meganuclease may also differ from those of wild-type or naturally occurring meganucleases. do.

[0049] The term "recombinant" as used herein with respect to a protein refers to a recombinant protein that encodes the protein. The application of genetic engineering techniques to nucleic acids results in changes in the amino acid sequence, and this "Recombinant" with respect to nucleic acids means that a cell or organism that expresses a protein is obtained. The term refers to a change in a nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR, transfection, DNA cloning techniques such as homologous recombination, transformation and other gene transfer techniques This definition includes site-directed mutagenesis and gene fusion. It has the same amino acid sequence as α-glucan, but is produced by cloning and expression in a heterologous host. Proteins produced from recombinant DNA are not considered "recombinant."

[0050] The term "modification" as used herein with respect to recombinant proteins refers to a modification of a reference sequence (e.g., What are the amino acid residues in the recombinant sequence compared to the wild-type? It is meant to include any insertion, deletion or substitution.

[0051] As used in this application, the term "genetically modified" means that the genomic DNA sequence has been modified in a manner that is consistent with the intended purpose. A cell, organism, or ancestor that has a genomic DNA sequence that has been intentionally modified by recombinant techniques As used in this application, the term "genetically modified" refers to a " includes the term ".

[0052] As used in this application, the term "wild-type" refers to any meganuclease The term "wild type" refers to all naturally occurring forms of the enzyme. It does not refer to a wild-type meganuclear gene, but rather to any mutations found in nature. Meganucleases are distinguished from recombinant or non-naturally occurring meganucleases.

[0053] As used in this application, a "recognition sequence half-site" or simply a "half-site" refers to a mono-LAGLIDA Monomer of DG meganuclease or one of di-LAGLIDADG meganuclease The nucleic acid sequence between double-stranded DNA molecules recognized by the LAGLIDADG subunit Taste.

[0054] The term "recognition sequence" as used in this application refers to a mono-LAGLIDADG meganuclear bound and cleaved by the dimer or di-LAGLIDADG meganuclease monomer The two half-sites are specifically recognized by this enzyme. I-CREI, I In the case of I-MSOI and I-CEUI, the recognition sequence half-site of each monomer is 9 base pairs wide. The two half-sites are not specifically recognized but are the actual cleavage site (4 base pairs). The I-CREI fragment is separated by a base pair overhang. The binding recognition sequences of I-MSOI and I-CEUI meganuclease dimers are generally 4 It has a width of 22 base pairs, including two 9-base pair cleavage sites flanking each half site. The base pairs are designated -9 to -1, with position -9 being the most distal from the cleavage site and position -1 being N It is flanked by four central base pairs, denoted 1-N4. Each half-site strand, oriented 5' to 3' in the direction of the cleavage site, The opposite strand is designated the "sense" strand, and the opposite strand is designated the "antisense strand." The other half of the ribosomal RNA does not encode a protein. The "sense" strand of one half-site is the antisense strand of the other half-site. In the case of I-SCEI meganuclease, which is a ganucleases monomer, the recognition sequence is approximately 18 It is a non-palindromic sequence of BP, and there is no central base pair that is not specifically recognized. Thus, one of the two strands is called the "sense" strand and the other the "antisense" strand. Neither chain encodes a protein.

[0055] The term "specificity" as used in this application refers to the ability of a meganuclease to bind to a specific sequence called a recognition sequence. It recognizes and cuts double-stranded DNA molecules only at the base pair sequence or a specific set of recognition sequences. This set of recognition sequences shares some conserved positions or sequence motifs. These may be degenerate at one or more positions. Nucleases are capable of cleaving only one or a very few recognition sequences. This can be determined by the cleavage assay described in Example 1. As used in this application, The nuclease is a nucleotide that is bound and cleaved by a reference meganuclease (e.g., wild-type). If the recognition sequence is not bound to and cleaved, or if the cleavage rate of the recognition sequence is lower than the reference meganuclear Specificity is determined when the level is increased or decreased by a statistically significant value (P<0.05) compared to the level of β-glucose. is considered to have been "changed."

[0056] The term "degenerate" as used in this application means the opposite of "specific." Nucleases are capable of cleaving a wide variety of recognition sequences. The enzyme can have sequence degeneracy within single, multiple, or all half-sites. Such sequence degeneracy can be achieved by (I) allowing for any amino acid sequence in the DNA-binding domain of the meganuclease. (II) the amino acid cannot contact any base at one or more positions within the recognition sequence; One or more amino acids in the DNA binding domain of the nuclease are located at one or more positions in the recognition sequence. or (III) capable of specifically contacting one or more bases at two or more positions; and / or ) activity, and have sufficient nonspecific DNA binding affinity. A "fully" degenerate position may be occupied by any four bases, and in the half-sites by "N". A "partially" degenerate position can be occupied by any two or three of the four bases. (e.g., occupied by purines (PU) or pyrimidines (PY), but not by G (not occupied by

[0057] As used herein with respect to meganucleases, "DNA-binding affinity" or "binding affinity" refers to a "Binding affinity" refers to the degree to which a meganuclease binds to a reference DNA molecule (e.g., a recognition sequence or an arbitrary sequence). The binding affinity is measured by the dissociation constant, KD. (For example, the KD of I-CREI for the WT recognition sequence is approximately 0.1 NM.) When used, the KD of the recombinant meganuclease for the reference recognition sequence is Binding was determined when the increase or decrease in binding activity was statistically significant (P<0.05) compared to that of the ATPase. This means that the affinity has been "changed."

[0058] As used herein with respect to meganuclease monomers, "affinity for dimer formation" refers to The term "degree" refers to the degree to which a meganuclease monomer does not coincide with a reference meganuclease monomer. The affinity for dimer formation is determined by, for example, the tendency of a reference wild-type The dimers may be composed of the same monomer (i.e., homodimer formation) or different monomers, such as ganucleases. The binding affinity can be measured by the dissociation constant (dissociation constant). As used in this application, a recombinant meganucleotide sequence for a reference recognition sequence is measured by KD. The KD of the enzyme was statistically significant (P<0.05) compared to the reference meganuclease. The binding affinity is "altered" if the binding affinity is increased or decreased by a factor of 1.

[0059] The term "palindromic" as used in this application includes inverted repeats of the same half-sites. However, in this case, the palindromic sequence is the one that is contacted by the enzyme. It is not necessary for the four missing central base pairs to be palindromic. In the case of DNA fragments, the palindromic DNA sequence is such that two monomers contact the same half-site. It is recognized by a homodimer that interacts with the ATP.

[0060] As used in this application, the term "quasi-palindromic" refers to non-identical or imperfect palindromic In this case, the recognition sequence is a pseudopalindromic sequence. The sequence need not be palindromic with respect to the four central base pairs, but rather between the two half-sites. It is also possible to deviate from the palindromic sequence between the two. The rows represent wild-type homodimers, where two identical enzyme monomers contact different half-sites. These are typically found at natural DNA sites recognized by mer-type meganucleases.

[0061] The term "non-palindromic" as used in this application refers to the separation of two, refers to a recognition sequence composed of unrelated half-sites. In this case, a non-palindromic sequence is Palindromic across the four central base pairs and across the two monomer half-sites The non-palindromic DNA sequence does not have to be a di-LAGLIDADG meganuclear sequence. enzymes, highly degenerate mono-LAGLIDADG meganucleases (e.g., I-CeuI), non- Heterodimers of mono-LAGLIDADG meganucleases that recognize identical half sequences be recognized by either

[0062] The term "activity" as used in this application refers to the ability of the meganuclease of the present invention to bind to a specific recognition sequence. This activity refers to the rate at which the enzyme breaks the phosphodiester bond in double-stranded DNA. It is a measurable enzyme reaction for hydrolysis. The activity of a meganuclease is determined by the affinity or binding strength of the meganuclease for its particular DNA substrate. is influenced by sequence-specific and non-sequence-specific interactions with DNA. do.

[0063] As used in this application, the term "homologous recombination" refers to the process by which a double-stranded DNA break acts as a repair template. Refers to the natural, cellular process of repair using homologous DNA sequences (e.g., CAHIL LET AL.(2006),FONT.BIOSCI.11:1958-1976 The homologous DNA sequence may be an endogenous chromosomal sequence or may be a sequence that has been delivered to the cell. It may be an exogenous nucleic acid. Thus, in one embodiment, rationally designed meganuclear The enzyme is used to cleave a recognition sequence within the target sequence, and the enzyme is then used to cleave a recognition sequence that is homologous or similar to the target sequence. An exogenous nucleic acid with approximately the same sequence similarity is delivered to the cell and repaired by homologous recombination. Therefore, the external sequence may be significantly different from the target sequence. The native DNA sequence is integrated into the chromosomal sequence. The process of homologous recombination is primarily found in eukaryotic organisms. In this application, the term "homology" is used equivalently to "sequence similarity." It is not intended to require identity by descent or lineage.

[0064] As used in this application, the term "non-homologous end joining" refers to the process by which a double-stranded DNA break is generated between two non-homologous ends. refers to the natural cellular process by which DNA is repaired by the direct joining of identical DNA segments (e.g., For example, CAHILL ET AL. (2006), FRONT.BIOSCI.11: 1958-1976). DNA repair by non-homologous end joining is error-prone, Repair often results in the addition or removal of non-template DNA sequences at the site of repair. Thus, in some embodiments, mutations (e.g., base insertions, base deletions, or frameshift mutations) are Disrupt the gene by non-homologous end joining (by performing a mutation) and create a megagene within the target sequence. Using rationally designed meganucleases to create double-strand breaks in nuclease recognition sequences In other embodiments, the target sequence lacks homology or is a target sequence. Exogenous nucleic acids lacking substantial sequence similarity to the sequence can be integrated into the membrane by non-homologous end joining. It may be incorporated at the site of a ganucleases-stimulated double-stranded DNA break (e.g., SALOMO N, ET AL. (1998), EMBO J. 17:6086-6095). The process of homologous end joining occurs in both eukaryotes and prokaryotes such as bacteria.

[0065] As used in this application, the term "sequence of interest" refers to a sequence that is synthesized using a meganuclease protein. A nucleic acid sequence that can be inserted into a genome or used to replace a genomic DNA sequence. Thus, proteins, RNA or regulatory elements (e.g., enhancers, silencers, or By "nucleotide sequence" is meant any nucleic acid sequence, regardless of whether it encodes a nucleic acid sequence (e.g., a promoter sequence). The sequence of interest may be a non-transferable molecule that allows for labeling of the protein or RNA expressed from the sequence of interest. It is also possible to have homologous DNA sequences, for example but not limited to: However, proteins may contain epitopes (e.g., C-MYC, FLAG) or other ligands. It is possible to label the protein with a tag containing a nucleotide (e.g., poly-HIS). The flavor sequences may encode fusion proteins according to techniques well known in the art (e.g., AUSUBEL ET AL., "Current Protocols in Molecular Biology ("CUR RENT PROTOCOLS IN MOLECULAR BIOLOGY,”) WILEY 1999). In one embodiment, the flanking sequences are incorporated into the recombinant meganuclease. The DNA is then cut and inserted appropriately into the genomic recombination sequence.

[0066] Thus, the flanking sequences are cleaved and the resulting fragments are cleaved by the recombinant meganuclease. This allows for the proper insertion of a sequence of interest into a genomic recognition sequence. In this case, all of the sequences of interest are homologous to the target sequence in the genome or are substantially identical to the target sequence. The target sequence is efficiently transformed by the sequence of interest through homologous recombination. In other embodiments, the sequence of interest has homology to the target sequence or is substantially It is flanked by DNA sequences with similarity to the target gene in the genome and is then transferred to the target gene by homologous recombination. In one embodiment, the sequence of interest is inserted at the target sequence locus. The target sequence is nearly identical except for mutations or other modifications in the cleavage enzyme recognition sequence. Therefore, meganucleases can be used to modify the target sequence with the sequence of interest after the target sequence has been modified. The target sequence cannot be cleaved.

[0067] As used herein with respect to amino acid and nucleic acid sequences, the terms "percent similarity" and " The term "sequence similarity" refers to the similarity between aligned amino acid residues or nucleotides. It is a measure of the similarity between two sequences based on sequences aligned to maximize similarity. This allows for the presence of gaps in the alignment sequence and the number of gaps, nucleic acids or residues. The total number of identical or similar nucleic acids or residues is a function of the number of identical or similar nucleic acids or residues. A variety of algorithms and computer programs are available to determine sequence similarity. In the present application, sequence similarity is measured using the BLASTP program in the case of amino acid sequences. For nucleic acid sequences, the BLASTN program is used. TIONAL CENTER FOR BIOTECHNOLOGY INFORMAT Available through ION (WWW.NCBI.NLM.NIH.GOV / ), e.g. ALTSCHUL ET AL. (1990), J.MOL.BIOL.215: 403-410;GISH AND STATES(1993), NATURE GE NET.3:266-272;MADDEN ET AL.(1996),METH.E NZYMOL.266:131-141;ALTSCHUL ET AL.(1997) ,NUCLEIC ACIDS RES.25:3389-3402);ZHANG E T AL. (2000) J.COMPUT.BIOL.7(1-2):203-14 In this application, the percent similarity of two amino acid sequences is determined using the BLASTP algorithm. The score is based on the following parameters in the rhythm: word size = 3; gap opening penalty = -11; gap extension penalty = -1; and scoring matrix In this application, the percent similarity of two nucleic acid sequences is calculated using the BLOSUM62 index. The score is based on the following parameters in the STN algorithm: Word size = 11 ;Gap opening penalty = -5;Gap extension penalty = -2;Match rating = 1 ; and mismatch penalty = -3.

[0068] As used herein in reference to modifications of two proteins or amino acid sequences, "corresponding" The term is used to generically identify two proteins (e.g., using the BLASTP program). When the sequences are aligned in a suitable manner, the modification of the first protein is more closely related to the modification of the second protein. the amino acid residue of the modification is the same as that of the modification in the first protein; This means that the position corresponds to or aligns with the amino acid position of the modification in the second protein. Thus, if residues X and Y correspond to each other in a sequence alignment, then the Even if the sequence numbers are different, residue "A" in the first protein is the amino acid "A " corresponds to the modification of residue "Y" to amino acid "A".

[0069] In this application, a numerical range for a variable is equal to any value within that range. It is intended that the present invention can be implemented using variables that are inherently discontinuous. If it is a number, this variable can be equal to any integer at either end of the numeric range. Similarly, if a variable is inherently continuous, it can be expressed as a function of the endpoints of its numerical range. can be equal to any real number, including, but not limited to: Although not a fixed value, a variable described as having a value between 0 and 2 is considered to be a fixed value if the variable is inherently non-interdependent. If the variable is inherently continuous, it can take on the values ​​0, 1, or 2. If the value is 0.0, 0.1, 0.01, 0.001 or any real number between 0 and 2, can take any real number.

[0070] In this application, unless specifically stated otherwise, the word "or" means "and / or." It is used in the inclusive sense, not in the exclusive sense of "either / or."

[0071] 2.1 Rationally designed meganucleases with altered sequence specificity In one embodiment of the present invention, recombinant LAGLIDADG family meganucleases are synthesized. In this embodiment, a method is provided for rationally designing the bases at each position of the half-site. By predicting amino acid substitutions that can alter targeting, we can develop a method for engineering recombinant meganucleases. These substitutions, individually or in combination, are experimentally tested to determine their effectiveness. The efficacy is verified to produce meganucleases with the desired cleavage specificity.

[0072] According to the present invention, amino acid substitutions that result in the desired change in base preference are made in the meganuclease. DNA recognition sequence of the enzyme and its interaction with the nucleobases of the DNA phosphodiester backbone A reference meganuclease (e.g., a wild-type meganuclease or a non-naturally occurring meganuclease) that can participate in the contact The spatial and chemical properties of the amino acid side chains and their contact points of the reference meganucleases were determined. These amino acids are predicted by contacting the canonical DNA half-site. Generally, this determination involves determining the side chains that contribute to the Knowledge of the structure of the complex between the cleavage enzyme and its double-stranded DNA recognition sequence, or The structure of the complex with the highest similarity (e.g., the same meganuclease and a different DNA recognition sequence) or an allelic or systematic variant of the meganuclease and its Knowledge of the complex structure between the target and the DNA recognition sequence is required.

[0073] A polypeptide or two or more polypeptides described as atomic coordinate data The three-dimensional structure of a complex can be obtained in several ways. Determination of the structure of the substance may be accomplished by, but is not limited to, X-ray crystallography, NMR, and spectroscopic techniques. Another method is to measure the amount of the meganuclei of interest using techniques including quantitative analysis. By analyzing the existing database of structural coordinates of nucleases or related nucleases, Such structural data is often obtained from databases in the form of three-dimensional coordinates. Often this data is available through online databases (e.g. For example, see the RCSB PROTEIN DATA at WWW.RCSB.ORG / PDB BANK) can be accessed.

[0074] Structural information is the ordered, two- or three-dimensional array of a protein or protein complex. obtained by analyzing the diffraction patterns of a sample (e.g., a crystal) e.g., X-ray or electron diffraction patterns It is possible to experimentally obtain diffraction data by converting it into three-dimensional atomic coordinates in space. For example, the field of X-ray crystallography uses computer methods to convert For generating three-dimensional structural information for many protein-DNA complexes, including nucleases (e.g., CHEVALIER ET AL. (2001), NUCL See EIC ACIDS RES.29(18):3757-3774).

