Optimization of engineered meganucleases for recognition sequences

Abstract

To provide an engineered I-CreI-derived meganuclease for recognition and cleavage of a recognition sequence having a specific center sequence.SOLUTION: One aspect is an engineered meganuclease that binds to and cleaves a recognition sequence comprising a center sequence consisting of ACAA, ACAG, ACAT, ACGA, ACGC, ACGG, ACGT, ATAA, ATAG, ATAT, ATGA, ATGG, TTGG, GCAA, GCAT, GCGA, GCAG, TCAA or TTAA, where the engineered meganuclease comprises a first subunit and a second subunit, where the first subunit and the second subunit each comprise a specific amino acid sequence with substitution at one or more positions corresponding to positions 48, 50, 71, 72, 73 and 74 of a specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the fields of molecular biology and recombinant nucleic acid technology. In particular, the present invention relates to the Optimization of engineered I-CreI-derived meganucleases for recognition sequences including the central sequence Regarding.

[0002] Reference to sequence listings submitted as text files via EFS-WEB This application contains a sequence listing submitted in ASCII format via EFS-Web, the entirety of which is hereby incorporated by reference. The ASCI created on May 7, 2020, is incorporated herein by reference. The copy is named P109070040WO00-SEQ-EPG and is 1 ,446 kilobytes. [Background technology]

[0003] Genome engineering involves inserting, deleting, substituting, or otherwise modifying specific gene sequences within a genome. Genome engineering is required and has many therapeutic and biotechnological applications. The development of effective tools for this remains dependent on gene therapy, agricultural technology, and synthetic biology. This is a major goal in the field (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3). One approach to this is to use meganucleases (i.e., homing endonucleases). The key to successful application of nucleases is to utilize site-specific, rare-cutting nucleases such as nucleases (e.g., nucleases).

[0004] Meganucleases generally fall into four families: the LAGLIDADG (SEQ ID NO: 2) family Milli, GIY-YIG family, His-Cys box family and HNH family These families affect catalytic activity and recognition sequences. They are characterized by a structural motif that includes the LAGLIDADG (SEQ ID NO: 2) motif. Family members contain one or more of the conserved LAGLIDADG (SEQ ID NO: 2) motifs. It is characterized by having one or two copies (see Non-Patent Document 4). LAGLIDADG (SEQ ID NO: 2) with a single copy of the IDADG (SEQ ID NO: 2) motif 2) Meganucleases form homodimers, but the LAGLIDADG (SEQ ID NO: 2) monomer Members with two copies of chief are found as monomers.

[0005] I-CreI (SEQ ID NO: 1) is a member of the LAGLIDADG (SEQ ID NO: 2) family It recognizes and cuts a 22-base pair recognition sequence in the chloroplast chromosome. It has been used to alter the preference of wild-type I-CreI recognition sites (Non-Patent Document 5, Non-patent literature 6, 7, 8). I-CreI is designed to target a wide variety of DNA sites, including sites within the virus genome. Methods for manipulating the same have been previously disclosed, for example, in US Pat. No. 6,213,999.

[0006] The DNA sequence recognized by I-CreI is 22 base pairs in length. An example of an I-CreI recognition site is provided in SEQ ID NO: 3, although the enzyme may recognize a variety of The wild-type I-CreI enzyme appears to bind to the tandem sequence with varying affinities. It binds to DNA as a homodimer that makes direct contact with base-paired "half sites." The two half sites are separated by a four base pair "center sequence." The two central bases are not directly contacted by the enzyme. After cleavage, wild-type I-CreI and The engineered I-CreI-derived meganuclease generates a staggered double-stranded cleavage site in the middle of the recognition sequence. This fragment fragments the DNA, generating a 4-base pair 3' overhang (Figure 1).

[0007] The present invention provides a method for cleaving a meganuclease recognition sequence comprising the central 4-amino acid residue of the meganuclease recognition sequence, which becomes a 3' overhang after cleavage. Regarding the base pair (i.e., the central sequence). Chlamydomonas reinhardtii 23S rRNA gene In the case of the natural I-CreI recognition sequence of IC, the central sequence is 5'-GTGA-3'. Many published studies on reI or its derivatives have shown that wild-type or genetically engineered The enzymes were selected from the natural 5´-GTGA-3´ central sequence or the palindromic sequence 5´-GT The results were evaluated using DNA substrates with either AC-3' or 'AC-3'. Reference 9) describes a set of engineered meganucleases derived from I-CreI, The substrate sequences are 5´-GTAC-3´, 5´-TTGA-3´, 5´-GAAA-3´, and binds to DNA substrates with varying efficiencies depending on whether the substrate has a central 5´-ACAC-3´. reported that the line had been disconnected.

[0008] Furthermore, Patent Document 2 (Publication '147) describes an engineered meganuclease that has a central sequence It was disclosed that different recognition sequences are cleaved with varying efficiencies depending on the recognition sequence. Targeting and cleavage of the recognition sequence of the engineered meganuclease based on the central sequence of the recognition sequence. It describes general rules for cleavage, as well as the efficiency with which such sequences can be cleaved. [Prior art documents] [Patent documents]

[0009]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

[0011] However, Patent Document 2 (Publication '147) describes a method for modifying meganuclease derived from I-CreI. and their activity and / or specificity for cleaving recognition sequences having specific central sequences. It was not stated whether the heterozygosity could be improved. and the subunits of I-CreI-derived meganuclease directly interact with the central sequence. Therefore, the present invention provides a method for detecting a recognition sequence having a specific central sequence. and specific cleavage sequences that allow for the optimization of I-CreI-derived meganucleases Identifying positions and residues advances the art. [Means for solving the problem]

[0012] One aspect is ACAA, ACAG, ACAT, ACGA, ACGC, ACGG, ACGT , ATAA, ATAG, ATAT, ATGA, ATGG, TTGG, GCAA, GCAT , GCGA, GCAG, TCAA, or TTAA. an engineered meganuclease that combines and cleaves a first subunit and a second subunit; subunits, wherein the first subunit and the second subunit each have SEQ ID NO: 1, each of which contains an amino acid sequence derived from positions 48, 50, 71, and 72 of SEQ ID NO: 1. The engineered methyltransferases contained substitutions at one or more positions corresponding to positions 72, 73, and 74. It is a ganucleases.

[0013] In some embodiments, the central sequence consists of ACAA.

[0014] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K or L residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K or L residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a C, R, T, K, or S residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) a G or R residue at a position corresponding to position 72 of SEQ ID NO: 1; and (e) an A or C residue at a position corresponding to position 73 of SEQ ID NO:1.

[0015] In some embodiments, the second subunit comprises one or more of the following residues: (a) a K, T, S, or A residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K, T, S, or A residue at a position corresponding to position 48 of SEQ ID NO: 1; (c) a C, R, E, K, or T residue at a position corresponding to position 50 of SEQ ID NO: 1; (d) a G or A residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) a V or at a position corresponding to position 73 of SEQ ID NO:1; an I residue; and (f) an S, T, or A residue at a position corresponding to position 74 of SEQ ID NO:1.

[0016] In some embodiments, the first subunit is any one of SEQ ID NOs: 11-33. In some embodiments, the residues correspond to residues 48, 50, 71, 72, and 73 of The second subunit is composed of residues 239 and 241 of any one of SEQ ID NOs: 11 to 33. , 262, 263, 264, and 265. In the present invention, the first subunit comprises one or more of the following residues: (a) 1 of SEQ ID NO: 1 (b) an A or G residue at a position corresponding to position 9 of SEQ ID NO: 1; (c) an A or G residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) a K or R residue at a position corresponding to position 139 of SEQ ID NO: 1; and (d) ) an S or G residue at a position corresponding to position 154 of SEQ ID NO:1.

[0017] In some embodiments, the second subunit comprises one or more of the following residues: (a) a G, A, or S residue at a position corresponding to position 19 of SEQ ID NO:1; (b) SEQ ID NO:1 (c) a Y or C residue at a position corresponding to position 80 of SEQ ID NO: 1; (d) a Q or E residue at a position corresponding to position 92 of SEQ ID NO: 1; (e) a Q or R residue at a position corresponding to position 92 of SEQ ID NO: 1; (f) an E or G residue at a position corresponding to position 117 of SEQ ID NO: 1; and (f) 139 of SEQ ID NO: 1. K or R residue at the position corresponding to position.

[0018] In some embodiments, the first subunit is any one of SEQ ID NOs: 11-33. The residues correspond to residues 19, 80, 139, and 154 of

[0019] In some embodiments, the second subunit is any one of SEQ ID NOs: 11-33. The residues correspond to residues 19, 66, 80, 92, 117, and 139 of

[0020] Another embodiment is a targeting moiety comprising a meganuclease recognition sequence comprising a central sequence consisting of ACAA. A method for cleaving double-stranded DNA at a target site, comprising: and contacting the engineered meganuclease described in the specification with the engineered meganuclease, The method comprises binding to and cleaving a recognition sequence.

[0021] In some embodiments, the central sequence consists of ACAG.

[0022] In some embodiments, the first subunit comprises one or more of the following residues: (a) an R residue at a position corresponding to position 50 of SEQ ID NO: 1; (b) an R residue at a position corresponding to position 71 of SEQ ID NO: 1 (c) a G or R residue at a position corresponding to position 72 of SEQ ID NO: 1; or a T residue; and (d) an A or C residue at a position corresponding to position 73 of SEQ ID NO:1.

[0023] In some embodiments, the second subunit comprises one or more of the following residues: (a) a C residue at a position corresponding to position 50 of SEQ ID NO: 1; (b) a C residue at a position corresponding to position 71 of SEQ ID NO: 1 (c) a G, S, or D residue at a position corresponding to position 72 of SEQ ID NO: 1; (d) a G residue; (d) an R residue at a position corresponding to position 73 of SEQ ID NO: 1; and optionally (e) a an R residue at the position following the position corresponding to position 73 in column number 1;

[0024] In some embodiments, the first subunit is any one of SEQ ID NOs: 36-43. The residues correspond to residues 50, 71, 72, and 73 of

[0025] In some embodiments, the first subunit is any one of SEQ ID NOs: 36-43. The residues correspond to residues 50, 71, 72, and 73 of

[0026] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A or G residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) a G residue at a position corresponding to position 54 of SEQ ID NO: 1; (c) an F, I, or L residue at a position corresponding to position 80 of SEQ ID NO: 1; a Q or E residue; and (d) an S or P residue at a position corresponding to position 158 of SEQ ID NO:1.

[0027] In some embodiments, the second subunit comprises one or more of the following residues: (a) a G, A, or S residue at a position corresponding to position 19 of SEQ ID NO:1; (b) SEQ ID NO:1 (c) a V or A residue at a position corresponding to position 66 of SEQ ID NO: 1; (d) a Y or H residue; (e) a Q residue at a position corresponding to position 80 of SEQ ID NO: 1; (f) an I or T residue at a position corresponding to position 81 of SEQ ID NO: 1; and (g) an I or T residue at a position corresponding to position 139 of SEQ ID NO: 1. K or R residue at the position.

[0028] In some embodiments, the first subunit is any one of SEQ ID NOs: 36-43. The residues correspond to residues 19, 54, 80, and 158 of

[0029] In some embodiments, the second subunit is any one of SEQ ID NOs: 36-43. The residues correspond to residues 19, 59, 66, 80, 81, and 139 of

[0030] In some embodiments, the second subunit is located at positions 73 and 74 of SEQ ID NO:1. It further contains R residues inserted between the corresponding positions.

[0031] Another embodiment is a targeting moiety comprising a meganuclease recognition sequence comprising a central sequence consisting of ACAG. A method for cleaving double-stranded DNA at a target site, comprising: and contacting the engineered meganuclease described in the specification with the engineered meganuclease, The method comprises binding to and cleaving a recognition sequence.

[0032] In some embodiments, the central sequence consists of ACAT.

[0033] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K, S, I, L, or N residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K, S, I, L, or N residue at a position corresponding to position 48 of SEQ ID NO: 1; (c) a Q, S, R, or K residue at a position corresponding to position 50 of SEQ ID NO: 1; (d) a G or R residue at a position corresponding to position 72 of SEQ ID NO: 1; is a T residue; and (e) an A or G residue at a position corresponding to position 73 of SEQ ID NO:1.

[0034] In some embodiments, the second subunit comprises one or more of the following residues: (a) an H, T, G, A, S, L, or K residue at a position corresponding to position 48 of SEQ ID NO:1; (b) an S, K, C, NR, G, or Q residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) an S, G, R, T, K, or E residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) a sequence (e) a T, K, A, S, R, H, G, or N residue at the position corresponding to position 72 in column number 1; an H, A, C, S, G, or R residue at a position corresponding to position 73 of SEQ ID NO: 1; and (f) an S, C, or A residue at a position corresponding to position 74 of SEQ ID NO:1;

[0035] In some embodiments, the first subunit is any one of SEQ ID NOs: 46-67. The residues correspond to residues 48, 50, 71, 72, and 73 of

[0036] In some embodiments, the second subunit is any one of SEQ ID NOs: 46-67. The residues correspond to residues 239, 241, 262, 263, 264, and 265 of

[0037] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A, G, or S residue at a position corresponding to position 19 of SEQ ID NO:1; (b) SEQ ID NO:1 (c) an F or I residue at a position corresponding to position 80 of SEQ ID NO: 1; Q or E residue; and (d) K, H, or R at a position corresponding to position 139 of SEQ ID NO:1. residue.

[0038] In some embodiments, the second subunit comprises one or more of the following residues: (a) an A, G, or S residue at a position corresponding to position 19 of SEQ ID NO:1; (b) SEQ ID NO:1 (c) a Q or E residue at a position corresponding to position 81 of SEQ ID NO: 1; (d) a P or H residue at a position corresponding to position 83 of SEQ ID NO: 1; (e) a P or H residue at a position corresponding to position 83 of SEQ ID NO: 1; (f) an E or G residue at a position corresponding to position 117 of SEQ ID NO: 1; and (f) 139 of SEQ ID NO: 1. K, R, T, or H residue at the position corresponding to position .

[0039] In some embodiments, the first subunit is any one of SEQ ID NOs: 46-67. The residues correspond to residues 19, 54, 80, and 139 of

[0040] In some embodiments, the second subunit is any one of SEQ ID NOs: 46-67. The residues correspond to residues 19, 80, 81, 83, 117, and 139 of

[0041] Another embodiment is a targeting moiety comprising a meganuclease recognition sequence comprising a central sequence consisting of ACAT. A method for cleaving double-stranded DNA at a target site, comprising: and contacting the engineered meganuclease described in the specification with the engineered meganuclease, The method comprises binding to and cleaving a recognition sequence.

[0042] In some embodiments, the central sequence consists of ACGA.

[0043] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K residue at a position corresponding to position 50 of SEQ ID NO: 1 (c) a V, R, T, W, or A residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) a G or P residue at a position corresponding to position 72 of SEQ ID NO: 1; and e) An A residue at a position corresponding to position 73 of SEQ ID NO:1.

[0044] In some embodiments, the second subunit comprises one or more of the following residues: (a) a K, H, T, A, G, or Q residue at a position corresponding to position 48 of SEQ ID NO: 1; ) an R, S, C, I, V, or G residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) the sequence (d) a G residue at a position corresponding to position 71 of SEQ ID NO: 1; (e) an R residue at a position corresponding to position 72 of SEQ ID NO: 1 or H residue; (e) an I or V residue at a position corresponding to position 73 of SEQ ID NO: 1; and (f ) an S or A residue at a position corresponding to position 74 of SEQ ID NO:1.

[0045] In some embodiments, the first subunit is any one of SEQ ID NOs: 70-89. The residues correspond to residues 48, 50, 71, 72, and 73 of

[0046] In some embodiments, the second subunit is any one of SEQ ID NOs: 70-89. The residues correspond to residues 239, 241, 262, 263, 264, and 265 of

[0047] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A, G, or S residue at a position corresponding to position 19 of SEQ ID NO:1; (b) SEQ ID NO:1 and (c) a Q or E residue at a position corresponding to position 80 of SEQ ID NO: 1; and R residue at the position.

[0048] In some embodiments, the second subunit comprises one or more of the following residues: (a) an A or G residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) an A or G residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) a Q or E residue at a position corresponding to position 139 of SEQ ID NO: 1; and to K or R residues.

[0049] In some embodiments, the first subunit is any one of SEQ ID NOs: 70-89. It contains residues corresponding to residues 19, 80, and 139 of

[0050] In some embodiments, the second subunit is any one of SEQ ID NOs: 70-89. It contains residues corresponding to residues 19, 80, and 139 of

[0051] Another embodiment is a targeting moiety comprising a meganuclease recognition sequence comprising a central sequence consisting of ACGA. A method for cleaving double-stranded DNA at a target site, comprising: and contacting the engineered meganuclease described in the specification with the engineered meganuclease, The method comprises binding to and cleaving a recognition sequence.

[0052] In some embodiments, the central sequence consists of ACGC.

[0053] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K, H, Q, L, A, or S residue at a position corresponding to position 48 of SEQ ID NO: 1; ) a Q, R, K, S, T, or C residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) the sequence (d) a G, R, or A residue at a position corresponding to position 71 of SEQ ID NO: 1; (e) an R, P, or H residue at a position corresponding to position 73 of SEQ ID NO:1; and A residue.

[0054] In some embodiments, the second subunit comprises one or more of the following residues: (a) an H, K, L, A, S, or N residue at a position corresponding to position 48 of SEQ ID NO: 1; ) an S, E, K, I, N, or V residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) the sequence (d) an S, G, K, A, or R residue at a position corresponding to position 71 of SEQ ID NO: 1; (e) a T, R, A, S, H, or G residue at a position corresponding to position 2 of SEQ ID NO: 1; an H, T, V, I, or C residue at the corresponding position; and (f) corresponding to position 74 of SEQ ID NO:1. S, A, or T residue at this position.

[0055] In some embodiments, the first subunit is any one of SEQ ID NOs: 92-118. The residues corresponding to residues 48, 50, 71, 72, and 73 are included.

[0056] In some embodiments, the second subunit is selected from the group consisting of any one of SEQ ID NOs: 92-118. Contains residues corresponding to residues 239, 241, 262, 263, 264, and 265 .

[0057] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A, G, or S residue at a position corresponding to position 19 of SEQ ID NO:1; and (b) the sequence Q or E residue at the position corresponding to position 80 of number 1.

[0058] In some embodiments, the second subunit comprises one or more of the following residues: (a) an A or G residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) an A or G residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) a Q or E residue at a position corresponding to position 87 of SEQ ID NO: 1; is an L residue; and (d) K, R, N, H, or a residue at a position corresponding to position 139 of SEQ ID NO:1. A residue.

[0059] In some embodiments, the first subunit is any one of SEQ ID NOs: 92-118. It contains residues corresponding to residues 19 and 80.

[0060] In some embodiments, the second subunit is selected from the group consisting of any one of SEQ ID NOs: 92-118. The residues corresponding to residues 19, 80, 87, and 139 are included.

[0061] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of ACGC. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0062] In some embodiments, the central sequence consists of ACGG.

[0063] In some embodiments, the first subunit comprises one or more of the following residues: (a) an R or K residue at a position corresponding to position 50 of SEQ ID NO: 1; (b) an R or K residue at a position corresponding to position 72 of SEQ ID NO: 1; and (c) an R residue at a position corresponding to position 73 of SEQ ID NO:1.

[0064] In some embodiments, the second subunit comprises one or more of the following residues: (a) a K residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K residue at a position corresponding to position 50 of SEQ ID NO: 1 (c) an R or P residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) a D residue at a position corresponding to position 71 of SEQ ID NO: 1; (e) a G residue at a position corresponding to position 72 of SEQ ID NO:1; and (f) a G residue at a position corresponding to position 73 of SEQ ID NO:1. R or G residue at the position.

[0065] In some embodiments, the first subunit is any of SEQ ID NOs: 121-135. One contains residues corresponding to residues 50, 72, and 73.

[0066] In some embodiments, the second subunit is any of SEQ ID NOs: 121-135. One contains residues corresponding to residues 239, 241, 262, 263, and 264.

[0067] In some embodiments, the first subunit comprises one or more of the following residues: (a) an F or L residue at a position corresponding to position 54 of SEQ ID NO:1; and (b) SEQ ID NO:1 A Q residue at the position corresponding to position 80 of

[0068] In some embodiments, the second subunit comprises one or more of the following residues: (a) an A residue at a position corresponding to position 19 of SEQ ID NO:1; and (b) an A residue at position 80 of SEQ ID NO:1. Q residue at the position corresponding to

[0069] In some embodiments, the first subunit is any of SEQ ID NOs: 121-135. One contains residues corresponding to residues 54 and 80.

[0070] In some embodiments, the second subunit is any of SEQ ID NOs: 121-135. One contains residues corresponding to residues 19 and 80.

[0071] In some embodiments, the second subunit is located at positions 73 and 74 of SEQ ID NO:1. It further contains R residues inserted between the corresponding positions.

[0072] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of ACGG. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0073] In some embodiments, the central sequence consists of ACGT.

[0074] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K, L, S, or H residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K, L, S, or H residue at a position corresponding to position 48 of SEQ ID NO: 1; (c) a Q, R, C, S, or V residue at a position corresponding to position 50 of SEQ ID NO: 1; (d) a G residue at a position corresponding to position 72 of SEQ ID NO: 1; and (e) an A residue at a position corresponding to position 73 of SEQ ID NO:1.

[0075] In some embodiments, the second subunit comprises one or more of the following residues: (a) an H, K, L, or S residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) an H, K, L, or S residue at a position corresponding to position 48 of SEQ ID NO: 1; (c) an S, C, Q, E, or A residue at a position corresponding to position 50 of SEQ ID NO: 1; (d) an S, P, G, T, A, R, or N residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) a T, R, K, or A residue at a position corresponding to position 73 of SEQ ID NO: 1; and (f) an H, C, A, or S residue at a position corresponding to position 74 of SEQ ID NO:1. , or T residue.

[0076] In some embodiments, the first subunit is any of SEQ ID NOs: 138-156. One contains residues corresponding to residues 48, 50, 71, 72, and 73.

[0077] In some embodiments, the second subunit is any of SEQ ID NOs: 138-156. Contains residues corresponding to residues 239, 241, 262, 263, 264, and 265 nothing.

[0078] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A or G residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) an A or G residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) a Q or E residue at a position corresponding to position 139 of SEQ ID NO: 1; and to K or R residues.

[0079] In some embodiments, the second subunit comprises one or more of the following residues: (a) an A or G residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) an A or G residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) a Q or E residue at a position corresponding to position 85 of SEQ ID NO: 1; is a Y residue; and (d) a K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0080] In some embodiments, the first subunit is any of SEQ ID NOs: 138-156. One contains residues corresponding to residues 19, 80, and 139.

[0081] In some embodiments, the second subunit is any of SEQ ID NOs: 138-156. One contains residues corresponding to residues 19, 80, 85, and 139.

[0082] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of ACGT. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0083] In some embodiments, the central sequence consists of ATAA.

[0084] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K, A, H, S, L, or Q residue at a position corresponding to position 48 of SEQ ID NO: 1; ) a Q, T, R, I, G, K, D, C, or V residue at a position corresponding to position 50 of SEQ ID NO:1 (c) a G, K, S, H, or N residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) a G, K, S, H, or N residue at a position corresponding to position 71 of SEQ ID NO: 1; an R, A, G, Q, H, L, or S residue at the position corresponding to position 72 in sequence number 1; (e) sequence (f) an A, T, or C residue at a position corresponding to position 73 of SEQ ID NO: 1; and (f) 74 of SEQ ID NO: 1. S or A residue at the position corresponding to position.

[0085] In some embodiments, the second subunit comprises one or more of the following residues: (a) an S, T, A, K, or N residue at a position corresponding to position 48 of SEQ ID NO: 1; an R, K, E, A, C, or T residue at a position corresponding to position 50 of sequence number 1; (c) SEQ ID NO: (d) an S, G, K, or R residue at a position corresponding to position 71 of SEQ ID NO:1; (e) a T, R, Q, G, A, Y, S, N, or K residue at the corresponding position of SEQ ID NO: 1; (f) an I, C, or V residue at a position corresponding to position 74 of SEQ ID NO:1; and S, A, or T residue at position.

[0086] In some embodiments, the first subunit is any of SEQ ID NOs: 159-183. One contains residues corresponding to residues 48, 50, 71, 72, 73, and 74.

[0087] In some embodiments, the second subunit is any of SEQ ID NOs: 159-183. Contains residues corresponding to residues 239, 241, 262, 263, 264, and 265 nothing.

[0088] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A, G, or S residue at a position corresponding to position 19 of SEQ ID NO:1; (b) SEQ ID NO:1 (c) a Q or E residue at a position corresponding to position 100 of SEQ ID NO: 1; (d) a K or E residue at a position corresponding to position 139 of SEQ ID NO: 1; (e (f) an S or G residue at a position corresponding to position 154 of SEQ ID NO: 1; and (g) an S or G residue at a position corresponding to position 154 of SEQ ID NO: 1. S or A residue at the position corresponding to position 72.

[0089] In some embodiments, the second subunit comprises one or more of the following residues: (a) a G, S, or A residue at a position corresponding to position 19 of SEQ ID NO:1; (b) SEQ ID NO:1 (c) a V or A residue at a position corresponding to position 78 of SEQ ID NO: 1; (d) an L residue at a position corresponding to position 79 of SEQ ID NO: 1; (e) an S residue at position 80 of SEQ ID NO: 1 (f) a Q or E residue at a position corresponding to position 118 of SEQ ID NO: 1; is an F residue; and (g) a K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0090] In some embodiments, the first subunit is any of SEQ ID NOs: 159-183. One containing residues corresponding to residues 19, 80, 100, 139, 154, and 172.

[0091] In some embodiments, the second subunit is any of SEQ ID NOs: 159-183. Contains residues corresponding to residues 19, 59, 78, 79, 80, 118, and 139 .

[0092] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of ATAA. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0093] In some embodiments, the central sequence consists of ATAG.

[0094] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K or H residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K or H residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) an R residue at a position corresponding to position 71 of SEQ ID NO: 1; an H residue; (d) an R, G, S, A, P, or Q residue at a position corresponding to position 72 of SEQ ID NO:1; and (e) an A or C residue at a position corresponding to position 73 of SEQ ID NO:1.

[0095] In some embodiments, the second subunit comprises one or more of the following residues: (a) a C or R residue at a position corresponding to position 50 of SEQ ID NO: 1; (b) a C or R residue at a position corresponding to position 72 of SEQ ID NO: 1; (c) a G or S residue at a position corresponding to position 73 of SEQ ID NO:1; and R residue.

[0096] In some embodiments, the first subunit is any of SEQ ID NOs: 186-199. One contains residues corresponding to residues 48, 50, 71, 72, and 73.

[0097] In some embodiments, the second subunit is any of SEQ ID NOs: 186-199. One contains residues corresponding to residues 241, 263, and 264.

[0098] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A or G residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) an A or G residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) a Q or E residue at a position corresponding to position 139 of SEQ ID NO: 1; and to K or R residues.

[0099] In some embodiments, the second subunit comprises one or more of the following residues: (a) a G or A residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) a G or A residue at a position corresponding to position 36 of SEQ ID NO: 1; (c) a K or R residue at a position corresponding to position 59 of SEQ ID NO: 1; (d) a Q residue at a position corresponding to position 80 of SEQ ID NO: 1; and (e) SEQ ID NO: 1 A K or R residue at the position corresponding to position 139 of

[0100] In some embodiments, the first subunit is any of SEQ ID NOs: 186-199. One contains residues corresponding to residues 19, 80, and 139.

[0101] In some embodiments, the second subunit is any of SEQ ID NOs: 186-199. One containing residues corresponding to residues 19, 36, 59, 80, and 139.

[0102] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of ATAG. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0103] In some embodiments, the central sequence consists of ATAT.

[0104] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K, H, C, A, S, D, or T residue at a position corresponding to position 48 of SEQ ID NO:1; (b) a Q, N, C, R, K, S, T, or V residue at a position corresponding to position 50 of SEQ ID NO:1 (c) a G, H, or I residue at a position corresponding to position 71 of SEQ ID NO:1; (d) SEQ ID NO:1 an R, A, N, or Q residue at a position corresponding to position 72 of SEQ ID NO: 1; and (e) an R, A, N, or Q residue at a position corresponding to position 73 of SEQ ID NO: 1. A, C, or S residue at the corresponding position.

[0105] In some embodiments, the second subunit comprises one or more of the following residues: (a) an H, K, A, S, R, or T residue at a position corresponding to position 48 of SEQ ID NO: 1; ) an S, C, K, R, Q, or N residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) the sequence (d) an S, K, E, I, G, or R residue at a position corresponding to position 71 of SEQ ID NO: 1; (e) a T, A, R, S, K, G, or N residue at a position corresponding to position 72 of SEQ ID NO: 1; (f) an H, C, A, S, or G residue at a position corresponding to position 73 of SEQ ID NO: 1; and S, C, or A residue at the position corresponding to position.

[0106] In some embodiments, the first subunit is any of SEQ ID NOs: 202-219. One contains residues corresponding to residues 48, 50, 71, 72, and 73.

[0107] In some embodiments, the second subunit is any of SEQ ID NOs: 202-219. Contains residues corresponding to residues 239, 241, 262, 263, 264, and 265 nothing.

[0108] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A or G residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) an A or G residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) a Q or E residue at a position corresponding to position 139 of SEQ ID NO: 1; and to K, R, or S residues.

[0109] In some embodiments, the second subunit comprises one or more of the following residues: (a) a G or A residue at a position corresponding to position 19 of SEQ ID NO:1; (b) a G or A residue at a position corresponding to position 59 of SEQ ID NO:1; (c) a V or A residue at a position corresponding to position 80 of SEQ ID NO: 1; or K residue; and (d) K, R, P, or is an N residue.

[0110] In some embodiments, the first subunit is any of SEQ ID NOs: 202-219. One contains residues corresponding to residues 19, 80, and 139.

[0111] In some embodiments, the second subunit is any of SEQ ID NOs: 202-219. One contains residues corresponding to residues 19, 59, 80, and 139.

[0112] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of ATAT. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0113] In some embodiments, the central sequence consists of ATGA.

[0114] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K, A, H, or L residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K, A, H, or L residue at a position corresponding to position 48 of SEQ ID NO: 1; (c) an R, T, E, S, C, or V residue at a position corresponding to position 50 of SEQ ID NO: 1; (d) an R, T, S, A, or K residue at a position corresponding to position 72 of SEQ ID NO: 1; and A or S residue at the position corresponding to position.

[0115] In some embodiments, the second subunit comprises one or more of the following residues: (a) an H, K, R, A, or S residue at a position corresponding to position 48 of SEQ ID NO: 1; an S, I, R, C, A, or Q residue at a position corresponding to position 50 of sequence number 1; (c) SEQ ID NO: (d) an R or H residue at a position corresponding to position 73 of SEQ ID NO: 1; and (e) an S, A, or T at a position corresponding to position 74 of SEQ ID NO:1. residue.