[0075] Nuclear magnetic resonance (NMR) is also used to determine interatomic distances in molecules in solution. Multidimensional NMR techniques combined with computer methods have enabled the identification of atomic coordinates for polypeptides of increasing size. has been successfully determined (e.g., TZAKOS ET AL. (2006), ANNU.RE V.BIOPHYS.BIOMOL.STRUCT.35:12-42).

[0076] Separately, the known primary structure of the protein / DNA and, if available, secondary and tertiary structures secondary and / or quaternary structure, as well as known properties of amino acid side chains, nucleobases and bonding interactions. By applying algorithms based on the physical and chemical properties of the known compounds, Such methods may optionally be iterative or experimentally derived. An example of such computer software is ADAMS ET AL. (1999), ACTA CRYSTALLOGR.D. BIOL.CRYSTALLOGR.55(PT 1):181-90 Other CNS programs include the spatial arrangement of amino acids in protein structures and Various computer programs are available to predict the interactions between amino acid side chains of proteins and various target molecules. A program has been developed (see, for example, US Pat. No. 6,988,041).

[0077] Thus, in one embodiment of the present invention, a compound that specifically interacts with DNA nucleobases, and / or specific phosphodiester backbone interactions that promote nonspecific phosphodiester backbone interactions. A computer model is used to identify amino acid residues. A computer model of the entire clease-DNA interaction can be constructed, for example, by limiting Although not intended to be used with MOLSCRIPT® 2.0 (AVATAR SOFTWARE), TWARE AB, STOCKHOLM, SWEDEN), Graphic Display (JONES ET AL.(1991), ACTA CRYSTALLOGRAPHY ,A47:110), graphic display program GRASP (registered trademark) NICHOLLS ET AL. (1991), PROTEINS, STRUCTURE ,FUNCTION AND GENETICS 11(4):281FF),Graphy Display program INSIGT (registered trademark) (TSI, INC., SHOR Produced using appropriate software programs, including EVIEW, Minnesota It is possible to produce, view, and manipulate three-dimensional structural images of protein-DNA complexes. Suitable computer hardware for producing the (e.g., SILICON GRAPHICS WORKSTATION, SILICON N GRAPHICS, INC., MOUNTAINVIEW, CA).

[0078] Specifically, the interaction between the meganuclease and its double-stranded DNA recognition sequence can be resolved using methods known in the art. Three-dimensional structural drawings, or models, of multi-component complex structures are produced using molecular replacement, or SIR / MIR (SINGLE / MULTIPLE ISOMORPHOUS R EPLACEMENT) (e.g., BRUNGER (1997), METH. ENZYM .276:558-580;NAVAZA AND SALUDIJIAN(1997) ,MATH.ENZYM.276:581-594;TONG AND ROSSMAN N(1997), METH.ENZYM.276:594-611; and BENTLE Y (1997), METH.ENZYM.276:611-619) For example, AMORE / MOSFLM(NAVAZA(1 994), ACTA CRYST.A50:157-163;CCP4(1994),A CTA CRYST.D50:760-763) or XPLOR(BRUENGE R ET AL.(1992),X-PLOR VERSION 3.1.A.SYST EM FOR X-RAY CRYSTALLOGRAPHY AND NMR,YAL University Press, New Haven, Connecticut) It can be implemented using software.

[0079] Determination of the protein structure and possible meganuclease-DNA interactions will allow for enzymatic activity and This allows for rational selection of amino acids that can be varied to affect activity and specificity. The determination is based on the amino acid side chain, the specific base or the DNA phosphodiester backbone. The appropriate amino acid substitutions are based on several factors related to the interaction with the target protein. Chemical interactions that may be used include, but are not limited to, van der Waals These include ionic forces, steric hindrance, ionic bonds, hydrogen bonds and hydrophobic interactions. Does the enzyme prefer a particular interaction with a particular base in a potential recognition sequence half-site? Amino acid substitutions that promote or repel the recognition of the target protein are selected. and to some extent to increase or decrease overall binding affinity and activity. Furthermore, due to increased or decreased overall activity and, to some extent, reduced specificity, to the phosphodiester backbone of double-stranded DNA to increase or decrease Amino acid substitutions can be selected that increase or decrease binding.

[0080] Thus, in certain embodiments, the three-dimensional structure of the meganuclease-DNA complex is determined. A "contact surface" is defined for each base pair in the DNA recognition sequence half-site. In some embodiments, the interface is such that the residues are identical to those in the wild-type meganuclease-DNA complex. The major groove hydrogen bonds in either base of the pair are formed regardless of whether they perform base contacts. In enzymes with a β-carbon less than 9.0 Å from the donor or acceptor In another embodiment, the residues comprise the amino acids of the wild-type meganuclease-DNA complex. If no contact is made in the Inclusion or exclusion of residues based on the designer's intent to vary the number or names of groups In one example described below, the wild-type I-CREI half site is located at base position - In 2, -7, -8, and -9, the contact surface is actually the same as in the wild-type enzyme-DNA complex. The interaction was limited to amino acid positions. However, positions -1, -3, -4, and -5 In -6 and -7, the contact surface is not involved in wild-type contacts but is replaced by different amino acids. If the base is contacted, the amino acid sequence is defined to include additional amino acid positions that may contact the base. Ta.

[0081] Recognition sequence half-sites are usually expressed on only one strand of DNA, but in meganuclear It should be noted that enzymes bind to the major groove of double-stranded DNA and contact the nucleobases of both strands. Furthermore, the designations "sense" and "antisense" strands are not The binding and recognition of the nucleotides is entirely ad hoc. Interaction with one member of the base pair or a combination of interactions with both members of the base pair Therefore, for example, at position X, is an A / T base pair, where the A base is on the "sense" strand and the T base is on the "antisense" strand. To favor the presence of Residues are selected that favor the presence of A and / or A bond to the antisense strand at position X. Residues that are close enough to be touchable and that favor the presence of a T are selected. If the β-carbon of the group is within 9 Å of the nearest atom of the relevant base, the residue are considered to be sufficiently close.

[0082] Thus, for example, within 9 angstroms of the DNA sense strand, but Amino acids with β-carbons more than 9 Å away from the sense strand Similarly, the antisense strand of DNA is considered to have the potential to interact only with the Within 9 Å, but more than 9 Å away from the sense strand Amino acids with β-carbons may interact only with the antisense strand. Which amino acids have β-carbons within 9 angstroms of each other on both DNA strands? It is considered that there is a possibility of interaction with both chains.

[0083] For each interface, one or more of the four DNA bases are selected for possible amino acid substitutions. Predict the ability of a target molecule to interact with other target molecules, and then select amino acid substitutions based on that prediction. This selection process is based on two main criteria: (I) different nucleobases and (II) the size of the amino acid side chains that affect the steric interactions of the It is the chemical properties of the amino acid side chains that influence electrostatic and bonding interactions.

[0084] Regarding the size of the side chain, the β-carbon of the amino acid at the interface is 6 oz from the base. If the distance is less than 1 / 3 of a tetramer, then amino acids with relatively short and / or relatively small side chains are preferred. It is possible to select an amino acid such that the β-carbon of the amino acid at the interface is 6 octadecyl groups from the base. If the distance is greater than 1 / 2 Angstrom, the side chains will be relatively long and / or relatively large. It is possible to select amino acids with intermediate size side chains at the interface. The amino acid β-carbon in the nucleotide sequence is preferably selected when it is 5-8 angstroms from the base. It is possible.

[0085] Amino acids with relatively short and relatively small side chains include glycine (G), alanine (A), Serine (S), threonine (T), cysteine ​​(C), valine (V), leucine (L), Isoleucine (I), aspartic acid (D), asparagine (N) and proline (P) However, proline is relatively less flexible and therefore Furthermore, glycine does not introduce unnecessary bending into the peptide backbone. To introduce a certain degree of affinity and because of its extremely small size, substitution of larger residues In this case, the probability of effective contact is thought to be low, so relatively frequent use is expected. On the other hand, glycine can be used to promote degenerate positions in some cases. Amino acids with side chains of relatively intermediate length and size include lysine (K), methionine ( They were divided into two groups containing glutamine (Q), arginine (R), glutamic acid (E), and glutamine (Q). Amino acids with relatively long and / or relatively large side chains are assigned ), methionine (M), arginine (R), histidine (H), phenylalanine (F) They are assigned to three groups, including α, tyrosine (Y), and tryptophan (W). However, tryptophan is relatively inflexible and is therefore expected to be used less frequently. Furthermore, the flexibility of the lysine, arginine, and methionine side chains allows these amino acids to be easily attached to the lysine, arginine, and methionine side chains. Acids can contact the base from long or intermediate distances, so they are divided into groups 2 and 3. These groups are further shown below in Table 1.

[0086] [Table 1]

[0087] Regarding the chemical nature of the side chains, different amino acids have different possibilities for interacting with different nucleobases. The properties of the bond have been evaluated (e.g., van der Waals forces, ionic bonds, hydrogen bonds, and Hydrophobic interactions), specific bases at specific positions in the double-stranded DNA recognition sequence half-site, and Residues are selected that favor or disfavor specific interactions with the ganucleases. It is desirable to create half-sites with one or more fully or partially degenerate positions. In such cases, residues that favor the presence of two or more bases or that dislike one or more bases may be present. For example, partially degenerate base recognition may be used to identify sense or antisense bases. This can be achieved by sterically hindering the pyrimidine in the sense position. .

[0088] The recognition of the guanine (G) base involves the formation of hydrogen bonds between the N7 and O6 of the base. This is achieved by using amino acids with a basic side chain. The specificity of cytosine (C) is achieved by using all amino acids except C. The negatively charged side, which is present on the base of the The recognition of thymine (T) is conferred by the hydrophobic side chain and the major groove methyl group above the base. The hydrophobic and van der Waals interactions between the , the adenine (A) base is connected to A via a pair of hydrogen bonds to N7 and N6 of the base. Recognition by the carboxamide side chain of SN and GLN or the hydroxyl side chain of TYR Finally, HIS binds to the purine by donating a hydrogen bond to N7. It can be used to confer specificity for the base (A or G). Using simple rules for DNA recognition, we can determine whether a base pair at a particular base pair position is a salt. One or both of the groups are identified through rational design contacts. It is possible to predict.

[0089] Therefore, preferred bases at positions that make binding interactions and contacts with various nucleobases. Based on this, each amino acid residue is assigned to the various bases it prefers (i.e., G, C, T, or A) can be assigned to one or more different groups corresponding to G Group C contains arginine (R), lysine (K), and histidine (H); The T group includes alanine (A), valine ( V), leucine (L), isoleucine (I), cysteine ​​(C), threonine (T), Group A includes asparagine ( N), glutamine (N), tyrosine (Y), and histidine (H). Cysteine ​​appears in both the G and A groups; serine (S) is not included in either group, but degenerate positions may be used as preferred; and proline, glycine, and thiamin It is noted that tryptophan is not included in any particular group, primarily due to steric considerations. These groups are also shown below in Table 2.

[0090] [Table 2]

[0091] Therefore, according to the present invention, at any position X, the recognition sequence half of the meganuclease is (1) wild-type, or reference, meganuclease-D to perform the desired changes at the site of division; At least a relevant portion of the three-dimensional structure of the NA complex and an amino acid that defines the contact surface at position X (2) determining the β-carbon and position of at least one residue that includes the contact surface; (3) determining the distance between at least one base of a base pair at position X; To facilitate the change, (A) for residues less than 6 angstroms from the base, Group 1 and / or Group 2 that are members of Group G, Group C, Group T or Group A as appropriate. (B) selecting residues from group 2 and / or (C) being more than 6 angstroms from the base For residues in the group G, C, T or A, as appropriate, The first step is to select residues from two and / or three groups that contain contact surfaces. Such residues can be selected for analysis and modification, and in some embodiments, such residues are Each residue is analyzed and multiple residues are modified. Similarly, the β-carbon of residues involved in the contact area is modified. It is possible to measure the distance between the base and each of the two bases of the base pair at position X. and if the residue is within 9 angstroms of both bases, then the It is possible to carry out different substitutions to affect two bases (e.g., one A group of residues is used to affect proximal bases on one strand, or distal bases on the other strand. In addition, residues that can interact with both bases in the pair Combinations of substitutions can affect specificity (e.g., antisense strand Residues in the T group contacting the sense strand in combination with residues in the A group contacting the T group select T / A. Finally, multiple candidate modifications of residues can be empirically tested (e.g., by testing recombinant meganucleases). by producing a sequence recognition algorithm and examining its sequence recognition, or computationally (e.g., by modifying The effectiveness of the enzyme was confirmed by computer modeling of the meganuclease-DNA complex. It is possible to verify the validity of the candidate and select from multiple candidates.

[0092] Once one or more desired amino acid modifications of a wild-type or reference nuclease have been selected, Rationally designed meganucleases are produced by recombinant methods and techniques well known in the art. In some embodiments, non-random sequencing is used to create specific sequence modifications. In some cases, site-directed mutagenesis techniques are used. Non-limiting examples of non-random mutagenesis techniques include: For example, overlapping primer PCR (e.g., WANG ET AL. (2006), NU CLEIC ACIDS RES.34(2):517-527), site-directed protrusion spontaneous mutagenesis (see, e.g., U.S. Patent No. 7,041,814), cassette mutagenesis (See, e.g., U.S. Patent No. 7,041,814), and PROMEGA BIOS From Sciences, Inc. (San Luis Obispo, California) Commercially available ALTERED SITES® II MUTAGENESIS SYS The manufacturer's protocol for the TEM kit is listed below.

[0093] The recognition and cleavage of specific DNA sequences by rationally designed meganucleases is well known to those skilled in the art. The amount of hydroxybenzoates can be quantified by any method known in the art (see, for example, U.S. Patent Application Publication No. 2006 In one embodiment, quantification of meganuclease cleavage is performed using an in vitro assay. Such assays are useful for determining the specificity of the meganuclei to be quantified. in vitro cleavage of a polynucleotide substrate containing the intended recognition sequence of the enzyme, In some embodiments, one or more bases in one or both half sites are changed to a different base. In vitro cleavage of polynucleotide substrates containing variants of the intended recognition sequence is employed. Typically, the polynucleotide substrate is a double stranded polynucleotide containing the target site that is synthesized and cloned into a vector. A polynucleotide substrate is a single stranded DNA molecule. The polynucleotide substrate can be linear or circular. Meganucleases, under appropriate conditions, can synthesize polynucleosides, but usually contain only one recognition sequence. The resulting polynucleotides are then incubated with the cleavage substrate and analyzed to identify the cleavage products. The two linear In the case of a single recognition sequence in a stranded DNA substrate, meganuclease activity is determined by the Detection is by the appearance of a band and the disappearance of the initial full-length substrate band. Meganuclease activity can be measured, for example, by the method described in WANG ET AL. (1997), NUCLEI C ACID RES., 25:3767-3776. It is possible.

[0094] In other embodiments, the cleavage pattern of the meganuclease is determined by in vivo cleavage assays. (See, e.g., U.S. Patent Application Publication No. 2006 / 0078552). In one embodiment, the in vivo assay is a single strand annealing recombination assay (SSA). Tests of this kind are known to those skilled in the art (RUDIN ET AL. (1989), GENE TICS 122:519-534;FISHMAN-LOBELL ET AL.(1 992),SCIENCE 258:480-4).

[0095] As will be apparent to those skilled in the art, the domains of the meganucleotides other than those involved in DNA recognition and binding may be included. The enzyme domain of the cleavage enzyme is further modified to produce new amino acids without completely losing its activity. Substitutions, insertions, or deletions may be made without structural or functional constraints. Conservative substitutions of similar amino acid residues at selected positions may be used, or may be structural or Non-conservative substitutions may also be made at positions that are relatively unconstrained functionally. Substitutions, insertions, or deletions can be made by one of ordinary skill in the art without undue effort and with routine experimentation. Thus, in one embodiment, the recombinant DNA of the present invention can be identified. The ganucleases have a random sequence identity between 85% and 99% of the reference meganuclease sequence. Percentage (e.g., 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 99%). Many N- and C-terminal sequences for the I-SCEI and I-CEUI proteins cannot be clearly seen in X-ray crystal experiments because these positions are structurally These residues are therefore not functionally constrained by sequence similarity. can be excluded from the calculation of , using the reference meganuclease sequence below For I-CREI, residues 2-153 of SEQ ID NO: 1 are possible; for I-MSOI, residues 2-153 of SEQ ID NO: 1 are possible; residues 6-160 of SEQ ID NO: 6 for I-SCEI; residues 3-18 of SEQ ID NO: 9 for I-SCEI 6; residues 5-211 of SEQ ID NO: 12 for I-CEUI.

[0096] 2.2 LAGLIDADG family meganucleases The LAGLIDADG meganuclease family originates from a diverse phylogenetic group of host organisms The family consists of over 200 members. All members of this family are specific D The highly conserved LAGLIDADG motif, along with other structural motifs involved in cleavage of the NA sequence Contains one or two copies of the LAGLIDADG motif. Contains one copy of the LAGLIDADG motif. The enzyme (i.e., mono-LAGLIDADG meganuclease) functions as a dimer. On the other hand, enzymes with two copies of this motif (i.e., di-LAGLIDADG Meganucleases) function as monomers.

[0097] All LAGLIDADG family members recognize relatively long sequences (>12 bp). These enzymes also cleave the nucleotides, leaving a four nucleotide 3' overhang. In addition to LAGLIDADG, antiparallel β-strands at the protein-DNA interface They share several structural motifs, including similar arrangements. These conserved structural motifs The amino acids in the family interact with DNA bases, conferring sequence specificity. Some members (e.g., I-CREI, I-MSOI, I-SCEI and I-CE UI ) have been shown by X-ray crystallography to have similar overall structures. Thus, members of this family have specific amino acid sequences within this structural motif. It is possible to modify the enzyme in various ways to alter its overall activity or sequence specificity, and to It is reasonable to expect that modifications of this kind will have similar consequences in other family members. For an overview, see CHEVALIERET AL. (2001), NUCLEIC See ACID RES. 29(18):3757-3774).

[0098] 2.2.1 Meganucleases derived from I-CREI In one aspect, the present invention provides a method for the preparation of a strain of CHLAMYDOMONAS REINHARDTII. Rationale based on or derived from I-CREI meganuclease Regarding the designed meganuclease, the wild-type amino acid sequence of I-CREI meganuclease is , shown in SEQ ID NO: 1, corresponding to GENBANK Accession #PO5725. Crystal structure The two recognition sequence halves of the wild-type I-CREI meganuclease in PDB#1BP7 The division sites are shown in Table 3 below.

[0099] [Table 3]

[0100] This natural recognition sequence is not completely palindromic, even outside the central four bases. Note that there are no two recognition sequence half-sites in bold on each sense strand. It is shown as follows.

[0101] Wild-type I-CREI further comprises the following bases (excluding the central N1-N4 bases) as shown in Table 4 below: k) It also recognizes and cleaves perfectly palindromic sequences.

[0102] [Table 4]

[0103] The palindromic sequences of SEQ ID NO: 4 and SEQ ID NO: 5 are more specific to wild-type I-CREI. It is believed to be a good substrate because the enzyme reacts more efficiently with the natural DNA sequence. This is because the enzyme binds to this site with high affinity and cleaves the site more efficiently. For purposes of illustration, and particularly in the context of the experimental results presented in this application, wild-type I- This palindromic sequence cleaved by CREI will be called "WT" (for example, (See Figure 2(A) for an example.) Two recognition sequence half-sites are shown in bold on each sense strand. can be.

[0104] Figure 1(A) shows the double-stranded DNA of the wild-type I-CREI meganuclease homodimer. Figure 1(B) depicts the interaction between the wild-type enzyme half-site and the wild-type recognition sequence. The specific interactions between the amino acid residues and bases of this enzyme at the -4 position are shown in Figure 1 ( Figures 1(C)-1(E) show three of the present invention's modified specificity at the -4 position of the half-site. The amino acid residues of the enzyme at the -4 position of one half site of the rationally designed meganuclease are It shows specific interactions between the bases.

[0105] Thus, the preference for a base at any given base position of a half-site is Using the methods disclosed in

[0046] , it is possible to rationally vary each of the other three bases. First, the wild-type recognition surface at a specified base position is determined (e.g., meganuclear By analyzing the co-crystal structure of the meganuclease-DNA complex, or by analyzing the meganuclease-DNA Second, the surrounding amino acids at a given base position are identified. Based on the distance between the β-carbon of the carboxylic acid position and the nucleobase in each DNA strand, Possible contact residues are determined, for example, but not limited to, the residues shown in Figure 1(A). As shown, the I-CREI wild-type meganuclease-DNA contact residue at position -4 is: It contains a glutamine at position 26, which hydrogen bonds with the A base of the antisense DNA strand. Furthermore, it is particularly noteworthy that residue 77 has the potential to contact base −4 of the DNA antisense strand. The β-carbon of residue 26 is 5.9 Å away from the N7 of the A base of the antisense DNA strand. The β-carbon of residue 77 is 7.15 Å from the C5 methyl of T in the sense strand. According to this distance and the base chemistry rules described in this application, the sense strand is This allows hydrogen bonding with glutamic acid at position 77, forming the antisense strand. If G is used, it can bind to glutamine at position 26 (wild-type I-CREI binding As seen in the crystal structure, the sense strand is G (see Figure 1(C)). This allows hydrogen bonding with the arginine at position 77, and the antisense strand is C. This allows it to hydrogen bond with glutamic acid at position 26 (see Figure 1(D)). If the sense strand is A, it can hydrogen bond with glutamine at position 77, and the anti If the sense strand is T, it can form hydrophobic contact with the alanine at position 26 ( (See Figure 1(E)). If the base specific contact is provided by position 77, then The wild-type contact Q26 was substituted (e.g., by a serine residue) to determine its effect on specificity. Alternatively, the phases at positions 26 and 77 can be reduced or eliminated. Complementary mutations can be combined to specify specific base pairs (e.g., A2 6 specifies a T in the antisense strand and Q77 specifies an A in the sense strand ( Figure 1(E)). All of these predicted residue substitutions were experimentally validated. .

[0106] Therefore, according to the present invention, the DNA recognition domain of I-CREI meganuclease A defined number of amino acid substitutions have been identified for the α-glucan derivatives of α-glucan. These may be used alone or in combination. Recombination specificity was altered at individual bases within the DNA recognition sequence half-sites. The meganucleases are therefore rationally designed meganucleases with half-sites that differ from the wild-type enzyme. It is possible to produce a CREI by amino acid modification of I-CREI and thereby The resulting changes in recognition sequence half-site specificity are shown in Table 5.

[0107] [Table 5]

[0108] The bolded entries are wild-type contact residues and do not constitute "modifications" as used in this application. An asterisk indicates that the residue contacts a base in the antisense strand.

[0109] 2.2.2. Meganucleases obtained from I-MSOLI In another aspect, the invention provides I-MSOI meganuclei of MONOMASTIX sp. The present invention relates to rationally designed meganucleases based on or derived from this nuclease. The wild-type amino acid sequence of I-MSOI meganuclease is available at GENBANK accession number AA L34387 and is shown in SEQ ID NO: 6. The wild type in the crystal structure PDB#1M5X The two recognition sequence half-sites of the Type I-MSOI meganucleases are shown in Table 6 below.

[0110] [Table 6]

[0111] The recognition sequence is not completely palindromic, even outside the central four bases. Note: The two recognition sequence half-sites are shown in bold on each sense strand.

[0112] According to the present invention, a certain targeting molecule for the DNA recognition domain of I-MSOI meganuclease is A defined number of amino acid substitutions were identified. These may be used alone or in combination to Engineered meganuclei with altered specificity at individual bases within the NA recognition sequence half-site and therefore rationally designed meganucleases with half-sites that differ from the wild-type enzyme. The amino acid modifications of I-MSOI and the predicted recognition sequences can be obtained. The changes in sequence half-site specificity are shown in Table 7.

[0113] [Table 7]

[0114] Bold indicates wild-type contact residues and does not constitute a "modification" as used in this application. An asterisk indicates that the residue contacts a base in the antisense strand.

[0115] 2.2.3. Meganucleases derived from I-SCEI In another aspect, the present invention provides an IS of SACCHAROMYCESCEREVISIAE. Rationally designed meganucleases based on or derived from CEI meganucleases The wild-type amino acid sequence of the I-SCEI meganuclease is Corresponding to NK accession number CAA09843 and shown in SEQ ID NO: 9. Crystal structure PDB#1 The recognition sequence of the wild-type I-SCEI meganuclease in R7M is shown in Table 8 below.

[0116] [Table 8]

[0117] The recognition sequence is non-palindromic and does not have four base pairs separating the half-sites Please note that.

[0118] According to the present invention, certain antibodies against the DNA recognition domain of I-SCEI meganuclease are A defined number of amino acid substitutions were identified. These may be used alone or in combination to Recombinant meganucleases with altered specificity at individual bases within the NA recognition sequence, This could lead to rationally designed meganucleases with recognition sequences different from those of wild-type enzymes. The amino acid modifications of I-SCEI and the changes in predicted recognition sequence specificity were investigated. , as shown in Table 9.

[0119] [Table 9]

[0120] Bold entries represent wild-type contact residues and do not constitute "modifications" as used in this application. indicates that the residue contacts a base in the antisense strand.

[0121] 2.2.4 Meganucleases derived from I-CEUI In another aspect, the present invention provides a method for the production of a medicament for the treatment of malaria. Rationally designed meganucleases based on or derived from UI meganucleases The wild-type amino acid sequence of the I-CEUI meganuclease is It corresponds to K accession number P32761 and is shown in SEQ ID NO: 12. Crystal structure PDB#2EX The two recognition sequence half-sites of the wild-type I-CEUI meganuclease in 5 are shown below: vinegar.

[0122] [Table 10]

[0123] Although I-CEUI is a homodimer, the recognition interface of I-CEUI is Due to the natural degeneracy in the CTURE 14:869-80), the recognition sequence is also non-parametric even outside the central four bases. Note that the two recognition sequence half-sites are phosphodiesterase-specific. Shown in bold on the chain.

[0124] According to the present invention, a certain targeting molecule for the DNA recognition domain of I-CEUI meganuclease is A defined number of amino acid substitutions were identified. These may be used alone or in combination to Engineered meganuclei with altered specificity at individual bases within the NA recognition sequence half-site and therefore rationally designed meganucleases with half-sites that differ from the wild-type enzyme. Amino acid modifications of I-CEUI and predicted recognition sequence specificity The changes in are shown in Table 11.

[0125] [Table 11]

[0126] Bold entries represent wild-type contact residues and do not constitute "modifications" as used in this application. indicates that the residue contacts a base in the antisense strand.

[0127] 2.2.5 Specifically Excluded Recombinant Meganucleases The present invention relates to several recombinant proteins that have been described in the prior art and developed by alternative methods. These excluded meganucleases and For example, Arnold et al. (2006), J. Mol. Biol. 355: 443-58;SUSSMAN ET AL.(2004),J.MOL.BIOL.3 42:31-41;CHAMES ET AL.(2005),NUCLEIC ACI DS RES.33:E178;SELIGMAN ET AL.(2002),NUC LEIC ACIDS RES.30:3870-9; and ASHWORTH ET Described by AL.(2006) NATURE 441(7093):656-659 The above disclosures are also applicable to C33, R33, A44, H33, K 32, F33, R32, A28, A70, E33, V33, A26 and R66 and recombinant meganucleases based on I-CREI with a single substitution, Included in this application are A68 / N70 / N75 and D44 / D70 / N75, three substitutions selected from K44 / T68 / G60 / N75 and R44 / A68 / T Recombinant meganucleases based on I-CREI with four substitutions selected from 70 / N75 Finally, specifically excluded is I with the pair of substitutions L28 and R83. - MSOI-based recombinant meganucleases. These modifications are referred to in this application as "exclusion modifications."

[0128] 2.2.6 Meganucleases with multiple alterations in the recognition sequence half-sites In another aspect, the invention provides a DNA recognition sequence comprising a half-site at two or more positions of the half-site. To change the orientation, two or more amino acid substitutions, as described in sections 2.2.1-2.2.4 above, may be used. The present invention relates to rationally designed meganucleases obtained by combining substitutions. As described in more detail below, the enzyme DJ1 includes, but is not limited to, , modifications R30 / E38 (which favors C at position -7), R40 (which favors C at position -6 ), R42 (which prefers G at position -5), and N32 (which prefers G at position -6). , which prefers full degeneracy at position -9) This rationally designed DJ1 meganuclease is -7 A -6 C -5 Wild-type orientation against Compared to gender, C is almost the same. -7 G -6 G -5Recognizes A at position -9 Increases resistance to

[0129] The ability to combine residue substitutions affecting different base positions is due, in part, to Due to the modular nature of LAGLIDADG meganucleases, LAGLIDADG recognition The majority of base contacts at the interface are formed by individual amino acid side chains, The interface is the interconnectivity, or hydrogen bond network, between side chains that interact with neighboring bases. This generally allows for a single base position to interact with the residue. This allows the group to be manipulated without affecting the side chain interactions of adjacent bases. Furthermore, the additive nature of the mutations listed in Sections 2.21-2.2.4 above is This is a direct result of the method used to identify the bases that interact directly with the single bases. Predict side chain substitutions that have utility. Generally, the interconnections between side chains, or hydrogen bond networks, are used. is avoided to maintain the independence of substitutions within the recognition interface.

[0130] Some combinations of side chain substitutions are completely or partially mismatched with each other. When a set of amino acids is incorporated into a rationally designed meganuclease, the catalytic activity of the resulting enzyme is These mismatches are usually due to the mismatch between the introduced amino acid side chains. This is due to steric interference, and activity can be restored by identifying and removing this interference. Specifically, two amino acids with large side chains (e.g., amino acids from groups 2 or 3) are When incorporated into adjacent amino acid positions in the ganucleases structure (e.g. , in the case of I-CREI-derived meganuclease, positions 32 and 33, 28 and 40, 28 and 42, 42 and 77, or 68 and 77), these two amino acids interfere with each other. This interference likely reduces enzyme activity by replacing one or both mismatched amino acids. Eliminate by substituting amino acids with smaller side chains (e.g., group 1 or group 2) For example, in the rationally designed meganuclease derived from I-CREI, K 28 interferes with both R40 and R42. To maximize enzyme activity, R40 and R42 can be combined with serine or aspartic acid at position 28 .

[0131] The wild-type meganuclear cells were grown using combinations of amino acid substitutions identified as described in this application. The specificity of a meganuclease (or a previously modified meganuclease) can be determined from the original recognition sequence by can be rationally altered to the desired recognition sequence present in the nucleic acid of interest (e.g., genome). For example, Figure 2(A) shows the I-CREI meganuclease recognition sequence WT (SEQ ID NO: If we create a rationally designed meganuclease for the "sense" strand of No. 4) Some other sequences that may be useful are shown below. Conserved bases between columns are shaded. Recombinant meganucleases based on these enzymes have been developed for each of these desired recognition sequences. In addition, for any other recognition sequence, suitable amino acid substitutions as described in this application may be used. This allows for rational design.

[0132] 3. Rationally designed meganucleases with altered DNA binding affinity As mentioned above, the DNA binding affinity of the recombinant meganuclease of the present invention is determined by the fragmentation of DNA. By changing some of the amino acids that form contact surfaces with the phosphodiester backbone, This interface is located 9 angstroms from the DNA backbone. The amino acid of the enzyme with a β-carbon less than 1000 nm away was chosen because this residue was the same as that of the wild-type meganuclear. This includes whether or not it contacts the DNA backbone in the Zeta-DNA complex. Since NA binding is a necessary prerequisite for enzyme activity, an increase / decrease in DNA binding affinity It has been shown that they increase or decrease enzyme activity, respectively. The increase / decrease in binding affinity may also result in a decrease / increase in the sequence specificity of the meganuclease. Therefore, both activity and specificity are comparable to those of the phosphodiester bacterium. It is possible to modify the carbon contacts.

[0133] For increased enzyme activity / decreased enzyme specificity:

[0134] (I) Eliminates electrostatic repulsion between the enzyme and the DNA backbone. The amino acids have negatively charged side chains that are expected to repel the negatively charged DNA backbone. When the amino acid has a charge (e.g., aspartic acid, glutamic acid), this repulsion causes the amino acid to Alternatively, it can be removed by substituting a positively charged side chain. In this case, it is affected by the effect of steric hindrance. This is the case when glutamic acid 80 is mutated to glutamine.

[0135] (II) Introduction of electrostatic attraction interactions between the enzyme and the DNA backbone. amino acids with positively charged side chains (e.g., lysine or arginine) at any of the positions ) is expected to increase binding affinity, although it is subject to steric interference.

[0136] (III) Hydrogen bonds are introduced between the enzyme and the DNA backbone. , due to the lack of suitable hydrogen-bonding functional groups or the inability to interact with the DNA backbone. , side chains that are too short or too long and / or lack flexibility to form hydrogen bonds. If this is not possible, try to create a suitable polymer with the appropriate length and flexibility that can donate hydrogen bonds. Polar amino acids (e.g., serine, threonine, tyrosine, histidine, glutamine, aspartate, It is possible to introduce a nucleotide sequence (paragine, lysine, cysteine ​​or arginine) with the proviso that: It is affected by steric hindrance.

[0137] Specifically, to decrease enzyme activity / increase enzyme specificity,

[0138] (I) Electrostatic repulsion is introduced between the enzyme and the DNA backbone. In these, amino acids with negatively charged side chains (e.g., glutamic acid, asparagine) The introduction of phosphates is expected to decrease the binding affinity. It is affected by use.

[0139] (II) Eliminates the electrostatic attraction between the enzyme and DNA. , which has positively charged side chains that interact with the negatively charged DNA backbone (e.g., lysine or arginine), this favorable interaction allows the amino acid to be uncharged or It can be eliminated by substituting negatively charged side chains. However, steric interference can occur. The experimentally confirmed example is that lysine 116 in I-CREI is When mutated to aspartic acid.

[0140] (III) Remove the hydrogen bonds between the enzyme and the DNA backbone. If an amino acid forms a hydrogen bond with the DNA backbone, then the amino acid's side chain must be properly aligned. They either do not have the necessary functional groups or lack the necessary length / flexibility to form similar hydrogen bonds. It is possible to substitute an amino acid that is not predicted to be present.

[0141] For example, in a recombinant meganuclease based on I-CREI, the activity is increased. Therefore, the glutamic acid at position 80 of the I-CREI meganuclease can be replaced with a lysine or glutamic acid. In another embodiment, the tyrosine at position 66 of I-CREI is changed to glutamine. The amino acid sequence is changed to guanine or lysine, which increases the activity of the meganuclease. In one embodiment, the lysine at position 34 of I-CREI is changed to aspartic acid and the amino acid at position 66 of I-CREI is changed to aspartic acid. and / or the lysine at position 116 is replaced with aspartic acid. By converting it to arginic acid, the enzyme activity is reduced.