[0116] In some embodiments, the first subunit is any of SEQ ID NOs: 222-243. One contains residues corresponding to residues 48, 50, 72, and 73.

[0117] In some embodiments, the second subunit is any of SEQ ID NOs: 222-243. One contains residues corresponding to residues 239, 241, 263, 264, and 265.

[0118] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A, G, or S residue at a position corresponding to position 19 of SEQ ID NO:1; (b) SEQ ID NO:1 (c) a Q or E residue at a position corresponding to position 87 of SEQ ID NO: 1; (d) a Q or R residue at a position corresponding to position 92 of SEQ ID NO: 1; and e) A K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0119] In some embodiments, the second subunit comprises one or more of the following residues: (a) a G, A, or S residue at a position corresponding to position 19 of SEQ ID NO:1; (b) SEQ ID NO:1 (c) a V or A residue at a position corresponding to position 80 of SEQ ID NO: 1; a Q or E residue; and (d) a K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0120] In some embodiments, the first subunit is any of SEQ ID NOs: 222-243. One contains residues corresponding to residues 19, 80, 87, 92, and 139.

[0121] In some embodiments, the second subunit is any of SEQ ID NOs: 222-243. One contains residues corresponding to residues 19, 59, 80, and 139.

[0122] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of ATGA. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0123] In some embodiments, the central sequence consists of ATGG.

[0124] In some embodiments, the first subunit comprises one or more of the following residues: (a) an R residue at a position corresponding to position 50 of SEQ ID NO: 1; (b) an R residue at a position corresponding to position 71 of SEQ ID NO: 1 (c) a G or S residue at a position corresponding to position 72 of SEQ ID NO: 1; (d) an A or C residue at a position corresponding to position 73 of SEQ ID NO:1; and (e) SEQ ID NO:1 S or C residue at the position corresponding to position 74 of

[0125] In some embodiments, the second subunit comprises one or more of the following residues: (a) an R residue at a position corresponding to position 50 of SEQ ID NO: 1; (b) an R residue at a position corresponding to position 71 of SEQ ID NO: 1 (c) a D or G residue at a position corresponding to position 72 of SEQ ID NO: 1; and (d) an R residue at a position corresponding to position 73 of SEQ ID NO:1.

[0126] In some embodiments, the first subunit is any of SEQ ID NOs: 246-247. One contains residues corresponding to residues 50, 71, 72, 73, and 74.

[0127] In some embodiments, the second subunit is any of SEQ ID NOs: 246-247. One contains residues corresponding to residues 239, 241, 262, 263, and 264.

[0128] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A or G residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) an A or G residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) an E or Q residue at a position corresponding to position 82 of SEQ ID NO: 1; is a K residue; and (d) an R or K residue at a position corresponding to position 139 of SEQ ID NO:1.

[0129] In some embodiments, the second subunit comprises one or more of the following residues: (a) an A or G residue at a position corresponding to position 19 of SEQ ID NO:1; (b) an A or G residue at a position corresponding to position 77 of SEQ ID NO:1; and (c) a Q or N residue at a position corresponding to position 80 of SEQ ID NO:1. R residue.

[0130] In some embodiments, the first subunit is any of SEQ ID NOs: 246-247. One contains residues corresponding to residues 19, 80, 82, and 139.

[0131] In some embodiments, the second subunit is any of SEQ ID NOs: 246-247. One contains residues corresponding to residues 19, 77, and 80.

[0132] In some embodiments, the second subunit is located at positions 73 and 74 of SEQ ID NO:1. It further contains R residues inserted between the corresponding positions.

[0133] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of ATGG. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0134] In some embodiments, the central sequence consists of TTGG.

[0135] In some embodiments, the first subunit comprises one or more of the following residues: (a) an R residue at a position corresponding to position 50 of SEQ ID NO: 1; (b) an R residue at a position corresponding to position 71 of SEQ ID NO: 1 (c) an S residue at a position corresponding to position 72 of SEQ ID NO: 1; and (d) a G residue at a position corresponding to position 73 of SEQ ID NO: 1. R residue at the position corresponding to position 73 in column no.

[0136] In some embodiments, the second subunit comprises one or more of the following residues: (a) a K or S residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K or S residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a C, T, E, K, or R residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) a G or K residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) an H, A, or S residue; (f) an I or V residue at a position corresponding to position 73 of SEQ ID NO: 1; and and (f) an S or A residue at a position corresponding to position 74 of SEQ ID NO:1.

[0137] In some embodiments, the first subunit is any of SEQ ID NOs: 250-266. One contains residues corresponding to residues 50, 71, 72, and 73.

[0138] In some embodiments, the second subunit is any of SEQ ID NOs: 250-266. Contains residues corresponding to residues 239, 241, 262, 263, 264, and 265 nothing.

[0139] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A or G residue at a position corresponding to position 19 of SEQ ID NO:1; and (b) SEQ ID NO:1 A Q residue at the position corresponding to position 80 of

[0140] In some embodiments, the second subunit comprises one or more of the following residues: (a) a G or A residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) a G or A residue at position 19 of SEQ ID NO: 1; (c) a Y or H residue at a position corresponding to position 66 of SEQ ID NO: 1; (d) a Q residue at a position corresponding to position 80 of SEQ ID NO: 1; (d) an H or R residue at a position corresponding to position 85 of SEQ ID NO: 1; and (e) a residue of SEQ ID NO: a K or R residue at the position corresponding to position 139 of 1;

[0141] In some embodiments, the first subunit is any of SEQ ID NOs: 250-266. One contains residues corresponding to residues 19 and 80.

[0142] In some embodiments, the second subunit is any of SEQ ID NOs: 250-266. One contains residues corresponding to residues 19, 66, 80, 85, and 139.

[0143] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of TTGG. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0144] In some embodiments, the central sequence consists of GCAA.

[0145] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K or H residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K or H residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) an R, C, K, T, or L residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) a G, N, T, R, S, or H residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) an R, P, S, N, Q, G, A, T, M, or V residue at position 73 of SEQ ID NO: 1; (f) a T or V residue at a position corresponding to position 74 of SEQ ID NO: 1; and , or A residue.

[0146] In some embodiments, the second subunit comprises one or more of the following residues: (a) an S, A, K, or T residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) an SEQ ID NO: (c) an R, C, T, K, or E residue at a position corresponding to position 50 of SEQ ID NO: 1; (d) a G, R, A, or H residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) a T, G, S, A, E, N, K, H, R, C, or Y residue at position 73 of SEQ ID NO: 1; (f) a C, V, or I residue at a position corresponding to position 74 of SEQ ID NO: 1; and S, A, or T residue at position.

[0147] In some embodiments, the first subunit is any of SEQ ID NOs: 269-291. One contains residues corresponding to residues 48, 50, 71, 72, 73, and 74.

[0148] In some embodiments, the second subunit is any of SEQ ID NOs: 269-291. Contains residues corresponding to residues 239, 241, 262, 263, 264, and 265 nothing.

[0149] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A, G, or S residue at a position corresponding to position 19 of SEQ ID NO:1; (b) SEQ ID NO:1 and (c) a Q or E residue at a position corresponding to position 80 of SEQ ID NO: 1; and K or R residue at the position.

[0150] In some embodiments, the second subunit comprises one or more of the following residues: (a) a G or A residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) a G or A residue at a position corresponding to position 31 of SEQ ID NO: 1; (c) a Q or P residue at a position corresponding to position 80 of SEQ ID NO: 1; is an E residue; and (d) a K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0151] In some embodiments, the first subunit is any of SEQ ID NOs: 269-291. One contains residues corresponding to residues 19, 80, and 139.

[0152] In some embodiments, the second subunit is any of SEQ ID NOs: 269-291. One contains residues corresponding to residues 19, 31, 80, and 139.

[0153] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of GCAA. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0154] In some embodiments, the central sequence consists of GCAT.

[0155] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K, A, H, or R residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K, A, H, or R residue at a position corresponding to position 48 of SEQ ID NO: 1; (c) a Q, V, R, K, or S residue at a position corresponding to position 50 of SEQ ID NO: 1; (d) a G, A, H, R, T, N, or S residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) an R, T, G, S, Q, N, or A residue at a position corresponding to position 73 of SEQ ID NO: 1; an A, T, V, or C residue at the corresponding position; and (f) a residue corresponding to position 74 of SEQ ID NO:1. S or A residue at position.

[0156] In some embodiments, the second subunit comprises one or more of the following residues: (a) an H, A, K, T, L, or I residue at a position corresponding to position 48 of SEQ ID NO: 1; ) an S, R, K, Q, H, or V residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) the sequence (d) an S, K, R, A, G, T, H, or Y residue at a position corresponding to position 71 of sequence number 1; A T, A, G, N, S, R, H, Q, or K residue at the position corresponding to position 72 in column number 1; e) an H, C, G, S, or A residue at a position corresponding to position 73 of SEQ ID NO: 1; and (f) an S, C, or A residue at a position corresponding to position 74 of SEQ ID NO:1;

[0157] In some embodiments, the first subunit is any of SEQ ID NOs: 294-313. One contains residues corresponding to residues 48, 50, 71, 72, 73, and 74.

[0158] In some embodiments, the second subunit is any of SEQ ID NOs: 294-313. Contains residues corresponding to residues 239, 241, 262, 263, 264, and 265 nothing.

[0159] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A or G residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) an A or G residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) a Q or E residue at a position corresponding to position 139 of SEQ ID NO: 1; an H, or R residue; and (d) a T or I residue at a position corresponding to position 143 of SEQ ID NO:1. .

[0160] In some embodiments, the second subunit comprises one or more of the following residues: (a) a G, S, or A residue at a position corresponding to position 19 of SEQ ID NO:1; (b) SEQ ID NO:1 (c) a Q or E residue at a position corresponding to position 125 of SEQ ID NO: 1; and (d) a V or A residue at a position corresponding to position 139 of SEQ ID NO:1; H residue.

[0161] In some embodiments, the first subunit is any of SEQ ID NOs: 294-313. One contains residues corresponding to residues 19, 80, 139, and 143.

[0162] In some embodiments, the second subunit is any of SEQ ID NOs: 294-313. One contains residues corresponding to residues 19, 80, 125, and 139.

[0163] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of GCAT. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0164] In some embodiments, the central sequence consists of GCGA.

[0165] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K or R residue at a position corresponding to position 50 of SEQ ID NO: 1; (b) a K or R residue at a position corresponding to position 71 of SEQ ID NO: 1; (c) a G, R, S, A, or N residue at a position corresponding to position 72 of SEQ ID NO: 1; (d) an R, N, G, A, or Q residue at a position corresponding to position 73 of SEQ ID NO: 1; , T, or I residue; and (e) an S or A residue at a position corresponding to position 74 of SEQ ID NO:1. .

[0166] In some embodiments, the second subunit comprises one or more of the following residues: (a) a K, T, S, A, or Q residue at a position corresponding to position 48 of SEQ ID NO: 1; (c) a C or R residue at the position corresponding to position 72 of SEQ ID NO: 1; (d) an R residue at a position corresponding to position 73 of SEQ ID NO: 1; and e) An S or A residue at the position corresponding to position 74 of SEQ ID NO:1.

[0167] In some embodiments, the first subunit is any of SEQ ID NOs: 316-325. One contains residues corresponding to residues 50, 71, 72, 73, and 74.

[0168] In some embodiments, the second subunit is any of SEQ ID NOs: 316-325. One contains residues corresponding to residues 239, 241, 263, 264, and 265.

[0169] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A, G, or S residue at a position corresponding to position 19 of SEQ ID NO: 1; and (b) a Q or E residue at the position corresponding to position 80 of No. 1.

[0170] In some embodiments, the second subunit comprises one or more of the following residues: (a) a G, S, or A residue at a position corresponding to position 19 of SEQ ID NO:1; (b) SEQ ID NO:1 and (c) a Q or E residue at a position corresponding to position 80 of SEQ ID NO: 1; and R residue at the position.

[0171] In some embodiments, the first subunit is any of SEQ ID NOs: 316-325. One contains residues corresponding to residues 19 and 80.

[0172] In some embodiments, the second subunit is any of SEQ ID NOs: 316-325. One contains residues corresponding to residues 19, 80, and 139.

[0173] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of GCGA. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0174] In some embodiments, the central sequence consists of GCAG.

[0175] In some embodiments, the first subunit comprises one or more of the following residues: (a) an R residue at a position corresponding to position 50 of SEQ ID NO: 1; (b) an R residue at a position corresponding to position 71 of SEQ ID NO: 1 (c) an S residue at a position corresponding to position 72 of SEQ ID NO: 1; and (d) a G residue at a position corresponding to position 73 of SEQ ID NO: 1. an R residue at the position corresponding to position 73 in column number 1;

[0176] In some embodiments, the second subunit comprises one or more of the following residues: (a) a K or H residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K or H residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a Q or R residue at a position corresponding to position 72 of SEQ ID NO: 1; is an R residue; and (d) a V or T residue at a position corresponding to position 73 of SEQ ID NO: 1; and

[0177] In some embodiments, the first subunit is any of SEQ ID NOs: 328-330. One contains residues corresponding to residues 50, 71, 72, 73, and 74.

[0178] In some embodiments, the second subunit is any of SEQ ID NOs: 328-330. One contains residues corresponding to residues 239, 241, 263, 264, and 265.

[0179] In some embodiments, the second subunit is located at a position corresponding to position 80 of SEQ ID NO:1. contains E residues.

[0180] In some embodiments, the second subunit is any of SEQ ID NOs: 328-330. Contains one residue corresponding to residue 80.

[0181] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of GCAG. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0182] In some embodiments, the central sequence consists of TCAA.

[0183] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K or S residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K or S residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) an R, T, or C residue at a position corresponding to position 71 of SEQ ID NO: 1; G, R, or T residue; and (d) R, S, P, or T, or G residue.

[0184] In some embodiments, the second subunit comprises one or more of the following residues: (a) an S or K residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) an S or K residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a K, R, C, or E residue at a position corresponding to position 72 of SEQ ID NO: 1; (d) an R, Q, N, or S residue at a position corresponding to position 73 of SEQ ID NO: 1; and and (e) an S or A residue at a position corresponding to position 74 of SEQ ID NO:1.

[0185] In some embodiments, the first subunit is any of SEQ ID NOs: 333-340. One contains residues corresponding to residues 48, 50, 71, and 72.

[0186] In some embodiments, the second subunit is any of SEQ ID NOs: 333-340. One contains residues corresponding to residues 239, 241, 263, 264, and 265.

[0187] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A or S residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) an A or S residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) a Q or E residue at a position corresponding to position 139 of SEQ ID NO: 1; and to K or R residues.

[0188] In some embodiments, the second subunit comprises one or more of the following residues: (a) a G or S residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) a G or S residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) a Q or E residue at a position corresponding to position 139 of SEQ ID NO: 1; and to R residue.

[0189] In some embodiments, the first subunit is any of SEQ ID NOs: 333-340. One contains residues corresponding to residues 19, 80, and 139.

[0190] In some embodiments, the second subunit is any of SEQ ID NOs: 333-340. One contains residues corresponding to residues 19, 80, and 139.

[0191] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of TCAA. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0192] In some embodiments, the central sequence consists of TTAA. (a) a K, N, S, or R residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K, N, S, or R residue at a position corresponding to position 48 of SEQ ID NO: 1; (c) an R, V, K, or S residue at a position corresponding to position 50 of SEQ ID NO: 1; (d) a G, R, N, S, or A residue at the corresponding position; (e) a G, R, N, S, or A residue at the corresponding position; (e) an R, T, S, N, D, Q, K, or A residue at position 74 of SEQ ID NO:1; and S or A residues at the corresponding positions.

[0193] In some embodiments, the second subunit comprises one or more of the following residues: (a) a K, S, A, or T residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K, S, A, or T residue at a position corresponding to position 48 of SEQ ID NO: 1; (c) a C, K, R, T, or E residue at a position corresponding to position 50 of SEQ ID NO: 1; (d) a C, K, R, T, or E residue at a position corresponding to position 72 of SEQ ID NO: 1; (d) a T, K, R, A, S, or Q residue at a position corresponding to position 73 of SEQ ID NO: 1; (e) an I or V residue at a position corresponding to position 74 of SEQ ID NO:1; is the A residue.

[0194] In some embodiments, the first subunit is any of SEQ ID NOs: 343-357. One contains residues corresponding to residues 48, 50, 71, 72, and 74.

[0195] In some embodiments, the second subunit is any of SEQ ID NOs: 343-357. One contains residues corresponding to residues 239, 241, 263, 264, and 265.

[0196] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A, G, or S residue at a position corresponding to position 19 of SEQ ID NO:1; (b) SEQ ID NO:1 and (c) a Q or E residue at a position corresponding to position 80 of SEQ ID NO: 1; and K or R residue at the position.

[0197] In some embodiments, the second subunit comprises one or more of the following residues: (a) a G, A, or S residue at a position corresponding to position 19 of SEQ ID NO:1; (b) SEQ ID NO:1 (c) a Y or H residue at a position corresponding to position 80 of SEQ ID NO: 1; and (d) an R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0198] In some embodiments, the first subunit is any of SEQ ID NOs: 343-357. One contains residues corresponding to residues 19, 80, and 139.

[0199] In some embodiments, the second subunit is any of SEQ ID NOs: 343-357. One contains residues corresponding to residues 19, 66, 80, and 139.

[0200] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of TTAA. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0201] Another embodiment is ACAA, ACAG, ACAT, ACGA, ACGC, ACGG, ACG T, ATAA, ATAG, ATAT, ATGA, ATGG, TTGG, GCAA, GCA Recognition sequences containing a central sequence consisting of T, GCGA, GCAG, TCAA, or TTAA The first subunit and the second subunit bind to and cleave the The unit and the second subunit each contain an amino acid sequence derived from SEQ ID NO: 1. A method for enhancing the cleavage activity of an engineered meganuclease, comprising: The first and second subunits are respectively located at positions 48, 50, 71, and 72 of SEQ ID NO: 1. Modified nucleic acids at one or more positions corresponding to positions 72, 73, and 74. The enzyme has enhanced cleavage activity when compared to a control engineered meganuclease. The method includes:

[0202] In some embodiments of this method, the central sequence consists of ACAA.

[0203] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K or L residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a K or S residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) a G or R residue at a position corresponding to position 71 of SEQ ID NO: 1; (e) an R or Q residue at a position corresponding to position 72 of SEQ ID NO:1; and (f) an R or Q residue at position 73 of SEQ ID NO:1. A or C residue at the corresponding position.

[0204] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, T, S, or A residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a G or A residue at a position corresponding to position 71 of SEQ ID NO:1; (d) a T, R, S, P, N, G, or A residue at a position corresponding to position 72 of SEQ ID NO:1 (e) a V or I residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) SEQ ID NO:1 an S, T, or A residue at a position corresponding to position 74 of

[0205] In some embodiments of this method, the first subunit is any of SEQ ID NOs: 8-30. modified to contain residues corresponding to one of residues 48, 50, 71, 72, and 73. can be.

[0206] In some embodiments of this method, the second subunit is any of SEQ ID NOs: 8-30. Residues corresponding to one of residues 239, 241, 262, 263, 264, and 265 is modified to include

[0207] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or G residue at the corresponding position; (b) a Q or E at the position corresponding to position 80 of SEQ ID NO:1 (c) a K or R residue at a position corresponding to position 139 of SEQ ID NO: 1; and (d) the sequence S or G residue at the position corresponding to position 154 of no. 1.

[0208] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) a G, A, or S residue at the corresponding position; (c) a Y or S residue at the position corresponding to position 66 of SEQ ID NO: 1; or a C residue; (c) a Q or E residue at a position corresponding to position 80 of SEQ ID NO: 1; (d) a Q or E residue at a position corresponding to position 80 of SEQ ID NO: 1; (e) a Q or R residue at a position corresponding to position 92 of SEQ ID NO: 1; (f) a Q or R residue at a position corresponding to position 117 of SEQ ID NO: 1 (f) an E or G residue at position 139 of SEQ ID NO:1; and (g) a K or R at position 139 of SEQ ID NO:1. residue.

[0209] In some embodiments of this method, the first subunit is any of SEQ ID NOs: 8-30. modified to contain residues corresponding to one of residues 19, 80, 139, and 154. do.

[0210] In some embodiments of this method, the second subunit is any of SEQ ID NOs: 8-30. containing residues corresponding to one of residues 19, 66, 80, 92, 117, and 139. It will be modified as follows.

[0211] In some embodiments of this method, the central sequence consists of ACAG.

[0212] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) an R residue at a position corresponding to position 71 of SEQ ID NO: 1; (c) a G or R residue at a position corresponding to position 72 of SEQ ID NO: 1; (c) an R, K, Q, P, or T residue at a position corresponding to position 72 of SEQ ID NO: 1; (d) an R, K, Q, P, or T residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) an A or C residue at a position corresponding to position 73 of SEQ ID NO: 1; and optionally (f) an A or C residue at a position corresponding to position 73 of SEQ ID NO: 1. an R residue at the position following the position corresponding to position 3;

[0213] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a C residue at a position corresponding to position 71 of SEQ ID NO:1; (b) a G, S, or D residue at a position corresponding to position 71 of SEQ ID NO:1 (c) an R or G residue at a position corresponding to position 72 of SEQ ID NO:1; and (d) SEQ ID NO:1 An R residue at the position corresponding to position 73 of

[0214] In some embodiments of this method, the first subunit is any of SEQ ID NOs: 33-40. modified to include residues corresponding to any one of residues 50, 71, 72, and 73 .

[0215] In some embodiments of this method, the second subunit is any of SEQ ID NOs: 33-40. The residues were modified to include one of residues 241, 262, 263, and 264. It will be changed.

[0216] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or G residue at the corresponding position; (c) an F, I, or or L residue; (c) a Q or E residue at a position corresponding to position 80 of SEQ ID NO: 1; and (d) an S or P residue at a position corresponding to position 158 of SEQ ID NO:1;

[0217] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) a G, A, or S residue at the corresponding position; (c) a V or S residue at the position corresponding to position 59 of SEQ ID NO:1; or A residue; (c) a Y or H residue at a position corresponding to position 66 of SEQ ID NO: 1; (d) a Y or H residue at a position corresponding to position 66 of SEQ ID NO: 1; (e) a Q residue at a position corresponding to position 80 of SEQ ID NO: 1; (f) an I or I residue at a position corresponding to position 81 of SEQ ID NO: 1; or T residue; and (f) a K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0218] In some embodiments of this method, the first subunit is any of SEQ ID NOs: 33-40. modified to include residues corresponding to any one of residues 19, 54, 80, and 158 do.

[0219] In some embodiments of this method, the second subunit is any of SEQ ID NOs: 33-40. containing residues corresponding to any one of residues 19, 59, 66, 80, 81, and 139. It will be modified as follows.

[0220] In some embodiments of this method, the second subunit is a nucleotide sequence corresponding to positions 73 and 74 of SEQ ID NO:1. It is further modified by inserting an R residue between the positions corresponding to positions 71 and 74.

[0221] In some embodiments of this method, the central sequence consists of ACAT.

[0222] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, S, I, L, or N residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a Q, S, R, or K residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) an R or T residue at a position corresponding to position 72 of SEQ ID NO: 1; and (e) SEQ ID NO: an A or G residue at the position corresponding to position 73 of 1;

[0223] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) an H, T, G, A, S, L, or K residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) an S, K, C, NR, G, or Q residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) an S, G, R, T, K, or E residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) a T, K, A, S, R, H, G, or N residue at a position corresponding to position 73 of SEQ ID NO: 1; (f) an H, A, C, S, G, or R residue at position 74 of SEQ ID NO:1; and S, C, or A residues at position.

[0224] In some embodiments of this method, the first subunit is any of SEQ ID NOs: 43-64. Modified to include residues corresponding to any one of residues 48, 50, 71, 72, and 73 will be done.

[0225] In some embodiments of this method, the second subunit is any of SEQ ID NOs: 43-64. Residues corresponding to any one of residues 239, 241, 262, 263, 264, and 265 The hydroxyl group is modified to include the hydroxyl group.

[0226] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A, G, or S residue at the corresponding position; (c) an F or S residue at the position corresponding to position 54 of SEQ ID NO:1; (c) a Q or E residue at a position corresponding to position 80 of SEQ ID NO: 1; and (d) A K, H, or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0227] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A, G, or S residue at the corresponding position; (c) a Q or S residue at the position corresponding to position 80 of SEQ ID NO:1; (c) an I or T residue at a position corresponding to position 81 of SEQ ID NO: 1; (d) an E residue at a position corresponding to position 81 of SEQ ID NO: 1; (e) a P or H residue at a position corresponding to position 83 of SEQ ID NO: 1; (f) a P or H residue at a position corresponding to position 117 of SEQ ID NO: 1 (f) an E or G residue at position 139 of SEQ ID NO:1; and (g) a K, R, T at position 139 of SEQ ID NO:1. , or H residue.

[0228] In some embodiments of this method, the first subunit is any of SEQ ID NOs: 43-64. modified to contain residues corresponding to any one of residues 19, 54, 80, and 139 do.

[0229] In some embodiments of this method, the second subunit is any of SEQ ID NOs: 43-64. Contains residues corresponding to any one of residues 19, 80, 81, 83, 117, and 139 It will be modified as follows.

[0230] In some embodiments of this method, the central sequence consists of ACGA.

[0231] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a V, R, T, W, or V residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a G or P residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) SEQ ID NO: (e) an R or P residue at a position corresponding to position 72 of SEQ ID NO:1; and (f) an R or P residue at a position corresponding to position 73 of SEQ ID NO:1. A residue at this position.

[0232] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, H, T, A, G, or Q residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) an R, S, C, I, V, or G residue at a position corresponding to position 71 of SEQ ID NO:1; (d) a G residue; (e) an R or H residue at a position corresponding to position 72 of SEQ ID NO: 1; and (f) an I or V residue at a position corresponding to position 74 of SEQ ID NO: 1. S or A residue at position.

[0233] In some embodiments of this method, the first subunit is any of SEQ ID NOs: 67-89. Modified to include residues corresponding to any one of residues 48, 50, 71, 72, and 73 will be done.

[0234] In some embodiments of this method, the second subunit is any of SEQ ID NOs: 67-89. Residues corresponding to any one of residues 239, 241, 262, 263, 264, and 265 The hydroxyl group is modified to include the hydroxyl group.

[0235] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A, G, or S residue at the corresponding position; (c) a Q or S residue at the position corresponding to position 80 of SEQ ID NO:1; or an E residue; and (c) an R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0236] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or G residue at the corresponding position; (b) a Q or E at the position corresponding to position 80 of SEQ ID NO:1 residue; and (c) a K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0237] In some embodiments of this method, the first subunit is any of SEQ ID NOs: 67-89. It is modified to include residues corresponding to any one of residues 19, 80, and 139.

[0238] In some embodiments of this method, the second subunit is any of SEQ ID NOs: 67-89. It is modified to include residues corresponding to any one of residues 19, 80, and 139.

[0239] In some embodiments of this method, the central sequence consists of ACGC.

[0240] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, H, Q, L, A, or S residue at position 50 of SEQ ID NO: 1; (c) a Q, R, K, S, T, or C residue at a position corresponding to position 71 of SEQ ID NO:1; (d) a G, R, or A residue at a position corresponding to position 72 of SEQ ID NO: 1; and (e) an A residue at a position corresponding to position 73 of SEQ ID NO:1.

[0241] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a residue of H, K, L, A, S, or N at a position corresponding to position 50 of SEQ ID NO: 1; (c) at a position corresponding to position 71 of SEQ ID NO:1; (d) an S, G, K, A, or R residue at a position corresponding to position 72 of SEQ ID NO: 1; , S, H, or G residue; (e) H, T, V, I, or G residue at a position corresponding to position 73 of SEQ ID NO: 1; or a C residue; and (f) an S, A, or T residue at a position corresponding to position 74 of SEQ ID NO:1. .

[0242] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 92-118. Modified to include residues corresponding to any one of residues 48, 50, 71, 72, and 73 It will be changed.

[0243] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 92-118. Corresponding to any one of residues 239, 241, 262, 263, 264, and 265 The residue is modified to include:

[0244] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 an A, G, or S residue at the corresponding position; and (b) a position corresponding to position 80 of SEQ ID NO:1. to Q or E residues.

[0245] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or G residue at the corresponding position; (b) a Q or E at the position corresponding to position 80 of SEQ ID NO:1 (c) an F or L residue at a position corresponding to position 87 of SEQ ID NO: 1; and (d) a a K, R, N, H, or A residue at the position corresponding to position 139 of No. 1;

[0246] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 92-118. It is modified to include residues corresponding to either one of residues 19 and 80.

[0247] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 92-118. modified to include residues corresponding to any one of residues 19, 80, 87, and 139 can be.

[0248] In some embodiments of this method, the central sequence consists of ACGG.

[0249] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) an R or K residue at a position corresponding to position 72 of SEQ ID NO: 1; and and (c) an A residue at a position corresponding to position 73 of SEQ ID NO:1.

[0250] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) an R or P residue at a position corresponding to position 50 of SEQ ID NO: 1; (d) a D residue at a position corresponding to position 72 of SEQ ID NO: 1; and (e) an R or G residue at a position corresponding to position 73 of SEQ ID NO:1.

[0251] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 121-135 The nucleotide sequence is modified to include residues corresponding to residues 50, 72, and 73 of any one of the sequences.

[0252] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 121-135 Residues corresponding to residues 239, 241, 262, 263, and 264 in any one of will be modified to include

[0253] In some embodiments of this method, the method comprises: (a) at position 54 of SEQ ID NO: 1 (b) an F or L residue at the corresponding position; and (b) a Q residue at the position corresponding to position 80 of SEQ ID NO:1. Base.

[0254] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 an A residue at the corresponding position; and (b) a Q residue at the position corresponding to position 80 of SEQ ID NO:1.

[0255] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 121-135 is modified to include residues corresponding to residues 54 and 80 of either one of:

[0256] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 121-135 is modified to include residues corresponding to residues 19 and 80 of either one of:

[0257] In some embodiments of this method, the second subunit is a nucleotide sequence corresponding to positions 73 and 74 of SEQ ID NO:1. It is further modified by inserting an R residue between the positions corresponding to positions 71 and 74.

[0258] In some embodiments of this method, the central sequence consists of ACGT.

[0259] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, L, S, or H residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a G residue at a position corresponding to position 71 of SEQ ID NO: 1; (d ) an R residue at a position corresponding to position 72 of SEQ ID NO:1; and (e) an R residue at a position corresponding to position 73 of SEQ ID NO:1. A residue at this position.

[0260] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) an H, K, L, or S residue at a position corresponding to position 50 of SEQ ID NO: 1; , C, Q, E, or A residue; (c) S, P, G, or (d) a T, A, R, or N residue at a position corresponding to position 72 of SEQ ID NO:1; or A residue; (e) an H, C, A, or S residue at a position corresponding to position 73 of SEQ ID NO: 1; and and (f) an S, A, or T residue at a position corresponding to position 74 of SEQ ID NO:1.