[0142] The activity of the recombinant meganuclease is determined by the activity of the recombinant enzyme with respect to a specific recognition sequence. It can be modified to have any activity level between no activity and extremely high activity. For example, the DJ engineered meganuclease may contain a glutamic acid at position 26. However, the glutamic acid substitution at position 26 completely abolishes the activity. The combination of the substitution and glutamine substitution at position 80 resulted in a substitution at -4 within the recognition sequence half-site. We will create a recombinant meganuclease with high specificity and activity for guanine ( See Figure 1(D)).

[0143] According to the present invention, acetylation at various positions near the phosphodiester DNA backbone is The amino acids can be altered to simultaneously affect both meganuclease activity and specificity. This "tuning" of enzyme specificity and activity is possible through the phosphodiester backbone. This is achieved by increasing or decreasing the number of contact points made by amino acids against the Various contacts to the phosphodiester backbone are made by amino acid side chains. In some embodiments, ionic bonds, salt bridges, hydrogen bonds, and steric bonds can be used to facilitate the formation of hydroxyl groups. The disorder affects the contact of the amino acid side chain with the phosphodiester backbone. For example, in the case of I-CREI meganuclease, the lysine at position 116 The change to aspartic acid creates a salt bridge between the nucleobases at positions -8 and -9. This reduces the enzyme's cleavage rate but increases its specificity.

[0144] Wild-type I-CREI (SEQ ID NO: 1), I-MSOI (SEQ ID NO: 6), I-SCEI (SEQ ID NO: The backbone of each meganuclease, I-CEUI (SEQ ID NO: 12), The residues that form the contact surface are identified in Table 12 below.

[0145] [Table 12]

[0146] To increase the affinity of the enzyme and therefore make it more active / less specific, (1) In the corresponding enzyme, negative charge (D or E), hydrophobicity (A, C, F, G, I, L) , M, P, V, W, Y) or uncharged / polar (H, N, Q, S, T) amino acids are listed in Table 1 Choose from 2. (2) If an amino acid is negatively charged or hydrophobic, it is replaced by an uncharged / polar ( Mutate the molecule to make it positively charged (K or R, less potent) or positively charged (K or R, more potent) . (3) If the amino acid is uncharged / polar, mutate it to positively charged.

[0147] To decrease the affinity of the enzyme and therefore make it less active / more specific, (1) In the corresponding enzyme, the positive charge (K or R), hydrophobicity (A, C, F, G, I, L) , M, P, V, W, Y) or uncharged / polar (H, N, Q, S, T) amino acids are listed in Table 1 Choose from 2. (2) If the amino acid is positively charged, change it to uncharged / polar (less effective) or Mutate it so that it becomes negatively charged (greater effect). (3) If the amino acid is hydrophobic or uncharged, mutate it to a negative charge.

[0148] 4. Heterodimeric meganucleases In another aspect, the invention provides two monomers, one of which is wild-type and one or both of which are nucleotides. The heterogeneity formed by contacts between non-native or recombinant monomers is Dimeric meganucleases are provided, e.g., wild-type I-CREI meganucleases. Each enzyme typically binds to one half-site in a pseudopalindromic recognition sequence. It is a homodimer consisting of two monomers. Nucleases can be synthesized by combining two meganucleases that recognize different half-sites. For example, by co-expressing two meganucleases in a cell. Alternatively, it can be produced by mixing two meganucleases in solution. In two monomers, the contact between them affects the formation of dimers. By changing each amino acid, the formation of heterodimers can be changed to homodimers. In certain embodiments, the interfacial formation of two monomers can be preferred. Some amino acids in the interface are negatively charged amino acids (D or E) to a positively charged amino acid (K or R), and in the second monomer, The amino acid can be changed to a negatively charged amino acid (see Table 13). For example, In the case of this meganuclease, the lysines at positions 7 and 57 are replaced by glutamic acid in the first monomer. In the second monomer, the glutamic acids at positions 8 and 61 were mutated to lysines. The result of this process is that the first monomer is mutated to a dimer interface. The second monomer has an excess of positively charged residues at the dimer interface. Therefore, the first and second Due to electrostatic interactions between the modified amino acids at the interface, It makes preferential contacts with the same monomer pair.

[0149] [Table 13]

[0150] Alternatively or additionally, some of the Some amino acids may be altered to sterically prevent homodimer formation. Specifically, the amino acids at the dimer interface of one monomer are separated into two parts: The second monomer is replaced by a relatively large or bulky residue that is sterically hindering. Amino acids at the mer interface were added to compensate for the bulky residues of the first monomer. It is possible to replace the heterodimer with a relatively small residue and eliminate the clash in the heterodimer. or may remain unmodified.

[0151] In yet another embodiment, ionic bridges or hydrogen bonds are introduced into the heterodimer interface. It is possible to embed the nanoparticles in the hydrophobic core of the interface. A hydrophobic residue in one monomer of the core can be replaced by a positively charged residue. Furthermore, in the wild-type homodimer, the hydrophobic residues substituted in the first monomer are The interacting hydrophobic residue of the second monomer can be replaced by a negatively charged residue Therefore, the two substituted residues can form ionic or hydrogen bonds. At the same time, the static charge of the unsatisfied charge buried in the hydrophobic interface can be Electrical repulsion discourages homodimer formation.

[0152] Finally, as mentioned above, each monomer in the heterodimer has a different DNA recognition domain. Each has a different DNA half-site and The combined dimeric DNA recognition sequences may be non-palindromic.

[0153] 5. Methods for Producing Recombinant Cells and Organisms Aspects of the present invention further include methods for producing recombinant, transgenic, and / or recombinant proteins using rationally designed meganucleases. and methods for producing nicked or otherwise genetically modified cells and organisms. Thus, in one embodiment, precise insertion of a sequence of interest by homologous recombination (singular or multiple sites of genomic DNA of a cell or organism, Recombinant meganucleases have been developed to specifically induce double-strand breaks at a small number of sites. In another embodiment, (A) rare insertion of a sequence of interest by non-homologous end joining (single (B) to enable the disruption of a target sequence by non-homologous end joining. To achieve this, a single site or a relatively small number of sites in the genomic DNA of a cell or organism may be A recombinant meganuclease has been developed to specifically induce double-strand breaks. As used herein with respect to homologous recombination or non-homologous end joining of sequences, "insertion" refers to a sequence that is inserted into a target gene. The term "transgenic" refers to the process by which a sequence of interest is integrated into a chromosome. In the case of homologous recombination, the inserted sequence replaces the endogenous sequence. Therefore, the original DNA is transformed into an exogenous DNA of equal length but with an altered nucleotide sequence. Alternatively, the inserted sequence may be more specific than the sequence it replaces. It is possible to include more or fewer bases.

[0154] Therefore, according to this aspect of the invention, recombinant organisms include monocotyledonous plant species, e.g. For example, rice, wheat, corn (maize) and rye, and dicotyledonous plant species, e.g., pulses (e.g., broad beans, soybeans, lentils, peanuts, peas), alfalfa, These include, but are not limited to, tobacco, tobacco, and Arabidopsis species. Additionally, recombinant organisms include animals, such as humans and non-human primates. Horses, cattle, goats, pigs, sheep, dogs, cats, guinea pigs, rats, mice, and toca insects, including but not limited to, gnats, fish, and insects, such as Drosophila species. In other embodiments, the organism is a fungus, e.g., Candida, Neurospora gracilis, or the like. Borre or Saccharomyces.

[0155] In one embodiment, the methods of the present invention involve the production of a recombinant organism capable of developing into a mature recombinant organism or a genetically modified organism. The genetically modified organism gives birth to offspring that carry the inserted sequence of interest in their genome. This includes, for example, introducing a sequence of interest into a cell, embryonic cell, or stem cell. nothing.

[0156] The meganuclease protein can be transported into cells to cleave genomic DNA. This allows the formation of a cleavage site by various mechanisms known in the art. This allows for homologous recombination with the sequence of interest or non-homologous end joining. Meganuclease proteins can be delivered, for example, by microinjection or liposomal transfection. Infection (e.g., LIPOFECTAMINE®, INVITROG EN CORP., CARLSBAD, CALIFORNIA Although the liposome formulation is not suitable for intracellular delivery, it can be introduced into cells by various techniques. It can be used to promote lipid bilayer fusion with target cells, and thus liposomes It allows the uptake of proteins that come into contact with the contents of the membrane or its surface into the cell. Alternatively, the enzyme may be coupled to a suitable uptake protein, such as the HIV TAT protein. It is also possible to fuse it to a target such as a head-up display (HUD) to direct cellular uptake. ECZ ET AL.(2005), MED.RES.REV.25:679-736 reference).

[0157] Alternatively, the gene sequences encoding the meganuclease proteins are known in the art. techniques (e.g., AUSUBEL ET AL., "Recent Protocols in Molecular Biology" (“CURRENTPROTOCOLS IN MOLECULAR BIOLOG Y,” (see Wiley 1999) The sequence of interest is transfected into the nucleus by the same vector, but not by a different vector. The introduction can be by means of a ion exchange or by other means known in the art.

[0158] Non-limiting examples of DNA transfection include viral vectors, plasmids, These include cosmid and YAC vectors. Transfection of DNA sequences is well known in the art. This can be achieved by a variety of methods known to those skilled in the art. For example, Liposomes and immunoliposomes are used for delivery to the vesicles (e.g., LAS See IC ET AL. (1995), SCIENCE 267:1275-76). Additionally, viruses can be used to introduce vectors into cells (e.g., (See, for example, U.S. Patent No. 7,037,492.) Alternatively, the vector may be naked DNA. It is also possible to use transfection strategies to introduce the vector into the host (e.g., RUI ET AL.(2002), LIFE SCI.71(15):1771-8 reference).

[0159] Common methods for delivering nucleic acids into cells include: (1) chemical methods (GRAHAM ET AL. (1973), VIROLOGY 54(2):536-539;ZAT LOUKAL ET AL.(1992),ANN.NYACAD.SCI.,66 0:136-153); (2) physical methods, such as microinjection (CAP) ECCHI(1980),CELL22(2):479-488), electroporation (WONG ET AL,(1982),BIOCHEM.BIOPHYS.RES.COMMUN .107(2):584-587;FROMM ET AL,(1985),PROC. NAT'L ACAD SCI.USA 82(17):5824-5828;U.S. Patent No. 5,384,253), and bullet injection method (JOHNSTON ET AL, (1 994),METHODS CELL.BIOL.43(A):353-365;FYN AN ET AL,(1993),PROC.NAT'L ACAD.SCI.USA 90(24):11478-11482); (3) viral vectors (CLAPP( 1993), CLIN. PERINATOL.20(1):155-168;LU E T AL,(1993),J.EXP.MED.178(6):2089-2096;E GLITIS ET AL,(1988),AVD.EXP.MED.BIOL.241 :19-27;EGLITIS ET AL.(1988),BIOTECHNIQUE S 6(7):608-614); and (4) receptor-mediated mechanisms (CURIEL E T AL.(1991),PROC.NAT'L ACAD.SCI.USA 88(1 9):8850-8854;CURIEL ET AL.(1992),HUM.GEN .THER.3(2):147-154;WAGNER ET AL.(1992),P ROC.NAT'L ACAD.SCI.USA 89(13):6099-6103) Examples include:

[0160] In one embodiment, a genetically modified plant containing a sequence of interest inserted into its genome is In one embodiment, the DNA sequence corresponding to the recombinant meganuclease and and / or the target sequence of the meganuclease. Transfecting and genetically modifying plant cells with a sequence of interest, which may or may not be flanked. In another embodiment, the genetically modified plant is produced by the recombinant Plant cells are transfected with DNA sequences corresponding only to the meganuclease. Cleavage by this promotes non-homologous end joining, destroying the target sequence containing the recognition sequence. In such an embodiment, the meganuclease sequence is produced in a host plant cell. These regulatory sequences and Examples include constitutive plant promoters, such as the NOS promoter, and chemically inducible genes. Gene promoters, such as dexamethasone-inducible promoters (e.g., GREMIL See LON ET AL.(2004), PLANT J.37:218-228). and plant tissue-specific promoters, such as the LGC1 promoter (e.g., SIN (See GH ET AL. (2003), FEBS LETT. 542:47-52) These include, but are not limited to:

[0161] A suitable method for introducing DNA into plant cells is to introduce DNA into the cells. Almost any method is acceptable as long as it is possible to IUM infection, PEG-mediated transformation of protoplasts (Omirulleh et al. L. (1993), PLANT MOLECULAR BIOLOGY, 21:415- 428), DESICCATION / INHIBITION MEDIATED DNA UPTAKE, electroporation, silicon carbide fiber agitation, bullet injection, or micro Examples include, but are not limited to, projectile firing.

[0162] In another embodiment, recombinant meganucleases are used to produce genetically modified animals. As in plant cells, nucleic acid sequences can be introduced into embryonic cells or ultimately into transgenic plants. In one embodiment, the cells are fertilized. eggs, and exogenous DNA molecules can be injected into the pronuclei of the fertilized eggs. The microinjected eggs are then transferred to the fallopian tubes of pseudopregnant surrogate mothers and allowed to grow. Recombinant meganucleases (e.g., 3-phosphoglycerate kinase) It is expressed in the fertilized egg (under the control of a constitutive promoter) and is expressed in a region of the genome or in a small, discontinuous region. It promotes homologous recombination of sequences of interest at several sites. ET AL.(1986),PROC.NATL.ACAD.SCI.USA 83:9 065 9069, recombinant embryos for the production of transgenic animals. Genetically modified animals can be obtained by utilizing embryonic stem ("ES") cells. It is possible to do this.

[0163] In some embodiments, the recombinant mammalian expression vector directs tissue-specific expression of the nucleic acid in a particular Tissue-specific regulatory elements can be used to preferentially target specific cell types. Non-limiting examples of suitable tissue-specific promoters include albumin promoters, ribosomal promoters, and ribosomal promoters known in the art. promoter (liver-specific, PINKERT ET AL. (1987), GENES D EV.1:268-277), lymphoid-specific promoter (CALAME AND E ATON (1988), ADV. IMMUNOL. 43: 235-275), especially T-cell The promoter of the cellular receptor (WINOTO AND BALTIMORE (1989), E MBO J.8:729-733), and immunoglobulin promoter (BANER JIET AL.(1983),CELL 33:729-740;QUEEN AND BALTIMORE (1983), CELL 33:741-748), Neuron Heterologous promoters (e.g., neurofilament promoter; BAYNE AND RUDDLE(1989),PROC.NATL.ACAD.SCI.USA 86: 5473-5477), pancreas-specific promoter (EDLUND ET AL. (198 5), SCIENCE 230:912-916), and mammary gland-specific promoters ( See, e.g., Milkfay Promoter, U.S. Pat. No. 4,873,316, and European Patent No. Developmentally regulated promoters, such as Mouse HOX promoter (KESSEL AND GRUSS (1990), SCI ENCE 249:374-379), and the α-fetoprotein promoter ( CAMPES AND TILGHMAN(1989),GENES DEV.3:53 7-546) are also included.

[0164] In some embodiments, the rationally-designed meganucleases are used to monitor expression levels or localization. To achieve this, peptide epitopes (e.g., HA, FLAG, or MYC epitopes) In some embodiments, the meganuclease may be labeled with a subcellular localization signal, e.g., For example, a nuclear localization signal (e.g., the SV40 nuclear localization signal) or a chloroplast or In another embodiment, the meganuclear The enzyme may be fused to a nuclear export signal that localizes it to the cytoplasm. The cleavage enzymes may be unrelated proteins or protein domains, e.g., DNA-repair or Proteins that stimulate recombination (e.g., RECA, RAD51, RAD52, RAD 54, RAD57 or BRCA2).

[0165] 6. Gene Therapy Aspects of the present invention enable the use of recombinant meganucleases for gene therapy. The term "gene therapy" as used in this application refers to the treatment of a gene that is defective in its structure and / or function. In a patient, at least one gene is functional copies of gene regulatory sequences, such as promoters, enhancers or silencers Furthermore, the term "gene therapy" refers to a therapeutic treatment that involves the introduction of a gene into a target organ. , directed against a harmful gene or regulatory element to reduce or eliminate expression of the gene. Gene therapy can refer to modifications that are made to specific genes throughout the patient's life. Treating conditions caused by mutations or lesions in genetic loci or by infectious organisms It is possible to do so.

[0166] In one aspect of the invention, the dysfunctional gene is located in a genomic region that affects gene expression. It is replaced or disabled by inserting an exogenous nucleic acid sequence. So, recombinant meganucleases can target specific genomic regions to be modified to alleviate pathology. This sequence may be an exon, an intron, a region within a promoter, or a gene. It may also be other regulatory regions that cause dysfunctional expression of a gene. The term "underexpression" refers to cells that produce too little gene product or cells that produce too much gene product. Too many cells or different functions, e.g., gene products that lack or have more functions than necessary It refers to the abnormal expression of a gene product by a cell that produces the product.

[0167] The exogenous nucleic acid sequence inserted into the modified region provides a "repair" sequence that normalizes the gene. Gene repair allows for proper function to be restored. This is achieved by introducing the appropriate gene sequence into the gene of interest. In this case, the inserted nucleic acid sequence may be the entire coding sequence of the protein or, in some embodiments, In some embodiments, the gene fragment may include only the region to be repaired. The nucleic acid sequence to be inserted may be selected so that the mutation causing the abnormal expression or regulation is repaired. In another embodiment, the inserted nucleic acid sequence includes a promoter sequence or other regulatory elements. The nucleic acid sequence includes the appropriate translation stop codon that is absent in the mutant gene. , a sequence for terminating translation in recombinant genes lacking an appropriate translation termination signal It is possible to have

[0168] Alternatively, nucleic acid sequences may be used to disrupt gene function by disrupting regulatory sequences of genes or By providing a silencer that removes In some embodiments, the exogenous nucleic acid sequence is transcribed to prevent expression of the gene product. In another embodiment, the exogenous nucleic acid sequence provides a stop codon for expression of the full-length RNA molecule. In yet another embodiment, the gene function is Induction of base insertions, deletions, and / or frameshift mutations via non-homologous end joining By inserting the protein into the target cell, it is directly destroyed by the meganuclease.

[0169] In many instances, the appropriate gene sequence is directed to the target cell or cell population responsible for the pathology. Such targeting of therapeutic agents is desirable because it allows healthy cells to react to the therapeutic agent. This increases the efficacy of the treatment, while preventing the treatment from affecting healthy individuals. Reduces potentially harmful effects that may be exerted on cells.

[0170] Transport of the recombinant meganuclease gene and the sequence of interest to be inserted into the cell of interest. Delivery can be achieved by a variety of mechanisms. In some embodiments, the nucleic acid is delivered via a viral via a virus that has specific viral genes inactivated to prevent the reproduction of the virus. Therefore, the virus is transported and maintained in the target cell. but not retain the ability to replicate in the target cell or tissue. This modified viral genome produces a viral genome that acts as a vector. It is possible to insert one or more DNA sequences, but these sequences must be inserted into the host's genome. It may be introduced into the host and then expressed, or it may not be introduced and then expressed. In some embodiments, the vectors are selected from retroviruses, including but not limited to retroviruses. However, this includes the adoption of MFG or PLJ vectors. MFG vectors are simplified moro The vector is a murine leukemia virus vector (MOMLV), which is replication-deficient. To achieve this, the DNA sequences encoding the POL and ENV proteins are deleted. Retroviral vectors are also a form of MOMLV (e.g., KORMAN ET AL.(1987), PROC.NAT'L ACAD.SCI.,84:2150- In another embodiment, the recombinant adenovirus or adeno-associated virus is used as a vector for delivery. It can be used as a delivery vector.

[0171] In another embodiment, the recombinant meganuclease protein and / or Delivery of recombinant meganuclease gene sequences is achieved by the use of liposomes. The production of liposomes containing nucleic acid and / or protein payloads is known in the art (e.g., For example, LASIC ET AL. (1995), SCIENCE 267:1275-7 6) Immunoliposomes contain antibodies against cell-associated antigens in the liposomes, and The DNA sequence of the meganuclease or the meganuclease itself is then administered to the cell type. It is possible to transport (e.g., Lasic et al. (1995), SCI ENCE 267:1275-76;YOUNG ET AL.(2005),J.CA LIF.DENT.ASSOC.33(12):967-71;PFEIFFER ET (See AL. (2006), J.VASC.SURG.43(5):1021-7). Methods for producing and using liposomal formulations are well known in the art (see, e.g., US Pat. No. 6,229,139). Patent No. 6,316,024, U.S. Patent No. 6,379,699, U.S. Patent No. 6,387 ,397, U.S. Patent No. 6,511,676, and U.S. Patent No. 6,593,308 (See, for example, the references cited therein.) In one embodiment, the liposome comprises: In addition to the sequence of interest, the recombinant meganuclease protein or recombinant meganuclease Used to transport gene sequences.

[0172] 7. Treatment of pathogen infections Aspects of the present invention further provide methods for treating infection by pathogens. , viruses, such as, but not limited to, herpes simplex virus 1, Herpes simplex virus 2, Human immunodeficiency virus 1, Human immunodeficiency virus 2, Smallpox Viruses, poliovirus, Epstein-Barr virus or human papillomavirus and bacterial organisms, such as, but not limited to, Bacillus ANTHRACIS, HAEMOPHILUS species, PNEUMOCOCCUS species, ST APHYLOCOCCUS AUREUS, STREPTOCOCCUS spp., methicillin Resistant Staphylococcus aureus and Mycoplasma tuberculosis Pathogenic organisms also include fungal organisms, e.g., These include, but are not limited to, CANDIDA, BLASTOMYCES, CRYPTOCOCCUS, and HISTOPLASMA species.

[0173] In some embodiments, the rationally-designed meganucleases target a recognition sequence within a pathogen genome, e.g., targeting genes or regulatory elements essential for the growth, reproduction, or virulence of the pathogen In some embodiments, the recognition sequence may be within a bacterial plasmid. Meganuclease-mediated cleavage of recognition sequences in pathogen genomes stimulates non-homologous end-joining This induces mutations in the target essential gene in the form of insertions, deletions, or frameshifts. Alternatively, cleavage of bacterial plasmids can be achieved by cleaving the genes encoded thereon. Any gene that is capable of infecting a host, such as a toxin gene (e.g., a B. anthracis lethal factor gene) This may result in the loss of the plasmid along with the gene or antibiotic resistance gene. Thus, meganucleases can be synthesized by the synthesis of proteins or nucleic acids using techniques common in the art. In one embodiment, the meganuclease gene may be delivered to an infected patient, animal, or plant in the form of a Genes can also be integrated into bacteriophage genomes for delivery to pathogenic bacteria. good.

[0174] Aspects of the invention further provide therapeutic agents for the treatment of certain forms of cancer. are often associated with tumor formation (e.g., Epstein-Barr virus and nasopharyngeal human papillomavirus and cervical cancer), the inactivation of these viral pathogens Alternatively, rationally designed meganucleases may inhibit the development or progression of cancer. This targets the genome of these tumor-associated viruses, using double-strand breaks in the DNA. Apoptosis may be induced via the damage response pathway. It may be possible to selectively induce apoptosis in tumor cells harboring .

[0175] 8. Genotyping and pathogen identification A further aspect of the invention is to provide tools for in vitro molecular biology research and development. Nucleic acids, such as plasmids, PCR products, BAC sequences, YAC sequences, viruses and For the isolation, cloning, and manipulation of nucleic acids, including genomic sequences from nuclear and prokaryotic organisms The use of site-specific endonucleases (e.g., restriction enzymes) is common in the art. (See, for example, Ausbel et al., "Recent Progress in Molecular Biology"). Protocols ("Current Protocols in Molecular Biology") (See "THE DIRECTOR OF PHILOSOPHY," Wiley 1999). Thus, in one embodiment In a similar manner, rationally designed meganucleases may be used for the manipulation of nucleic acids in vitro. For example, rationally designed meganucleases that recognize a pair of recognition sequences within the same DNA molecule are for subsequent manipulation, e.g., contacting a bacterial plasmid, BAC, or YAC It can be used to isolate the intervening DNA segment of the

[0176] In another aspect, the present invention provides tools for identifying pathogenic genes and organisms. In this study, rationally designed meganucleases distinguish diseased alleles from healthy alleles. to cleave the corresponding recognition site in the disease-correlated polymorphic gene region. It is possible to recognize the ΔF-508 allele of the human CFTR gene (for example, (See Example 4.) In this embodiment, the human patient or In some cases, DNA sequences isolated from other organisms can be synthesized by rationally designed meganucleases. In some cases, the resulting DNA fragments are digested with additional site-specific nucleases. The pattern may be analyzed by gel electrophoresis, capillary electrophoresis, mass spectrometry or other techniques known in the art. The fragmentation patterns and specifically the rationally designed meganucleases are analyzed by the method. The presence or absence of cleavage by α-glucan can be determined by determining whether or not a recognition sequence exists in the genome. In another embodiment, the rationally-designed meganuclease is capable of detecting the genotype of a pathogenic virus. targeting polymorphic regions in the genome of viruses, fungi, or bacteria to identify the organisms In this embodiment, the rationally designed meganuclease is used to identify a recognition sequence that is unique to the pathogen. The sequence is cut (e.g., the space between the 16S and 23S RNA genes in bacteria). Sir region; e.g., VAN DER GIESSEN ET AL. (1994), MI CROBIOLOGY 140:1103-1108), which can be used to Endonuclease digestion followed by electrophoresis, mass spectrometry, or other techniques known in the art. from other closely related organisms based on analysis of fragmentation patterns by other methods. It is possible.

[0177] 9. Production of custom DNA-binding domains In another aspect, the present invention provides rationally designed DNA-binding proteins that have eliminated endonuclease cleavage activity. The catalytic activity of the rationally designed meganuclease is eliminated by removing the amino acid residues involved in the catalysis. This can be achieved by mutating the amino acid (e.g., I-CREI) Q47 to E mutation in CHEVALIER ET AL. (2001), BI OCHEMISTRY, 43:14015-14026); in I-SCEI Mutation of D44 or hD145 to N; mutation of E66 to Q in I-CREI (D22 to N mutation in I-MSOI). Then, this inactivated meganuclear The enzyme may be coupled to another protein, such as, but not limited to, a transcription activator. transactivation domain (e.g., GAL4 transactivation domain, or VP16 transactivation domain) activation domain), transcriptional repressors (e.g., K of KRUPPEL protein) RAB domain), DNA methylase domain (e.g., M.CVIPI, or MS SSI), or histone acetyltransferase domains (e.g., HDAC1 It is possible to fuse these to effector domains such as ribosomal kinase (RIK) or HDAC2. Also prominent are engineered zinc finger domains and engineered DNA binding. Chimeric proteins consisting of a domain and an effector domain are known in the art. (e.g., PAPWORTH ET AL. (2006), GENE 366:27-3 8).

[0178] The present invention will be further illustrated by the following examples. should not be considered limiting. Those skilled in the art will be able to determine the It is understood that the present invention is not limited to the foregoing and is not intended to be limiting unless expressly stated otherwise. Such equivalents may be found in the claims preceding the examples below. Examples 1-4 below are intended to be included in the rational design method based on I-CREI. It specifically refers to ganucleases, but also to I-SCEI, I-MSOI, I-CE UI-based rationally designed meganucleases and other LAGLIDADG meganucleases Ze can similarly be produced and used as described herein. Example 1

[0179] Rational design of meganucleases that recognize the HIV-1 TAT gene 1. Meganuclease design A pair of meganucleases target the DNA site 5'- found in the HIV-1 TAT gene. Recognizes and cleaves GAAGAGCTCATCAGAACAGTCA-3' (SEQ ID NO: 15) According to Table 5, two meganucleases, TAT1 and TAT2, were designed to The following base contacts (non-WT contacts are shown in bold) were used to identify the 5'-half sites, respectively: GAAGAGCTC-3' (SEQ ID NO: 16), and 5'-TGACTGTTC-3' ( It was designed to bind to the nucleotide sequence of SEQ ID NO: 17.

[0180] TAT1: [Table 14]

[0181] TAT2: [Table 15]

[0182] The two enzymes were cloned and expressed in E. coli and the corresponding ATPases were isolated as described below. In both cases, the enzyme activity against the DNA recognition sequence was quantified. The nuclease was found to be inactive. To improve this, E80 was mutated to Q, producing a second generation of each. The first generation TAT2 enzyme was found to have activity against its intended recognition sequence, but On the other hand, the TAT1 enzyme remained inactive. Visual inspection of the co-crystal structure with wild-type I-CREI. This suggests that TAT1 is inactive due to steric hindrance between R40 and K28. To alleviate this clash, K28 was replaced with an amino acid with a smaller side chain (A, S, T, or C) while maintaining the Q80 mutation. These enzymes were produced in E. coli and quantified. Both TAT1 variants with the -7 position are active against the desired recognition sequence, while the It was found that the desired base directivity was maintained.

[0183] 2. Construction of recombinant meganucleases Mutagenesis in an overlapping PCR strategy to induce mutations for the redesigned I-CREI enzyme The recombinant DNA fragment of I-CREI generated by primary PCR was introduced using primers The pieces were joined in a secondary PCR to produce the full-length recombinant nucleic acid. All I constructs contain a PET gene with a six-histidine tag fused to the 3' end of the purification gene. 21A Vector (NOVAGEN CORP., SAN DIEGO, CA) ). All nucleic acid sequences were analyzed by Sanger dideoxynucleotide sequencing (S ANGER ET AL.(1977),PROC.NATL.ACAD.SCI.US A.74(12):5463-7).

[0184] Wild-type I-CREI and all engineered meganucleases were expressed using the following method: The construct cloned into the PET21A vector was made chemically competent. Transformed in BL21(DE3) PLYSS and treated with 200 μg / mL of carbapenicillin After overnight growth, transformed bacterial colonies were plated onto 2X YT plates containing 100% ethanol. The cells were scraped off the plate and used to inoculate 50 ml of 2XYT broth. The mixture was incubated at 37°C with shaking until the optical turbidity reached 0.9 at a wavelength of 600 nm. The growth temperature was then lowered from 37°C to 22°C. Protein expression was confirmed by 1M I Induce by adding PTG and incubate cells for 2.5 hours with shaking The cells were then pelleted by centrifugation at 6000 xg for 10 minutes. Add 1 mL of binding buffer (20 mM TRIS-HCl, pH 8.0, 500 mM The cells were resuspended in 10 mM NACL (10 mM imidazole) by vortexing. The cells were disrupted by sonication with 12 pulses at 50% power and the cell debris was removed by 15 min. The cells were pelleted by centrifugation at 14,000 x g for 1 hour. The cell supernatant was transferred to a 4 mL binding buffer. Dilute with PHA and load onto 200 μL of a nickel-loaded metal chelating Sepharose column (PHA The test was loaded onto a MRI machine.

[0185] The column was then washed with 4 mL of wash buffer (20 mM TRIS HCl, pH 8. 0.500 mM NaCl, 60 mM imidazole) and 0.2 mL of elution buffer ( 20MM TRIS-HCl, pH 8.0, 500MM NACL, 400MM imidazoline The meganuclease enzyme was washed with 0.6 mL of elution buffer. The solution was eluted by VIVOSPIN disposable concentrator (ISC, INC. The enzyme was concentrated to 50-130 μL using a PBS (Kaysville, Utah). , ZEBA spin desalting column (PIERCE BIOTECHNOLOGY, INC., ROCKFORD, IL) was used for quantification and storage in SA buffer (25 MM TRIS-HCL,PH8.0,100MM NACL,5MM MGCL2,5 The enzyme concentration was determined using an extinction coefficient of 23,590 M-1 cm-1. The purity and molecular weight of the enzyme were then determined by the absorbance at 280 nm. Confirmation was performed by I-TOF mass spectrometry.

[0186] Heterodimeric enzymes can be synthesized by purifying the two proteins independently and mixing them in vitro. or constructing an artificial operon for tandem expression of two proteins in E. coli In the former case, purified meganuclease was mixed 1:1 in solution. In the latter case, the mixture was pre-incubated at 42°C for 20 minutes before the addition of the DNA substrate. The genes were sequentially PET-transcribed using NDEI / ECORI and ECORI / HINDIII. The 12 base pair nucleic acid spacer and the PET21 vector were cloned into the PET21A expression vector. A Shine-Delgarno sequence derived from β-actin separated the first and second genes in the artificial operon. .

[0187] 3. Cleavage Assay All of the purified enzymes described above have two linear strands containing the recognition sequence of the meganuclease. Activity was quantified by incubation with a single strand DNA substrate. Synthetic oligonucleotides corresponding to both the sense and antisense strands were annealed and blunt-ended. The clone was cloned into the SMAI site of the PUC19 plasmid by end contact. The sequence of the identified binding site was confirmed by Sanger dideoxynucleotide sequencing. All plasmid substrates were subjected to meganuclease digestion and simultaneous amplification with XMNI, SCAI, and The enzyme digest was linearized with 5 μL of 0.05 μM D NA substrate, 2.5 μL of 5 μM recombinant I-CREI meganuclease, 9.5 μL of S The digest contained 0.5 μL of XMNI, SCAI, or BPMI. , and incubated for 4 hours at 37°C or 42°C for some meganuclease enzymes. Digestion was carried out by adding 0.3 mg / ml proteinase K and 0.5% SDS. The digest was stopped by 1000 kJ / min and incubated at 37°C for 1 hour. The cells were analyzed by filtration and visualized by ethidium bromide staining.

[0188] To assess the directionality of the meganuclease half-sites, the rationally designed meganucleases were A set of DNA substrates corresponding to the perfect palindrome of the intended half-site, as well as The fragments were incubated with one of 27 possible single base pair substitutions at the site.

[0189] 4. Recognition sequence - specificity Purified recombinant TAT1 and TAT2 meganucleases were synthesized using wild-type meganuclease sequences. The wild-type I-CREI meganuclease recognized a different DNA sequence from the wild-type I-CREI meganuclease (Figure 2(B)). , cleaves the WT recognition sequence but not the sequence intended for TAT1, intended for TAT2 Similarly, TAT1 and TAT2 do not cleave any of the sequences intended for their recognition. These meganucleases then cleave the recognition sequence but not the wild-type sequence. The site specificity and overall specificity of wild-type I-CREI were evaluated (Figure 3). Single base pair substitutions at the natural half-sites were found to be highly tolerated. On the other hand, TAT1 and TAT2 are highly specific. In the case of TAT1, positions -1 and -2 , -3, -6 and -8, and in the case of TAT2, salts at positions -1, -2 and -6. It was not completely tolerant to group substitution. Example 2

[0190] Rational design of meganucleases with altered DNA-binding affinities 1. Meganucleases with increased affinity and activity Meganucleases CCR1 and BRP2 were each cloned with the half-site 5'-AACCCT CTC-3' (SEQ ID NO: 18) and 5'-CTCCGGGTC-3' (SEQ ID NO: 19) These enzymes were produced according to Table 1, as in Example 1. did.