[0261] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 138-156 to include residues corresponding to residues 48, 50, 71, 72, and 73 in any one of It will be modified.

[0262] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 138-156 corresponding to residues 239, 241, 262, 263, 264, and 265 of any one of The amino acid sequence is modified to include a residue that

[0263] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or G residue at the corresponding position; (b) a Q or E at the position corresponding to position 80 of SEQ ID NO:1 residue; and (c) a K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0264] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or G residue at the corresponding position; (b) a Q or E at the position corresponding to position 80 of SEQ ID NO:1 (c) an H or Y residue at a position corresponding to position 85 of SEQ ID NO: 1; and (d) a SEQ ID NO: K or R residue at the position corresponding to position 139 of No. 1.

[0265] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 138-156 is modified to include residues corresponding to residues 19, 80, and 139 of any one of .

[0266] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 138-156 modified to include residues corresponding to residues 19, 80, 85, and 139 in any one of will be done.

[0267] In some embodiments of this method, the central sequence consists of ATAA.

[0268] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, A, H, S, L, or Q residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a Q, T, R, I, G, K, D, C, or V residue at position 71 of SEQ ID NO: 1; (d) a G, K, S, H, or N residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) an R, A, G, Q, H, L, or S residue at a position corresponding to position 73 of SEQ ID NO: 1; (f) an A, T, or C residue; and (f) an S or A residue at a position corresponding to position 74 of SEQ ID NO:1. Base.

[0269] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) an S, T, A, K, or N residue at a position corresponding to position 50 of SEQ ID NO:1; (c) an R, K, E, A, C, or T residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) a G, K, or R residue; (e) a T, R, Q, G, A residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) an I, C, or a Y, S, N, or K residue at a position corresponding to position 73 of SEQ ID NO:1; is a V residue; and (f) an S, A, or T residue at a position corresponding to position 74 of SEQ ID NO:1.

[0270] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 159-183 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 in any one of It will be modified as follows.

[0271] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 159-183 corresponding to residues 239, 241, 262, 263, 264, and 265 of any one of The amino acid sequence is modified to include a residue that

[0272] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A, G, or S residue at the corresponding position; (c) a Q or S residue at the position corresponding to position 80 of SEQ ID NO:1; or E residue; (c) a K or E residue at a position corresponding to position 100 of SEQ ID NO: 1; (d) the sequence (e) a K or R residue at a position corresponding to position 139 of SEQ ID NO: 1; (f) a K or R residue at a position corresponding to position 154 of SEQ ID NO: 1 (f) an S or G residue at a position corresponding to position 172 of SEQ ID NO: 1; and is the A residue.

[0273] In some embodiments of this method, the method further comprises: (a) at position 19 of SEQ ID NO: 1 (b) a G, S, or A residue at the corresponding position; (c) a V or A residue at the position corresponding to position 59 of SEQ ID NO: 1; or an A residue; (c) an L residue at a position corresponding to position 78 of SEQ ID NO: 1; (d) an L residue at a position corresponding to position 79 of SEQ ID NO: 1; (e) an S residue at a position corresponding to position 9 of SEQ ID NO: 1; (f) a Q or E residue at a position corresponding to position 80 of SEQ ID NO: 1 (f) an S or F residue at a position corresponding to position 118 of SEQ ID NO: 1; and (g) a K or R residue at the position corresponding to position 139 of No. 1.

[0274] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 159-183 Residues corresponding to residues 19, 80, 100, 139, 154, and 172 in any one of The hydroxyl group is modified to include the hydroxyl group.

[0275] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 159-183 corresponding to residues 19, 59, 78, 79, 80, 118, and 139 in any one of The residue is modified to include:

[0276] In some embodiments of this method, the central sequence consists of ATAG.

[0277] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K or H residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) an R residue at a position corresponding to position 50 of SEQ ID NO: 1; (d) a G, R, or H residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) an R, G, S, A, P, or Q residue at a position corresponding to position 73 of SEQ ID NO: 1; and A or C residue at the position corresponding to position.

[0278] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a C or R residue at a position corresponding to position 72 of SEQ ID NO: 1; and (c) an R residue at a position corresponding to position 73 of SEQ ID NO:1.

[0279] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 186-199 to include residues corresponding to residues 48, 50, 71, 72, and 73 in any one of It will be modified.

[0280] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 186-199 modified to include residues corresponding to residues 241, 263, and 264 of any one of can be.

[0281] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or G residue at the corresponding position; (b) a Q or E at the position corresponding to position 80 of SEQ ID NO:1 residue; and (c) a K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0282] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) a G or A residue at the corresponding position; (b) a K or R at the position corresponding to position 36 of SEQ ID NO:1 (c) a V or A residue at a position corresponding to position 59 of SEQ ID NO: 1; (d) a V or A residue at a position corresponding to position 59 of SEQ ID NO: 1; (e) a Q residue at a position corresponding to position 80 of SEQ ID NO:1; and (f) a K residue at a position corresponding to position 139 of SEQ ID NO:1. or R residue.

[0283] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 186-199 is modified to include residues corresponding to residues 19, 80, and 139 of any one of .

[0284] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 186-199 containing residues corresponding to residues 19, 36, 59, 80, and 139 in any one of will be changed to.

[0285] In some embodiments of this method, the central sequence consists of ATAT.

[0286] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, H, C, A, S, D, or T residue at the corresponding position; (c) a K, H, C, A, S, D, or T residue at the corresponding position; (d) a K, H, C, A, S, D, or T residue at the corresponding position; (e) a K, H, C, A, S, D, or T residue at the corresponding position; (f) a K, H, C, A, S, D, or T residue at the corresponding position; (g ... (c) a Q, N, C, R, K, S, T, or V residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) a G, H, or I residue at a position corresponding to position 72 of SEQ ID NO: 1; , N, or Q residue; and (e) an A, C, or S residue.

[0287] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a residue of H, K, A, S, R, or T at a position corresponding to position 50 of SEQ ID NO: 1; (c) an S, C, K, R, Q, or N residue at a position corresponding to position 71 of SEQ ID NO:1; (d) an S, K, E, I, G, or R residue; (e) a T, A, or B residue at a position corresponding to position 72 of SEQ ID NO:1; , R, S, K, G, or N residue; (e) H, C, or N at a position corresponding to position 73 of SEQ ID NO: 1; (f) an A, S, or G residue at a position corresponding to position 74 of SEQ ID NO:1; is the A residue.

[0288] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 202-219 to include residues corresponding to residues 48, 50, 71, 72, and 73 in any one of It will be modified.

[0289] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 202-219 corresponding to residues 239, 241, 262, 263, 264, and 265 of any one of The amino acid sequence is modified to include a residue that

[0290] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or G residue at the corresponding position; (b) a Q or E at the position corresponding to position 80 of SEQ ID NO:1 residue; and (c) a K, R, or S residue at a position corresponding to position 139 of SEQ ID NO:1.

[0291] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) a G or A residue at the corresponding position; (b) a V or A at the position corresponding to position 59 of SEQ ID NO:1 (c) a Q, E, or K residue at a position corresponding to position 80 of SEQ ID NO: 1; and (d) A K, R, P, or N residue at a position corresponding to position 139 of SEQ ID NO:1.

[0292] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 202-219 is modified to include residues corresponding to residues 19, 80, and 139 of any one of .

[0293] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 202-219 modified to include residues corresponding to residues 19, 59, 80, and 139 in any one of will be done.

[0294] In some embodiments of this method, the central sequence consists of ATGA.

[0295] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, A, H, or L residue at a position corresponding to position 50 of SEQ ID NO: 1; , T, E, S, C, or V residue; (c) an R, T, or V residue at a position corresponding to position 72 of SEQ ID NO: 1; an S, A, or K residue; and (d) an A or S residue at a position corresponding to position 73 of SEQ ID NO:1. Base.

[0296] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a position corresponding to position 50 of SEQ ID NO: 1; (c) an S, I, R, C, A, or Q residue at a position corresponding to position 72 of SEQ ID NO: 1; or H residue; (d) an I or V residue at a position corresponding to position 73 of SEQ ID NO: 1; and (e) an S, A, or T residue at a position corresponding to position 74 of SEQ ID NO:1;

[0297] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 222-243 modified to include residues corresponding to residues 48, 50, 72, and 73 of any one of can be.

[0298] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 222-243 Residues corresponding to residues 239, 241, 263, 264, and 265 in any one of will be modified to include

[0299] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A, G, or S residue at the corresponding position; (c) a Q or S residue at the position corresponding to position 80 of SEQ ID NO:1; or E residue; (c) an F or L residue at a position corresponding to position 87 of SEQ ID NO: 1; (d) an F or L residue at a position corresponding to position 87 of SEQ ID NO: 1; (e) a Q or R residue at a position corresponding to position 92 of SEQ ID NO:1; and (f) a Q or R residue at a position corresponding to position 139 of SEQ ID NO:1. K or R residue at the corresponding position.

[0300] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) a G, A, or S residue at the corresponding position; (c) a V or S residue at the position corresponding to position 59 of SEQ ID NO:1; or an A residue; (c) a Q or E residue at a position corresponding to position 80 of SEQ ID NO: 1; and (d) A K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0301] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 222-243 containing residues corresponding to residues 19, 80, 87, 92, and 139 in any one of will be changed to.

[0302] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 222-243 modified to include residues corresponding to residues 19, 59, 80, and 139 in any one of will be done.

[0303] In some embodiments of this method, the central sequence consists of ATGG.

[0304] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) an R residue at a position corresponding to position 71 of SEQ ID NO: 1; (c) a G or S residue at a position corresponding to position 72 of SEQ ID NO: 1; (d) a P or G residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) a P or G residue at a position corresponding to position 73 of SEQ ID NO: 1; (e) an A or C residue at a position corresponding to position 74 of SEQ ID NO:1; is a C residue.

[0305] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) an R residue at a position corresponding to position 71 of SEQ ID NO: 1; (c) a D or G residue at a position corresponding to position 72 of SEQ ID NO: 1; ) a G residue at a position corresponding to position 72 of SEQ ID NO:1; and (d) a G residue at a position corresponding to position 73 of SEQ ID NO:1. R residue at the position.

[0306] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 246-247 modified to include residues corresponding to residues 50, 71, 72, and 73 of any one of can be.

[0307] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 246-247 containing residues corresponding to residues 241, 262, 263, and 264 in any one of will be changed to.

[0308] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or G residue at the corresponding position; (b) an E or Q at the position corresponding to position 80 of SEQ ID NO: 1 (c) an E or K residue at a position corresponding to position 82 of SEQ ID NO: 1; and (d) a R or K residue at the position corresponding to position 139 of No. 1.

[0309] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or G residue at the corresponding position; (b) an N residue at the position corresponding to position 77 of SEQ ID NO: 1; and and (c) a Q or R residue at a position corresponding to position 80 of SEQ ID NO:1.

[0310] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 246-247 modified to include residues corresponding to residues 19, 80, 82, and 139 in any one of will be done.

[0311] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 246-247 The modified nucleotide sequence is modified to include residues corresponding to residues 19, 77, and 80 of any one of the following:

[0312] In some embodiments of this method, the second subunit is a nucleotide sequence corresponding to positions 73 and 74 of SEQ ID NO:1. It is further modified by inserting an R residue between the positions corresponding to positions 71 and 74.

[0313] In some embodiments of this method, the central sequence consists of TTGG.

[0314] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) an R residue at a position corresponding to position 71 of SEQ ID NO: 1; (c) an S residue at a position corresponding to position 72 of SEQ ID NO: 1; (d) a G residue at a position corresponding to position 72 of SEQ ID NO:1; and (e) a G residue at a position corresponding to position 73 of SEQ ID NO:1. to R residue.

[0315] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K or S residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a K or R residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) (e) a T, Q, K, R, H, A, or S residue at a position corresponding to position 72 of SEQ ID NO: 1; (f) an I or V residue at a position corresponding to position 73 of SEQ ID NO: 1; and (f) an I or V residue at position 74 of SEQ ID NO: 1. S or A residues at the corresponding positions.

[0316] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 250-266 modified to include residues corresponding to residues 50, 71, 72, and 73 of any one of can be.

[0317] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 250-266 corresponding to residues 239, 241, 262, 263, 264, and 265 of any one of The amino acid sequence is modified to include a residue that

[0318] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or G residue at the corresponding position; and (b) a Q residue at the position corresponding to position 80 of SEQ ID NO:1. Base.

[0319] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) a G or A residue at the corresponding position; (b) a Y or H at the position corresponding to position 66 of SEQ ID NO:1 (c) a Q residue at a position corresponding to position 80 of SEQ ID NO: 1; (d) a Q residue at position 85 of SEQ ID NO: 1 an H or R residue at the corresponding position; and (e) a K at the position corresponding to position 139 of SEQ ID NO:1. or R residue.

[0320] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 250-266 is modified to include residues corresponding to residues 19 and 80 of either one of:

[0321] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 250-266 containing residues corresponding to residues 19, 66, 80, 85, and 139 in any one of will be changed to.

[0322] In some embodiments of this method, the central sequence consists of GCAA.

[0323] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K or H residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a G, N, T, R, S, or L residue at a position corresponding to position 71 of SEQ ID NO: 1. or H residue; (d) R, P, S, N, Q, G, A, or H residue at a position corresponding to position 72 of SEQ ID NO: 1. (e) a T or V residue at a position corresponding to position 73 of SEQ ID NO: 1; and and (f) an S, C, or A residue at a position corresponding to position 74 of SEQ ID NO:1.

[0324] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) an S, A, K, or T residue at a position corresponding to position 50 of SEQ ID NO: 1; , C, T, K, or E residue; (c) G, R, A, or E residue at a position corresponding to position 71 of SEQ ID NO: 1; or H residue; (d) T, G, S, A, E, N, K at a position corresponding to position 72 of SEQ ID NO: 1 (e) a C, V, or Y residue at a position corresponding to position 73 of SEQ ID NO: 1; is an I residue; and (f) an S, A, or T residue at a position corresponding to position 74 of SEQ ID NO:1.

[0325] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 269-291 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 in any one of It will be modified as follows.

[0326] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 269-291 corresponding to residues 239, 241, 262, 263, 264, and 265 of any one of The amino acid sequence is modified to include a residue that

[0327] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A, G, or S residue at the corresponding position; (c) a Q or S residue at the position corresponding to position 80 of SEQ ID NO:1; or an E residue; and (c) a K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0328] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or G residue at the corresponding position; (b) a Q or P at the position corresponding to position 31 of SEQ ID NO:1 (c) a Q or E residue at a position corresponding to position 80 of SEQ ID NO: 1; and (d) a Q or E residue at a position corresponding to position 80 of SEQ ID NO: 1. K or R residue at the position corresponding to position 139 of No. 1.

[0329] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 269-291 is modified to include residues corresponding to residues 19, 80, and 139 of any one of .

[0330] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 269-291 modified to include residues corresponding to residues 19, 31, 80, and 139 in any one of will be done.

[0331] In some embodiments of this method, the central sequence consists of GCAT.

[0332] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, A, H, or R residue at a position corresponding to position 50 of SEQ ID NO: 1; , V, R, K, or S residue; (c) G, A, H, or (d) an R, T, N, or S residue at a position corresponding to position 72 of SEQ ID NO:1; (e) an A, T, V, or N residue at a position corresponding to position 73 of SEQ ID NO:1; is a C residue; and (f) an S or A residue at a position corresponding to position 74 of SEQ ID NO:1.

[0333] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a residue H, A, K, T, L, or I at a position corresponding to position 50 of SEQ ID NO: 1; (c) an S, R, K, Q, H, or V residue at a position corresponding to position 71 of SEQ ID NO:1; an S, K, R, A, G, T, H, or Y residue; (d) a position corresponding to position 72 of SEQ ID NO:1 (e) a T, A, G, N, S, R, H, Q, or K residue at position 73 of SEQ ID NO: 1; (f) an H, C, G, S, or A residue at a position corresponding to position 74 of SEQ ID NO:1; and S, C, or A residues at position.

[0334] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 294-313 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 in any one of It will be modified as follows.

[0335] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 294-313 corresponding to residues 239, 241, 262, 263, 264, and 265 of any one of The amino acid sequence is modified to include a residue that

[0336] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or G residue at the corresponding position; (b) a Q or E at the position corresponding to position 80 of SEQ ID NO:1 (c) a K, H, or R residue at a position corresponding to position 139 of SEQ ID NO: 1; and (d ) A T or I residue at a position corresponding to position 143 of SEQ ID NO:1.

[0337] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) a G, S, or A residue at the corresponding position; (c) a Q or A residue at the position corresponding to position 80 of SEQ ID NO: 1; (c) a V or A residue at a position corresponding to position 125 of SEQ ID NO: 1; and (d) ) a K, R, or H residue at a position corresponding to position 139 of SEQ ID NO:1.

[0338] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 294-313 The modified nucleotide sequence was modified to include residues corresponding to residues 19, 80, 139, and 143 in one of It will be changed.

[0339] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 294-313 The modified nucleotide sequence was modified to include residues corresponding to residues 19, 80, 125, and 139 in one of the It will be changed.

[0340] In some embodiments of this method, the central sequence consists of GCGA.

[0341] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K or R residue at a position corresponding to position 71 of SEQ ID NO: 1; (c) an R, N, G, A, or N residue at a position corresponding to position 72 of SEQ ID NO: 1 (d) a V, T, or I residue at a position corresponding to position 73 of SEQ ID NO: 1; and (e ) an S or A residue at a position corresponding to position 74 of SEQ ID NO:1.

[0342] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, T, S, A, or Q residue at a position corresponding to position 50 of SEQ ID NO:1; (c) a C or R residue at a position corresponding to position 72 of SEQ ID NO: 1; (d) a C or R residue at a position corresponding to position 73 of SEQ ID NO: 1; (e) a V or I residue at a position corresponding to position 73 of SEQ ID NO: 1; and (f) a V or I residue at a position corresponding to position 74 of SEQ ID NO: 1. S or A residue at the position.

[0343] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 316-325 to include residues corresponding to any one of residues 50, 71, 72, 73, and 74 It will be modified.

[0344] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 316-325 Residues corresponding to residues 239, 241, 263, 264, and 265 in any one of will be modified to include

[0345] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 an A, G, or S residue at the corresponding position; and (b) a position corresponding to position 80 of SEQ ID NO:1. to Q or E residues.

[0346] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) a G, S, or A residue at the corresponding position; (c) a Q or A residue at the position corresponding to position 80 of SEQ ID NO: 1; or an E residue; and (c) an R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0347] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 316-325 is modified to include residues corresponding to residues 19 and 80 of either one of:

[0348] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 316-325 is modified to include residues corresponding to residues 19, 80, and 139 of any one of .

[0349] In some embodiments of this method, the central sequence consists of GCAG.

[0350] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) an R residue at a position corresponding to position 71 of SEQ ID NO: 1; (c) an S residue at a position corresponding to position 72 of SEQ ID NO: 1; (d) a G residue at a position corresponding to position 72 of SEQ ID NO:1; and (e) a G residue at a position corresponding to position 73 of SEQ ID NO:1. to an R residue; and

[0351] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K or H residue at a position corresponding to position 50 of SEQ ID NO: 1; and (c) an S or R residue at a position corresponding to position 72 of SEQ ID NO:1;

[0352] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 328-330. to include residues corresponding to any one of residues 50, 71, 72, 73, and 74 It will be modified.

[0353] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 328-330. Residues corresponding to residues 239, 241, 263, 264, and 265 in any one of will be modified to include

[0354] In some embodiments of this method, the method further comprises: to contain a Q or E residue at a position corresponding to position 80 of

[0355] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 328-330. is modified to include a residue corresponding to residue 80 of any one of:

[0356] In some embodiments of this method, the central sequence consists of TCAA.

[0357] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K or S residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a G, R, or T residue at a position corresponding to position 71 of SEQ ID NO: 1; and d) An R, S, P, T, or G residue at a position corresponding to position 72 of SEQ ID NO:1.

[0358] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, R, C, or S residue at a position corresponding to position 50 of SEQ ID NO: 1; or an E residue; (c) an R, Q, N, or S residue at a position corresponding to position 72 of SEQ ID NO:1; (d) a I residue at a position corresponding to position 73 of SEQ ID NO:1; and (e) a I residue at position 74 of SEQ ID NO:1. S or A residues at the corresponding positions.

[0359] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 333-340 modified to include residues corresponding to residues 48, 50, 71, and 72 of any one of can be.

[0360] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 333-340. Residues corresponding to residues 239, 241, 263, 264, and 265 in any one of will be modified to include

[0361] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or S residue at the corresponding position; (b) a Q or E at the position corresponding to position 80 of SEQ ID NO:1 residue; and (c) a K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0362] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) a G or S residue at the corresponding position; (b) a Q or E at the position corresponding to position 80 of SEQ ID NO:1 residue; and (c) an R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0363] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 333-340 is modified to include residues corresponding to residues 19, 80, and 139 of any one of .

[0364] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 333-340. is modified to include residues corresponding to residues 19, 80, and 139 of any one of .

[0365] In some embodiments of this method, the central sequence consists of TTAA.

[0366] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, N, S, or R residue at a position corresponding to position 50 of SEQ ID NO: 1; , V, K, or S residue; (c) G, R, N, S, or or A residue; (d) R, T, S, N, D, Q, K at a position corresponding to position 72 of SEQ ID NO: 1 or an A residue; and (e) an S or A residue at a position corresponding to position 74 of SEQ ID NO:1.

[0367] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, S, A, or T residue at a position corresponding to position 50 of SEQ ID NO: 1; , K, R, T, or E residue; (c) T, K, R, or E residue at a position corresponding to position 72 of SEQ ID NO: 1; (d) an A, S, or Q residue; (e) an I or V residue at a position corresponding to position 73 of SEQ ID NO: 1; and and (e) an S or A residue at a position corresponding to position 74 of SEQ ID NO:1.

[0368] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 343-357 to include residues corresponding to residues 48, 50, 71, 72, and 74 in any one of It will be modified.

[0369] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 343-357 Residues corresponding to residues 239, 241, 263, 264, and 265 in any one of will be modified to include

[0370] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A, G, or S residue at the corresponding position; (c) a Q or S residue at the position corresponding to position 80 of SEQ ID NO:1; or an E residue; and (c) a K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0371] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) a G, A, or S residue at the corresponding position; (c) a Y or S residue at the position corresponding to position 66 of SEQ ID NO: 1; or an H residue; (c) a Q residue at a position corresponding to position 80 of SEQ ID NO: 1; and (d) a Q residue at a position corresponding to position 80 of SEQ ID NO: 1. an R residue at the position corresponding to position 139 of 1;

[0372] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 343-357 is modified to include residues corresponding to residues 19, 80, and 139 of any one of .

[0373] In some embodiments of this method, the second subunit is selected from the group consisting of SEQ ID NOs: 343-357 modified to include residues corresponding to residues 19, 66, 80, and 139 in any one of will be done.

[0374] Another embodiment is GTAA, GTAG, GTAT, GTGA, GTGC, GTGG, or Engineered meganuclei that bind and cleave recognition sequences containing a central sequence consisting of GTGT The enzyme comprises a first subunit and a second subunit, the first subunit comprises an amino acid sequence derived from SEQ ID NO: 1, and the second subunit comprises an amino acid sequence derived from SEQ ID NO: One or more positions corresponding to positions 48, 50, 71, 72, 73, and 74 of 1 The present invention relates to an engineered meganuclease comprising a substitution at

[0375] In some embodiments, the central sequence consists of GTAA.

[0376] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K, S, A, R, N, or T residue at a position corresponding to position 48 of SEQ ID NO: 1; ) a T, R, A, K, or C residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) SEQ ID NO: (d) a G, R, S, T, A, N, H, or K residue at a position corresponding to position 71 of SEQ ID NO:1; R, S, C, N, K, A, H, G, T, D, Y, P at the position corresponding to position 72 of No. 1, or (e) a V, C, I, or T residue at a position corresponding to position 73 of SEQ ID NO: 1; and and (f) an S, A, or T residue at a position corresponding to position 74 of SEQ ID NO:1.

[0377] In some embodiments, the first subunit is any of SEQ ID NOs: 360-389. One contains residues corresponding to residues 48, 50, 71, 72, 73, and 74.

[0378] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A or S residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) an A or S residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) a Q or E residue at a position corresponding to position 139 of SEQ ID NO: 1; and to K or R residues.

[0379] In some embodiments, the first subunit is any of SEQ ID NOs: 360-389. One contains residues corresponding to residues 19, 80, and 139.

[0380] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of GTAA. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0381] In some embodiments, the central sequence consists of GTAG.

[0382] In some embodiments, the first subunit comprises one or more of the following residues: (a) an R or C residue at a position corresponding to position 50 of SEQ ID NO: 1; (b) an R or C residue at a position corresponding to position 71 of SEQ ID NO: 1; (c) an S or D residue at a position corresponding to position 72 of SEQ ID NO: 1; is an N residue; and (d) an R residue at a position corresponding to position 73 of SEQ ID NO:1.

[0383] In some embodiments, the first subunit is any of SEQ ID NOs: 392-399. One contains residues corresponding to residues 50, 71, 72, and 73.

[0384] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A or S residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) an A or S residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) a Q residue at a position corresponding to position 139 of SEQ ID NO:1; and is the R residue.

[0385] In some embodiments, the first subunit is any of SEQ ID NOs: 392-399. One contains residues corresponding to residues 19, 80, and 139.

[0386] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of GTAG. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0387] In some embodiments, the central sequence consists of GTAT.

[0388] In some embodiments, the first subunit comprises one or more of the following residues: (a) K, G, T, A, M, H, S, L, or a residue at a position corresponding to position 48 of SEQ ID NO: 1 R residue; (b) Q, V, R, S, T, G, K, C, or a residue corresponding to position 50 of SEQ ID NO: 1 or L residue; (c) G, T, A, K, H, R, Y at a position corresponding to position 71 of SEQ ID NO: 1 , L, S, or N residue; (d) R, K, S, Y, or N, T, G, W, H, or A residue; (e) A, C at the position corresponding to position 73 of SEQ ID NO: 1 , S, or T residue; and (f) an S, A, or C residue.

[0389] In some embodiments, the first subunit is any of SEQ ID NOs: 402-433. One contains residues corresponding to residues 48, 50, 71, 72, 73, and 74.

[0390] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A or S residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) an A or S residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) a Q or E residue at a position corresponding to position 139 of SEQ ID NO: 1; and to K, R, T, or H residues.

[0391] In some embodiments, the first subunit is any of SEQ ID NOs: 402-433. One contains residues corresponding to residues 19, 80, and 139.

[0392] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of GTAT. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0393] In some embodiments, the central sequence consists of GTGA.

[0394] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K, A, G, R, S, or H residue at a position corresponding to position 48 of SEQ ID NO: 1; (c) an R, V, C, or S residue at a position corresponding to position 50 of SEQ ID NO: 1; a G, R, V, S, A, T, N, D, or H residue at a position corresponding to position 71 of sequence; (d) sequence (e) an R, T, S, G, H, K, or Y residue at a position corresponding to position 72 of SEQ ID NO:1; (f) an A, V, or T residue at a position corresponding to position 73 of SEQ ID NO: 1; and (f) position 74 of SEQ ID NO: 1. S, T, A, or G residues at the corresponding positions.

[0395] In some embodiments, the first subunit is any of SEQ ID NOs: 436-462. One contains residues corresponding to residues 48, 50, 71, 72, 73, and 74.

[0396] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A or S residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) an A or S residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) a Q or E residue at a position corresponding to position 139 of SEQ ID NO: 1; and to K or R residues.

[0397] In some embodiments, the first subunit is any of SEQ ID NOs: 436-462. One contains residues corresponding to residues 19, 80, and 139.

[0398] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of GTGA. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0399] In some embodiments, the central sequence consists of GTGC.

[0400] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K, L, H, A, R, N, or S residue at a position corresponding to position 48 of SEQ ID NO:1; (b) an R, S, V, K, I, or G residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) G, S, N, I, R, A, E, Q, Y, T, K, F at the position corresponding to position 71 of SEQ ID NO: 1 or V residue; (d) R, K, G, H, P, S, at a position corresponding to position 72 of SEQ ID NO: 1 a C, N, T, A, M, D, or Q residue; (e) an A at a position corresponding to position 73 of SEQ ID NO:1; , V, T, N, C, or L residue; and (f) S at a position corresponding to position 74 of SEQ ID NO:1. , A, or T residues.

[0401] In some embodiments, the first subunit is any of SEQ ID NOs: 465-495. One contains residues corresponding to residues 48, 50, 71, 72, 73, and 74.

[0402] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A or S residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) an A or S residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) a Q or E residue at a position corresponding to position 139 of SEQ ID NO: 1; and to K, T, S, R, H, or V residues.

[0403] In some embodiments, the first subunit is any of SEQ ID NOs: 465-495. One contains residues corresponding to residues 19, 80, and 139.

[0404] Another embodiment includes a meganuclease recognition sequence that includes a central sequence consisting of GTGC. A method for cleaving double-stranded DNA at a target site comprising: A is contacted with an engineered meganuclease as described herein, and the engineered meganuclease The method comprises the step of: a polypeptide chain reaction (e.g., a polypeptide chain reaction) in which the polypeptide chain is cleaved by binding to a recognition sequence;

[0405] In some embodiments, the central sequence consists of GTGG.

[0406] In some embodiments, the first subunit comprises one or more of the following residues: (a) an R residue at a position corresponding to position 50 of SEQ ID NO: 1; (b) an R residue at a position corresponding to position 71 of SEQ ID NO: 1 (c) an S residue at a position corresponding to position 72 of SEQ ID NO: 1; and (d) a G residue at a position corresponding to position 73 of SEQ ID NO: 1. R residue at the position corresponding to position 73 in column no.

[0407] In some embodiments, the first subunit comprises residue 50 of SEQ ID NOs: 498-501. , 71, 72, and 73.

[0408] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) an A residue at a position corresponding to position 62 of SEQ ID NO: 1 and (c) a Q residue at a position corresponding to position 80 of SEQ ID NO:1.

[0409] In some embodiments, the first subunit comprises residue 19 of SEQ ID NOs: 498-501. , 62, and 80.

[0410] Another embodiment is a targeting moiety comprising a meganuclease recognition sequence comprising a central sequence consisting of GTGG. A method for cleaving double-stranded DNA at a target site, comprising: and contacting the engineered meganuclease described in the specification with the engineered meganuclease, The method comprises binding to and cleaving a recognition sequence.

[0411] In some embodiments, the central sequence consists of GTGT.