[0191] CCR1: [Table 16]

[0192] BRP2: [Table 17]

[0193] Both enzymes were expressed in E. coli, purified, and quantified as in Example 1. The proenzyme acts at a rate significantly slower than that of wild-type I-CREI with the natural recognition sequence. It was found that cleavage of the intended recognition sequence of ATP was achieved. To evaluate the DNA-binding affinities of CCR1 and BRP2, we investigated the E80-Q binding affinity of both enzymes. These second-generation variants of CCR1 and BRP2 were increased by natural mutation. John was able to cleave its intended recognition sequence with a significant increase in catalytic rate. It was recognized.

[0194] 2. Meganucleases with reduced DNA-binding affinity, reduced activity, but increased specificity Crease Wild-type I-CREI is known to be highly tolerant to substitutions at its half-sites. In an attempt to further enhance the specificity of this enzyme, The lysine at position 116 of the enzyme forms a salt bridge with the phosphate in the DNA backbone, and the asparagus The wild-type recognition sequence of this rationally designed enzyme was mutated to a phosphate to reduce its DNA-binding affinity. Although the cleavage of the target sequence was clearly reduced, the specificity of this recombinant enzyme was greater than that of the wild type. The targeting of the half site of the K116D variant was evaluated in the same manner as in Example 1. However, this enzyme tolerates deviations from the native half-site at positions -1, -2, and -3. The remaining six positions in the half-site showed at least partial resistance. It showed excellent base specificity (Figure 3(B)). Example 3

[0195] Rationally designed meganuclease heterodimers 1. Non-palindromic meganuclease heterodimers formed in solution DNA site cleavage Two meganucleases, LAM1 and LAM2, each contain the half-site 5'-TGCG GTGTC-3' (SEQ ID NO: 20) and 5'-CAGGCTGTC-3' (SEQ ID NO: 21) The heterodimer consisting of these two enzymes was designed to cleave the bacterium The DNA sequence 5'-TGCGGTGTCCGGCG found in the phage λ P05 gene It was predicted to recognize ACAGCCTG-3' (SEQ ID NO: 22).

[0196] LAM1: [Table 18]

[0197] LAM2: [Table 19]

[0198] LAM1 and LAM2 were cloned and expressed in E. coli as described in Example 1. The two enzymes were then mixed in a 1:1 ratio and incubated at 42°C for 20 minutes. The enzymes were then allowed to exchange subunits and reach equilibrium again. Mixture of AM1 homodimer, LAM2 homodimer, and LAM1 / LAM2 heterodimer The resulting enzyme solution, which is expected to be a mixture of the perfect palindrome of the LAM1 half site, LAM Perfect palindromes of two half-sites and non-palins found in the genome of bacteriophage .LAMBDA.. The hybridization sites were incubated with three different recognition sequences, each corresponding to a different target site. Purified LAM1 enzyme alone cleaves the LAM1 palindromic site, but not the LAM2 palindromic site. It does not cleave either the dromic site or the LAM1 / LAM2 hybrid site. The M2 enzyme alone cleaves the LAM2 palindromic site, but also the LAM1 site. However, the 1: / LAM2 hybrid site is not cleaved. One mixture cuts all three DNA sites. Cleavage is achieved by mixing two different redesigned meganucleases in solution to form non-palindole fragments. It is possible to form heterodimeric enzymes that can cleave specific DNA sites. Indicates that there is something.

[0199] 2. Non-palindole-mediated cytotoxicity by meganuclease heterodimers formed by syngeneic expression Cleavage of DNA fragments The genes encoding the LAM1 and LAM2 enzymes were prepared as described in Example 1. The co-expressed enzymes were arranged in an operon for co-expression in E. coli. The enzyme mixture was purified as in Example 1, and the three potential recognition sequences were identified. The co-expressed enzymes were then incubated with the LAM1 / LAM2 hybrid site. It was found that the cleavage of all three sites, including Meganucleases can be co-expressed to cleave non-palindromic DNA sites We show that it forms a heterodimeric enzyme.

[0200] 3. Meganuclease heterodimers with modified protein-protein interfaces Preferential cleavage of non-palindromic DNA sites by For applications requiring cleavage of non-palindromic DNA sites, different (palindromic) (mechanical) DNA site recognition and cleavage while minimizing the formation of homodimers. To this end, it is desirable to enhance the formation of dimers at positions 7, 57, and 96. A variant of the LAM1 enzyme was produced in which the lysine in the enzyme was changed to glutamic acid. This was co-expressed with a LAM2 variant in which glutamic acid at positions 8 and 61 was changed to lysine, as described above. In this case, the formation of LAM1 homodimers was due to the presence of a β-amyloid in one of the monomers. Electrostatic interactions between E7, E57, and E96 in one monomer and E8 and E61 in the other Similarly, the formation of LAM2 homodimers was predicted to be suppressed by electrochemical repulsion. K7, K57, and K96 in one monomer and K8 and K61 in the other monomer It was predicted that the electrostatic repulsion between LAM1 and LAM2 would decrease the binding affinity. The telodimers consist of E7, E57, and E96 in LAM1 and E96 in LAM. It was expected that the interface would be favored by electrostatic attraction between K8 and K61. The two modified meganucleases were co-expressed and quantified as described above. The LAM1 / LAM2 hybrid site is preferentially expressed over the two palindromic sites. This is due to the protein-protein interaction of the meganuclease. We show that substitutions at the β-interface can drive preferential formation of heterodimers. . Example 4

[0201] Additional meganuclease heterodimers that cleave physiological DNA sequences 1. Meganuclease heterodimers that cleave DNA sequences relevant to gene therapy The sequence 5'- in the human FGR3 gene, a mutation that causes achondroplasia CTGGGAGTCTCAGGACAGCCTG-3' (SEQ ID NO: 23), It is possible to produce a synthetically designed meganuclease heterodimer (ACH1 / ACH2) For example, as described above, the following conjugates can be prepared based on the I-CREI meganuclease: Meganucleases with catalytic residues and recognition sequence half-sites were designed.

[0202] ACH1: [Table 20]

[0203] ACH2: [Table 21]

[0204] The sequence 5'-CCAGGTGTCTC in the promoter of the human growth hormone gene A rationally designed meganuclease that cleaves TGGACTCCTCC-3' (SEQ ID NO: 24) It is possible to produce heterodimers (HGH1 / HGH2). Based on the I-CREI meganuclease, the contact residues and recognition sequence half are as follows: Meganucleases with sites have been designed.

[0205] HGH1: [Table 22]

[0206] HGH2: [Table 23]

[0207] 5'-GAAAATATCAT in the ΔF508 allele of the human CFTR gene A rationally designed meganuclease that cleaves TGGTGTTTCCT-3' (SEQ ID NO: 25) It is possible to produce heterodimers (CF1 / CF2). For example, as mentioned above, Based on the I-CREI meganuclease, the following contact residues and recognition sequence half-sites were determined: A meganuclease with

[0208] CF1: [Table 24]

[0209] CF2: [Table 25]

[0210] The sequence of the human CCR5 gene (HIV coreceptor) is 5'-AACCCTCTCCAG Rationally designed meganuclease cleaving TGAGATGCCT-3' (SEQ ID NO: 26) It is possible to produce a heterodimer (CCR1 / CCR2). For example, Based on the I-CREI meganuclease, the following contact residues and recognition sequence halves were identified: A meganuclease with the position has been designed.

[0211] CCR1: [Table 26]

[0212] CCR2: [Table 27]

[0213] 5'-GACCTCGTCCTC in the 3' untranslated region of the human DM kinase gene A rationally designed meganuclease that cleaves CGACTCGCTG-3' (SEQ ID NO: 27) It is possible to produce heterodimers (MYD1 / MYD2). For example, Based on the I-CREI meganuclease, the following contact residues and recognition sequence halves were identified: A meganuclease with the position has been designed.

[0214] MYD1: [Table 28]

[0215] MYD1: [Table 29]

[0216] 2. Meganuclease heterodimers that cleave DNA sequences in pathogen genomes Sequences of the UL36 genes of herpes simplex virus-1 and herpes simplex virus-2. Cleave '-CTCGATGTCGGACGACACGGCA-3' (SEQ ID NO: 28) Rationally designed meganuclease heterodimer (HSV1 / HSV2) can be produced. For example, as described above, the following method is based on the I-CREI meganuclease: Meganucleases with contact residues and recognition sequence half-sites were designed.

[0217] HSV1: [Table 30]

[0218] HSV2: [Table 31]

[0219] BACILLUS ANTHRACIS genome sequence 5'-ACAAGTGTCTAT Rationally designed meganuclease cleaving GGACAGTTTA-3' (SEQ ID NO: 29) It is possible to produce a heterodimer (ANT1 / ANT2). For example, Based on the I-CREI meganuclease, the following contact residues and recognition sequence halves were identified: A meganuclease with the position has been designed.

[0220] ANT1: [Table 32]

[0221] ANT2: [Table 33]

[0222] Variola virus GP009 gene sequence: 5'-AAAACTGTCAAATG Rationally designed meganuclease heterodimer cleaving ACATCGCA-3' (SEQ ID NO: 30) It is possible to produce a dimer (POX1 / POX2). For example, as mentioned above, Based on the I-CREI meganuclease, the following contact residues and recognition sequence half-sites were determined: A meganuclease with the nucleotide sequence nucleotide sequence was designed.

[0223] POX1: [Table 34]

[0224] POX2: [Table 35]

[0225] Epstein-Barr virus BALF2 ​​gene sequence 5'-CGGGGTCTCGTGC A rationally designed meganuclease that cleaves GAGGCCTCC-3' (SEQ ID NO: 31) It is possible to produce a dimer (EBB1 / EBB2). For example, as mentioned above, Based on the I-CREI meganuclease, the following contact residues and recognition sequence half-sites were determined: A meganuclease with

[0226] EBB1: [Table 36]

[0227] EBB1: [Table 37]

[0228] 3. Meganuclease heterodimers that cleave DNA sequences in plant genomes Arabidopsisthaliana GL2 gene sequence 5'-CACTAA A rationally designed meganucleotide that cleaves CTCGTATGAGTCGGTG-3' (SEQ ID NO: 32) It is possible to produce a heterodimer of GLA1 / GLA2. As mentioned above, based on the I-CREI meganuclease, the following contact residues and recognition residues were determined: Meganucleases with recognition sequence half-sites have been designed.

[0229] GLA1: [Table 38]

[0230] GLA2: [Table 39]

[0231] ARABIDOPSIS THALIANNA BP1 gene sequence 5'-TGCCT A rationally designed meganucleotide that cleaves CCTCTAGAGACCCGGAG-3' (SEQ ID NO: 33) It is possible to produce a cleavage heterodimer (BRP1 / BRP2). For example, based on the I-CREI meganuclease as described above, the following contact residues and Meganucleases with recognition sequence half-sites have been designed.

[0232] BRP1: [Table 40]

[0233] BRP2: [Table 41]

[0234] Sequence of the NICOTIANA tabacum magnesium chelatase gene 5'-TA Rational design to cleave AAATCTCTAAGGTCTGTGCA-3' (SEQ ID NO: 34) It is possible to produce meganuclease heterodimers (MGC1 / MGC2) For example, based on the I-CREI meganuclease described above, the following contact residues and meganucleases with recognition sequence half-sites were designed.

[0235] MGC1: [Table 42]

[0236] MGC2: [Table 43]

[0237] NICOTIANA TABACUM CYP82E4 gene sequence 5'-CAAGA A rationally designed meganucleotide that cleaves ATTCAAGCGAGCATTAA-3' (SEQ ID NO: 35) It is possible to produce a cytochrome P450 / HGH2 heterodimer. As mentioned above, based on the I-CREI meganuclease, the following contact residues and recognition residues were determined: Meganucleases with recognition sequence half-sites have been designed.

[0238] CYP: [Table 44]

[0239] HGH2: [Table 45]

[0240] 4. Meganuclease heterodimers cleaving DNA sequences in the yeast genome SACCHAROMYCES CEREVISIAE URA3 gene sequence 5'-T Rational design for cleaving TAGATGACAAGGGAGACGCAT-3' (SEQ ID NO: 36) It is possible to produce a total meganuclease heterodimer (URA1 / URA2) For example, as described above, based on the I-CREI meganuclease, the following contact residues Meganucleases with base and recognition sequence half-sites have been designed.

[0241] URA1: [Table 46]

[0242] URA2: [Table 47]

[0243] 5. Recognition sequence specificity The rationally designed meganucleases outlined above in this example were synthesized in the same manner as in Example 1, Each purified meganuclease was then cloned, expressed in E. coli, and purified. Heterodimerization partners (e.g., ACH1 with ACH2, HGH1 with HGH2) and, etc.) are intended for each meganuclease heterodimer. The enzyme was incubated with a linearized DNA substrate containing a non-palindromic DNA recognition sequence. As shown in Figure 1, each rationally designed meganuclease heterodimer binds to its intended DNA. Cut the part. [Brief explanation of the drawings]

[0244] [Figure 1]Figure 1(A) shows the interaction of the I-CREI homodimer and its native double-stranded recognition sequence based on crystallographic data. The schematic depicts the recognition sequences (SEQ ID NO: 2 and SEQ ID NO: 3), shown unwound for illustrative purposes only, bound by the homodimer, shown as two ovals. The bases of each DNA half-site are numbered from -1 to -9, and the amino acid residues of I-CREI that form the recognition surface are represented by single-letter amino acid designations and numbers indicating the residue positions. Solid lines: hydrogen bonds to DNA bases. Dashed lines: amino acid positions that form new contact points in the enzyme design but do not contact DNA in the wild-type complex. Arrows: residues that interact with the DNA backbone and affect cleavage activity. Figure 1(B) also shows the wild-type contact between the A-T base pair at the -4 position of the cleavage half-site on the right side of Figure 1(A). Specifically, residue Q26 is shown interacting with the A base. Residue I77 is close to the base pair but does not have a specific interaction. Furthermore, Figure 1(C) shows the interaction between a rationally designed variant of I-CREI meganuclease in which residue I77 has been modified to E77. This modification results in a preference for a GC base pair at the -4 position. The interaction between Q26 and the G base is mediated by a water molecule, as observed crystallographically in the left cleavage half-site of Figure 1(A). Furthermore, Figure 1(D) shows the interaction between a rationally designed variant of I-CREI meganuclease in which residue Q26 has been modified to E26 and residue I77 has been modified to R77. This modification results in a preference for a GC base pair at the -4 position. And further, Figure 1(E) shows the interaction between a rationally designed variant of I-CREI meganuclease in which residue Q26 has been modified to A26 and residue I77 has been modified to Q77. This modification results in a preference for a TA base pair at the -4 position. [Figure 2]Figure 2(A) shows a comparison of one recognition sequence between the wild-type I-CREI meganuclease (WT) and each of the 11 rationally designed meganuclease heterodimers of the present invention. Bases conserved relative to the WT recognition sequence are shaded. The 9BP half-site is shown in bold. WT: wild type (SEQ ID NO: 4); CF: ΔF508 allele of the human CFTR gene (SEQ ID NO: 25), which accounts for many cases of cystic fibrosis; MYD: human DM kinase gene associated with myotonic dystrophy; CCR: human CCR5 gene (large HIV coreceptor) (SEQ ID NO: 26); ACH: human FGFR3 gene correlated with achondroplasia (SEQ ID NO: 23); TAT: HIV-1 TAT / REV gene (SEQ ID NO: 15); HSV: HSV-1 UL36 gene (SEQ ID NO: 28); LAM: bacteriophage lambda P05 gene (SEQ ID NO: 22); POX: variola (smallpox) virus GP009 gene (SEQ ID NO: 30); URA: SACCHAROMYCES CERVISIAE URA3 gene (SEQ ID NO: 36); GLA: ARABIDOPSIS THALIANA GL2 gene (SEQ ID NO: 32); BRP: ARABIDOPSIS THALIANA BP-1 gene (SEQ ID NO: 33). Figure 2(B) shows the results of incubation of wild-type I-CREI (WT) and each of the 11 rationally designed meganuclease heterodimers with a plasmid carrying the recognition sites for all 12 enzymes at 37°C for 6 hours. The percentage of cleavage is shown in each box. [Figure 3]Figure 3 shows the cleavage patterns of wild-type and rationally designed I-CREI homodimers. (A) Wild-type I-CREI. (B) I-CREIK116D. (CL) Rationally designed meganuclease of the present invention. The enzymes were incubated with a set of plasmids carrying the intended cleavage half-site palindrome with 27 corresponding single base pair variations. Black bars: predicted cleavage patterns based on Table 1. Gray bars: DNA sites that deviate from the predicted cleavage pattern. Circles indicate bases in the intended recognition site. Also shown is the time course of cleavage over 2 hours. The time course plots of open circles in C and L correspond to cleavage by CCR1 and BRP2 enzymes lacking the E80Q mutation. The cleavage sites correspond to the 5' (left column) and 3' (right column) half-sites of the heterodimeric enzyme described in Figure 2(A).

number

Claims

1. Compared to wild-type I-CREI meganuclease, A recombinant meganuclease with altered specificity for At least 85% relative to residues 2-153 of the I-CREI meganuclease of SEQ ID NO: 1 and I-CR selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 A recognition sequence half that differs by at least one base pair from a half-site within the EI meganuclease recognition sequence. It has specificity for the site, A recombinant meganuclease comprising at least one modification in Table 5 that is not an exclusive modification.