[0412] In some embodiments, the first subunit comprises one or more of the following residues: (a) a K, S, L, V, G, R, or N residue at a position corresponding to position 48 of SEQ ID NO:1; (b) a Q, V, R, S, K, A, E, or C residue at a position corresponding to position 50 of SEQ ID NO:1 (c) a G, R, N, H, A, or T residue at a position corresponding to position 71 of SEQ ID NO: 1; (d ) an R, P, A, Q, K, T, G, or V residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) an A, S, C, or T residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) the sequence an S, A, or T residue at the position corresponding to position 74 of no. 1;

[0413] In some embodiments, the first subunit is any of SEQ ID NOs: 504-529. One contains residues corresponding to residues 48, 50, 71, 72, 73, and 74.

[0414] In some embodiments, the first subunit comprises one or more of the following residues: (a) an A or S residue at a position corresponding to position 19 of SEQ ID NO: 1; (b) an A or S residue at a position corresponding to position 80 of SEQ ID NO: 1; (c) a Q or E residue at a position corresponding to position 139 of SEQ ID NO: 1; and to K or R residues.

[0415] In some embodiments, the first subunit is any of SEQ ID NOs: 504-529. One contains residues corresponding to residues 19, 80, and 139.

[0416] Another embodiment is a targeting moiety comprising a meganuclease recognition sequence comprising a central sequence consisting of GTGT. A method for cleaving double-stranded DNA at a target site, comprising: and contacting the engineered meganuclease described in the specification with the engineered meganuclease, The method comprises binding to and cleaving a recognition sequence.

[0417] Another embodiment is GTAA, GTAG, GTAT, GTGA, GTGC, GTGG, or The first subunit and the second subunit bind to and cleave the recognition sequence containing the central sequence GTGT. and a second subunit, wherein the first subunit comprises an amino acid sequence derived from SEQ ID NO: 1. A method for enhancing the cleavage activity of an engineered meganuclease comprising the sequence The first subunit is located at positions 48, 50, 71, 72, 73, and and one or more positions corresponding to positions 74 and 75, and the modified nuclease is and having enhanced cleavage activity when compared to the meganucleases produced by the method. It is the law.

[0418] In some embodiments of this method, the central sequence consists of GTAA.

[0419] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, S, A, R, N, or T residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a T, R, A, K, or C residue at position 71 of SEQ ID NO:1; , R, S, T, A, N, H, or K residue; (d) at a position corresponding to position 72 of SEQ ID NO:1. (e) an R, S, C, N, K, A, H, G, T, D, Y, P, or Q residue of SEQ ID NO: 1; (f) a V, C, I, or T residue at a position corresponding to position 73 of SEQ ID NO: 1; and S, A, or T residues at the corresponding positions.

[0420] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 360-389 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 in any one of It will be modified as follows.

[0421] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or S residue at the corresponding position; (b) a Q or E at the position corresponding to position 80 of SEQ ID NO:1 residue; and (c) a K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0422] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or S residue at the corresponding position; (b) a Q or E at the position corresponding to position 80 of SEQ ID NO:1 residue; and (c) a K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0423] In some embodiments of this method, the central sequence consists of GTAG.

[0424] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) an R or C residue at a position corresponding to position 71 of SEQ ID NO: 1; (c) a G or N residue at a position corresponding to position 72 of SEQ ID NO: 1; and (d) SEQ ID NO: an R residue at the position corresponding to position 73 of 1;

[0425] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 392-399 modified to include residues corresponding to residues 50, 71, 72, and 73 of any one of can be.

[0426] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or S residue at the corresponding position; (b) a Q residue at the position corresponding to position 80 of SEQ ID NO: 1; and and (c) a K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0427] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 392-399 is modified to include residues corresponding to residues 19, 80, and 139 of any one of .

[0428] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 360-389 is modified to include residues corresponding to residues 19, 80, and 139 of any one of .

[0429] In some embodiments of this method, the central sequence consists of GTAT.

[0430] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, G, T, A, M, H, S, L, or R residue at the corresponding position of SEQ ID NO: 1; (c) a Q, V, R, S, T, G, K, C, or L residue at a position corresponding to SEQ ID NO: 1; (d) a G, T, A, K, H, R, Y, L, S, or N residue at a position corresponding to position 71 of R, K, S, Y, N, T, G, W, H, A residues at the position corresponding to position 72 of SEQ ID NO: 1; (e) an A, C, S, or T residue at a position corresponding to position 73 of SEQ ID NO: 1; and (f) the sequence an S, A, or C residue at the position corresponding to position 74 of no. 1;

[0431] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 402-433 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 in any one of It will be modified as follows.

[0432] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or S residue at the corresponding position; (b) a Q or E at the position corresponding to position 80 of SEQ ID NO:1 residue; and (c) a K, R, T, or H residue at a position corresponding to position 139 of SEQ ID NO:1.

[0433] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 402-433 is modified to include residues corresponding to residues 19, 80, and 139 of any one of .

[0434] In some embodiments of this method, the central sequence consists of GTGA.

[0435] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, A, G, R, S, or H residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) an R, V, C, or S residue at position 71 of SEQ ID NO: 1; V, S, A, T, N, D, or H residue; (d) R at a position corresponding to position 72 of SEQ ID NO: 1 , T, S, G, H, K, or Y residue; (e) A at a position corresponding to position 73 of SEQ ID NO: 1; V, or T residue; and (f) S, T, A, or is a G residue.

[0436] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 436-462 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 in any one of It will be modified as follows.

[0437] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or S residue at the corresponding position; (b) a Q or E at the position corresponding to position 80 of SEQ ID NO:1 residue; and (c) a K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0438] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 436-462 is modified to include residues corresponding to residues 19, 80, and 139 of any one of .

[0439] In some embodiments of this method, the central sequence consists of GTGC.

[0440] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, L, H, A, R, N, or S residue at the corresponding position; ...c) a K, L, H, A, R, N, or S residue at the corresponding position; (d) a K, L, H, A, R, N, or S residue at the corresponding position; (e) a K, L, H, A, R, N, or S residue at the corresponding position; (f) a K, L (c) an R, S, V, K, I, or G residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) a G, S, N, I, R, A, E, Q, Y, T, K, F, or V residue at each position; R, K, G, H, P, S, C, N, T, A, M, D at the position corresponding to position 72 of 1, or Q residue; (e) an A, V, T, N, C, or L residue at a position corresponding to position 73 of SEQ ID NO:1 and (f) an S, A, or T residue at a position corresponding to position 74 of SEQ ID NO:1.

[0441] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 465-495 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 in any one of It will be modified as follows.

[0442] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or S residue at the corresponding position; (b) a Q or E at the position corresponding to position 80 of SEQ ID NO:1 residue; and (c) K, T, S, R, H, or a residue at a position corresponding to position 139 of SEQ ID NO:1. V residue.

[0443] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 465-495 is modified to include residues corresponding to residues 19, 80, and 139 of any one of .

[0444] In some embodiments of this method, the central sequence consists of GTGG.

[0445] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) an R residue at a position corresponding to position 71 of SEQ ID NO: 1; (c) an S residue at a position corresponding to position 72 of SEQ ID NO: 1; (d) a G residue at a position corresponding to position 72 of SEQ ID NO:1; and (e) a G residue at a position corresponding to position 73 of SEQ ID NO:1. to R residue.

[0446] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 498-501 The nucleotide sequence is modified to include residues corresponding to residues 50, 71, 72, and 73 of the nucleotide sequence ...

[0447] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A residue at the corresponding position; (b) an I residue at the position corresponding to position 62 of SEQ ID NO: 1; and (c) ) a Q residue at a position corresponding to position 80 of SEQ ID NO:1.

[0448] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 498-501 The nucleotide sequence is modified to include residues corresponding to residues 19, 62, and 80 of the nucleotide sequence ...

[0449] In some embodiments of this method, the middle sequence consists of GTGT.

[0450] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a K, S, L, V, G, R, or N residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a Q, V, R, S, K, A, E, or C residue at position 71 of SEQ ID NO: 1; (d) a G, R, N, H, A, or T residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) an R, P, A, Q, K, T, G, or V residue at position 73 of SEQ ID NO: 1; (f) an A, S, C, or T residue at a position corresponding to position 74 of SEQ ID NO:1; and S, A, or T residues.

[0451] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 504-529 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 in any one of It will be modified as follows.

[0452] In some embodiments of this method, the method comprises: (a) at position 19 of SEQ ID NO: 1 (b) an A or S residue at the corresponding position; (b) a Q or E at the position corresponding to position 80 of SEQ ID NO:1 residue; and (c) a K or R residue at a position corresponding to position 139 of SEQ ID NO:1.

[0453] In some embodiments of this method, the first subunit is selected from the group consisting of SEQ ID NOs: 504-529 is modified to include residues corresponding to residues 19, 80, and 139 of any one of .

[0454] Another embodiment is ACAA, ACAG, ACAT, ACGA, ACGC, ACGG, ACG T, ATAA, ATAG, ATAT, ATGA, ATGG, TTGG, GCAA, GCA Recognition sequences containing a central sequence consisting of T, GCGA, GCAG, TCAA, or TTAA An engineered meganuclease derived from I-CreI that binds and cleaves the first subunit a first subunit and a second subunit; Each of the subunits comprises an amino acid sequence derived from SEQ ID NO: 1, and the first and second subunits The two subunits are located at positions 48, 50, 71, 72, and 73 of SEQ ID NO: 1, respectively. and an engineered meganuclease comprising substitutions at one or more positions corresponding to position 74. be.

[0455] Another embodiment is ACAA, ACAG, ACAT, ACGA, ACGC, ACGG, ACG T, ATAA, ATAG, ATAT, ATGA, ATGG, TTGG, GCAA, GCA Recognition sequences containing a central sequence consisting of T, GCGA, GCAG, TCAA, or TTAA The first subunit and the second subunit bind to and cleave the The unit and the second subunit each contain an amino acid sequence derived from SEQ ID NO: 1. Improved I-CreI derived engineered meganucleases, wherein the improvement is ACA A, ACAG, ACAT, ACGA, ACGC, ACGG, ACGT, ATAA, ATA G, ATAT, ATGA, ATGG, TTGG, GCAA, GCAT, GCGA, GCA Engineered I-Cre against recognition sequences containing G, TCAA, or TTAA central sequences any amino acid substitution described herein that improves the cleavage activity of I-derived meganucleases. , an improved I-CreI-derived engineered meganuclease.

[0456] In some embodiments, the first subunit comprises one or more of the following residues: (a) A, C, D, G, H, I, K, L, N, Q at the position corresponding to position 48 of SEQ ID NO: 1 , R, S, or T residue; (b) A, C, D, E, or (c) a G, I, K, L, N, Q, R, S, T, V, or W residue at position 71 of SEQ ID NO:1; an A, C, G, H, I, K, N, P, R, S, or T residue at the corresponding position; (d) sequence no. A, D, G, H, K, L, M, N, P, Q, R, S, T at the position corresponding to position 72 of No. 1 or V residue; (e) A, C, G, I, S, T, or V residue at a position corresponding to position 73 of SEQ ID NO:1. or V residue; and (f) an A, C, T, or S residue at a position corresponding to position 74 of SEQ ID NO:1. Base.

[0457] In some embodiments, the second subunit comprises one or more of the following residues: (a) A, C, G, H, I, K, L, N, Q, R at the position corresponding to position 48 of SEQ ID NO: 1 , S, or T residue; (b) A, C, E, G, H, or (c) a residue I, K, N, P, Q, R, S, T, or V corresponding to position 71 of SEQ ID NO: 1; (d) residues at positions A, D, E, G, H, I, K, N, P, Q, R, S, T, or Y; A, C, E, G, H, I, K, M, N, P, Q, R, at the position corresponding to the 72nd position in column number 1. (e) an S, T, V, or Y residue; (f) an A, C, G, or H residue at a position corresponding to position 73 of SEQ ID NO:1; , I, R, S, T, or V residue; and (f) A at a position corresponding to position 74 of SEQ ID NO:1. , C, S, or T residues.

[0458] In some embodiments, the central sequence is ACAA, ACAG, ACAT, ACGC, A CGG, or ACGT, and the first subunit consists of one or more of the following residues: (a) A, C, G, H, I, K, L, N at a position corresponding to position 48 of SEQ ID NO: 1, (b) A, C, K, Q, R, S at the position corresponding to position 50 of SEQ ID NO: 1 (c) an A, G, P, or T residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) an H, K, P, Q, R, or T residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) an A, C, G, or V residue at a position corresponding to position 73 of SEQ ID NO: 1; and (f ) an S residue at a position corresponding to position 74 of SEQ ID NO:1.

[0459] In some embodiments, the central sequence is ATAA, ATAG, ATAT, ATGA, A TGG, and the first subunit contains one or more of the following residues: (a) the sequence A, C, D, G, H, K, L, N, Q, S, or T in the position corresponding to the 48th position in column number 1 Residues (b) C, D, E, G, I, K, N, R, S at the position corresponding to position 50 of SEQ ID NO: 1 , T, or V residue; (c) a G, H, I, K, N, or V residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) A, G, H, K, L, N at the position corresponding to position 72 of SEQ ID NO: 1 , P, Q, R, S, or T residue; (e) A, C, or S, or T residue; and (f) A, C, or S at a position corresponding to position 74 of SEQ ID NO:1. residue.

[0460] In some embodiments, the central sequence is GCAA, GCAT, GCGA, or GCA G, and the first subunit comprises one or more of the following residues: (a) SEQ ID NO: (b) an A, H, K, or R residue at a position corresponding to position 48 of SEQ ID NO: 1; a C, K, L, Q, R, S, T, or V residue at the corresponding position; (c) position 71 of SEQ ID NO: 1; (d) an A, G, H, N, R, S, or T residue at position 72 of SEQ ID NO: 1; an A, G, H, M, N, P, Q, R, S, T, or V residue at the corresponding position; (e) sequence no. (f) an A, C, I, T, or V residue at a position corresponding to position 73 of SEQ ID NO:1; and an A or S residue at the position corresponding to position 74 of

[0461] In some embodiments, the central sequence consists of TTGG or TTAA, and the first subsequence The unit comprises one or more of the following residues: (a) corresponding to position 48 of SEQ ID NO: 1 (b) a K, N, R, or S residue at position 50 of SEQ ID NO: 1; (c) a K, R, S, T, or V residue at a position corresponding to position 71 of SEQ ID NO: 1; , N, R, or S residue; (d) A, D, H, K, or S residue at a position corresponding to position 72 of SEQ ID NO: 1; an N, Q, R, S, or T residue; (e) an I or V at a position corresponding to position 73 of SEQ ID NO: 1; residue; and (f) an A, S, or T residue at a position corresponding to position 74 of SEQ ID NO:1.

[0462] In some embodiments, the central sequence consists of TCAA and the first subunit consists of Contains one or more of the following residues: (a) A, G, or a nucleotide at a position corresponding to position 48 of SEQ ID NO:1; H, K, N, Q, R, or S residue; (b) C, R at the position corresponding to position 50 of SEQ ID NO: 1 (c) a G, R, S, or T residue at a position corresponding to position 71 of SEQ ID NO:1 (d) a G, H, P, R, S, or T residue at a position corresponding to position 72 of SEQ ID NO:1; (e) an I or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) a residue of SEQ ID NO:1 an A or S residue at the position corresponding to position 74;

[0463] In some embodiments, the central sequence is ACAA, ACAG, ACAT, ACGC, A CGG, or ACGT, and the second subunit consists of one or more of the following residues: (a) A, C, G, H, K, L, N, Q at a position corresponding to position 48 of SEQ ID NO: 1, (b) an R, S, or T residue; (b) an A, C, G, H, K at a position corresponding to position 50 of SEQ ID NO:1 , L, N, Q, R, S, or T residue; (c) A at a position corresponding to position 71 of SEQ ID NO: 1, D, E, G, H, K, N, P, R, S, or T residue; (d) corresponding to position 72 of SEQ ID NO: 1 (e) a sequence number of an A, G, H, K, M, N, P, P, Q, R, S, or T residue at a position corresponding to the sequence number; (f) an A, C, G, H, I, R, S, T, or V residue at a position corresponding to position 73 of No. 1; Optionally, an R residue at position (73B) immediately following the position corresponding to position 73 of SEQ ID NO: 1; and and (g) an A, C, S, or T residue at a position corresponding to position 74 of SEQ ID NO:1.

[0464] In some embodiments, the central sequence is ATAA, ATAG, ATAT, ATGA, or or ATGG, and the second subunit contains one or more of the following residues: a) A, C, G, H, K, N, Q, R, S, or a residue at the position corresponding to position 48 of SEQ ID NO: 1 T residue; (b) A, C, E, I, K, N, Q, R, or a residue corresponding to position 50 of SEQ ID NO: 1 (c) an S, C, E, I, K, or N residue at a position corresponding to position 71 of SEQ ID NO: 1; , Q, R, S, or T residue; (d) A, G, H, or a K, N, Q, R, S, T, V, or Y residue; (e) a position corresponding to position 73 of SEQ ID NO: 1 (f) optionally, an A, C, G, H, I, R, S, or V residue at position 73 of SEQ ID NO: 1; and (g) an R residue at position (73B) immediately following the position corresponding to position 74 of SEQ ID NO: 1; A, C, S, or T residue at the corresponding position.

[0465] In some embodiments, the central sequence is GCAA, GCAT, GCGA, or GCA G, and the second subunit comprises one or more of the following residues: (a) SEQ ID NO: A, C, G, H, I, K, L, N, Q, R, S, or T at the position corresponding to position 48 of No. 1 (b) residues C, E, H, K, Q, R, S, T, or or V residue; (c) A, G, H, K, R, S, T, or V residue at a position corresponding to position 71 of SEQ ID NO: 1. or Y residue; (d) A, C, E, G, H, K, N at a position corresponding to position 72 of SEQ ID NO: 1 , Q, R, S, T, or Y residue; (e) A, C, or a G, H, I, R, S, or V residue; and (f) a position corresponding to position 74 of SEQ ID NO:1. A, S, or T residues.

[0466] In some embodiments, the central sequence consists of TTGG or TTAA, and the second subsequence The unit comprises one or more of the following residues: (a) corresponding to position 48 of SEQ ID NO: 1 (b) an A, K, S, or T residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a K, R, or T residue at a position corresponding to position 71 of SEQ ID NO: 1; , R, S, or T residue; (d) G, I, R, S, or T residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) an I, R, or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an A, S, or T residue at a position corresponding to position 74 of SEQ ID NO:1.

[0467] In some embodiments, the central sequence consists of TCAA and the second subunit consists of (a) a K or S at a position corresponding to position 48 of SEQ ID NO: 1; (b) a C, K, R, or T residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a C, K, R, or T residue at a position corresponding to position 50 of SEQ ID NO: 1; (d) a G, R, or T residue at position 72 of SEQ ID NO:1; (e) a G, P, R, S, or T residue at the corresponding position; (f) a G, P, R, S, or T residue at the corresponding position; and (f) an I or V residue at a position corresponding to position 74 of SEQ ID NO:1. T residue.

[0468] In some embodiments: (a) the central sequence is ACAA and the first subunit is Residues 48, 50, 71, 72, 73, and 74 in any one of columns 11 to 33 (b) the central sequence is ACAG and the first subunit contains residues corresponding to SEQ ID NO:3 Residues corresponding to any one of residues 48, 50, 71, 72, 73, and 74 in any one of 6 to 43 (c) the central sequence is ACAT, and the first subunit is a subunit selected from SEQ ID NOs: 46 to 67 Residues corresponding to any one of residues 48, 50, 71, 72, 73, and 74 of (d) the central sequence is ACGA, and the first subunit is any one of SEQ ID NOs: 70 to 89; or one of residues 48, 50, 71, 72, 73, and 74; (e) The central sequence is ACGC, and the first subunit is any one of SEQ ID NOs: 92 to 118. (f) residues corresponding to residues 48, 50, 71, 72, 73, and 74 of the central sequence The sequence is ACGG, and the first subunit is any one of residues of SEQ ID NOs: 121 to 135. containing residues corresponding to groups 48, 50, 71, 72, 73, and 74, and (g) the central sequence ACGT, and the first subunit is residue 4 of any one of SEQ ID NOS: 138 to 156 (h) The central sequence contains residues corresponding to 8, 50, 71, 72, 73, and 74, and AT AA, and the first subunit is residue 48 of any one of SEQ ID NOs: 159 to 183; It contains residues corresponding to 50, 71, 72, 73, and 74, and (i) the central sequence is ATAG and the first subunit is a sequence of residues 48, 50 of any one of SEQ ID NOs: 186 to 199. (j) the central sequence is ATAT; The first subunit is composed of residues 48, 50, and 7 of any one of SEQ ID NOs: 202 to 219. (k) containing residues corresponding to 1, 72, 73, and 74, and the central sequence is ATGA; The first subunit is composed of residues 48, 50, 71, and 52 of any one of SEQ ID NOs: 222 to 243. (l) the central sequence is ATGG, and the first The subunit is a sequence of residues 48, 50, 71, and 72 of any one of SEQ ID NOs: 246 to 247. (m) the central sequence is TTGG, and the first subunit contains residues corresponding to 1, 73, and 74; The subunit is selected from residues 48, 50, 71, 72, and 73 of any one of SEQ ID NOs: 250 to 266. 3, and 74, (n) the central sequence is GCAA, and the first subunit The unit is a sequence of any one of residues 48, 50, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, and 74, (o) the central sequence is GCAT, and the first subunit The amino acid sequence is residues 48, 50, 71, 72, 73, and 54 of any one of SEQ ID NOs: 294 to 313. and 74, (p) the central sequence is GCGA, and the first subunit is Residues 48, 50, 71, 72, 73, and 7 of any one of SEQ ID NOs: 316 to 325 4, (q) the central sequence is GCAG, and the first subunit contains residues corresponding to the sequence Residues 48, 50, 71, 72, 73, and 74 of any one of the sequences 328 to 330 (r) the central sequence is TCAA and the first subunit is SEQ ID NO: 333–340, corresponding to residues 48, 50, 71, 72, 73, and 74 or (s) the central sequence is TTAA and the first subunit comprises residues Residues 48, 50, 71, 72, 73, and 74 of any one of the sequences 343 to 357 Contains the corresponding residues.

[0469] In some embodiments: (a) the central sequence is ACAA and the second subunit is Residues 48, 50, 71, 72, 73, and 74 in any one of columns 11 to 33 (b) the central sequence is ACAG and the second subunit contains residues corresponding to SEQ ID NO:3 Residues corresponding to any one of residues 48, 50, 71, 72, 73, and 74 in any one of 6 to 43 (c) the central sequence is ACAT, and the second subunit is a subunit selected from the group consisting of SEQ ID NOs: 46 to 67 Residues corresponding to any one of residues 48, 50, 71, 72, 73, and 74 of (d) the central sequence is ACGA, and the second subunit is any of SEQ ID NOs: 70 to 89; or one of residues 48, 50, 71, 72, 73, and 74; (e) The central sequence is ACGC, and the second subunit is any one of SEQ ID NOs: 92 to 118. (f) residues corresponding to residues 48, 50, 71, 72, 73, and 74 of the central sequence The sequence is ACGG, and the second subunit is any one of residues of SEQ ID NOs: 121 to 135. containing residues corresponding to groups 48, 50, 71, 72, 73, and 74, and (g) the central sequence ACGT, and the second subunit is residue 4 of any one of SEQ ID NOS: 138 to 156. (h) The central sequence contains residues corresponding to 8, 50, 71, 72, 73, and 74, and AT AA, and the second subunit is residue 48 of any one of SEQ ID NOs: 159 to 183; It contains residues corresponding to 50, 71, 72, 73, and 74, and (i) the central sequence is ATAG and the second subunit is a sequence of residues 48, 50 of any one of SEQ ID NOs: 186 to 199. (j) the central sequence is ATAT; The second subunit is composed of residues 48, 50, and 7 of any one of SEQ ID NOs: 202 to 219. (k) containing residues corresponding to 1, 72, 73, and 74, and the central sequence is ATGA; The second subunit is composed of residues 48, 50, 71, and 52 of any one of SEQ ID NOs: 222 to 243. (l) the central sequence is ATGG, and the second The subunit is a sequence of residues 48, 50, 71, and 72 of any one of SEQ ID NOs: 246 to 247. (m) the central sequence is TTGG, and the second subunit contains residues corresponding to 73, 74, and 75; The subunit is selected from residues 48, 50, 71, 72, and 73 of any one of SEQ ID NOs: 250 to 266. 3, and 74, and (n) the central sequence is GCAA, and the second subunit The unit is a sequence of any one of residues 48, 50, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, and 74, (o) the central sequence is GCAT, and the second subunit The amino acid sequence is residues 48, 50, 71, 72, 73, and 54 of any one of SEQ ID NOs: 294 to 313. and 74, (p) the central sequence is GCGA, and the second subunit is Residues 48, 50, 71, 72, 73, and 7 of any one of SEQ ID NOs: 316 to 325 4, (q) the central sequence is GCAG, and the second subunit contains residues corresponding to the sequence Residues 48, 50, 71, 72, 73, and 74 of any one of the sequences 328 to 330 (r) the central sequence is TCAA and the second subunit is SEQ ID NO: 333–340, corresponding to residues 48, 50, 71, 72, 73, and 74 or (s) the central sequence is TTAA and the second subunit comprises residues Residues 48, 50, 71, 72, 73, and 74 of any one of the sequences 343 to 357 Contains the corresponding residues.

[0470] Another embodiment is ACAA, ACAG, ACAT, ACGA, ACGC, ACGG, ACG T, ATAA, ATAG, ATAT, ATGA, ATGG, TTGG, GCAA, GCA Meganucleotides containing a central sequence consisting of T, GCGA, GCAG, TCAA, or TTAA A method for cleaving double-stranded DNA at a target site containing a cleavage enzyme recognition sequence, comprising: contacting double-stranded DNA having the structure with an engineered meganuclease described herein; wherein the selected meganuclease binds to and cleaves the recognition sequence.

[0471] Another embodiment is ACAA, ACAG, ACAT, ACGA, ACGC, ACGG, ACG T, ATAA, ATAG, ATAT, ATGA, ATGG, TTGG, GCAA, GCA Meganucleotides containing a central sequence consisting of T, GCGA, GCAG, TCAA, or TTAA A double-stranded DNA having a target site containing a cleavage enzyme recognition sequence is mixed with the double-stranded DNA and a first sample. a first subunit and a second subunit, Each kit contains an engineered I-CreI-derived amino acid sequence derived from SEQ ID NO:1. and an improved method of cleaving at said target site by contacting said target site with a meganuclease. wherein the improvement is achieved by using an engineered I-Cr as described herein that binds to and cleaves the recognition sequence. A method comprising the use of an eI-derived meganuclease.

[0472] Another embodiment is ACAA, ACAG, ACAT, ACGA, ACGC, ACGG, ACG T, ATAA, ATAG, ATAT, ATGA, ATGG, TTGG, GCAA, GCA Recognition sequences containing a central sequence consisting of T, GCGA, GCAG, TCAA, or TTAA The first subunit and the second subunit bind to and cleave the The unit and the second subunit each contain an amino acid sequence derived from SEQ ID NO: 1. A method for enhancing the cleavage activity of an I-CreI engineered meganuclease, comprising: The first subunit and the second subunit were respectively located at positions 48, 50, and 7 of SEQ ID NO: 1. Modifications at one or more positions corresponding to positions 1, 72, 73, and 74 were made to the modified nucleic acid. Enhanced cleavage activity of the nuclease compared to the control engineered meganuclease. a.

[0473] Another embodiment is ACAA, ACAG, ACAT, ACGA, ACGC, ACGG, ACG T, ATAA, ATAG, ATAT, ATGA, ATGG, TTGG, GCAA, GCA Recognition sequences containing a central sequence consisting of T, GCGA, GCAG, TCAA, or TTAA The first subunit and the second subunit bind to and cleave the The unit and the second subunit each contain an amino acid sequence derived from SEQ ID NO: 1. An improved method to enhance the cleavage activity of engineered I-CreI-derived meganucleases wherein the improvement is achieved by using an engineered I-Cr as described herein that binds to and cleaves the recognition sequence. A method comprising the use of an eI-derived meganuclease.

[0474] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) an A, C, D, G, H, I, K, L, N, Q, R, S, or T residue at the corresponding position; A, C, D, E, G, I, K, L, N, Q, R, S at the position corresponding to position 50 of SEQ ID NO: 1 , T, V, or W residue; (c) A, C, G, H, or (d) an I, K, N, P, R, S, or T residue; (d) an A at a position corresponding to position 72 of SEQ ID NO:1 , D, G, H, K, L, M, N, P, Q, R, S, T, or V residue; (e) SEQ ID NO: 1 an A, C, G, I, S, T, or V residue at a position corresponding to position 73 of SEQ ID NO: A, C, T, or S residue at the position corresponding to position 74 of No. 1.

[0475] In some embodiments of this method, the modifying step comprises modifying the second subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) a sequence of A, C, G, H, I, K, L, N, Q, R, S, or T residues at the corresponding positions; A, C, E, G, H, I, K, N, P, Q, R, S, T in the position corresponding to the 50th place of number 1 or V residue; (c) A, D, E, G, H, I, or V residue at the position corresponding to position 71 of SEQ ID NO: 1 a K, N, P, Q, R, S, T, or Y residue; (d) a position corresponding to position 72 of SEQ ID NO: 1 to A, C, E, G, H, I, K, M, N, P, Q, R, S, T, V, or Y residues; (e ) an A, C, G, H, I, R, S, T, or V residue at a position corresponding to position 73 of SEQ ID NO:1 and (f) an A, C, S, or T residue at a position corresponding to position 74 of SEQ ID NO:1.

[0476] In some embodiments of this method, the central sequence is ACAA, ACAG, ACAT, A CGC, ACGG, or ACGT, and the modifying step comprises modifying the first subunit by (a) 48 of SEQ ID NO: 1 to include one or more of the following residues: an A, C, G, H, I, K, L, N, Q, or S residue at a position corresponding to position (b) of sequence no. (c) an A, C, K, Q, R, S, T, V, or W residue at a position corresponding to position 50 of No. 1; (d) an A, G, P, or R residue at a position corresponding to position 71 of SEQ ID NO: 1; (e) an H, K, P, Q, R, or T residue at a position corresponding to position 2 of SEQ ID NO: 1; an A, C, G, or V residue at the corresponding position; and (f) a residue corresponding to position 74 of SEQ ID NO:1. S residue at position.

[0477] In some embodiments of this method, the central sequence is ATAA, ATAG, ATAT, A TGA, or ATGG, and the modifying step comprises modifying the first subunit to the following residues: (a) a sequence corresponding to position 48 of SEQ ID NO: 1; (b) an A, C, D, G, H, K, L, N, Q, S, or T residue at a position corresponding to SEQ ID NO: 1; a C, D, E, G, I, K, N, R, S, T, or V residue at the position corresponding to position 50; (d) a G, H, I, K, N, R, or S residue at a position corresponding to position 71 of SEQ ID NO:1; A, G, H, K, L, N, P, Q, R, S, or (e) an A, C, S, or T residue at a position corresponding to position 73 of SEQ ID NO: 1; and (f) an A, C, or S residue at a position corresponding to position 74 of SEQ ID NO:1.

[0478] In some embodiments of this method, the central sequence is GCAA, GCAT, GCGA, or or GCAG, and the modifying step comprises modifying the first subunit by replacing the first subunit with one of the following residues: (a) modifying the nucleotide sequence of the present invention to include one or more of the following: (a) A at a position corresponding to position 48 of SEQ ID NO: 1; , H, K, or R residue; (b) a C, K, L, Q, or R residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) an R, S, T, or V residue; or (d) an A, G, H, N at a position corresponding to position 71 of SEQ ID NO: 1. , R, S, or T residue; (d) A, G, H, M, or an N, P, Q, R, S, T, or V residue; (e) an A at a position corresponding to position 73 of SEQ ID NO: 1; (f) an A or C residue at a position corresponding to position 74 of SEQ ID NO:1; is an S residue.

[0479] In some embodiments of this method, the central sequence consists of TTGG or TTAA; The modifying step modifies the first subunit to include one or more of the following residues: (a) adding a K, N, R, or S residue at a position corresponding to position 48 of SEQ ID NO:1 (b) a C, E, K, R, S, T, or V residue at a position corresponding to position 50 of SEQ ID NO:1; (c) an A, G, K, N, R, or S residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) an A, D, H, K, N, Q, R, S, or T residue at a position corresponding to position 72 of SEQ ID NO:1; (e) an I or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) a residue of SEQ ID NO:1 an A, S, or T residue at the position corresponding to position 74;

[0480] In some embodiments of this method, the central sequence consists of TCAA and the modifying step comprises: The first subunit is modified to include one or more of the following residues: (a) an A, G, H, K, N, Q, R, or S residue at a position corresponding to position 48 of SEQ ID NO:1 (b) a C, R, S, or T residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a C, R, S, or T residue at a position corresponding to position 50 of SEQ ID NO: 1; (d) a G, R, S, or T residue at a position corresponding to position 71 of SEQ ID NO: 1; (e) a G, H, P, R, S, or T residue at the corresponding position; (f) a G, H, P, R, S, or T residue at the corresponding position; (f) an I or V residue at a position corresponding to position 74 of SEQ ID NO: 1; and residue.

[0481] In some embodiments of this method, the central sequence is ACAA, ACAG, ACAT, A CGC, ACGG, or ACGT, and the modifying step comprises modifying the second subunit to: (a) 48 of SEQ ID NO: 1 to include one or more of the following residues: (b) an A, C, G, H, K, L, N, Q, R, S, or T residue at a position corresponding to the amino acid sequence; A, C, G, H, K, L, N, Q, R, S, or T in the position corresponding to the 50th position in column number 1 Residues; (c) A, D, E, G, H, K, N, P, R at the position corresponding to position 71 of SEQ ID NO: 1 , S, or T residue; (d) A, G, H, K, M, or T residue at a position corresponding to position 72 of SEQ ID NO: 1; an N, P, P, Q, R, S, or T residue; (e) an A at a position corresponding to position 73 of SEQ ID NO: 1; , C, G, H, I, R, S, T, or V residue; (f) optionally, position 73 of SEQ ID NO: 1 and (g) an R residue at position (73B) immediately following the position corresponding to position 74 of SEQ ID NO: 1; A, C, S, or T residue at the corresponding position.

[0482] In some embodiments of this method, the central sequence is ATAA, ATAG, ATAT, A TGA, or ATGG, and the modifying step modifies the second subunit to the following residues: (a) a sequence corresponding to position 48 of SEQ ID NO: 1; (b) an A, C, G, H, K, N, Q, R, S, or T residue at a position corresponding to 50 of SEQ ID NO: 1; an A, C, E, I, K, N, Q, R, S, or T residue at a position corresponding to position (c) of sequence no. A, C, E, I, K, N, Q, R, S, or T residue at the position corresponding to position 71 of No. 1; d) A, G, H, K, N, Q, R, S, T, V, or or Y residue; (e) A, C, G, H, I, R, S, or Y residue at a position corresponding to position 73 of SEQ ID NO: 1; or V residue; (f) optionally, a position immediately following the position corresponding to position 73 of SEQ ID NO: 1 (7 3B) an R residue; and (g) an A, C, S, or T residue.

[0483] In some embodiments of this method, the central sequence is GCAA, GCAT, GCGA, or or GCAG, and the modifying step modifies the second subunit by modifying it to one of the following residues: (a) modifying the nucleotide sequence of the present invention to include one or more of the following: (a) A at a position corresponding to position 48 of SEQ ID NO: 1; , C, G, H, I, K, L, N, Q, R, S, or T residue; (b) position 50 of SEQ ID NO: 1; (c) a C, E, H, K, Q, R, S, T, or V residue at a position corresponding to SEQ ID NO: 1; (d) an A, G, H, K, R, S, T, or Y residue at a position corresponding to position 71; A, C, E, G, H, K, N, Q, R, S, T, or Y residue at the position corresponding to position 72 of (e) an A, C, G, H, I, R, S, or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an A, S, or T residue at a position corresponding to position 74 of SEQ ID NO:1.

[0484] In some embodiments of this method, the central sequence consists of TTGG or TTAA; The modifying step modifies the second subunit to include one or more of the following residues: (a) an A, K, S, or T residue at a position corresponding to position 48 of SEQ ID NO:1 (b) a C, E, K, R, or T residue at a position corresponding to position 50 of SEQ ID NO: 1; (c) a C, E, K, R, or T residue at a position corresponding to position 50 of SEQ ID NO: 1; (d) an A, D, G, K, Q, R, S, or T residue at the position corresponding to position 71 in column number 1; (e) a G, I, R, S, T, or V residue at a position corresponding to position 72 of SEQ ID NO: 1; (f) an I, R, or V residue at a position corresponding to position 73 of SEQ ID NO: 1; and (f) position 74 of SEQ ID NO: 1. A, S, or T residue at the corresponding position.

[0485] In some embodiments of this method, the central sequence consists of TCAA and the modifying step comprises: The second subunit is modified to include one or more of the following residues: (a) a K or S residue at a position corresponding to position 48 of SEQ ID NO:1; (b) a K or S residue at position 50 of SEQ ID NO:1 (c) a C, K, R, or T residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) a G, P, R, S, or T residue at a position corresponding to position 72 of SEQ ID NO: 1; or T residue; (e) an I or V residue at a position corresponding to position 73 of SEQ ID NO: 1; and (f ) an A, S, or T residue at a position corresponding to position 74 of SEQ ID NO:1.

[0486] In some embodiments of this method: (a) the central sequence is ACAA and the first subunit is The unit is composed of residues 48, 50, 71, 72, 73, and 54 of any one of SEQ ID NOs: 11 to 33. and 74, (b) the central sequence is ACAG, and the first subunit is Residues 48, 50, 71, 72, 73, and 74 of any one of SEQ ID NOs: 36 to 43 (c) the central sequence is ACAT and the first subunit is SEQ ID NO: 46 to 67, corresponding to residues 48, 50, 71, 72, 73, and 74 (d) the central sequence is ACGA, and the first subunit is SEQ ID NO: 70-8 9, containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of (e) the central sequence is ACGC, and the first subunit is any of SEQ ID NOs: 92 to 118 containing residues corresponding to any one of residues 48, 50, 71, 72, 73, and 74, f) The central sequence is ACGG, and the first subunit is any of SEQ ID NOs: 121 to 135. or one of residues 48, 50, 71, 72, 73, and 74; (g) The central sequence is ACGT, and the first subunit is any one of SEQ ID NOs: 138 to 156. (h) the central residues corresponding to residues 48, 50, 71, 72, 73, and 74; The sequence is ATAA, and the first subunit is any one of SEQ ID NOs: 159 to 183. containing residues corresponding to residues 48, 50, 71, 72, 73, and 74, and (i) the central sequence is ATAG, and the first subunit is any one of residues of SEQ ID NOs: 186 to 199 (j) the central sequence contains residues corresponding to 48, 50, 71, 72, 73, and 74; TAT, the first subunit being residue 48 of any one of SEQ ID NOS: 202 to 219 , 50, 71, 72, 73, and 74, and (k) the central sequence is ATG A, and the first subunit is a sequence of residues 48, 5 of any one of SEQ ID NOs: 222 to 243. (l) contains residues corresponding to 0, 71, 72, 73, and 74, and the central sequence is ATGG. The first subunit is a sequence of residues 48, 50, and 60 of any one of SEQ ID NOs: 246 to 247. (m) containing residues corresponding to 71, 72, 73, and 74, with the central sequence TTGG The first subunit is a sequence of residues 48, 50, 71 of any one of SEQ ID NOs: 250 to 266. , 72, 73, and 74, (n) the central sequence is GCAA, and The first subunit is composed of residues 48, 50, 71, and 72 of any one of SEQ ID NOs: 269 to 291. (o) the central sequence is GCAT, and the first The subunit is composed of residues 48, 50, 71, 72, or any one of SEQ ID NOs: 294 to 313. (p) The central sequence is GCGA, and the first subunit contains residues corresponding to 73 and 74. The unit is composed of residues 48, 50, 71, 72, and 73 of any one of SEQ ID NOs: 316 to 325. , and residues corresponding to 74, (q) the central sequence is GCAG, and the first subunit The set is composed of residues 48, 50, 71, 72, 73, and 54 of any one of SEQ ID NOs: 328 to 330. and 74, (r) the central sequence is TCAA, and the first subunit residues 48, 50, 71, 72, 73, and any one of SEQ ID NOs: 333 to 340; 74, or (s) the central sequence is TTAA and the first subunit The target is residues 48, 50, 71, 72, 73, and 54 of any one of SEQ ID NOs: 343 to 357. and 74.

[0487] In some embodiments of this method: (a) the central sequence is ACAA and the second subunit is The unit is composed of residues 48, 50, 71, 72, 73, and 54 of any one of SEQ ID NOs: 11 to 33. and 74, (b) the central sequence is ACAG, and the second subunit is Residues 48, 50, 71, 72, 73, and 74 of any one of SEQ ID NOs: 36 to 43 (c) the central sequence is ACAT and the second subunit is SEQ ID NO: 46 to 67, corresponding to residues 48, 50, 71, 72, 73, and 74 (d) the central sequence is ACGA, and the second subunit is SEQ ID NO: 70-8 9, containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of (e) the central sequence is ACGC, and the second subunit is any of SEQ ID NOs: 92 to 118 containing residues corresponding to any one of residues 48, 50, 71, 72, 73, and 74, f) The central sequence is ACGG, and the second subunit is any of SEQ ID NOs: 121 to 135. or one of residues 48, 50, 71, 72, 73, and 74; (g) The central sequence is ACGT, and the second subunit is any one of SEQ ID NOs: 138 to 156. (h) the central residues corresponding to residues 48, 50, 71, 72, 73, and 74; The sequence is ATAA, and the second subunit is any one of SEQ ID NOs: 159 to 183. containing residues corresponding to residues 48, 50, 71, 72, 73, and 74, and (i) the central sequence is ATAG, and the second subunit is any one of residues 186 to 199 (j) the central sequence contains residues corresponding to 48, 50, 71, 72, 73, and 74; TAT, and the second subunit is residue 48 of any one of SEQ ID NOs: 202 to 219. , 50, 71, 72, 73, and 74, and (k) the central sequence is ATG A, and the second subunit is a sequence of residues 48, 5 of any one of SEQ ID NOs: 222 to 243. (l) contains residues corresponding to 0, 71, 72, 73, and 74, and the central sequence is ATGG. the second subunit is a sequence of residues 48, 50, and 60 of any one of SEQ ID NOs: 246-247; (m) containing residues corresponding to 71, 72, 73, and 74, with the central sequence TTGG The second subunit is a sequence of residues 48, 50, 71 of any one of SEQ ID NOs: 250 to 266. , 72, 73, and 74, (n) the central sequence is GCAA, and The second subunit is composed of residues 48, 50, 71, and 7 of any one of SEQ ID NOs: 269 to 291. (o) the central sequence is GCAT, and the second The subunit is composed of residues 48, 50, 71, 72, or any one of SEQ ID NOs: 294 to 313. The second subunit contains residues corresponding to 73 and 74, and the central sequence is GCGA. The unit is composed of residues 48, 50, 71, 72, and 73 of any one of SEQ ID NOs: 316 to 325. , and residues corresponding to 74, (q) the central sequence is GCAG, and the second subunit The set is composed of residues 48, 50, 71, 72, 73, and 54 of any one of SEQ ID NOs: 328 to 330. and 74, (r) the central sequence is TCAA, and the second subunit residues 48, 50, 71, 72, 73, and any one of SEQ ID NOs: 333 to 340; 74, or (s) the central sequence is TTAA and the second subunit The target is residues 48, 50, 71, 72, 73, and 54 of any one of SEQ ID NOs: 343 to 357. and 74.

[0488] Another embodiment is GTAA, GTAG, GTAT, GTGA, GTGC, GTGG, or I-CreI-derived recombinant polypeptides with specificity for recognition sequences containing the central sequence GTGT A meganuclease constructed according to the present invention, comprising a first subunit and a second subunit. the first subunit comprises an amino acid sequence derived from SEQ ID NO: 1, The fragments correspond to positions 48, 50, 71, 72, 73, and 74 of SEQ ID NO: 1. Engineered meganucleases derived from I-CreI containing substitutions at one or more positions .

[0489] Another embodiment is GTAA, GTAG, GTAT, GTGA, GTGC, GTGG, or The first subunit and the second subunit bind to and cleave the recognition sequence containing the central sequence GTGT. and a second subunit, wherein the first subunit and the second subunit are each an improved I-CreI-derived engineered membrane comprising an amino acid sequence derived from SEQ ID NO: 1; The improved ganucleases are GTAA, GTAG, GTAT, GTGA, GTGC Any of the amino acids described herein that improve the cleavage activity of the GTGG, GTGT, or GTGT central sequences. An improved I-CreI-derived engineered meganuclease containing an amino acid substitution.

[0490] In some embodiments, the first subunit comprises one or more of the following residues: (a) A, C, G, H, K, L, M, N, Q, R at the position corresponding to position 48 of SEQ ID NO: 1 , S, T, or V residue; (b) A, C, E, G, or V at a position corresponding to position 50 of SEQ ID NO:1; an I, K, L, Q, R, S, T, or V residue; (c) a position corresponding to position 71 of SEQ ID NO: 1 to A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, or Y residues; (d ) A, C, D, G, H, K, M, N, P, Q, R, or (e) an S, T, V, W, or Y residue; (f) an A, C, I residue at a position corresponding to position 73 of SEQ ID NO: 1; , L, N, R, S, T, or V residue; and (f) a position corresponding to position 74 of SEQ ID NO:1. A, C, G, S, or T residues.

[0491] In some embodiments, the second subunit comprises one or more of the following residues: (a) a K residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K residue at a position corresponding to position 50 of SEQ ID NO: 1 (c) a Q residue at a position corresponding to position 71 of SEQ ID NO: 1; (d) a G residue at a position corresponding to position 72 of SEQ ID NO: 1; (e) an S residue at a position corresponding to position 72 of SEQ ID NO: 1; (f) a V residue at a position corresponding to position 73 of SEQ ID NO: 1 and (f) an S residue at a position corresponding to position 74 of SEQ ID NO:1.

[0492] In some embodiments: (a) the central sequence is GTAA and the first subunit is Residues 48, 50, 71, 72, 73, and 74 in any one of columns 360 to 389 (b) the central sequence is GTAG and the first subunit contains residues corresponding to sequence no. Residues 48, 50, 71, 72, 73, and 74 of any one of Nos. 392 to 399 (c) the central sequence is GTAT and the first subunit contains residues corresponding to SEQ ID NO:4 Residues 48, 50, 71, 72, 73, and 74 of any one of 02 to 433 (d) the central sequence is GTGA and the first subunit is SEQ ID NO: 436 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of ~462 (e) the central sequence is GTGC, and the first subunit is SEQ ID NOs: 465-464 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 95 (f) the central sequence is GTGG, and the first subunit is a sequence selected from SEQ ID NOs: 498 to 501. Residues corresponding to any one of residues 48, 50, 71, 72, 73, and 74 of or (g) the central sequence is GTGT and the first subunit is SEQ ID NOs: 504-505 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 29 include.

[0493] Another embodiment is GTAA, GTAG, GTAT, GTGA, GTGC, GTGG, or Double-stranded DNA at the target site containing a meganuclease recognition sequence containing the central sequence GTGT A method for cleaving A, comprising: subjecting a double-stranded DNA having a target site to any of the methods described herein; and contacting the engineered meganuclease with any engineered meganuclease, wherein the engineered meganuclease recognizes the recognition sequence. and cleaving the

[0494] Another embodiment is GTAA, GTAG, GTAT, GTGA, GTGC, GTGG, or Two target sites containing meganuclease recognition sequences containing the central sequence GTGT The double-stranded DNA is then subjected to a step of synthesizing the double-stranded DNA, the step comprising a first subunit and a second subunit. The first subunit and the second subunit each comprise an amino acid sequence derived from SEQ ID NO: 1. by contacting the I-CreI-derived meganuclease with an engineered I-CreI-derived meganuclease containing the amino acid sequence an improved method of cleaving at said target site by binding to said recognition sequence, and methods comprising the use of the engineered I-CreI-derived meganucleases described herein to cleave the I-CreI gene. It is the law.

[0495] Another embodiment is GTAA, GTAG, GTAT, GTGA, GTGC, GTGG, or The first subunit and the second subunit bind to and cleave the recognition sequence containing the central sequence GTGT. and a second subunit, wherein the first subunit has an amino acid sequence derived from SEQ ID NO: 1. A method for enhancing the cleavage activity of an engineered meganuclease derived from I-CreI, comprising: The first subunit is composed of the amino acids at positions 48, 50, 71, 72, 73, and 74 of SEQ ID NO: 1. and 74, and the modified nuclease was and having enhanced cleavage activity when compared to the meganucleases produced by the method. It is the law.

[0496] Another embodiment is GTAA, GTAG, GTAT, GTGA, GTGC, GTGG, or The first subunit and the second subunit bind to and cleave the recognition sequence containing the central sequence GTGT. and a second subunit, wherein the first subunit and the second subunit are each enhances the cleavage activity of an engineered meganuclease comprising an amino acid sequence derived from SEQ ID NO: 1. an improved method for enhancing the recognition of a target gene, wherein the improvement comprises the step of: A method comprising the use of the engineered I-CreI-derived meganucleases described.

[0497] In some embodiments of this method, the modifying step comprises modifying the first subunit by: (a) modifying the nucleotide sequence of SEQ ID NO: 1 to include one or more of the following residues: (b) an A, C, G, H, K, L, M, N, Q, R, S, T, or V residue at the corresponding position; A, C, E, G, I, K, L, Q, R, S, T, or or V residue; (c) A, D, E, F, G, H, I, or V residue at a position corresponding to position 71 of SEQ ID NO: 1. (d) a K, L, N, Q, R, S, T, V, or Y residue; (d) a residue corresponding to position 72 of SEQ ID NO: 1 A, C, D, G, H, K, M, N, P, Q, R, S, T, V, W, or Y residue at position; (e) A, C, I, L, N, R, S, T, or V at the position corresponding to position 73 of SEQ ID NO: 1 residue; and (f) an A, C, G, S, or T residue at a position corresponding to position 74 of SEQ ID NO:1. .

[0498] In some embodiments of this method, the second subunit contains one of the following residues: (a) a K residue at a position corresponding to position 48 of SEQ ID NO: 1; (b) a K residue at a position corresponding to position 5 of SEQ ID NO: 1; (c) a Q residue at a position corresponding to position 0 of SEQ ID NO: 1; (d) a G residue at a position corresponding to position 71 of SEQ ID NO: 1; (e) an S residue at a position corresponding to position 73 of SEQ ID NO: 1; and (f) a V residue at a position corresponding to position 74 of SEQ ID NO:1.

[0499] In some embodiments of this method: (a) the central sequence is GTAA and the first subunit is The unit is a sequence of any one of residues 48, 50, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, and 74, (b) the central sequence is GTAG, and the first subunit The amino acid sequence is residues 48, 50, 71, 72, 73, and 54 of any one of SEQ ID NOs: 392 to 399. and 74, (c) the central sequence is GTAT, and the first subunit is Residues 48, 50, 71, 72, 73, and 7 of any one of SEQ ID NOs: 402 to 433 4, (d) the central sequence is GTGA, and the first subunit contains residues corresponding to the sequence Residues 48, 50, 71, 72, 73, and 74 of any one of the sequences 436 to 462 (e) the central sequence is GTGC and the first subunit contains the corresponding residues; 465–495, corresponding to residues 48, 50, 71, 72, 73, and 74 (f) the central sequence is GTGG and the first subunit comprises residues corresponding to SEQ ID NO: 49 8 to 501, corresponding to residues 48, 50, 71, 72, 73, and 74 or (g) the central sequence is GTGT and the first subunit comprises residues 504 to 529, corresponding to residues 48, 50, 71, 72, 73, and 74 Contains residues that

[0500] Another embodiment is ACAA, ACAG, ACAT, ACGA, ACGC, ACGG, ACG T, ATAA, ATAG, ATAT, ATGA, ATGG, TTGG, GCAA, GCA a central sequence selected from the group consisting of T, GCGA, GCAG, TCAA, or TTAA It was an engineered I-CreI-derived meganuclease that binds to and cleaves a recognition sequence containing and a first subunit and a second subunit, at least one of the second subunits is at positions 48, 50, 71, Except for amino acid substitutions at one or more positions corresponding to positions 72, 73, and 74, the sequence Column number 1 and at least 75%, at least 80%, at least 85%, at least 88% , at least 90%, at least 92%, at least 94%, at least 96%, less and engineered sequences containing at least 97%, at least 98%, or at least 99% sequence identity. It is an I-CreI-derived meganuclease.

[0501] In some embodiments, a small amount of the first subunit or the second subunit At least one of the amino acids at positions 48, 50, 71, 72, 73, and 74 of SEQ ID NO: 1 A sequence identical to SEQ ID NO: 1, except for amino acid substitutions at one or more positions corresponding to SEQ ID NO: 1, with at least 85% similarity. Another aspect is to compare any of the engineered meganucleases described herein with In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding the The protease is mRNA.

[0502] Another aspect is a nucleic acid sequence encoding any of the engineered meganucleases described herein. In some embodiments, the recombinant DNA construct comprises a polynucleotide comprising a sequence. The recombinant DNA construct encodes a recombinant virus containing the polynucleotide. In this embodiment, the recombinant virus is a recombinant adenovirus, a recombinant lentivirus, a recombinant Recombinant retroviruses, or recombinant adeno-associated viruses (AAV). In an embodiment, the recombinant virus is a recombinant AAV.

[0503] Another aspect is a nucleic acid sequence encoding any of the engineered meganucleases described herein. In some embodiments, the recombinant virus comprises a polynucleotide comprising the sequence. The viruses include recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, or a recombinant AAV. In some embodiments, the recombinant virus is a recombinant AAV is.

[0504] Another aspect is a gene having a target sequence disrupted in the chromosome of a genetically modified eukaryotic cell. A method for producing a modified eukaryotic cell, comprising administering to a eukaryotic cell a gene encoding a gene encoding a target gene, the gene being expressed in the eukaryotic cell. a polypeptide comprising a nucleic acid sequence encoding any of the engineered meganucleases described herein, and introducing a nucleotide into the recognition sequence of the engineered meganuclease. It generates a break site in the chromosome and the target sequence is destroyed by non-homologous end joining at the break site. This is a method.

[0505] In some embodiments of this method, the nucleic acid is delivered by mRNA or a recombinant virus. In some embodiments of this method, the eukaryotic cell is a mammalian cell. In some embodiments of this method, the eukaryotic cell is a human cell. In some embodiments, the eukaryotic cell is a plant cell.

[0506] Another aspect is a gene having a target sequence disrupted in the chromosome of a genetically modified eukaryotic cell. A method for producing a modified eukaryotic cell, comprising: subjecting a eukaryotic cell to any of the procedures described herein. and introducing an engineered meganuclease into the target gene, wherein the engineered meganuclease has a recognition sequence. and the target sequence is inserted into the chromosome by non-homologous end joining at the break site. It is a way to be destroyed.

[0507] In some embodiments of this method, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a human cell. The eukaryotic cell is a plant cell.

[0508] Another aspect is a gene comprising an exogenous sequence of interest inserted into the chromosome of a genetically modified eukaryotic cell. A method for producing a modified eukaryotic cell, comprising: (a) injecting into a eukaryotic cell a gene encoding a gene that is expressed in said eukaryotic cell; a first nucleic acid encoding any of the engineered meganucleases described herein, and (b) a second nucleic acid sequence comprising the sequence of interest. The engineered meganuclease cleaves the chromosome at the recognition sequence, A site is created and the sequence of interest is inserted into the chromosome at the cleavage site.

[0509] In some embodiments of this method, the second nucleic acid sequence is complementary to the sequence adjacent to the cleavage site. The desired sequence is inserted into the cleavage site by homologous recombination. In some embodiments of the method, the first nucleic acid sequence is expressed by mRNA or a recombinant virus. In some embodiments of this method, the second nucleic acid is introduced into the eukaryotic cell via recombinant In some embodiments of this method, the eukaryotic cell is introduced by a virus. is a mammalian cell. In some embodiments of this method, the eukaryotic cell is a human cell. In some embodiments of this method, the eukaryotic cell is a plant cell.

[0510] Another aspect is a gene comprising an exogenous sequence of interest inserted into the chromosome of a genetically modified eukaryotic cell. A method for producing a modified eukaryotic cell, comprising: (a) any engineered eukaryotic cell described herein; (b) introducing the meganuclease into a eukaryotic cell; and (b) introducing a nucleic acid sequence containing the sequence of interest into the eukaryotic cell. introducing into a eukaryotic cell a polynucleotide comprising the engineered meganuclease; creates a cleavage site in the chromosome at the recognition sequence, and the target sequence is inserted into the chromosome at the cleavage site. The way it is inserted.

[0511] In some embodiments of this method, the polynucleotide is The sequence of interest further comprises a homologous sequence, and is inserted into the cleavage site by homologous recombination. In some embodiments of the method, the polynucleotide is delivered to a eukaryotic cell by a recombinant virus. In some embodiments of this method, the eukaryotic cell is a mammalian cell. In some embodiments of this method, the eukaryotic cell is a human cell. In an embodiment, the eukaryotic cell is a plant cell.

[0512] Another aspect is a method for preparing a genetically modified cell, prepared by any of the methods for preparing a genetically modified cell described herein. It is a genetically modified eukaryotic cell.

[0513] Another aspect is a method for producing a medicament comprising administering to a subject therapies ... a pharmaceutically acceptable carrier and any of the engineered meganucleases described herein. a nucleic acid encoding a meganuclease or any of the engineered meganucleases described herein and a polynucleotide comprising the sequence. The nucleotide is mRNA. In some embodiments, the mRNA is incorporated into a lipid nanoparticle. In some embodiments, the pharmaceutical composition comprises a polynucleotide. In some embodiments, the pharmaceutical composition comprises a recombinant DNA construct containing a polynucleotide. In some embodiments, the recombinant virus comprises a recombinant A It's AV.

[0514] These and other aspects and embodiments of the present invention are described in the following detailed description, figures, and drawings. and the appended claims will be apparent to one skilled in the art. [Brief explanation of the drawings]

[0515] [Figure 1] Schematic diagram of the 22-base pair wild-type I-CreI recognition sequence. The bases in each DNA half-site are numbered from -1 to -9. One of each strand of the four base pairs that make up the central sequence is numbered from +1 to +4. [Figure 2]

[0023] Figure 1 illustrates an engineered meganuclease described herein that comprises two subunits. The first subunit comprises a first hypervariable (HVR1) region that binds to a first recognition half-site of the recognition sequence. Similarly, the second subunit comprises a second hypervariable (HVR2) region that binds to a second recognition half-site of the recognition sequence. In embodiments where the recombinant meganuclease is a single-chain meganuclease, the first subunit comprising the HVR1 region can be positioned as either an N- or C-terminal subunit. Similarly, the second subunit comprising the HVR2 region can be positioned as either an N- or C-terminal subunit. [Figure 3]Schematic diagram of a reporter assay in CHO cells for evaluating recombinant meganucleases targeting test recognition sequences with different four-base pair central sequences. For the recombinant meganucleases described herein, CHO cell lines were generated in which a reporter cassette was stably integrated into the cell's genome. This reporter cassette, from 5' to 3', consists of: the SV40 early promoter; the 5' two-thirds of the GFP gene; the recognition sequence of an engineered meganuclease described herein (e.g., LOX 3-4; SEQ ID NO: 6); the recognition sequence of the CHO-23 / 24 meganuclease (WO / 2012 / 167192); and the 3' two-thirds of the GFP gene. Cells stably transfected with this cassette do not express GFP in the absence of a DNA cleavage-inducing agent. Meganucleases are introduced by transduction of mRNA encoding each meganuclease. Upon induction of DNA cleavage at either of the meganuclease recognition sequences, the duplicated regions of the GFP gene recombine with each other to generate a functional GFP gene. The percentage of GFP-expressing cells can then be determined by flow cytometry as an indirect measure of the frequency of genome cleavage by the meganuclease. [Figure 4] Crystal structure of a modified I-CreI-derived meganuclease (light) overlaid on wild-type I-CreI meganuclease (dark). The mutant meganuclease has modified residues Q50R, G71S, S72G, and V73R, which enhance the cleavage activity of the mutant meganuclease against a recognition sequence containing the four-base-pair central sequence GCAG. The nucleotide G from the mutant I-CreI meganuclease and the nucleotide A from the wild-type I-CreI meganuclease are shown. An overlaid alignment of positions 47, 48, 49, 50, 71, 72, and 73, which are positioned around the central four-base-pair central sequence, is also presented. Finally, a small sphere represents the overlaid metal cofactor, which is thought to be at least partially coordinated by residues 48, 50, 71, 72, 73, and 74.

[0516] A brief description of arrays

[0517] SEQ ID NO: 1 shows the amino acid sequence of wild-type I-CreI.

[0518] SEQ ID NO: 2 shows the amino acid sequence of the LAGLIDADG motif.

[0519] SEQ ID NO: 3 shows the nucleic acid sequence of the wild-type I-CreI recognition sequence (sense).

[0520] SEQ ID NO: 4 shows the nucleic acid sequence of the wild-type I-CreI recognition sequence (antisense). .

[0521] SEQ ID NO: 5 (gtga) shows the nucleic acid sequence of the central sequence of the wild-type I-CreI recognition sequence.

[0522] SEQ ID NO: 6 shows the nucleic acid sequence of the LOX3-4 recognition sequence (sense).

[0523] SEQ ID NO: 7 shows the nucleic acid sequence of the LOX3-4 recognition sequence (antisense).

[0524] SEQ ID NO: 8 shows the amino acid sequence of LOX3-4x.109 meganuclease .

[0525] SEQ ID NO: 9 shows the nucleic acid of the LOX3-4 recognition sequence (sense) having the ACAA central sequence. is doing.