2. Compared to wild-type I-MSOI meganuclease, A recombinant meganuclease with altered specificity for At least 85% relative to residues 6-160 of the I-MSOI meganuclease of SEQ ID NO: 6 and I-MSOI meganuclease recognition selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8 have specificity for a recognition sequence half-site that differs by at least one base pair from the half-site in the sequence 、 A recombinant meganuclease comprising at least one modification in Table 7 that is not an exclusive modification.

3. A set of I-SCEI meganucleases with altered specificity for recognition sequences compared to wild-type I-SCEI meganucleases.

1. A recombinant meganuclease comprising: At least 85% relative to residues 3-186 of the I-SCEI meganuclease of SEQ ID NO: 9 and I-SCEI meganuclease selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 11 having specificity for a recognition sequence that differs from the recognition sequence by at least one base pair; A recombinant meganuclease comprising at least one modification in Table 9 that is not an exclusive modification.

4. Compared to wild-type I-CEUI meganuclease, A recombinant meganuclease with altered specificity for at least 85 to residues 5-211 of the I-CEUI meganuclease of SEQ ID NO: 12 % sequence similarity, and I-CEUI meganuclease selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14 Specificity for a recognition sequence half-site that differs by at least one base pair from the recognition sequence half-site. Has, Recombinant meganucleases comprising at least one modification of Table 11 that is not an exclusive modification. 。

5. Compared to wild-type I-CREI meganuclease, A recombinant meganuclease with altered specificity for At least 85% relative to residues 2-153 of the I-CREI meganuclease of SEQ ID NO: 1 and I-CR selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 A recognition sequence half that differs by at least one base pair from a half-site within the EI meganuclease recognition sequence. It has specificity for the site, (1) The specificity at position −1 is (A) Q70, C70, L70, Y75, Q75, H75, H139, Q46 and H 46 to T in the sense strand by a modification selected from the group consisting of: (B) selected from the group consisting of Y75, L75, C75, Y139, C46, ​​and A46 to A in the sense strand by modification with (C) a modification selected from the group consisting of K70, E70, E75, E46, and D46; and is changed to G in the sense strand, (D) a modification selected from the group consisting of H75, R75, H46, K46, and R46; and thus changed to C in the sense strand, or (E) a modification selected from the group consisting of G70, A70, S70, and G46; modified to any base in the base strand; and / or (2) The specificity at position -2 is (A) from the group consisting of Q70, T44, A44, V44, I44, L44 and N44 Depending on the modification chosen, it is changed to A in the sense strand; (B) a modification selected from the group consisting of E70, D70, K44, and R44; changed to C in the ance strand; (C) a modification selected from the group consisting of H70, D44, and E44 in the sense strand; or (D) modified to A or T in the sense strand by a modification involving C44; and / or (3) The specificity at position -3 is (A) A modification selected from the group consisting of Q68 and C24 in the sense strand Changed to; (B) a modification selected from the group consisting of E68, F68, K24, and R24; changed to C in the ance strand; (C) a modification selected from the group consisting of M68, C68, L68, and F68; changed to T in the ance strand; (D) H68 is changed to A or C in the sense strand by a modification including H68; (E) modified to C or T in the sense strand by a modification involving Y68; (F) Modifications including K68 changed to G or T in the sense strand; and / or (4) The specificity at position -4 is (A) C in the sense strand by a modification selected from the group consisting of E77 and K26 Changed to; (B) A modification selected from the group consisting of E26 and R77 results in a G in the sense strand. Changed to; (C) modified to C or T in the sense strand by a modification involving S77; or (D) modified to any base in the sense strand by a modification involving S26; and / or (5) The specificity at position -5 is (A) Changed to C in the sense strand by a modification involving E42; (B) a modification involving R42 changed to G in the sense strand; (C) A modification selected from the group consisting of C28 and Q42 in the sense strand or changed to C; or (D) any of the modifications in the sense strand selected from the group consisting of M66 and K66; changed to the desired base; and / or (6) The specificity at position -6 is (A) From the group consisting of C40, I40, V40, C79, I79, V79 and Q28 Depending on the modification chosen, it is changed to T in the sense strand; (B) C in the sense strand by a modification selected from the group consisting of E40 and R28 Changed to; (C) modified to G in the sense strand by a modification involving R40; and / or (7) The specificity at position -7 is (A) a modification selected from the group consisting of E38, K30, and R30 in the sense strand; Changed to C in (B) a modification selected from the group consisting of K38, R38, and E30 in the sense strand; Changed to G in (C) a modification selected from the group consisting of I38 and L38, resulting in a T in the sense strand; Changed to; (D) modified to A or G in the sense strand by a modification involving C38; (E) a modification selected from the group consisting of H38, N38, and Q30 in the sense strand; is changed to any base in and / or (8) The specificity at position -8 is (A) a modification selected from the group consisting of L33, V33, I33, F33, and C33; to T in the sense strand; (B) a modification selected from the group consisting of E33 and D33, resulting in a C in the sense strand; Changed to; (C) a modification consisting of K33 changed to G in the sense strand; (D) changed to A or C in the sense strand by a modification involving R32; (E) modified to A or G in the sense strand by a modification involving R33; and / or (9) The specificity at position -9 is (A) Changed to C in the sense strand by a modification involving E32; (B) a modification selected from the group consisting of R32 and K32, resulting in a G in the sense strand; Changed to; (C) a modification selected from the group consisting of L32, V32, A32, and C32, changed to T in the ance strand; (D) a modification selected from the group consisting of D32 and I32, which provides C in the sense strand; or changed to T; or (E) a modification selected from the group consisting of S32, N32, H32, Q32, and T32; Thus, the base is changed to any base in the sense strand. A recombinant meganuclease characterized by:

6. Compared to wild-type I-MSOI meganuclease, A recombinant meganuclease with altered specificity for At least 85% relative to residues 6-160 of the I-MSOI meganuclease of SEQ ID NO: 6 and I-MSOI meganuclease recognition selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8 have specificity for a recognition sequence half-site that differs by at least one base pair from the half-site in the sequence 、 (1) The specificity at position −1 is (A) a modification selected from the group consisting of K75, Q77, A49, C49, and K79; to A in the sense strand; (B) a modification selected from the group consisting of C77, L77, and Q79 in the sense strand; It was changed to T in (C) a modification selected from the group consisting of K77, R77, E49, and E79; changed to G in the ance strand; and / or (2) The specificity at position -2 is (A) a modification selected from the group consisting of Q75, K81, C47, I47, and L47; to A in the sense strand; (B) selected from the group consisting of E75, D75, R47, K47, K81, and R81 The modification changes it to C in the sense strand; (C) a modification selected from the group consisting of K75, E47, and E81 in the sense strand; Changed to G in and / or (3) The specificity at position -3 is (A) selected from the group consisting of Q72, C26, L26, V26, A26 and I26 The modification changes it to A in the sense strand; (B) a modification selected from the group consisting of E72, Y72, H26, K26, and R26; to C in the sense strand; (C) a modification selected from the group consisting of K72, Y72, and H26 in the sense strand; It was changed to T in and / or (4) The specificity at position -4 is (A) a modification selected from the group consisting of K28, K83, and Q28 in the sense strand; It was changed to T in (B) A modification selected from the group consisting of R83 and K83 results in a G in the sense strand. Changed to; (C) A modification selected from the group consisting of K28 and Q83 in the sense strand Changed to; and / or (5) The specificity at position -5 is (A) A modification selected from the group consisting of R45 and E28 results in a G in the sense strand. Changed to; (B) modified to T in the sense strand by a modification involving Q28; (C) changed to C in the sense strand by a modification involving R28; and / or (6) The specificity at position -6 is (A) a modification selected from the group consisting of K43, V85, L85, and Q30; changed to T in the ance strand; (B) a modification selected from the group consisting of E43, E85, K30, and R30; or, (C) selected from the group consisting of R43, K43, K85, R85, E30, and D30 The modification changes it to G in the sense strand; and / or (7) The specificity at position -7 is (A) a modification selected from the group consisting of E32 and E41, resulting in a C in the sense strand; Changed to; (B) a modification selected from the group consisting of R32, R41, and K41 in the sense strand; Changed to G in (C) a modification selected from the group consisting of K32, M41, L41, and I41; changed to T in the ance strand; and / or (8) The specificity at position -8 is (A) A modification selected from the group consisting of K32 and K35 results in a T in the sense strand. Changed to; (B) changed to C in the sense strand by a modification involving E32; (C) a modification selected from the group consisting of K32, K35, and R35 on the sense strand; Changed to G in and / or (9) The specificity at position -9 is (A) A modification selected from the group consisting of N34 and H34 in the sense strand Changed to; (B) a modification selected from the group consisting of S34, C34, V34, T34, and A34; to T in the sense strand; (C) a modification selected from the group consisting of K34, R34, and H34 on the sense strand; In this case, it is changed to G. A recombinant meganuclease characterized by:

7. A set of I-SCEI meganucleases with altered specificity for recognition sequences compared to wild-type I-SCEI meganucleases.

1. A recombinant meganuclease comprising: At least 85% relative to residues 3-186 of the I-SCEI meganuclease of SEQ ID NO: 9 and I-SCEI meganucleases of SEQ ID NO: 10 and SEQ ID NO: 11 and at least one base have specificity for different recognition sequences, (1) The specificity at position 4 is (A) changed to A in the sense strand by a modification involving K50; (B) a modification selected from the group consisting of K57, M57, and Q50 in the sense strand; It was changed to T in (C) a modification selected from the group consisting of E50, R57, and K57 in the sense strand; and / or (2) The specificity at position 5 is (A) A in the sense strand by a modification selected from the group consisting of K48 and Q102 Changed to; (B) a modification selected from the group consisting of E48, K102, and R102, changed to G in the chain; (C) by a modification selected from the group consisting of Q48, C102, L102 and V102 to T in the sense strand; and / or (3) The specificity at position 6 is (A) changed to A in the sense strand by a modification containing K59; (B) a modification selected from the group consisting of R59 and K59, resulting in a C in the sense strand; Changed to; (B) A modification selected from the group consisting of K84 and E59 results in a G in the sense strand. Changed to; and / or (4) The specificity at position 7 is (A) a modification selected from the group consisting of R46, K46, and E86 in the sense strand; Changed to C in (B) a modification selected from the group consisting of K86, R86, and E46 in the sense strand; Changed to G in (C) a modification selected from the group consisting of C46, ​​L46, and V46 in the sense strand; In this case, it was changed to A; and / or (5) The specificity at position 8 is (A) a modification selected from the group consisting of E88, R61, and H61 in the sense strand; Changed to C in (B) a modification selected from the group consisting of K88, Q61, and H61 in the sense strand; It was changed to T in (C) a modification selected from the group consisting of K61, S61, V61, A61, and L61; to A in the sense strand; and / or (6) The specificity at position 9 is (A) a modification selected from the group consisting of C98, V98, and L98 in the sense strand; In this case, it was changed to A; (B) C in the sense strand by a modification selected from the group consisting of R98 and K98 or (C) a modification selected from the group consisting of E98 and D98, resulting in a G in the sense strand; Changed to; and / or (7) The specificity at position 10 is (A) A modification selected from the group consisting of K96 and R96 results in a C in the sense strand. Changed to; (B) a modification selected from the group consisting of D96 and E96, resulting in a G in the sense strand; Changed to; (C) A96 in the sense strand by a modification selected from the group consisting of C96 and A96 Changed to; and / or (8) The specificity at position 11 is (A) a modification involving Q90 changed to T in the sense strand; (B) C in the sense strand by a modification selected from the group consisting of K90 and R90 Changed to; (C) a modification involving E90 changed to G in the sense strand; and / or (9) The specificity at position 12 is (A) changed to A in the sense strand by a modification involving Q193; (B) a modification selected from the group consisting of E165, E193, and D193, Changed to C in the sulphur chain; (C) in the sense strand by a modification selected from the group consisting of K165 and R165; and changed to G; and / or (10) The specificity at position 13 is (A) a modification selected from the group consisting of Q193, C163, and L163, changed to T in the β-strand; (B) a modification selected from the group consisting of E193, D193, K163, and R192; to G in the sense strand; (C) in the sense strand by a modification selected from the group consisting of C193 and L193 and changed to A; and / or (11) The specificity at position 14 is (A) In the sense strand, a modification selected from the group consisting of K161 and Q192 and changed to T; (B) in the sense strand by a modification selected from the group consisting of L192 and C192 and changed to A; (C) the group consisting of K147, K161, R161, R197, D192 and E192 to G in the sense strand by a modification selected from: (D) in the sense strand by a modification selected from the group consisting of K161 and Q192; and changed to T; and / or (12) The specificity at position 15 is (A) a modification selected from the group consisting of C151, L151, and K151; changed to T in the β-strand; (B) modified to G in the sense strand by a modification involving K151; or (C) changed to C in the sense strand by a modification involving E151; and / or (13) The specificity at position 17 is (A) In the sense strand, a modification selected from the group consisting of G152 and Q150 and changed to T; (B) In the sense strand, a modification selected from the group consisting of K152 and K150 and changed to C; (C) selected from the group consisting of N152, S152, D152, D150 and E150 modified to G in the sense strand; and / or (14) The specificity at position 18 is (A) in the sense strand by a modification selected from the group consisting of H155 and Y155 Changed to T; (B) in the sense strand by a modification selected from the group consisting of R155 and K155; and changed to C; (C) in the sense strand by a modification selected from the group consisting of K155 and C155; and changed to A A recombinant meganuclease characterized by:

8. Compared to wild-type I-CEUI meganuclease, A recombinant meganuclease with altered specificity for at least 85 to residues 5-211 of the I-CEUI meganuclease of SEQ ID NO: 12 % sequence similarity, and I-CEUI meganuclease selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14 Specificity for a recognition sequence half-site that differs by at least one base pair from the recognition sequence half-site. Has, (1) The specificity at position −1 is (A) a modification selected from the group consisting of C92, A92, and V92 in the sense strand; In this case, it was changed to A; (B) in the sense strand by a modification selected from the group consisting of Q116 and Q92 Changed to T; (C) in the sense strand by a modification selected from the group consisting of E116 and E92 Changed to G; and / or (2) The specificity at position -2 is (A) by a modification selected from the group consisting of Q117, C90, L90, and V90; changed to A in the sense strand; (B) from the group consisting of K117, R124, K124, E124, E90, and D90 Depending on the modification chosen, it is changed to G in the sense strand; (C) from the group consisting of E117, D117, R174, K124, K90, and K68 Depending on the modification chosen, it is changed to C in the sense strand; and / or (3) The specificity at position -3 is (A) a modification selected from the group consisting of C70, V70, T70, L70, and K70; to A in the sense strand; (B) a modification involving Q70 changed to T in the sense strand; (B) modified to C in the sense strand by a modification involving K70; and / or (4) The specificity at position -4 is (A) a modification selected from the group consisting of E126, D126, R88, K88, and K72 to C in the sense strand by; (B) a modification selected from the group consisting of K126, L126, and Q88, resulting in a sense changed to T in the chain; (C) the group consisting of Q126, N126, K88, L88, C72, L72, and V72 to A in the sense strand by a modification selected from: and / or (5) The specificity at position -5 is (A) a modification selected from the group consisting of E74, K128, R128, and E128; and changed to G in the sense strand; (B) a modification selected from the group consisting of C128, L128, V128, and T128; to a T in the sense strand; or (C) a modification selected from the group consisting of C74, L74, V74, and T74; changed to A in the ance strand; and / or (6) The specificity at position -6 is (A) a modification selected from the group consisting of K86, C86, and L86 in the sense strand; It was changed to T in (B) a modification selected from the group consisting of D86, E86, R84, and K84; or, (C) a modification selected from the group consisting of K128, R128, R86, K86, and E84 to G in the sense strand by; and / or (7) The specificity at position -7 is (A) a modification selected from the group consisting of R76, K76, and H76 in the sense strand; Changed to C in (B) A modification selected from the group consisting of E76 and R84 results in a G in the sense strand. Changed to; (C) a modification selected from the group consisting of H76 and Q76, resulting in a T in the sense strand; Changed to; and / or (8) The specificity at position -8 is (A) a modification selected from the group consisting of Y79, R79, and Q76 in the sense strand; In this case, it was changed to A; (B) a modification selected from the group consisting of D79, E79, D76, and E76; changed to C in the ance strand; (C) a modification selected from the group consisting of R79, K79, K76, and R76; changed to G in the ance strand; and / or (9) The specificity at position -9 is (A) a modification selected from the group consisting of K78, V78, L78, C78, ​​and T78; to T in the sense strand; (B) a modification selected from the group consisting of D78 and E78, resulting in a C in the sense strand; or (C) a modification selected from the group consisting of R78, K78, and H78 in the sense strand; In this case, it is changed to G. A recombinant meganuclease characterized by:

9. Altered binding affinity to double-stranded DNA compared to wild-type I-CREI meganuclease a recombinant meganuclease comprising: at least 85 to residues 2-153 of the I-CREI meganuclease of SEQ ID NO: 1 % sequence similarity; DNA binding affinity is (A) E80, D137, I81, L112, P by H, N, Q, S, T, K or R 29, substitution of V64 or Y66; Or, (B) substitution of T46, T140 or T143 by K or R; is increased by at least one modification corresponding to one substitution selected from the group consisting of A recombinant meganuclease characterized by:

10. Altered binding affinity to double-stranded DNA compared to wild-type I-CREI meganuclease a recombinant meganuclease comprising: at least 85 to residues 2-153 of the I-CREI meganuclease of SEQ ID NO: 1 % sequence similarity; DNA binding affinity is (A) K34, K48, R51, K82, K11 by H, N, Q, S, T, D or E 6, or substitution of K139; Or, (B) I81, L112, P29, V64, Y66, T46, T140 by D or E or at least one modification corresponding to one substitution selected from the group consisting of substitutions in T143 A recombinant meganuclease characterized by being reduced by

11. Altered binding affinity to double-stranded DNA compared to wild-type I-MSOI meganuclease a recombinant meganuclease comprising: at least 85 to residues 6-160 of the I-MSOI meganuclease of SEQ ID NO: 6 % sequence similarity; DNA binding affinity is (A) E147, I85, G86 or Y118 by H, N, Q, S, T, K or R Replacement; Or, (B) Q41, N70, S87, T88, H89, Q122, Q139 by K or R , S150 or N152 A recombinant meganuclease characterized by an increase in activity due to a single modification.