[0526] SEQ ID NO: 10 is a LOX3-4 recognition sequence (antisense) having an ACAA central sequence. Nucleic acids are shown.

[0527] SEQ ID NO: 11 shows the amino acid sequence of LOX3-4m.680 meganuclease. do.

[0528] SEQ ID NO: 12 shows the amino acid sequence of LOX3-4m.683 meganuclease. do.

[0529] SEQ ID NO: 13 shows the amino acid sequence of LOX3-4m.684 meganuclease. do.

[0530] SEQ ID NO: 14 shows the amino acid sequence of LOX3-4m.691 meganuclease. do.

[0531] SEQ ID NO: 15 shows the amino acid sequence of LOX3-4m.693 meganuclease. do.

[0532] SEQ ID NO: 16 shows the amino acid sequence of LOX3-4m.701 meganuclease. do.

[0533] SEQ ID NO: 17 shows the amino acid sequence of LOX3-4m.708 meganuclease. do.

[0534] SEQ ID NO: 18 shows the amino acid sequence of LOX3-4m.714 meganuclease. do.

[0535] SEQ ID NO: 19 shows the amino acid sequence of LOX3-4m.731 meganuclease. do.

[0536] SEQ ID NO: 20 shows the amino acid sequence of LOX3-4m.739 meganuclease. do.

[0537] SEQ ID NO: 21 shows the amino acid sequence of LOX3-4m.741 meganuclease. do.

[0538] SEQ ID NO: 22 shows the amino acid sequence of LOX3-4m.742 meganuclease. do.

[0539] SEQ ID NO: 23 shows the amino acid sequence of LOX3-4m.743 meganuclease. do.

[0540] SEQ ID NO: 24 shows the amino acid sequence of LOX3-4m.744 meganuclease. do.

[0541] SEQ ID NO: 25 shows the amino acid sequence of LOX3-4m.747 meganuclease. do.

[0542] SEQ ID NO: 26 shows the amino acid sequence of LOX3-4m.750 meganuclease. do.

[0543] SEQ ID NO: 27 shows the amino acid sequence of LOX3-4m.756 meganuclease. do.

[0544] SEQ ID NO: 28 shows the amino acid sequence of LOX3-4m.757 meganuclease. do.

[0545] SEQ ID NO: 29 shows the amino acid sequence of LOX3-4m.759 meganuclease. do.

[0546] SEQ ID NO: 30 shows the amino acid sequence of LOX3-4m.762 meganuclease. do.

[0547] SEQ ID NO: 31 shows the amino acid sequence of LOX3-4m.765 meganuclease. do.

[0548] SEQ ID NO: 32 shows the amino acid sequence of LOX3-4m.770 meganuclease. do.

[0549] SEQ ID NO: 33 shows the amino acid sequence of LOX3-4m.771 meganuclease. do.

[0550] SEQ ID NO: 34 is a nucleic acid of the LOX3-4 recognition sequence (sense) having an ACAG central sequence. It shows.

[0551] SEQ ID NO: 35 is a LOX3-4 recognition sequence (antisense) with an ACAG central sequence. Nucleic acids are shown.

[0552] SEQ ID NO: 36 shows the amino acid sequence of LOX3-4m.775 meganuclease. do.

[0553] SEQ ID NO: 37 shows the amino acid sequence of LOX3-4m.776 meganuclease. do.

[0554] SEQ ID NO: 38 shows the amino acid sequence of LOX3-4m.785 meganuclease. do.

[0555] SEQ ID NO: 39 shows the amino acid sequence of LOX3-4m.788 meganuclease. do.

[0556] SEQ ID NO: 40 shows the amino acid sequence of LOX3-4m.815 meganuclease. do.

[0557] SEQ ID NO: 41 shows the amino acid sequence of LOX3-4m.831 meganuclease. do.

[0558] SEQ ID NO: 42 shows the amino acid sequence of LOX3-4m.856 meganuclease. do.

[0559] SEQ ID NO: 43 shows the amino acid sequence of LOX3-4m.863 meganuclease. do.

[0560] SEQ ID NO: 44 represents the nucleic acid of the LOX3-4 recognition sequence (sense) having the ACAT central sequence. It shows.

[0561] SEQ ID NO: 45 is a LOX3-4 recognition sequence (antisense) having an ACAT central sequence. Nucleic acids are shown.

[0562] SEQ ID NO: 46 shows the amino acid sequence of LOX3-4m.869 meganuclease. do.

[0563] SEQ ID NO: 47 shows the amino acid sequence of LOX3-4m.873 meganuclease. do.

[0564] SEQ ID NO: 48 shows the amino acid sequence of LOX3-4m.877 meganuclease. do.

[0565] SEQ ID NO: 49 shows the amino acid sequence of LOX3-4m.883 meganuclease. do.

[0566] SEQ ID NO: 50 shows the amino acid sequence of LOX3-4m.885 meganuclease. do.

[0567] SEQ ID NO: 51 shows the amino acid sequence of LOX3-4m.886 meganuclease. do.

[0568] SEQ ID NO: 52 shows the amino acid sequence of LOX3-4m.893 meganuclease. do.

[0569] SEQ ID NO: 53 shows the amino acid sequence of LOX3-4m.901 meganuclease. do.

[0570] SEQ ID NO: 54 shows the amino acid sequence of LOX3-4m.910 meganuclease. do.

[0571] SEQ ID NO: 55 shows the amino acid sequence of LOX3-4m.917 meganuclease. do.

[0572] SEQ ID NO: 56 shows the amino acid sequence of LOX3-4m.919 meganuclease. do.

[0573] SEQ ID NO: 57 shows the amino acid sequence of LOX3-4m.922 meganuclease. do.

[0574] SEQ ID NO: 58 shows the amino acid sequence of LOX3-4m.925 meganuclease. do.

[0575] SEQ ID NO: 59 shows the amino acid sequence of LOX3-4m.929 meganuclease. do.

[0576] SEQ ID NO: 60 shows the amino acid sequence of LOX3-4m.930 meganuclease. do.

[0577] SEQ ID NO: 61 shows the amino acid sequence of LOX3-4m.933 meganuclease. do.

[0578] SEQ ID NO: 62 shows the amino acid sequence of LOX3-4m.937 meganuclease. do.

[0579] SEQ ID NO: 63 shows the amino acid sequence of LOX3-4m.941 meganuclease. do.

[0580] SEQ ID NO: 64 shows the amino acid sequence of LOX3-4m.942 meganuclease. do.

[0581] SEQ ID NO: 65 shows the amino acid sequence of LOX3-4m.945 meganuclease. do.

[0582] SEQ ID NO: 66 shows the amino acid sequence of LOX3-4m.949 meganuclease. do.

[0583] SEQ ID NO: 67 shows the amino acid sequence of LOX3-4m.950 meganuclease. do.

[0584] SEQ ID NO: 68 is a nucleic acid of the LOX3-4 recognition sequence (sense) having an ACGA central sequence. It shows.

[0585] SEQ ID NO: 69 is a LOX3-4 recognition sequence (antisense) with an ACGA central sequence. Nucleic acids are shown.

[0586] SEQ ID NO: 70 shows the amino acid sequence of LOX3-4m.956 meganuclease. do.

[0587] SEQ ID NO: 71 shows the amino acid sequence of LOX3-4m.961 meganuclease. do.

[0588] SEQ ID NO: 72 shows the amino acid sequence of LOX3-4m.962 meganuclease. do.

[0589] SEQ ID NO: 73 shows the amino acid sequence of LOX3-4m.963 meganuclease. do.

[0590] SEQ ID NO: 74 shows the amino acid sequence of LOX3-4m.969 meganuclease. do.

[0591] SEQ ID NO: 75 shows the amino acid sequence of LOX3-4m.971 meganuclease. do.

[0592] SEQ ID NO: 76 shows the amino acid sequence of LOX3-4m.977 meganuclease. do.

[0593] SEQ ID NO: 77 shows the amino acid sequence of LOX3-4m.982 meganuclease. do.

[0594] SEQ ID NO: 78 shows the amino acid sequence of LOX3-4m.986 meganuclease. do.

[0595] SEQ ID NO: 79 shows the amino acid sequence of LOX3-4m.993 meganuclease. do.

[0596] SEQ ID NO: 80 shows the amino acid sequence of LOX3-4m.994 meganuclease. do.

[0597] SEQ ID NO: 81 shows the amino acid sequence of LOX3-4m.1001 meganuclease. There are.

[0598] SEQ ID NO: 82 shows the amino acid sequence of LOX3-4m.1013 meganuclease. There are.

[0599] SEQ ID NO: 83 shows the amino acid sequence of LOX3-4m.1017 meganuclease. There are.

[0600] SEQ ID NO: 84 shows the amino acid sequence of LOX3-4m.1018 meganuclease. There are.

[0601] SEQ ID NO: 85 shows the amino acid sequence of LOX3-4m.1021 meganuclease. There are.

[0602] SEQ ID NO: 86 shows the amino acid sequence of LOX3-4m.1029 meganuclease. There are.

[0603] SEQ ID NO: 87 shows the amino acid sequence of LOX3-4m.1036 meganuclease. There are.

[0604] SEQ ID NO: 88 shows the amino acid sequence of LOX3-4m.1041 meganuclease. There are.

[0605] SEQ ID NO: 89 shows the amino acid sequence of LOX3-4m.1044 meganuclease. There are.

[0606] SEQ ID NO: 90 is a nucleic acid of the LOX3-4 recognition sequence (sense) having an ACGC central sequence. It shows.

[0607] SEQ ID NO: 91 is a LOX3-4 recognition sequence (antisense) having an ACGC central sequence. Nucleic acids are shown.

[0608] SEQ ID NO: 92 shows the amino acid sequence of LOX3-4m.1049 meganuclease. There are.

[0609] SEQ ID NO: 93 shows the amino acid sequence of LOX3-4m.1050 meganuclease. There are.

[0610] SEQ ID NO: 94 shows the amino acid sequence of LOX3-4m.1052 meganuclease. There are.

[0611] SEQ ID NO: 95 shows the amino acid sequence of LOX3-4m.1068 meganuclease. There are.

[0612] SEQ ID NO: 96 shows the amino acid sequence of LOX3-4m.1069 meganuclease. There are.

[0613] SEQ ID NO: 97 shows the amino acid sequence of LOX3-4m.1074 meganuclease. There are.

[0614] SEQ ID NO: 98 shows the amino acid sequence of LOX3-4m.1085 meganuclease. There are.

[0615] SEQ ID NO: 99 shows the amino acid sequence of LOX3-4m.1093 meganuclease. There are.

[0616] SEQ ID NO: 100 shows the amino acid sequence of LOX3-4m.1095 meganuclease. are.

[0617] SEQ ID NO: 101 shows the amino acid sequence of LOX3-4m.1098 meganuclease. are.

[0618] SEQ ID NO: 102 shows the amino acid sequence of LOX3-4m.1100 meganuclease. are.

[0619] SEQ ID NO: 103 shows the amino acid sequence of LOX3-4m.1101 meganuclease. are.

[0620] SEQ ID NO: 104 shows the amino acid sequence of LOX3-4m.1107 meganuclease. are.

[0621] SEQ ID NO: 105 shows the amino acid sequence of LOX3-4m.1109 meganuclease. are.

[0622] SEQ ID NO: 106 shows the amino acid sequence of LOX3-4m.1111 meganuclease. are.

[0623] SEQ ID NO: 107 shows the amino acid sequence of LOX3-4m.1113 meganuclease. are.

[0624] SEQ ID NO: 108 shows the amino acid sequence of LOX3-4m.1116 meganuclease. are.

[0625] SEQ ID NO: 109 shows the amino acid sequence of LOX3-4m.1117 meganuclease. are.

[0626] SEQ ID NO: 110 shows the amino acid sequence of LOX3-4m.1118 meganuclease. are.

[0627] SEQ ID NO: 111 shows the amino acid sequence of LOX3-4m.1123 meganuclease. are.

[0628] SEQ ID NO: 112 shows the amino acid sequence of LOX3-4m.1125 meganuclease. are.

[0629] SEQ ID NO: 113 shows the amino acid sequence of LOX3-4m.1126 meganuclease. are.

[0630] SEQ ID NO: 114 shows the amino acid sequence of LOX3-4m.1127 meganuclease. are.

[0631] SEQ ID NO: 115 shows the amino acid sequence of LOX3-4m.1129 meganuclease. are.

[0632] SEQ ID NO: 116 shows the amino acid sequence of LOX3-4m.1131 meganuclease. are.

[0633] SEQ ID NO: 117 shows the amino acid sequence of LOX3-4m.1133 meganuclease. are.

[0634] SEQ ID NO: 118 shows the amino acid sequence of LOX3-4m.1137 meganuclease. are.

[0635] SEQ ID NO: 119 is a nucleic acid of the LOX3-4 recognition sequence (sense) with an ACGG central sequence. This shows:

[0636] SEQ ID NO: 120 is a LOX3-4 recognition sequence (antisense) with an ACGG central sequence. The nucleic acid shown is

[0637] SEQ ID NO: 121 shows the amino acid sequence of LOX3-4m.1876 meganuclease. are.

[0638] SEQ ID NO: 122 shows the amino acid sequence of LOX3-4m.1894 meganuclease. are.

[0639] SEQ ID NO: 123 shows the amino acid sequence of LOX3-4m.1898 meganuclease. are.

[0640] SEQ ID NO: 124 shows the amino acid sequence of LOX3-4m.1904 meganuclease. are.

[0641] SEQ ID NO: 125 shows the amino acid sequence of LOX3-4m.1910 meganuclease. are.

[0642] SEQ ID NO: 126 shows the amino acid sequence of LOX3-4m.1914 meganuclease. are.

[0643] SEQ ID NO: 127 shows the amino acid sequence of LOX3-4m.1930 meganuclease. are.

[0644] SEQ ID NO: 128 shows the amino acid sequence of LOX3-4m.1938 meganuclease. are.

[0645] SEQ ID NO: 129 shows the amino acid sequence of LOX3-4m.1941 meganuclease. are.

[0646] SEQ ID NO: 130 shows the amino acid sequence of LOX3-4m.1944 meganuclease. are.

[0647] SEQ ID NO: 131 shows the amino acid sequence of LOX3-4m.1946 meganuclease. are.

[0648] SEQ ID NO: 132 shows the amino acid sequence of LOX3-4m.1947 meganuclease. are.

[0649] SEQ ID NO: 133 shows the amino acid sequence of LOX3-4m.1950 meganuclease. are.

[0650] SEQ ID NO: 134 shows the amino acid sequence of LOX3-4m.1952 meganuclease. are.

[0651] SEQ ID NO: 135 shows the amino acid sequence of LOX3-4m.1960 meganuclease. are.

[0652] SEQ ID NO: 136 is a nucleic acid of the LOX3-4 recognition sequence (sense) with an ACGT central sequence. This shows:

[0653] SEQ ID NO: 137 is a LOX3-4 recognition sequence (antisense) with an ACGT central sequence The nucleic acid shown is

[0654] SEQ ID NO: 138 shows the amino acid sequence of LOX3-4m.1145 meganuclease. are.

[0655] SEQ ID NO: 139 shows the amino acid sequence of LOX3-4m.1149 meganuclease. are.

[0656] SEQ ID NO: 140 shows the amino acid sequence of LOX3-4m.1152 meganuclease. are.

[0657] SEQ ID NO: 141 shows the amino acid sequence of LOX3-4m.1153 meganuclease. are.

[0658] SEQ ID NO: 142 shows the amino acid sequence of LOX3-4m.1157 meganuclease. are.

[0659] SEQ ID NO: 143 shows the amino acid sequence of LOX3-4m.1158 meganuclease. are.

[0660] SEQ ID NO: 144 shows the amino acid sequence of LOX3-4m.1176 meganuclease. are.

[0661] SEQ ID NO: 145 shows the amino acid sequence of LOX3-4m.1191 meganuclease. are.

[0662] SEQ ID NO: 146 shows the amino acid sequence of LOX3-4m.1198 meganuclease. are.

[0663] SEQ ID NO: 147 shows the amino acid sequence of LOX3-4m.1201 meganuclease. are.

[0664] SEQ ID NO: 148 shows the amino acid sequence of LOX3-4m.1205 meganuclease. are.

[0665] SEQ ID NO: 149 shows the amino acid sequence of LOX3-4m.1206 meganuclease. are.

[0666] SEQ ID NO: 150 shows the amino acid sequence of LOX3-4m.1208 meganuclease. are.

[0667] SEQ ID NO: 151 shows the amino acid sequence of LOX3-4m.1212 meganuclease. are.

[0668] SEQ ID NO: 152 shows the amino acid sequence of LOX3-4m.1218 meganuclease. are.

[0669] SEQ ID NO: 153 shows the amino acid sequence of LOX3-4m.1224 meganuclease. are.

[0670] SEQ ID NO: 154 shows the amino acid sequence of LOX3-4m.1225 meganuclease. are.

[0671] SEQ ID NO: 155 shows the amino acid sequence of LOX3-4m.1226 meganuclease. are.

[0672] SEQ ID NO: 156 shows the amino acid sequence of LOX3-4m.1227 meganuclease. are.

[0673] SEQ ID NO: 157 is a nucleic acid of the LOX3-4 recognition sequence (sense) having an ATAA central sequence. This shows:

[0674] SEQ ID NO: 158 is a LOX3-4 recognition sequence (antisense) with an ATAA central sequence The nucleic acid shown is

[0675] SEQ ID NO: 159 shows the amino acid sequence of LOX3-4m.1232 meganuclease. are.

[0676] SEQ ID NO: 160 shows the amino acid sequence of LOX3-4m.1235 meganuclease. are.

[0677] SEQ ID NO: 161 shows the amino acid sequence of LOX3-4m.1236 meganuclease. are.

[0678] SEQ ID NO: 162 shows the amino acid sequence of LOX3-4m.1237 meganuclease. are.

[0679] SEQ ID NO: 163 shows the amino acid sequence of LOX3-4m.1240 meganuclease. are.

[0680] SEQ ID NO: 164 shows the amino acid sequence of LOX3-4m.1250 meganuclease. are.

[0681] SEQ ID NO: 165 shows the amino acid sequence of LOX3-4m.1253 meganuclease. are.

[0682] SEQ ID NO: 166 shows the amino acid sequence of LOX3-4m.1255 meganuclease. are.

[0683] SEQ ID NO: 167 shows the amino acid sequence of LOX3-4m.1256 meganuclease. are.

[0684] SEQ ID NO: 168 shows the amino acid sequence of LOX3-4m.1260 meganuclease. are.

[0685] SEQ ID NO: 169 shows the amino acid sequence of LOX3-4m.1261 meganuclease. are.

[0686] SEQ ID NO: 170 shows the amino acid sequence of LOX3-4m.1262 meganuclease. are.

[0687] SEQ ID NO: 171 shows the amino acid sequence of LOX3-4m.1268 meganuclease. are.

[0688] SEQ ID NO: 172 shows the amino acid sequence of LOX3-4m.1269 meganuclease. are.

[0689] SEQ ID NO: 173 shows the amino acid sequence of LOX3-4m.1278 meganuclease. are.

[0690] SEQ ID NO: 174 shows the amino acid sequence of LOX3-4m.1284 meganuclease. are.

[0691] SEQ ID NO: 175 shows the amino acid sequence of LOX3-4m.1293 meganuclease. are.

[0692] SEQ ID NO: 176 shows the amino acid sequence of LOX3-4m.1300 meganuclease. are.

[0693] SEQ ID NO: 177 shows the amino acid sequence of LOX3-4m.1301 meganuclease are.

[0694] SEQ ID NO: 178 shows the amino acid sequence of LOX3-4m.1308 meganuclease. are.

[0695] SEQ ID NO: 179 shows the amino acid sequence of LOX3-4m.1309 meganuclease. are.

[0696] SEQ ID NO: 180 shows the amino acid sequence of LOX3-4m.1311 meganuclease. are.

[0697] SEQ ID NO: 181 shows the amino acid sequence of LOX3-4m.1317 meganuclease. are.

[0698] SEQ ID NO: 182 shows the amino acid sequence of LOX3-4m.1319 meganuclease. are.

[0699] SEQ ID NO: 183 shows the amino acid sequence of LOX3-4m.1322 meganuclease. are.

[0700] SEQ ID NO: 184 is a nucleic acid of the LOX3-4 recognition sequence (sense) with an ATAG central sequence. This shows:

[0701] SEQ ID NO: 185 is a LOX3-4 recognition sequence (antisense) with an ATAG central sequence The nucleic acid shown is

[0702] SEQ ID NO: 186 shows the amino acid sequence of LOX3-4m.1329 meganuclease. are.

[0703] SEQ ID NO: 187 shows the amino acid sequence of LOX3-4m.1338 meganuclease. are.

[0704] SEQ ID NO: 188 shows the amino acid sequence of LOX3-4m.1343 meganuclease. are.

[0705] SEQ ID NO: 189 shows the amino acid sequence of LOX3-4m.1345 meganuclease. are.

[0706] SEQ ID NO: 190 shows the amino acid sequence of LOX3-4m.1347 meganuclease. are.

[0707] SEQ ID NO: 191 shows the amino acid sequence of LOX3-4m.1353 meganuclease. are.

[0708] SEQ ID NO: 192 shows the amino acid sequence of LOX3-4m.1361 meganuclease. are.

[0709] SEQ ID NO: 193 shows the amino acid sequence of LOX3-4m.1369 meganuclease. are.

[0710] SEQ ID NO: 194 shows the amino acid sequence of LOX3-4m.1391 meganuclease. are.

[0711] SEQ ID NO: 195 shows the amino acid sequence of LOX3-4m.1392 meganuclease. are.

[0712] SEQ ID NO: 196 shows the amino acid sequence of LOX3-4m.1394 meganuclease. are.

[0713] SEQ ID NO: 197 shows the amino acid sequence of LOX3-4m.1396 meganuclease are.

[0714] SEQ ID NO: 198 shows the amino acid sequence of LOX3-4m.1405 meganuclease are.

[0715] SEQ ID NO: 199 shows the amino acid sequence of LOX3-4m.1415 meganuclease. are.

[0716] SEQ ID NO: 200 is a nucleic acid of the LOX3-4 recognition sequence (sense) with an ATAT central sequence. This shows:

[0717] SEQ ID NO: 201 is a LOX3-4 recognition sequence (antisense) with an ATAT central sequence The nucleic acid shown is

[0718] SEQ ID NO: 202 shows the amino acid sequence of LOX3-4m.2244 meganuclease. are.

[0719] SEQ ID NO: 203 shows the amino acid sequence of LOX3-4m.2248 meganuclease. are.

[0720] SEQ ID NO: 204 shows the amino acid sequence of LOX3-4m.2254 meganuclease. are.

[0721] SEQ ID NO: 205 shows the amino acid sequence of LOX3-4m.2263 meganuclease. are.

[0722] SEQ ID NO: 206 shows the amino acid sequence of LOX3-4m.2273 meganuclease. are.

[0723] SEQ ID NO: 207 shows the amino acid sequence of LOX3-4m.2274 meganuclease. are.

[0724] SEQ ID NO: 208 shows the amino acid sequence of LOX3-4m.2313 meganuclease. are.

[0725] SEQ ID NO: 209 shows the amino acid sequence of LOX3-4m.2316 meganuclease. are.

[0726] SEQ ID NO: 210 shows the amino acid sequence of LOX3-4m.2327 meganuclease. are.

[0727] SEQ ID NO: 211 shows the amino acid sequence of LOX3-4m.2318 meganuclease. are.

[0728] SEQ ID NO: 212 shows the amino acid sequence of LOX3-4m.2319 meganuclease. are.

[0729] SEQ ID NO: 213 shows the amino acid sequence of LOX3-4m.2320 meganuclease. are.

[0730] SEQ ID NO: 214 shows the amino acid sequence of LOX3-4m.2322 meganuclease. are.

[0731] SEQ ID NO: 215 shows the amino acid sequence of LOX3-4m.2324 meganuclease. are.

[0732] SEQ ID NO: 216 shows the amino acid sequence of LOX3-4m.2326 meganuclease. are.

[0733] SEQ ID NO: 217 shows the amino acid sequence of LOX3-4m.2329 meganuclease. are.

[0734] SEQ ID NO: 218 shows the amino acid sequence of LOX3-4m.2330 meganuclease. are.

[0735] SEQ ID NO: 219 shows the amino acid sequence of LOX3-4m.2258 meganuclease. are.

[0736] SEQ ID NO: 220 is a nucleic acid of the LOX3-4 recognition sequence (sense) with an ATGA central sequence. This shows:

[0737] SEQ ID NO: 221 is a LOX3-4 recognition sequence (antisense) with an ATGA central sequence The nucleic acid shown is

[0738] SEQ ID NO: 222 shows the amino acid sequence of LOX3-4m.1417 meganuclease. are.

[0739] SEQ ID NO: 223 shows the amino acid sequence of LOX3-4m.1421 meganuclease. are.

[0740] SEQ ID NO: 224 shows the amino acid sequence of LOX3-4m.1432 meganuclease. are.

[0741] SEQ ID NO: 225 shows the amino acid sequence of LOX3-4m.1436 meganuclease. are.

[0742] SEQ ID NO: 226 shows the amino acid sequence of LOX3-4m.1437 meganuclease. are.

[0743] SEQ ID NO: 227 shows the amino acid sequence of LOX3-4m.1441 meganuclease. are.

[0744] SEQ ID NO: 228 shows the amino acid sequence of LOX3-4m.1450 meganuclease. are.

[0745] SEQ ID NO: 229 shows the amino acid sequence of LOX3-4m.1451 meganuclease. are.

[0746] SEQ ID NO: 230 shows the amino acid sequence of LOX3-4m.1453 meganuclease. are.

[0747] SEQ ID NO: 231 shows the amino acid sequence of LOX3-4m.1468 meganuclease. are.

[0748] SEQ ID NO: 232 shows the amino acid sequence of LOX3-4m.1469 meganuclease. are.

[0749] SEQ ID NO: 233 shows the amino acid sequence of LOX3-4m.1477 meganuclease. are.

[0750] SEQ ID NO: 234 shows the amino acid sequence of LOX3-4m.1478 meganuclease. are.

[0751] SEQ ID NO: 235 shows the amino acid sequence of LOX3-4m.1485 meganuclease. are.

[0752] SEQ ID NO: 236 shows the amino acid sequence of LOX3-4m.1486 meganuclease. are.

[0753] SEQ ID NO: 237 shows the amino acid sequence of LOX3-4m.1488 meganuclease. are.

[0754] SEQ ID NO: 238 shows the amino acid sequence of LOX3-4m.1491 meganuclease. are.

[0755] SEQ ID NO: 239 shows the amino acid sequence of LOX3-4m.1500 meganuclease. are.

[0756] SEQ ID NO: 240 shows the amino acid sequence of LOX3-4m.1501 meganuclease. are.

[0757] SEQ ID NO: 241 shows the amino acid sequence of LOX3-4m.1502 meganuclease. are.

[0758] SEQ ID NO: 242 shows the amino acid sequence of LOX3-4m.1505 meganuclease. are.

[0759] SEQ ID NO: 243 shows the amino acid sequence of LOX3-4m.1506 meganuclease. are.

[0760] SEQ ID NO: 244 shows the nucleic acid sequence of the ATGG LOX3-4 recognition sequence (sense). do.

[0761] SEQ ID NO: 245 shows the nucleic acid sequence of the ATGG LOX3-4 recognition sequence (antisense). is doing.

[0762] SEQ ID NO: 246 shows the amino acid sequence of LOX3-4m.1508 meganuclease. are.

[0763] SEQ ID NO: 247 shows the amino acid sequence of LOX3-4m.1515 meganuclease are.

[0764] SEQ ID NO: 248 is the nucleic acid of the LOX3-4 recognition sequence (sense) with a TTGG central sequence. This shows:

[0765] SEQ ID NO: 249 is a LOX3-4 recognition sequence (antisense) with a TTGG central sequence The nucleic acid shown is

[0766] SEQ ID NO: 250 shows the amino acid sequence of LOX3-4m.1970 meganuclease. are.

[0767] SEQ ID NO: 251 shows the amino acid sequence of LOX3-4m.1973 meganuclease. are.

[0768] SEQ ID NO: 252 shows the amino acid sequence of LOX3-4m.1974 meganuclease. are.

[0769] SEQ ID NO: 253 shows the amino acid sequence of LOX3-4m.1975 meganuclease are.

[0770] SEQ ID NO: 254 shows the amino acid sequence of LOX3-4m.1979 meganuclease are.

[0771] SEQ ID NO: 255 shows the amino acid sequence of LOX3-4m.1980 meganuclease. are.

[0772] SEQ ID NO: 256 shows the amino acid sequence of LOX3-4m.1981 meganuclease. are.

[0773] SEQ ID NO: 257 shows the amino acid sequence of LOX3-4m.1982 meganuclease. are.

[0774] SEQ ID NO: 258 shows the amino acid sequence of LOX3-4m.1986 meganuclease. are.

[0775] SEQ ID NO: 259 shows the amino acid sequence of LOX3-4m.1995 meganuclease are.

[0776] SEQ ID NO: 260 shows the amino acid sequence of LOX3-4m.1997 meganuclease. are.

[0777] SEQ ID NO: 261 shows the amino acid sequence of LOX3-4m.2045 meganuclease are.

[0778] SEQ ID NO: 262 shows the amino acid sequence of LOX3-4m.2050 meganuclease. are.

[0779] SEQ ID NO: 263 shows the amino acid sequence of LOX3-4m.2051 meganuclease. are.

[0780] SEQ ID NO: 264 shows the amino acid sequence of LOX3-4m.2052 meganuclease. are.

[0781] SEQ ID NO: 265 shows the amino acid sequence of LOX3-4m.2053 meganuclease. are.

[0782] SEQ ID NO: 266 shows the amino acid sequence of LOX3-4m.2059 meganuclease are.

[0783] SEQ ID NO: 267 is a nucleic acid of the LOX3-4 recognition sequence (sense) with a GCAA central sequence. This shows:

[0784] SEQ ID NO: 268 is a LOX3-4 recognition sequence (antisense) with a GCAA central sequence The nucleic acid shown is

[0785] SEQ ID NO: 269 shows the amino acid sequence of LOX3-4m.1784 meganuclease. are.

[0786] SEQ ID NO: 270 shows the amino acid sequence of LOX3-4m.1785 meganuclease. are.

[0787] SEQ ID NO: 271 shows the amino acid sequence of LOX3-4m.1787 meganuclease are.

[0788] SEQ ID NO: 272 shows the amino acid sequence of LOX3-4m.1789 meganuclease are.

[0789] SEQ ID NO: 273 shows the amino acid sequence of LOX3-4m.1798 meganuclease are.

[0790] SEQ ID NO: 274 shows the amino acid sequence of LOX3-4m.1805 meganuclease are.

[0791] SEQ ID NO: 275 shows the amino acid sequence of LOX3-4m.1809 meganuclease. are.

[0792] SEQ ID NO: 276 shows the amino acid sequence of LOX3-4m.1812 meganuclease. are.

[0793] SEQ ID NO: 277 shows the amino acid sequence of LOX3-4m.1814 meganuclease are.

[0794] SEQ ID NO: 278 shows the amino acid sequence of LOX3-4m.1820 meganuclease. are.

[0795] SEQ ID NO: 279 shows the amino acid sequence of LOX3-4m.1827 meganuclease are.

[0796] SEQ ID NO: 280 shows the amino acid sequence of LOX3-4m.1836 meganuclease. are.

[0797] SEQ ID NO: 281 shows the amino acid sequence of LOX3-4m.1837 meganuclease are.

[0798] SEQ ID NO: 282 shows the amino acid sequence of LOX3-4m.1838 meganuclease. are.

[0799] SEQ ID NO: 283 shows the amino acid sequence of LOX3-4m.1846 meganuclease. are.

[0800] SEQ ID NO: 284 shows the amino acid sequence of LOX3-4m.1853 meganuclease. are.

[0801] SEQ ID NO: 285 shows the amino acid sequence of LOX3-4m.1854 meganuclease are.

[0802] SEQ ID NO: 286 shows the amino acid sequence of LOX3-4m.1858 meganuclease. are.

[0803] SEQ ID NO: 287 shows the amino acid sequence of LOX3-4m.1862 meganuclease are.

[0804] SEQ ID NO: 288 shows the amino acid sequence of LOX3-4m.1868 meganuclease. are.

[0805] SEQ ID NO: 289 shows the amino acid sequence of LOX3-4m.1870 meganuclease. are.

[0806] SEQ ID NO: 290 shows the amino acid sequence of LOX3-4m.1873 meganuclease. are.

[0807] SEQ ID NO: 291 shows the amino acid sequence of LOX3-4m.1875 meganuclease are.

[0808] SEQ ID NO: 292 is a nucleic acid of the LOX3-4 recognition sequence (sense) with a GCAT central sequence. This shows:

[0809] SEQ ID NO: 293 is a LOX3-4 recognition sequence (antisense) with a GCAT central sequence The nucleic acid shown is

[0810] SEQ ID NO: 294 shows the amino acid sequence of LOX3-4m.1600 meganuclease. are.

[0811] SEQ ID NO: 295 shows the amino acid sequence of LOX3-4m.1601 meganuclease are.

[0812] SEQ ID NO: 296 shows the amino acid sequence of LOX3-4m.1605 meganuclease are.

[0813] SEQ ID NO: 297 shows the amino acid sequence of LOX3-4m.1606 meganuclease are.

[0814] SEQ ID NO: 298 shows the amino acid sequence of LOX3-4m.1623 meganuclease. are.

[0815] SEQ ID NO: 299 shows the amino acid sequence of LOX3-4m.1660 meganuclease. are.

[0816] SEQ ID NO: 300 shows the amino acid sequence of LOX3-4m.1661 meganuclease. are.

[0817] SEQ ID NO: 301 shows the amino acid sequence of LOX3-4m.1665 meganuclease. are.

[0818] SEQ ID NO: 302 shows the amino acid sequence of LOX3-4m.1667 meganuclease. are.

[0819] SEQ ID NO: 303 shows the amino acid sequence of LOX3-4m.1669 meganuclease. are.

[0820] SEQ ID NO: 304 shows the amino acid sequence of LOX3-4m.1672 meganuclease. are.

[0821] SEQ ID NO: 305 shows the amino acid sequence of LOX3-4m.1674 meganuclease are.

[0822] SEQ ID NO: 306 shows the amino acid sequence of LOX3-4m.1676 meganuclease. are.

[0823] SEQ ID NO: 307 shows the amino acid sequence of LOX3-4m.1677 meganuclease are.

[0824] SEQ ID NO: 308 shows the amino acid sequence of LOX3-4m.1679 meganuclease are.

[0825] SEQ ID NO: 309 shows the amino acid sequence of LOX3-4m.1684 meganuclease. are.

[0826] SEQ ID NO: 310 shows the amino acid sequence of LOX3-4m.1685 meganuclease. are.

[0827] SEQ ID NO: 311 shows the amino acid sequence of LOX3-4m.1687 meganuclease are.

[0828] SEQ ID NO: 312 shows the amino acid sequence of LOX3-4m.1689 meganuclease. are.

[0829] SEQ ID NO: 313 shows the amino acid sequence of LOX3-4m.1691 meganuclease. are.

[0830] SEQ ID NO: 314 is a nucleic acid of the LOX3-4 recognition sequence (sense) with a GCGA central sequence. This shows:

[0831] SEQ ID NO: 315 is a LOX3-4 recognition sequence (antisense) with a GCGA central sequence The nucleic acid shown is

[0832] SEQ ID NO: 316 shows the amino acid sequence of LOX3-4m.1694 meganuclease are.

[0833] SEQ ID NO: 317 shows the amino acid sequence of LOX3-4m.1745 meganuclease are.

[0834] SEQ ID NO: 318 shows the amino acid sequence of LOX3-4m.1752 meganuclease are.

[0835] SEQ ID NO: 319 shows the amino acid sequence of LOX3-4m.1753 meganuclease. are.

[0836] SEQ ID NO: 320 shows the amino acid sequence of LOX3-4m.1765 meganuclease. are.

[0837] SEQ ID NO: 321 shows the amino acid sequence of LOX3-4m.1770 meganuclease. are.

[0838] SEQ ID NO: 322 shows the amino acid sequence of LOX3-4m.1774 meganuclease. are.

[0839] SEQ ID NO: 323 shows the amino acid sequence of LOX3-4m.1780 meganuclease. are.

[0840]

[0841] SEQ ID NO: 324 shows the amino acid sequence of LOX3-4m.1781 meganuclease. are.

[0842] SEQ ID NO: 325 shows the amino acid sequence of LOX3-4m.1782 meganuclease. are.

[0843] SEQ ID NO: 326 shows the nucleic acid sequence of the GCAG LOX3-4 recognition sequence (sense). do.

[0844] SEQ ID NO: 327 shows the nucleic acid sequence of the GCAG LOX3-4 recognition sequence (antisense). is doing.

[0845] SEQ ID NO: 328 shows the amino acid sequence of LOX3-4m.494 meganuclease. There are.

[0846] SEQ ID NO: 329 shows the amino acid sequence of LOX3-4m.509 meganuclease. There are.

[0847] SEQ ID NO: 330 shows the amino acid sequence of LOX3-4m.524 meganuclease. There are.

[0848] SEQ ID NO: 331 is a nucleic acid of the LOX3-4 recognition sequence (sense) with a TCAA central sequence. This shows:

[0849] SEQ ID NO: 332 is a LOX3-4 recognition sequence (antisense) with a TCAA central sequence The nucleic acid shown is

[0850] SEQ ID NO: 333 shows the amino acid sequence of LOX3-4m.2157 meganuclease are.

[0851] SEQ ID NO: 334 shows the amino acid sequence of LOX3-4m.2165 meganuclease. are.

[0852] SEQ ID NO: 335 shows the amino acid sequence of LOX3-4m.2189 meganuclease are.

[0853] SEQ ID NO: 336 shows the amino acid sequence of LOX3-4m.2207 meganuclease. are.

[0854] SEQ ID NO: 337 shows the amino acid sequence of LOX3-4m.2225 meganuclease. are.

[0855] SEQ ID NO: 338 shows the amino acid sequence of LOX3-4m.2229 meganuclease are.

[0856] SEQ ID NO: 339 shows the amino acid sequence of LOX3-4m.2235 meganuclease are.

[0857] SEQ ID NO: 340 shows the amino acid sequence of LOX3-4m.2238 meganuclease. are.

[0858] SEQ ID NO: 341 is a nucleic acid of the LOX3-4 recognition sequence (sense) with a TTAA central sequence. This shows:

[0859] SEQ ID NO: 342 is a LOX3-4 recognition sequence (antisense) with a TTAA central sequence The nucleic acid shown is

[0860] SEQ ID NO: 343 shows the amino acid sequence of LOX3-4m.2071 meganuclease are.

[0861] SEQ ID NO: 344 shows the amino acid sequence of LOX3-4m.2077 meganuclease are.

[0862] SEQ ID NO: 345 shows the amino acid sequence of LOX3-4m.2082 meganuclease. are.

[0863] SEQ ID NO: 346 shows the amino acid sequence of LOX3-4m.2086 meganuclease. are.

[0864] SEQ ID NO: 347 shows the amino acid sequence of LOX3-4m.2087 meganuclease are.

[0865] SEQ ID NO: 348 shows the amino acid sequence of LOX3-4m.2102 meganuclease are.

[0866] SEQ ID NO: 349 shows the amino acid sequence of LOX3-4m.2111 meganuclease. are.

[0867] SEQ ID NO: 350 shows the amino acid sequence of LOX3-4m.2116 meganuclease. are.

[0868] SEQ ID NO: 351 shows the amino acid sequence of LOX3-4m.2125 meganuclease are.

[0869] SEQ ID NO: 352 shows the amino acid sequence of LOX3-4m.2132 meganuclease. are.

[0870] SEQ ID NO: 353 shows the amino acid sequence of LOX3-4m.2138 meganuclease are.

[0871] SEQ ID NO: 354 shows the amino acid sequence of LOX3-4m.2141 meganuclease are.

[0872] SEQ ID NO: 355 shows the amino acid sequence of LOX3-4m.2142 meganuclease are.

[0873] SEQ ID NO: 356 shows the amino acid sequence of LOX3-4m.2145 meganuclease are.

[0874] SEQ ID NO: 357 shows the amino acid sequence of LOX3-4m.2151 meganuclease are.

[0875] SEQ ID NO: 358 is the nucleic acid of the LOX3-4 recognition sequence (sense) with a GTAA central sequence. This shows:

[0876] SEQ ID NO: 359 is a LOX3-4 recognition sequence (antisense) with a GTAA central sequence The nucleic acid shown is

[0877] SEQ ID NO: 360 shows the amino acid sequence of LOX3-4m.1 meganuclease .

[0878] SEQ ID NO: 361 shows the amino acid sequence of LOX3-4m.2 meganuclease .

[0879] SEQ ID NO: 362 shows the amino acid sequence of LOX3-4m.3 meganuclease .

[0880] SEQ ID NO: 363 shows the amino acid sequence of LOX3-4m.4 meganuclease .

[0881] SEQ ID NO: 364 shows the amino acid sequence of LOX3-4m.5 meganuclease .

[0882] SEQ ID NO: 365 shows the amino acid sequence of LOX3-4m.6 meganuclease .

[0883] SEQ ID NO: 366 shows the amino acid sequence of LOX3-4m.7 meganuclease .

[0884] SEQ ID NO: 367 shows the amino acid sequence of LOX3-4m.8 meganuclease .

[0885] SEQ ID NO: 368 shows the amino acid sequence of LOX3-4m.9 meganuclease .

[0886] SEQ ID NO: 369 shows the amino acid sequence of LOX3-4m.10 meganuclease. do.

[0887] SEQ ID NO: 370 shows the amino acid sequence of LOX3-4m.11 meganuclease. do.

[0888] SEQ ID NO: 371 shows the amino acid sequence of LOX3-4m.12 meganuclease. do.

[0889] SEQ ID NO: 372 shows the amino acid sequence of LOX3-4m.13 meganuclease. do.

[0890] SEQ ID NO: 373 shows the amino acid sequence of LOX3-4m.14 meganuclease. do.

[0891] SEQ ID NO: 374 shows the amino acid sequence of LOX3-4m.15 meganuclease. do.

[0892] SEQ ID NO: 375 shows the amino acid sequence of LOX3-4m.16 meganuclease. do.

[0893] SEQ ID NO: 376 shows the amino acid sequence of LOX3-4m.17 meganuclease. do.

[0894] SEQ ID NO: 377 shows the amino acid sequence of LOX3-4m.18 meganuclease. do.

[0895] SEQ ID NO: 378 shows the amino acid sequence of LOX3-4m.19 meganuclease. do.

[0896] SEQ ID NO: 379 shows the amino acid sequence of LOX3-4m.20 meganuclease. do.

[0897] SEQ ID NO: 380 shows the amino acid sequence of LOX3-4m.21 meganuclease. do.

[0898] SEQ ID NO: 381 shows the amino acid sequence of LOX3-4m.22 meganuclease. do.

[0899] SEQ ID NO: 382 shows the amino acid sequence of LOX3-4m.23 meganuclease. do.

[0900] SEQ ID NO: 383 shows the amino acid sequence of LOX3-4m.24 meganuclease. do.

[0901] SEQ ID NO: 384 shows the amino acid sequence of LOX3-4m.25 meganuclease. do.

[0902] SEQ ID NO: 385 shows the amino acid sequence of LOX3-4m.26 meganuclease. do.

[0903] SEQ ID NO: 386 shows the amino acid sequence of LOX3-4m.27 meganuclease. do.

[0904] SEQ ID NO: 387 shows the amino acid sequence of LOX3-4m.28 meganuclease. do.

[0905] SEQ ID NO: 388 shows the amino acid sequence of LOX3-4m.29 meganuclease. do.

[0906] SEQ ID NO: 389 shows the amino acid sequence of LOX3-4m.30 meganuclease. do.

[0907] SEQ ID NO: 390 is the nucleic acid of the LOX3-4 recognition sequence (sense) with a GTAG central sequence. This shows:

[0908] SEQ ID NO: 391 is a LOX3-4 recognition sequence (antisense) with a GTAG central sequence The nucleic acid shown is

[0909] SEQ ID NO: 392 shows the amino acid sequence of LOX3-4m.95 meganuclease. do.

[0910] SEQ ID NO: 393 shows the amino acid sequence of LOX3-4m.96 meganuclease. do.

[0911] SEQ ID NO: 394 shows the amino acid sequence of LOX3-4m.97 meganuclease. do.

[0912] SEQ ID NO: 395 shows the amino acid sequence of LOX3-4m.102 meganuclease. There are.

[0913] SEQ ID NO: 396 shows the amino acid sequence of LOX3-4m.108 meganuclease. There are.

[0914] SEQ ID NO: 397 shows the amino acid sequence of LOX3-4m.111 meganuclease. There are.

[0915] SEQ ID NO: 398 shows the amino acid sequence of LOX3-4m.114 meganuclease. There are.

[0916] SEQ ID NO: 399 shows the amino acid sequence of LOX3-4m.123 meganuclease. There are.

[0917] SEQ ID NO: 400 is the nucleic acid of the LOX3-4 recognition sequence (sense) with a GTAT central sequence. This shows:

[0918] SEQ ID NO: 401 is a LOX3-4 recognition sequence (antisense) with a GTAT central sequence The nucleic acid shown is

[0919] SEQ ID NO: 402 shows the amino acid sequence of LOX3-4m.124 meganuclease. There are.

[0920] SEQ ID NO: 403 shows the amino acid sequence of LOX3-4m.125 meganuclease. There are.

[0921] SEQ ID NO: 404 shows the amino acid sequence of LOX3-4m.126 meganuclease. There are.

[0922] SEQ ID NO: 405 shows the amino acid sequence of LOX3-4m.127 meganuclease. There are.

[0923] SEQ ID NO: 406 shows the amino acid sequence of LOX3-4m.128 meganuclease. There are.

[0924] SEQ ID NO: 407 shows the amino acid sequence of LOX3-4m.129 meganuclease. There are.

[0925] SEQ ID NO: 408 shows the amino acid sequence of LOX3-4m.130 meganuclease. There are.

[0926] SEQ ID NO: 409 shows the amino acid sequence of LOX3-4m.131 meganuclease. There are.

[0927] SEQ ID NO: 410 shows the amino acid sequence of LOX3-4m.132 meganuclease. There are.

[0928] SEQ ID NO: 411 shows the amino acid sequence of LOX3-4m.133 meganuclease. There are.

[0929] SEQ ID NO: 412 shows the amino acid sequence of LOX3-4m.134 meganuclease. There are.

[0930] SEQ ID NO: 413 shows the amino acid sequence of LOX3-4m.135 meganuclease. There are.

[0931] SEQ ID NO: 414 shows the amino acid sequence of LOX3-4m.136 meganuclease. There are.

[0932] SEQ ID NO: 415 shows the amino acid sequence of LOX3-4m.137 meganuclease. There are.

[0933] SEQ ID NO: 416 shows the amino acid sequence of LOX3-4m.138 meganuclease. There are.

[0934] SEQ ID NO: 417 shows the amino acid sequence of LOX3-4m.139 meganuclease. There are.

[0935] SEQ ID NO: 418 shows the amino acid sequence of LOX3-4m.140 meganuclease. There are.

[0936] SEQ ID NO: 419 shows the amino acid sequence of LOX3-4m.141 meganuclease. There are.

[0937] SEQ ID NO: 420 shows the amino acid sequence of LOX3-4m.142 meganuclease. There are.

[0938] SEQ ID NO: 421 shows the amino acid sequence of LOX3-4m.143 meganuclease. There are.

[0939] SEQ ID NO: 422 shows the amino acid sequence of LOX3-4m.144 meganuclease. There are.

[0940] SEQ ID NO: 423 shows the amino acid sequence of LOX3-4m.145 meganuclease. There are.

[0941] SEQ ID NO: 424 shows the amino acid sequence of LOX3-4m.146 meganuclease. There are.

[0942] SEQ ID NO: 425 shows the amino acid sequence of LOX3-4m.147 meganuclease. There are.

[0943] SEQ ID NO: 426 shows the amino acid sequence of LOX3-4m.148 meganuclease. There are.

[0944] SEQ ID NO: 427 shows the amino acid sequence of LOX3-4m.149 meganuclease. There are.

[0945] SEQ ID NO: 428 shows the amino acid sequence of LOX3-4m.150 meganuclease. There are.

[0946] SEQ ID NO: 429 shows the amino acid sequence of LOX3-4m.151 meganuclease. There are.

[0947] SEQ ID NO: 430 shows the amino acid sequence of LOX3-4m.152 meganuclease. There are.

[0948] SEQ ID NO: 431 shows the amino acid sequence of LOX3-4m.153 meganuclease. There are.

[0949] SEQ ID NO: 432 shows the amino acid sequence of LOX3-4m.154 meganuclease. There are.

[0950] SEQ ID NO: 433 shows the amino acid sequence of LOX3-4m.155 meganuclease. There are.

[0951] SEQ ID NO: 434 is the nucleic acid of the LOX3-4 recognition sequence (sense) with a GTGA central sequence. This shows:

[0952] SEQ ID NO: 435 is a LOX3-4 recognition sequence (antisense) with a GTGA central sequence The nucleic acid shown is

[0953] SEQ ID NO: 436 shows the amino acid sequence of LOX3-4m.31 meganuclease. do.

[0954] SEQ ID NO: 437 shows the amino acid sequence of LOX3-4m.32 meganuclease. do.

[0955] SEQ ID NO: 438 shows the amino acid sequence of LOX3-4m.33 meganuclease. do.

[0956] SEQ ID NO: 439 shows the amino acid sequence of LOX3-4m.35 meganuclease. do.

[0957] SEQ ID NO: 440 shows the amino acid sequence of LOX3-4m.36 meganuclease. do.

[0958] SEQ ID NO: 441 shows the amino acid sequence of LOX3-4m.37 meganuclease. do.

[0959] SEQ ID NO: 442 shows the amino acid sequence of LOX3-4m.38 meganuclease. do.

[0960] SEQ ID NO: 443 shows the amino acid sequence of LOX3-4m.39 meganuclease. do.

[0961] SEQ ID NO: 444 shows the amino acid sequence of LOX3-4m.40 meganuclease. do.

[0962] SEQ ID NO: 445 shows the amino acid sequence of LOX3-4m.41 meganuclease. do.

[0963] SEQ ID NO: 446 shows the amino acid sequence of LOX3-4m.42 meganuclease. do.

[0964] SEQ ID NO: 447 shows the amino acid sequence of LOX3-4m.43 meganuclease. do.

[0965] SEQ ID NO: 448 shows the amino acid sequence of LOX3-4m.44 meganuclease. do.

[0966] SEQ ID NO: 449 shows the amino acid sequence of LOX3-4m.46 meganuclease. do.

[0967] SEQ ID NO: 450 shows the amino acid sequence of LOX3-4m.47 meganuclease. do.

[0968] SEQ ID NO: 451 shows the amino acid sequence of LOX3-4m.48 meganuclease. do.

[0969] SEQ ID NO: 452 shows the amino acid sequence of LOX3-4m.49 meganuclease. do.

[0970] SEQ ID NO: 453 shows the amino acid sequence of LOX3-4m.50 meganuclease. do.

[0971] SEQ ID NO: 454 shows the amino acid sequence of LOX3-4m.51 meganuclease. do.

[0972] SEQ ID NO: 455 shows the amino acid sequence of LOX3-4m.52 meganuclease. do.

[0973] SEQ ID NO: 456 shows the amino acid sequence of LOX3-4m.53 meganuclease. do.

[0974] SEQ ID NO: 457 shows the amino acid sequence of LOX3-4m.54 meganuclease. do.

[0975] SEQ ID NO: 458 shows the amino acid sequence of LOX3-4m.56 meganuclease. do.

[0976] SEQ ID NO: 459 shows the amino acid sequence of LOX3-4m.57 meganuclease. do.

[0977] SEQ ID NO: 460 shows the amino acid sequence of LOX3-4m.58 meganuclease. do.

[0978] SEQ ID NO: 461 shows the amino acid sequence of LOX3-4m.59 meganuclease. do.

[0979] SEQ ID NO: 462 shows the amino acid sequence of LOX3-4m.61 meganuclease. do.

[0980] SEQ ID NO: 463 is the nucleic acid of the LOX3-4 recognition sequence (sense) with a GTGC central sequence. This shows:

[0981] SEQ ID NO: 464 is a LOX3-4 recognition sequence (antisense) with a GTGC central sequence The nucleic acid shown is

[0982] SEQ ID NO: 465 shows the amino acid sequence of LOX3-4m.156 meganuclease. There are.

[0983] SEQ ID NO: 466 shows the amino acid sequence of LOX3-4m.157 meganuclease. There are.

[0984] SEQ ID NO: 467 shows the amino acid sequence of LOX3-4m.158 meganuclease. There are.

[0985] SEQ ID NO: 468 shows the amino acid sequence of LOX3-4m.159 meganuclease. There are.

[0986] SEQ ID NO: 469 shows the amino acid sequence of LOX3-4m.160 meganuclease. There are.

[0987] SEQ ID NO: 470 shows the amino acid sequence of LOX3-4m.161 meganuclease. There are.

[0988] SEQ ID NO: 471 shows the amino acid sequence of LOX3-4m.162 meganuclease. There are.

[0989] SEQ ID NO: 472 shows the amino acid sequence of LOX3-4m.163 meganuclease. There are.

[0990] SEQ ID NO: 473 shows the amino acid sequence of LOX3-4m.164 meganuclease. There are.

[0991] SEQ ID NO: 474 shows the amino acid sequence of LOX3-4m.165 meganuclease. There are.

[0992] SEQ ID NO: 475 shows the amino acid sequence of LOX3-4m.166 meganuclease. There are.

[0993] SEQ ID NO: 476 shows the amino acid sequence of LOX3-4m.167 meganuclease. There are.

[0994] SEQ ID NO: 477 shows the amino acid sequence of LOX3-4m.168 meganuclease. There are.

[0995] SEQ ID NO: 478 shows the amino acid sequence of LOX3-4m.169 meganuclease. There are.

[0996] SEQ ID NO: 479 shows the amino acid sequence of LOX3-4m.170 meganuclease. There are.

[0997] SEQ ID NO: 480 shows the amino acid sequence of LOX3-4m.171 meganuclease. There are.

[0998] SEQ ID NO: 481 shows the amino acid sequence of LOX3-4m.172 meganuclease. There are.

[0999] SEQ ID NO: 482 shows the amino acid sequence of LOX3-4m.173 meganuclease. There are.

[1000] SEQ ID NO: 483 shows the amino acid sequence of LOX3-4m.174 meganuclease. There are.

[1001] SEQ ID NO: 484 shows the amino acid sequence of LOX3-4m.175 meganuclease. There are.

[1002] SEQ ID NO: 485 shows the amino acid sequence of LOX3-4m.176 meganuclease. There are.

[1003] SEQ ID NO: 486 shows the amino acid sequence of LOX3-4m.177 meganuclease. There are.

[1004] SEQ ID NO: 487 shows the amino acid sequence of LOX3-4m.178 meganuclease. There are.

[1005] SEQ ID NO: 488 shows the amino acid sequence of LOX3-4m.179 meganuclease. There are.

[1006] SEQ ID NO: 489 shows the amino acid sequence of LOX3-4m.180 meganuclease. There are.

[1007] SEQ ID NO: 490 shows the amino acid sequence of LOX3-4m.181 meganuclease. There are.

[1008] SEQ ID NO: 491 shows the amino acid sequence of LOX3-4m.182 meganuclease. There are.

[1009] SEQ ID NO: 492 shows the amino acid sequence of LOX3-4m.183 meganuclease. There are.

[1010] SEQ ID NO: 493 shows the amino acid sequence of LOX3-4m.184 meganuclease. There are.

[1011] SEQ ID NO: 494 shows the amino acid sequence of LOX3-4m.185 meganuclease. There are.

[1012] SEQ ID NO: 495 shows the amino acid sequence of LOX3-4m.186 meganuclease. There are.

[1013] SEQ ID NO: 496 is the nucleic acid of the LOX3-4 recognition sequence (sense) with a GTGG central sequence. This shows:

[1014] SEQ ID NO: 497 is a LOX3-4 recognition sequence (antisense) with a GTGG central sequence The nucleic acid shown is

[1015] SEQ ID NO: 498 shows the amino acid sequence of LOX3-4m.187 meganuclease. There are.

[1016] SEQ ID NO: 499 shows the amino acid sequence of LOX3-4m.192 meganuclease. There are.

[1017] SEQ ID NO: 500 shows the amino acid sequence of LOX3-4m.201 meganuclease. There are.

[1018] SEQ ID NO: 501 shows the amino acid sequence of LOX3-4m.203 meganuclease. There are.

[1019] SEQ ID NO: 502 is the nucleic acid of the LOX3-4 recognition sequence (sense) with a central sequence of GTGT This shows:

[1020] SEQ ID NO: 503 is a LOX3-4 recognition sequence (antisense) with a central sequence of GTGT The nucleic acid shown is

[1021] SEQ ID NO: 504 shows the amino acid sequence of LOX3-4m.63 meganuclease. do.

[1022] SEQ ID NO: 505 shows the amino acid sequence of LOX3-4m.64 meganuclease. do.

[1023] SEQ ID NO: 506 shows the amino acid sequence of LOX3-4m.65 meganuclease. do.

[1024] SEQ ID NO: 507 shows the amino acid sequence of LOX3-4m.66 meganuclease. do.

[1025] SEQ ID NO: 508 shows the amino acid sequence of LOX3-4m.67 meganuclease. do.

[1026] SEQ ID NO: 509 shows the amino acid sequence of LOX3-4m.68 meganuclease. do.

[1027] SEQ ID NO: 510 shows the amino acid sequence of LOX3-4m.69 meganuclease. do.

[1028] SEQ ID NO: 511 shows the amino acid sequence of LOX3-4m.70 meganuclease. do.

[1029] SEQ ID NO: 512 is the amino acid sequence of a meganuclease having the central sequence of LOX3-4m.71. This indicates acetic acid.

[1030] SEQ ID NO: 513 is the amino acid sequence of a meganuclease having the central sequence of LOX3-4m.73. This indicates acetic acid.

[1031] SEQ ID NO: 514 shows the amino acid sequence of LOX3-4m.74 meganuclease. do.

[1032] SEQ ID NO: 515 shows the amino acid sequence of LOX3-4m.75 meganuclease. do.

[1033] SEQ ID NO: 516 shows the amino acid sequence of LOX3-4m.77 meganuclease. do.

[1034] SEQ ID NO: 517 shows the amino acid sequence of LOX3-4m.78 meganuclease. do.

[1035] SEQ ID NO: 518 shows the amino acid sequence of LOX3-4m.80 meganuclease. do.

[1036] SEQ ID NO: 519 shows the amino acid sequence of LOX3-4m.83 meganuclease. do.

[1037] SEQ ID NO: 520 shows the amino acid sequence of LOX3-4m.84 meganuclease. do.

[1038] SEQ ID NO: 521 shows the amino acid sequence of LOX3-4m.85 meganuclease. do.

[1039] SEQ ID NO: 522 shows the amino acid sequence of LOX3-4m.86 meganuclease. do.

[1040] SEQ ID NO: 523 shows the amino acid sequence of LOX3-4m.87 meganuclease. do.

[1041] SEQ ID NO: 524 shows the amino acid sequence of LOX3-4m.88 meganuclease. do.

[1042] SEQ ID NO: 525 shows the amino acid sequence of LOX3-4m.89 meganuclease. do.

[1043] SEQ ID NO: 526 shows the amino acid sequence of LOX3-4m.90 meganuclease. do.

[1044] SEQ ID NO: 527 shows the amino acid sequence of LOX3-4m.91 meganuclease. do.

[1045] SEQ ID NO: 528 shows the amino acid sequence of LOX3-4m.92 meganuclease. do.

[1046] SEQ ID NO: 529 shows the amino acid sequence of LOX3-4m.93 meganuclease. do.

[1047] SEQ ID NO: 530 shows the amino acid sequence of the polypeptide linker. DETAILED DESCRIPTION OF THE INVENTION

[1048] Detailed Description of the Invention 1.1 References and Definitions

[1049] The patent and scientific literature referred to herein establishes knowledge that is available to those skilled in the art. Issued U.S. patents, allowed applications, published foreign applications, etc., cited herein are not intended to be limiting unless otherwise specified. and GenBank database sequences are incorporated by reference. and are incorporated herein by reference to the same extent as if specifically and individually indicated to be incorporated herein by reference. do.

[1050] This invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments should not be construed as an endorsement by any person skilled in the art that this disclosure will be thorough and complete. As such, these embodiments are provided so that they will fully convey the scope of the present invention to those skilled in the art. Features illustrated with respect to one embodiment may be incorporated into other embodiments and may be used interchangeably with respect to a particular embodiment. Any feature illustrated as such may be omitted from that embodiment. Numerous variations and additions to the contemplated embodiments that do not depart from the invention are possible in light of this disclosure. It is believed that this will be apparent to one skilled in the art.

[1051] Unless otherwise defined, all technical and scientific terms used herein are defined by the It has the same meaning as commonly understood by a person skilled in the art to which the invention pertains. The terminology used in the description of the invention herein is intended to describe particular embodiments only. and is not intended to limit the present invention.

[1052] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. No. 6,239,999, filed on Oct. 1, 2003, which is incorporated herein in its entirety.

[1053] As used herein, "a," "an," or "this" refers to "The" can mean one or more. For example, "a" cell means a single It may refer to a cell or a plurality of cells.

[1054] As used herein, unless otherwise indicated, the term "or" means "and" and / or" and not the exclusive "either / or" sense. stomach.