12. Altered binding affinity to double-stranded DNA compared to wild-type I-MSOI meganuclease a recombinant meganuclease comprising: at least 85 to residues 6-160 of the I-MSOI meganuclease of SEQ ID NO: 6 % sequence similarity; DNA binding affinity is (A) K36, R51, K123, K143, or K36 by H, N, Q, S, T, D, or E or substitution of R144; or (B) I85, G86, Y118, Q41, N70, S87, T88 by D or E, one selected from the group consisting of substitutions at H89, Q122, Q139, S150 or N152 The recombinant DNA molecule is characterized by being reduced by at least one modification corresponding to the substitution of Ganuclease.

13. Altered binding affinity for double-stranded DNA compared to wild-type I-SCEI meganuclease a recombinant meganuclease comprising: at least 85 to residues 3-186 of the I-SCEI meganuclease of SEQ ID NO: 9 % sequence similarity; DNA binding affinity is (A) D201, L19, L80, L92, Y1 by H, N, Q, S, T, K or R 51, Y188, I191, Y199 or Y222 substitution; or (B) N15, N17, S81, H84, N94, N120, T156 by K or R , N157, S159, N163, Q165, S166, N194 or S202 substitution and the increase is due to at least one modification corresponding to one substitution selected from the group consisting of: A recombinant meganuclease characterized by:

14. Altered binding affinity for double-stranded DNA compared to wild-type I-SCEI meganuclease a recombinant meganuclease comprising: at least 85 to residues 3-186 of the I-SCEI meganuclease of SEQ ID NO: 9 % sequence similarity; DNA binding affinity is (A) K20, K23, K63, K122, K1 by H, N, Q, S, T, D or E 48, substitution of K153, K190, K193, K195 or K223; or (B) L19, L80, L92, Y151, Y188, I191, Y1 by D or E 99, Y222, N15, N17, S81, H84, N94, N120, T156, N1 57, S159, N163, Q165, S166, N194 or S202 substitutions and characterized by being reduced by at least one modification corresponding to one substitution selected from the group This is a recombinant meganuclease.

15. Altered binding affinity for double-stranded DNA compared to wild-type I-CEUI meganuclease a recombinant meganuclease comprising: at least 8 to residues 5-211 of the I-CEUI meganuclease of SEQ ID NO: 12 comprising a polypeptide having 5% sequence similarity; DNA binding affinity is (A) substitution of D25 or D128 by H, N, Q, S, T, K or R; or (B) S68, N70, H94, S117, N120, N129 by K or R, or at least one modification corresponding to one substitution selected from the group consisting of substitutions at H172 A recombinant meganuclease characterized by its increased activity.

16. Altered binding affinity for double-stranded DNA compared to wild-type I-CEUI meganuclease a recombinant meganuclease comprising: at least 8 to residues 5-211 of the I-CEUI meganuclease of SEQ ID NO: 12 comprising a polypeptide having 5% sequence similarity; DNA binding affinity is (A) K21, K28, K31, R112, R1 by H, N, Q, S, T, D or E 14, or substitution of R130; or (B) S68, N70, H94, S117, N120, N129 or H by D or E with at least one modification corresponding to one substitution selected from the group consisting of 172 substitutions A recombinant meganuclease characterized by reduced activity.

17. Modified affinity for dimer formation with reference meganuclease monomers a meganuclease monomer, at least 85 to residues 2-153 of the I-CREI meganuclease of SEQ ID NO: 1 % sequence similarity, The affinity for dimer formation is (A) substitution of K7, K57, or K96 by D or E; or (B) a minor amino acid corresponding to a substitution selected from the group consisting of substitution of E8 or E61 by K or R; Recombinant meganuclease mononucleotides characterized by being altered by at least one modification Mah.

18. A recombinant meganuclease heterodimer, comprising: at least 85 to residues 2-153 of the I-CREI meganuclease of SEQ ID NO: 1 a first polypeptide having a sequence similarity of 1 to 10%; The affinity for dimer formation is (A) for a substitution selected from the group consisting of substitution of K7, K57, or K96 by D or E; a first polypeptide that is altered by at least one corresponding modification; and at least 85 to residues 2-153 of the I-CREI meganuclease of SEQ ID NO: 1 a second polypeptide having a sequence similarity of 100% to 100% of the sequence of The affinity for dimer formation is (B) a minor amino acid sequence corresponding to a substitution selected from the group consisting of substitution of E8 or E61 by K or R; a second polypeptide that is altered by at least one modification, A recombinant meganuclease heterodimer, characterized in that:

19. Modified affinity for dimer formation with reference meganuclease monomers a meganuclease monomer, at least 85 to residues 6-160 of the I-MSOI meganuclease of SEQ ID NO: 6 % sequence similarity, The affinity for dimer formation is (A) replacement of R302 with D or E; or (B) a substitution selected from the group consisting of substitution of D20, E11 or Q64 by K or R; Recombinant meganucleic acid, characterized in that it is altered by at least one corresponding modification. Rease monomer.

20. A recombinant meganuclease heterodimer, comprising: at least 85 to residues 6-160 of the I-MSOI meganuclease of SEQ ID NO: 6 a first polypeptide having a sequence similarity of 1 to 10%; The affinity for dimer formation is (A) at least one corresponding to a substitution selected from the group consisting of substitution of R302 by D or E a first polypeptide that is altered by another modification; and at least 85 to residues 6-160 of the I-MSOI meganuclease of SEQ ID NO: 6 a second polypeptide having a sequence similarity of 100% to 100% of the sequence of The affinity for dimer formation is (B) a substitution selected from the group consisting of substitution of D20, E11 or Q64 by K or R; a second polypeptide that is altered by at least one corresponding modification, A recombinant meganuclease heterodimer, characterized in that:

21. Modified affinity for dimer formation with reference meganuclease monomers a meganuclease monomer, at least 8 to residues 5-211 of the I-CEUI meganuclease of SEQ ID NO: 12 comprising a polypeptide having 5% sequence similarity, The affinity for dimer formation is (A) substitution of R93 by D or E; or (B) at least one corresponding to a substitution selected from the group consisting of substitution of E152 by K or R The recombinant meganuclease monomer is characterized in that it is altered by one modification. -.

22. A recombinant meganuclease heterodimer, comprising: at least 8 to residues 5-211 of the I-CEUI meganuclease of SEQ ID NO: 12 a first polypeptide having 5% sequence similarity, The affinity for dimer formation is (A) at least one corresponding to a substitution selected from the group consisting of substitution of R93 by D or E a first polypeptide that is altered by a single modification; and at least 8 to residues 5-211 of the I-CEUI meganuclease of SEQ ID NO: 12 a second polypeptide having 5% sequence similarity, The affinity for dimer formation is (B) at least one corresponding to a substitution selected from the group consisting of substitution of E152 by K or R a second polypeptide that is altered by another modification, A recombinant meganuclease heterodimer characterized by:

23. and further including at least one modification selected from Table 1 that is not an exclusive modification. characterized by: The recombinant meganuclease monomer or heterodimer according to claim 17 or claim 18. Mah.

24. and further including at least one modification selected from Table 2 that is not an exclusive modification. characterized by: The recombinant meganuclease monomer or heterodimer according to claim 19 or 20. Mah.

25. and further comprising at least one modification selected from Table 4 that is not an exclusive modification. characterized by: The recombinant meganuclease monomer or heterodimer of claim 21 or claim 22. Mah.

26. A genetically modified eukaryotic cell containing an exogenous sequence of interest inserted into a chromosome of the eukaryotic cell.

1. A method for producing Eukaryotic cells, (I) a first nucleic acid sequence encoding a meganuclease; and (II) a second nucleic acid sequence comprising the sequence of interest. transfecting the cells with one or more nucleic acids comprising The meganuclease produces a cleavage site in the chromosome and the sequence of interest is inserted into the chromosome at the break site, The meganuclease is a recombinant meganuclease according to any one of claims 1 to 25. A method characterized in that the

27. the second nucleic acid further comprises sequences homologous to sequences flanking the cleavage site, A flavor sequence is inserted at the cleavage site by homologous recombination.

27. The method of claim 26.

28. The second nucleic acid lacks substantial homology to the cleavage site and the sequence of interest is a non-homologous and the fragment is inserted into the chromosome by selective end joining.

27. The method of claim 26.

29. A genetically modified eukaryotic cell containing an exogenous sequence of interest inserted into the chromosome of the eukaryotic cell.

1. A method for producing cells, comprising: Introducing the meganuclease protein into a eukaryotic cell; and, transfecting said eukaryotic cell with a nucleic acid comprising said sequence of interest; The meganuclease generates a cleavage site in the chromosome, and the sequence of interest is inserted into the inserted into the chromosome at the break site; and, The meganuclease is a recombinant meganuclease according to any one of claims 1 to 25. A method characterized in that the

30. the nucleic acid further comprises sequences homologous to sequences flanking the cleavage site, a sequence is inserted at the cleavage site by homologous recombination, 30. The method of claim 29.

31. The nucleic acid lacks substantial homology to the cleavage site and the sequence of interest is is inserted into the chromosome by end joining, 30. The method of claim 29.

32. Genetically modified eukaryotic cells by disrupting a target sequence in the chromosome of the eukaryotic cell 1. A method for producing The method includes transfecting a eukaryotic cell with a nucleic acid encoding a meganuclease. fruit, The meganuclease produces a cleavage site in the chromosome, and the target sequence is destroyed by non-homologous end joining at the cleavage site; and, The meganuclease is a recombinant meganuclease according to any one of claims 1 to 25. A method characterized in that the enzyme is

33. 1. A method for producing a genetically modified organism, comprising: Producing a genetically modified eukaryotic cell according to the method of any one of claims 26 to 32. and and, Cultivating the genetically modified eukaryotic cell to produce a genetically modified organism. Steps and A method comprising:

34. The eukaryotic cells are derived from gametes, zygotes, blastocyst cells, embryonic stem cells and protoplast cells. selected from the group consisting of 34. The method of claim 33.

35. 1. A method for treating disease by gene therapy in eukaryotes, comprising: at least one cell of said eukaryotic organism, (I) a first nucleic acid sequence encoding a meganuclease; and (II) a second nucleic acid sequence comprising the sequence of interest; transfecting the vector by The meganuclease generates a cleavage site in the chromosome, and the sequence of interest is inserted into the inserted into the chromosome at the break site; and, The meganuclease is a recombinant meganuclease according to any one of claims 1 to 25. A method characterized in that the enzyme is

36. the second nucleic acid sequence further comprises sequences homologous to sequences flanking the cleavage site, The sequence of interest is inserted at the cleavage site by homologous recombination. Ru, 36. The method of claim 35.

37. The second nucleic acid sequence lacks substantial homology to the cleavage site and the sequence of interest is a non- inserted into the chromosome by homologous end joining, 36. The method of claim 35.

38. 1. A method for treating disease by gene therapy in eukaryotes, comprising: introducing the meganuclease protein into at least one cell of said eukaryotic organism; Steps and transfecting said eukaryotic cell with a nucleic acid comprising a sequence of interest. fruit, The meganuclease generates a cleavage site in the chromosome, and the sequence of interest is inserted into the inserted into the chromosome at the break site; The meganuclease is a recombinant meganuclease according to any one of claims 1 to 25. Ze; and, the insertion of said sequence of interest provides said gene therapy for said disease. method.

39. the nucleic acid further comprises sequences homologous to sequences flanking the cleavage site, a sequence is inserted at the cleavage site by homologous recombination, 39. The method of claim 38.

40. The nucleic acid lacks substantial homology to the cleavage site and the sequence of interest is is inserted into the chromosome by end joining, 39. The method of claim 38.

41. In eukaryotes, gene therapy by disruption of a target sequence in the chromosome of said eukaryote.

1. A method for treating a disease by administering at least one cell of said eukaryotic organism is transfected with a nucleic acid encoding a meganuclease; transfecting the The meganuclease produces a cleavage site in the chromosome, and the target sequence is destroyed by non-homologous end joining at the cleavage site; The meganuclease is a recombinant meganuclease according to any one of claims 1 to 25. -ze; and, Disruption of said target sequence provides said gene therapy for said disease. Law.

42. Infection of a viral pathogen in a eukaryotic host is controlled by determining the presence of a gene in the genome of the viral pathogen.

1. A method of treating by disrupting a target sequence, comprising: At least one infected cell of said eukaryotic host is infected with a nucleic acid encoding a meganuclease. transfecting the vector by The meganuclease generates a cleavage site in the viral genome and the target sequence is disrupted by non-homologous end joining at the cleavage site, The meganuclease is a recombinant meganuclease according to any one of claims 1 to 25. -ze; and, wherein disruption of said target sequence provides a treatment for said infection.

43. Infection of a viral pathogen in a eukaryotic host is controlled by determining the presence of a gene in the genome of the viral pathogen.

1. A method of treating by disrupting a target sequence, comprising: At least one infected cell of the eukaryotic host is infected with a first gene encoding a meganuclease. transfecting the nucleic acid with a second nucleic acid; The meganuclease generates a cleavage site in the viral genome and the target sequence is formed at the cleavage site by homologous recombination between the viral genome and the second nucleic acid. and destroyed; The meganuclease is a recombinant meganuclease according to any one of claims 1 to 25. -ze; the second nucleic acid comprises sequences homologous to sequences flanking the cleavage site; and, wherein disruption of said target sequence provides a treatment for said infection.

44. Infection of a prokaryotic pathogen in a eukaryotic host is characterized by the presence of a target gene in the genome of said prokaryotic pathogen.

1. A method of treating by disrupting a target sequence, comprising: At least one cell of the prokaryotic pathogen that infects the eukaryotic host is subjected to a meganuclear transformation. transfecting the cell with a nucleic acid encoding a lyase; The meganuclease produces a cleavage site in the prokaryotic cell genome, and the target sequence is disrupted at the cleavage site by non-homologous end joining; The meganuclease is a recombinant meganuclease according to any one of claims 1 to 25. -ze; and, wherein disruption of said target sequence provides a treatment for said infection.

45. Infection of a prokaryotic pathogen in a eukaryotic host is characterized by the presence of a target gene in the genome of said prokaryotic pathogen.

1. A method of treating by disrupting a target sequence, comprising: At least one cell of the prokaryotic pathogen that infects the eukaryotic host is subjected to a meganuclear transformation. a first nucleic acid encoding a nucleic acid encoding a protease, and a second nucleic acid. fruit; The meganuclease produces a cleavage site in the prokaryotic cell genome, and the target sequence is formed at the cleavage site by homologous recombination between the prokaryotic cell genome and the second nucleic acid. and destroyed; The meganuclease is a recombinant meganuclease according to any one of claims 1 to 25. -ze; the second nucleic acid comprises sequences homologous to sequences flanking the cleavage site; and, wherein disruption of said target sequence provides a treatment for said infection.

46. At least one base position of the recognition sequence is selected to contain at least one desired change.

1. A method for rationally designing recombinant meganucleases with altered specificity, comprising: (1) determining at least a portion of the three-dimensional structure of a reference meganuclease-DNA complex; thing; (2) identifying amino acid residues that form a base contact surface at the base position; (3) the β-carbon of at least the first residue comprising said contact surface and at least one of the base positions; determining the distance between at least the first base; and, (4) selecting an amino acid substitution that promotes the desired change from the group shown in Table 2, (A) For a first residue that is less than 6 angstroms from the first base, , C, T, or A, as appropriate to promote the desired change. selecting group 1 and / or group 2 substitutions; Or, (B) for a first residue that is more than 6 angstroms from the first base, , G, C, T or A, as appropriate to promote the desired change. by selecting group 2 and / or group 3 substitutions that are A method comprising:

47. A method for rationally designing recombinant meganucleases with increased DNA-binding affinity There was, (1) determining at least a portion of the three-dimensional structure of a reference meganuclease-DNA complex; thing; (2) identifying amino acid contact residues that form the backbone interface; (3) The amino acid substitution that increases the DNA-binding affinity is (A) For contact residues with negatively charged or hydrophobic side chains, uncharged / polar, or select substitutions with positively charged side chains; or (B) For contact residues with uncharged / polar side chains, select substitutions with positively charged side chains. Identifying by A method comprising:

48. A method for rationally designing recombinant meganucleases with reduced DNA-binding affinity There was, (1) determining at least a portion of the three-dimensional structure of a reference meganuclease-DNA complex; thing; (2) identifying amino acid contact residues that form the backbone interface; (3) The amino acid substitution that reduces the DNA-binding affinity is (A) For contact residues with positively charged side chains, uncharged / polar, or negatively charged side chains Selecting a permutation with or (B) For contact residues with hydrophobic or uncharged / polar side chains, negatively charged side chains are used. by selecting a substitution that has A method comprising:

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