[1055] As used herein, the terms "nuclease" and "endonuclease" The terms are used interchangeably and refer to naturally occurring enzymes that cleave phosphodiester bonds within a polynucleotide chain. Refers to existing or engineered enzymes.

[1056] As used herein, the term "cleave" or "cleavage" refers to a fragment within a target sequence. refers to the hydrolysis of a phosphodiester bond within the recognition sequence backbone, which is referred to herein as a "cleavage site." In some embodiments described herein, In this embodiment, the amino acids at positions 48, 50, 71, 72, and 73 of I-CreI (i.e., SEQ ID NO: 1) are Modifications or substitutions at one or more positions corresponding to positions 3, 73B, and 74 were engineered. This enhances the cleavage activity of the meganuclease.

[1057] As used herein, the term "meganuclease" refers to a nucleic acid having more than 12 base pairs. It refers to an endonuclease that binds to double-stranded DNA at a recognition sequence. The recognition sequence of the meganuclease of the present disclosure is 22 base pairs. -CreI (SEQ ID NO: 1), e.g., a DNA-binding endonuclease derived from The natural I- It can refer to an engineered variant of I-CreI that is modified compared to CreI. Methods for generating such engineered variants of -CreI are known in the art. It is known (for example, International Publication No. 2007 / 047859, the document is incorporated herein by reference). (The entirety of which is incorporated by reference.) As used herein, meganucleases are heterologous. Binds to double-stranded DNA as a dimer. Meganucleases also contain a pair of DNA-binding domains. "Single-chain meganucleotides" in which the polypeptides are linked into a single polypeptide using a peptide linker The term "homing endonuclease" refers to a "meganuclease." The term "meganuclease" is synonymous with the term "meganuclease." The meganucleases of the present disclosure are capable of targeting the targets described herein. are substantially non-toxic when expressed in cells and can be produced using the methods described herein. No adverse effects on cell viability or significant meganuclease cleavage activity, if measured Cells can be transfected and maintained at 37°C without any observed degradation. can.

[1058] As used herein, the term "single-chain meganuclease" refers to a single-chain meganuclease It refers to a polypeptide comprising a pair of nuclease subunits linked by a single chain. The enzyme has the following structure: N-terminal subunit-linker-C-terminal subunit. The two meganuclease subunits generally do not have identical amino acid sequences; Therefore, single-chain meganucleases are usually called pseudo-palindole-binding enzymes. The engineered single-chain meganuclease cleaves palindromic or non-palindromic recognition sequences. I-CreI-derived meganucleases and methods for producing them are disclosed in International Publication No. WO 2005 / 024990. No. 2009 / 059195, which is incorporated herein by reference. The single-chain meganucleases are not actually dimers, but are "single-chain heteronucleases." These are sometimes referred to as "single-chain heterodimeric meganucleases" or "single-chain heterodimeric meganucleases." Therefore, unless otherwise specified, the term "meganuclease" refers to a dimer or a monomer. It can refer to a chain meganuclease.

[1059] As used herein, the term "linker" refers to a linker that connects two nuclease subunits. It refers to an exogenous peptide sequence used to link multiple fragments into a single polypeptide. Cars may have sequences found in natural proteins or may be non-naturally occurring proteins. The linker may be an artificial sequence not found in other sequences. The linker is flexible and lacks secondary structure. They may have a tendency to form specific three-dimensional structures under physiological conditions. Examples of linkers include, but are not limited to, those described in U.S. Patent No. 8,445,251, Nos. 9,340,777, 9,434,931, and 10,041,053 No. 6,293,797, each of which is incorporated herein by reference in its entirety. In some embodiments, the linker is selected from the group consisting of SEQ ID NOs: 11-33, 36-37, 38-39, 39-40, 41-42, 42-43, 43-44, 44-45, 45-46, 46-47, 47-48, 48-49, 49-50, 51-52, 53-54, 5 43, 46-67, 70-89, 92-118, 121-135, 138-156, 15 9-183, 186-199, 202-219, 222-243, 246-247, 25 0~266, 269~291, 294~313, 316~325, 328~330, 33 3~340, 343~357, 360~389, 392~399, 402~433, 43 residues 154–1 of 6–462, 465–495, 498–501, and 504–529; 95 and at least 80%, at least 85%, at least 9 0%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least There may be 99% or more sequence identity.

[1060] As used herein, the term "hypervariable region" refers to a region having a relatively high degree of variability. Refers to a localized sequence within a meganuclease monomer or subunit that contains amino acids The hypervariable region may consist of about 50-60 consecutive residues, about 53-57 consecutive residues, or The hypervariable region preferably comprises about 56 residues. The residues are those of SEQ ID NOs: 11 to 33, 36 to 43, 46 to 67, 70 to 89, 92 to 118, 121-135, 138-156, 159-183, 186-199, 202-219, 222-243, 246-247, 250-266, 269-291, 294-313, 316~325, 328~330, 333~340, 343~357, 360~389, 392~399, 402~433, 436~462, 465~495, 498~501, and 504 to 529, which may correspond to positions 24 to 79 or 215 to 270. Positions 48, 50, 71, 72, 73, and 74 are located within the hypervariable region. These positions affect cleavage of the central sequence but do not necessarily affect the specific recognition sequence site. Therefore, it is unlikely that the same central sequence will affect the binding of the meganuclease to the When designing two meganucleases that target two different recognition sequences with Positions 48, 50, 71, 72, 73, and 74 were identified between the two meganucleases. The hypervariable region is a single region that contacts the DNA bases in the recognition sequence. or more residues, so as to alter the base preference of the monomer or subunit. The hypervariable region can also be modified to allow the meganuclease to recognize double-stranded DNA. It may contain one or more residues that bind to the DNA backbone when bound. Such residues may be D Modifications were made to alter the binding affinity of the meganuclease to the NA scaffold and target recognition sequence. In a different embodiment of the invention, the hypervariable region exhibits variability and can vary from base to base. can be modified to affect preference and / or DNA binding affinity In certain embodiments, the hypervariable region exhibits variability and base preference. Approximately 15-fold can be modified to affect the potency and / or DNA binding affinity In some embodiments, the variable residues in the hypervariable region comprise 20 residues. 1-33, 36-43, 46-67, 70-89, 92-118, 121-135, 13 8~156, 159~183, 186~199, 202~219, 222~243, 24 6~247, 250~266, 269~291, 294~313, 316~325, 32 8~330, 333~340, 343~357, 360~389, 392~399, 40 2-433, 436-462, 465-495, 498-501, and 504-529 Any one of 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46 , 68, 70, 75, and 77. The variable residues in the variable region are SEQ ID NOs: 11-33, 36-43, 46-67, 70-89, 92-118, 121-135, 138-156, 159-183, 186-199, 2 02~219, 222~243, 246~247, 250~266, 269~291, 2 94~313, 316~325, 328~330, 333~340, 343~357, 3 60~389, 392~399, 402~433, 436~462, 465~495, 4 215, 217, 219, 221 of any one of 98-501 and 504-529 , 223, 224, 229, 231, 233, 235, 237, 259, 261, 266 , and corresponds to one or more of positions 268.

[1061] As used herein, the terms "recombinant" or "engineered" with respect to a protein The term refers to the nucleic acid encoding the protein and the cell or organism that expresses the protein. It means having an altered amino acid sequence as a result of the application of genetic engineering techniques. With respect to an acid, the terms "recombinant" or "engineered" refer to an organism that has been produced as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, the following: PCR and DNA cloning techniques; transfection, transformation and other gene transfer techniques; homologous recombination; site-directed mutagenesis; and gene By this definition, any protein that has the same amino acid sequence as a naturally occurring protein is included. However, proteins produced by cloning and expression in a heterologous host are considered recombinant. shall not be considered to have been manipulated or altered.

[1062] As used herein, the term "wild type" refers to an allele of the same type of gene. The most common naturally occurring alleles (i.e., polynucleotide sequences) in a population The wild-type allele refers to a polypeptide that has its original function. The term "wild-type" also refers to a polypeptide encoded by a wild-type allele. Wild-type alleles (i.e., polynucleotides) and polypeptides refer to the wild-type alleles. Mutants or variants containing one or more mutations and / or substitutions relative to the native sequence A wild-type allele or polypeptide is distinguishable from other alleles and polypeptides. Tides can confer a normal phenotype to an organism, but mutant or variant alleles The gene or polypeptide may, in some cases, confer an altered phenotype. A nuclease is distinguishable from a recombinant or non-naturally occurring nuclease. The term "type" also refers to cells, organisms, and / or cells that have a wild-type allele of a particular gene. or subject, or referring to cells, organisms, and / or subjects used for comparative purposes. can be done.

[1063] As used herein, the term "genetic modification" refers to a process in which a genomic DNA sequence is recombined. Cells or organisms that have been deliberately modified by technology, or that have been modified in their ancestors As used herein, the term "genetically modified" refers to a cell or organism that has been genetically modified. The term encompasses the term "transgenic."

[1064] As used herein, the term "modified" with respect to a recombinant protein means a modification of the reference sequence. Any insertion of an amino acid residue in a recombinant sequence relative to a sequence (e.g., a wild-type or native sequence) , deletion, or substitution.

[1065] As used herein, the term "recognition sequence" refers to the wild-type I-CreI of the present disclosure. or DNA that is bound and cleaved by engineered I-CreI-derived meganucleases A sequence cleaved by I-CreI and the disclosed engineered meganucleases The disclosed recognition sequences are typically 22 nucleotides in length. are bounded by a four base pair central sequence (numbered +1, +2, +3, and +4). A pair of inverted 9-base pair "half sites" (ranging from -1 to -9, respectively) separated by In the case of single-chain meganucleases, the protein The N-terminal domain of the protein recognizes one half-site and interacts and / or contacts it. The C-terminal domain of the protein recognizes the other half-site and interacts with it. Cleavage by meganucleases occurs at the 4 base pair 3´ “overhang” "Overhangs" or "sticky ends" are formed by cleaving the end of a double-stranded DNA sequence. It is a short single-stranded DNA segment that can be generated by protease cleavage with I-CreI. In the case of derived meganucleases and single-chain meganucleases, the overhangs are 22 It contains 10 to 13 bases of the base pair recognition sequence. Therefore, I-CreI meganuclease The recognition sequence can be defined according to Formula I:

[1066] X -9 X -8 X -7 X -6 X -5 X -4 X -3 X -2 X -1 N +1 N +2 N +3 N +4 X -1 X -2 X -3 X -4 X -5 X -6 X -7 X -8 X -9 ,

[1067] In the formula, X and N each independently represent an adenine nucleotide, a cytosine nucleotide, or nucleotides selected from the group consisting of nucleotides, guanine nucleotides, and thymine nucleotides. In the formula, N +1 N +2 N +3 N +4 is the 4 base pair central sequence.

[1068] As used herein, the term "center sequence" refers to the sequence of a meganuclease recognition sequence. Refers to the four base pairs that separate the half sites. These bases are numbered +1 through +4. The central sequence is the 3' single-stranded oligonucleotide that is formed after meganuclease cleavage (Fig. 1 and Equation 1). The "center sequence" contains four bases that serve as an overhang. The meganuclease is symmetrical, with a central sequence of sense strand and The sequence A on the sense strand is recognized equally on both the sense strand and the antisense strand. + 1A +2 A +3 A +4 on the antisense strand is T +1 T +2 T +3 T +4 As Meganu are recognized, interacted with and / or contacted by the cleavage enzyme, and thus +1 A +2 A +3 A +4 and T +1 T +2 T +3 T +4 are functionally equivalent (e.g., both (which can be cleaved by a given meganuclease). Thus, the sequence C +1 T +2 G +3 C +4 The fact that meganucleases bind to their recognition sequences as symmetric homodimers The reverse strand sequence, G +1 C +2 A +3 G +4 In most cases, it is equivalent to The first subunit of the nuclease binds the first two base pairs of the sense strand of a given central sequence and and antisense, and the second two base pairs on the For example, the central array is A +1 A+2 A +3 A +4 Then, the first subunit is 2 salts Base pair A +1 A +2 recognize, interact with, and / or contact the second subunit To is T +4 T +3 Antisense strand 2 base pairs A on the antisense strand +3 A +4 Recognize Recognize, interact with, and / or come into contact with

[1069] As used herein, "recognition half-site," "recognition sequence half-site," or The term "half-site" simply refers to the structure of a homodimeric or heterodimeric meganuclease. The monomers bind to (e.g., recognize) or bind to one subunit of a single-chain meganuclease. "DNA fragment" refers to a sequence of nucleic acids in a double-stranded DNA molecule that are bound by nucleotides.

[1070] As used herein, the term "central sequence half-site" or simply "central half-site" refers to a " refers to the 5' two base pairs of the four base pair central sequence of the recognition sequences described herein. For example, for the central sequence ACAG, The 5' two base pairs of the sequence (i.e., the 5' central half site) are "AC" and the 3' two base pairs are "AC". The base pair (i.e., the 3' central half site) is "AG" (the reverse complement is "CT" ).

[1071] As used herein, a meganuclease "derived from I-CreI" or "I-CreI-derived" The term "CreI-derived meganuclease" refers to the DNA binding specificity, DNA cleavage activity, and / or affect one or more of DNA binding affinity and / or dimerization properties. Naturally occurring proteins that have been modified by the insertion, deletion, and / or substitution of one or more amino acids. refers to a recombinant mutant of the existing I-CreI homing endonuclease (SEQ ID NO: 1). Several engineered meganucleases are known in the art (e.g., Porteus et al. (2005), Nat. Biotechnol. 23:9 67-73;Sussman et al.(2004),J.Mol.Biol.34 2:31-41;Epinat et al. (2003), Nucleic Acid s Res.31:2952-62), and rationally design such mutants. A general method for this is described, for example, in WO 2007 / 047859. The I-CreI-derived meganuclease is a direct modification of I-CreI. The engineered protein, I-CreI-derived meganuclease, was further modified to Proteins derived from I-CreI and / or synthetically generated proteins based on I-CreI derived sequences As used herein, the term "variant" refers to a substantially similar A "variant" polypeptide is intended to mean a sequence similar to that of a native protein. deletion or addition of one or more amino acids at one or more internal sites, and / or natural "Natural" polypeptides are those that are produced by substitution of one or more amino acids at one or more sites in the polypeptide. As used herein, the term "polypeptide" is intended to mean a polypeptide derived from a peptide. In this case, a "naturally occurring" polynucleotide or polypeptide includes the parent sequence from which the variant is derived. In some embodiments, the "I-CreI-derived meganuclease" is a nuclease derived from the I-CreI-derived meganuclease described in WO 2004 / 024444. 007 / 047859, International Publication No. 2009059195, International Publication No. 2010 / 0 09147, WO 2012 / 167192, WO 2015 / 13873 No. 9, International Publication No. 2016 / 179112, International Publication No. 2017 / 044649, International Publication No. 2017 / 062439, International Publication No. 2017 / 062451, International Publication No. 2017 / 112859, International Publication No. 2017 / 192741, International Publication No. 2018 / 071849, International Publication No. 2018 / 195449, International Publication No. 2019 / 005 957, International Publication No. 2019 / 089913, International Publication No. 2019 / 200122 and International Publication No. PCT / US2019 / 068186 and PCT / US2020 / 013198 Specifically, any engineered meganuclease within the scope of any published claim of and JP 2003-1026634, each of which is incorporated herein by reference in its entirety. In some embodiments, the "I-CreI-derived meganuclease" is a nuclease derived from a nucleotide sequence similar to that described in U.S. Pat. No. 8,021, 867, U.S. Patent No. 8,119,361, U.S. Patent No. 8,119,381, U.S. Patent No. No. 8,124,369, U.S. Patent No. 8,129,134, U.S. Patent No. 8,133, 697, U.S. Patent No. 8,143,015, U.S. Patent No. 8,143,016, U.S. Patent No. No. 8,148,098, U.S. Patent No. 8,163,514, U.S. Patent No. 8,304, 222, U.S. Patent No. 8,377,674, U.S. Patent No. 8,445,251, U.S. Patent No. No. 9,340,777, U.S. Patent No. 9,434,931, U.S. Patent No. 10,041 ,053, U.S. Patent No. 9,683,257, U.S. Patent No. 10,287,626, U.S. Patent No. US Patent No. 10,273,524, US Patent No. 9,683,257, US Patent No. 10, 287,626, U.S. Patent No. 10,273,524, U.S. Patent No. 9,822,381 No., U.S. Patent No. 10,603,363, U.S. Patent No. 9,889,160, U.S. Patent No. No. 9,889,161, U.S. Pat. No. 9,993,501, U.S. Pat. No. 9,993,50 No. 2, U.S. Patent No. 9,950,010, U.S. Patent No. 9,950,011, U.S. Patent No. Nos. 9,969,975, 10,093,899, and 10,0 Any engineered megapixels within the scope of any issued claim of No. 93,900. Specifically included are nucleases, each of which is incorporated herein by reference. In some embodiments, the engineered I-CreI derived meganuclease is described in U.S. Pat. No. 8,021,867, U.S. Patent No. 8,119,361, U.S. Patent No. 8,119, 381, U.S. Patent No. 8,124,369, U.S. Patent No. 8,129,134, U.S. Patent No. No. 8,133,697, U.S. Patent No. 8,143,015, U.S. Patent No. 8,143, 016, U.S. Patent No. 8,148,098, U.S. Patent No. 8,163,514, U.S. Patent No. The respective issued U.S. Patent Nos. 8,304,222 and 8,377,674 Residues 2 to 153 of the I-CreI meganuclease of SEQ ID NO: 1 of the claimed invention and a small amount of In some embodiments, the polypeptides include those having at least 85% sequence identity. The constructed I-CreI-derived meganuclease is the I-CreI meganuclease of SEQ ID NO: 1. Residues 2 to 153 of the enzyme and at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity The polypeptide includes a polypeptide having the formula:

[1072] As used herein, the term "DNA binding affinity" or "binding affinity" refers to The nuclease binds non-covalently to a reference DNA molecule (e.g., a recognition sequence or any sequence). The binding affinity is measured by the dissociation constant Kd. If the Kd of the nuclease for the reference recognition sequence is higher than that of the reference nuclease, A nuclease is "altered" if it increases or decreases by a statistically significant percent change. It has a "good" binding affinity.

[1073] As used herein, the term "specificity" refers to a specific sequence, called a recognition sequence. binds to double-stranded DNA molecules only at base pairs or at a specific set of recognition sequences (e.g. The set of recognition sequences refers to the ability of a nuclease to recognize and cleave a specific protein. They share a conserved position or sequence motif, but may be degenerate at one or more positions. Highly specific nucleases cleave only one or a very few recognition sequences. Specificity can be determined by any method known in the art.

[1074] As used herein, the term "activity" refers to the ability of a meganuclease of the invention to produce a particular This activity is measured by the rate at which the enzyme cleaves the phosphodiesterase (PDS) in double-stranded DNA. It is a measurable enzymatic reaction involving bond hydrolysis. The activity of a meganuclease is determined by the affinity or binding of the meganuclease to its specific DNA substrate. are influenced by forces, which in turn interact with DNA in both sequence-specific and non-sequence-specific ways. is affected by.

[1075] As used herein, the term "altered specificity" in reference to meganucleases The term refers to a nuclease that binds to and binds to a reference nuclease (e.g., wild-type) under physiological conditions. It means that the enzyme binds to and cleaves the recognition sequence and does not cleave it, or cleaves the recognition sequence. The rate is increased by a biologically significant amount (e.g., at least 2-fold or This means an increase or decrease (or 2-fold to 10-fold) in the amount of

[1076] As used herein, with respect to both amino acid and nucleic acid sequences, "identity pattern" refers to a sequence of amino acids or nucleic acids. Terms such as "sequence identity," "percent similarity," and "sequence similarity" are used to describe Alignment of sequences that maximizes similarity between aligned amino acid residues or nucleotides. A measure of the degree of similarity between two sequences based on alignment, and is based on the number of identical or similar residues or the number of residues or nucleotides, the total number of residues or nucleotides, and the number of residues or nucleotides in the sequence alignment The presence and length of gaps between sequences is a function of the sequence similarity. Various algorithms and computer programs can be used to determine As used herein, sequence similarity is measured using the BLASTp protocol for amino acid sequences. The BLASTn program was used for gram and nucleic acid sequences, and these were both National Center for Biotechnology Information available through iotechnology information (www .ncbi.nlm.nih.gov / ), e.g., Altschul et al. 1990), J.Mol.Biol.215:403-410;Gish and St. ates(1993),Nature Genet.3:266-272;Madden et al(1996),Meth.Enzymol.266:131-141;Al tschul et al. (1997), Nucleic Acids Res.25 :33 89-3402);Zhang et al.(2000),J.Comput Biol. 7(1-2):203-14. The percent similarity of two amino acid sequences is given below for the BLASTp algorithm: The score is based on the following parameters: word size = 3; gap opening penalty gap extension penalty = -1; and score matrix = BLOSUM62. As used herein, the percent similarity between two nucleic acid sequences is determined by the BLASTn algorithm. The score is based on the following parameters for the rhythm: word size = 1; gap Opening penalty = 5; Gap extension penalty = 2; Match reward = 1; and and mismatch penalty = -3.

[1077] As used herein, "to" refers to a modification of two proteins or amino acid sequences. The term "corresponding" means that a specified modification of a first protein is the same as a modification of a second protein. The substitutions are identical and the two proteins are aligned in a standard sequence alignment. When subjected to a BLASTp program (e.g., using the BLASTp program), the first protein the amino acid positions of the modifications correspond to or align with the amino acid positions of the modifications in the second protein; Therefore, the amino acid at residue "X" in the first protein The modification to "A" is such that when residues X and Y correspond to each other in a sequence alignment, in the second protein, despite the fact that X and Y can be different numbers It corresponds to the modification of residue "Y" to amino acid "A".

[1078] As used herein, "recombinant DNA construct," "recombinant construct," "expression cassette," "Flag," "Expression Construct," "Chimeric Construct," "Construct," and "Recombinant DNA Fragment Frame" The terms "fragment" and "fragment" are used interchangeably herein and refer to either single-stranded or double-stranded polynucleotides. Recombinant constructs include, but are not limited to, nucleic acids that are not found together in nature. This includes any artificial combination of nucleic acid fragments containing different regulatory and coding sequences. A DNA construct may contain regulatory and coding sequences derived from different sources, or from the same source. regulatory and coding sequences derived from a source and arranged in a manner different from that found in nature Such constructs may be used alone or in combination with a vector. They may also be used in combination.

[1079] As used herein, the term "vector" or "recombinant DNA vector" A replication system capable of transcribing and translating a polypeptide coding sequence in a given host cell. If a vector is used, the choice of vector is determined by The method used to transform the host cell will depend on the method well known to those skilled in the art. Examples include, but are not limited to, plasmid vectors and recombinant AAV vectors. or any other vector known in the art suitable for delivering genes to target cells. Those skilled in the art can easily identify any of the isolated nucleotide or nucleic acid sequences of the present invention. present on the vector for successful transformation, selection and propagation of host cells, including In some embodiments, the genetic elements that must be "Vector" also refers to a recombinant viral vector (e.g., a recombinant virus). Viral vectors (e.g., recombinant viruses) include, but are not limited to, retroviruses. viral vectors (e.g., retroviruses), lentiviral vectors (e.g., lentiviruses), viruses), adenoviral vectors (e.g., adenoviruses), and adeno-associated viruses Examples of such vectors include viral vectors (e.g., adeno-associated viruses) (AAV).

[1080] As used herein, the recitation of a numerical range for a variable does not restrict the present disclosure to any value within that range. The intent is to convey that the method can be implemented with variables equivalent to either For a discrete variable, the variable can be equal to any integer value within the numeric range, including the endpoints of the range. Similarly, for a variable that is inherently continuous, the variable can be any number, including the endpoints of the range. It can be equal to any real value in the range, for example and without limitation, 0 to 2 A variable that is described as having a value between 0 and 1 is a variable that is discrete in nature. , or 2, and if the variable is continuous in nature it can take the values ​​0.0, 0.1, 0. It can take on the value 0.01, 0.001, or any other real value between 0 and 2 inclusive.

[1081] 2.1 Principles of the invention

[1082] The present invention, in part, improves cleavage activity for recognition sequences containing specific four-base pair central sequences. This is based on the identification of positions and residues within I-CreI that can be modified to enhance the activity of the four DNA fragments. There are A bases (A, C, G, and T), resulting in a candidate DNA sequence that is 4 base pairs in length. There are 256. As described in the pamphlet of International Publication No. 2010 / 009147 Thus, these candidate sequences were differentiated by the engineered I-CreI-derived meganuclease. Previously, wild-type I-CreI was only able to recognize the four-base pair central sequence. Therefore, the residues in I-CreI were not thought to contact or interact with the The modification may affect the cleavage efficiency and / or activity of the meganuclease for a recognition sequence having a given central sequence. It was not previously thought that this could improve the specificity or accuracy of the assay.

[1083] However, as described herein, in I-CreI derived meganucleases By modifying specific residues, cleavage of a recognition sequence with a specific four-base pair central sequence can be achieved. It was discovered that the cleavage efficiency can be improved by using I-CreI-derived meganucleotides that cleave the central sequence. Positions found to affect ase activity include positions 48 and 50 in I-CreI; , 71, 72, 73, 73B, and 74. Without constraint, these sequences bind to DNA duplexes within the meganuclease binding pocket. It is thought to assist in the placement of the nuclei, water molecules, and / or necessary metal cofactors (Figure 1). (See the crystal structure shown in Figure 4.) The modification of residues within the hypervariable regions of the meganucleases is These hypervariable region residues primarily interact with the DNA backbone to allow the meganuclease to bind specific 2 To be able to bind to the two-base pair recognition sequence, it is possible to recognize the cleavage of the central sequence described herein. It should be understood that the binding does not necessarily affect the However, the cleavage activity of meganuclease is not necessarily imparted. For example, Recognition sequences with CAA include position 48, which corresponds to I-CreI described herein; Meganuclear polypeptides in which residues 50, 71, 72, 73, 73B, and 74 are unmodified. The cleavage enzyme binds to its recognition sequence but does not cleave the TCAA central sequence. Residues 48, 50, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 3B, and one or more modifications of 74 to its central sequence (e.g., TCAA) Cleavage activity is conferred or improved.

[1084] As shown herein, modification of these specific residues results in previously difficult to cleave proteins. The cleavage efficiency of the recognition sequence having a specific central sequence was significantly increased. The sequences TTGA (reverse complement TCAA) and CCGT (reverse complement ACGG) were engineered It has previously been described that meganucleases have low cleavage efficiency (Arnould, et al.(2007).J.Mol.Biol.371:49-65 and International Publication No. W However, in accordance with the present invention, By performing the conversion, the novel engineered meganucleases are able to convert TCAA (i.e., TTAA) GA) central sequence, and a 38-fold enhancement in cleavage of the recognition sequence containing the ACGG (i.e., CC GT) showed a 21-fold enhancement in cleavage of the recognition sequence containing the central sequence (Examples 23 and 24, respectively). and 7). Thus, the present invention provides specific compounds having substitutions at specific positions. I-Cr has enhanced nuclease activity against recognition sequences containing the four-base pair central sequence of The present invention also provides engineered meganucleases derived from eI. The present invention also provides a method for cleaving double-stranded DNA using the meganuclease. Furthermore, the activity of engineered meganucleases against recognition sequences containing specific four-base pair central sequences was investigated. This invention provides a method for improving the performance of

[1085] 2.2 Engineered meganucleases optimized for specific central sequences

[1086] Site-specific nucleases can be used to make DNA breaks in the genome of living cells. Such DNA breaks can then be transduced into the genome via homologous recombination with the transgenic DNA sequence. It is known in the art that the use of nucleases can result in permanent modifications of the gene. Induction of double-strand breaks at the target locus by ribosomal RNA induces homologous recombination, specifically cleavage of a target gene homologous to the genomic target. It is known that homologous recombination of transgenic DNA sequences adjacent to the sequence is stimulated. In this way, the exogenous nucleic acid sequence can be inserted into the target locus.

[1087] Site-specific nucleases can be used to make DNA breaks in the genome of living cells. Such DNA breaks are likely to occur due to mutagenic NHEJ repair or transgenic DNA repair. It is well known in the art that this can result in permanent modification of the genome through homologous recombination with NA sequences. It is known that NHEJ causes mutagenesis at the cleavage site, resulting in allele inactivation. NHEJ-associated mutagenesis results in the generation of premature stop codons, abnormal non-functional transcription factors, and Inactivating the allele via a frameshift mutation that produces a protein or This may lead to mechanisms such as sense-mediated mRNA decay. The use of nucleases to induce mutagenesis by removing the nucleotides present in the wild-type allele. It can be used to target specific mutations or sequences that are present in the nucleotide sequence. Induction of double-strand breaks at target loci using cleavage enzymes allows for homologous recombination, particularly genomic DNA fragments. Homologous recombination of transgenic DNA sequences flanked by sequences homologous to the target is stimulated In this way, it is possible to insert an exogenous nucleic acid sequence into a target gene locus. Such exogenous nucleic acid can encode any sequence or polypeptide of interest. can be done.

[1088] As disclosed herein, the nucleases used to practice the present invention include In some embodiments, the nucleases used to practice the present invention are The nuclease is a single-chain meganuclease. The single-chain meganuclease is a single-chain meganuclease that does not contain a linker peptide. It contains an N-terminal subunit and a C-terminal subunit linked by a domain. Each of the DNA fragments recognizes and binds to half of the recognition sequence (i.e., recognition half-site), forming a DNA fragment. The A cleavage site is located in the center of the recognition sequence near the interface between the two subunits. The DNA cleavage by the meganuclease generates a pair of four-base pair 3´ single-stranded overhangs. In some embodiments, the manipulations of the invention are off-center by four base pairs to form a The engineered meganuclease binds to and cleaves a recognition sequence with a specific central sequence. is being manipulated.

[1089] The engineered meganuclease of the present invention comprises a first hypervariable (HVR1) region. subunit, and a second subunit comprising a second hypervariable (HVR2) region. Furthermore, the first subunit binds to the first recognition half-site of the recognition sequence and the second subunit binds to the first recognition half-site of the recognition sequence. The subunit binds to the second recognition half-site of the recognition sequence. In embodiments where the enzyme is a single-chain meganuclease, the first subunit and the second subunit The first subunit contains the HVR1 region and binds to the first half-site. The first half-site is arranged as a terminal subunit and contains the HVR2 region. The second subunit can be oriented so that it is positioned as the C-terminal subunit. In one embodiment, the first subunit and the second subunit comprise an HVR1 region and The first subunit that binds to the half-site of H is positioned as the C-terminal subunit, The second subunit, which contains the VR2 region and binds to the second half-site, is the N-terminal subunit. As disclosed herein, meganuclei can be oriented so as to be arranged as a Specific modifications to the nucleotide sequence (e.g., positions 48, 50, 71, 72, 73, 73B, and 74) results in increased cleavage of recognition sequences with a specific four base pair central sequence. Exemplary engineered megakaryons of the invention demonstrating improved cleavage of recognition sequences containing specific central sequences. Nucleases are those of SEQ ID NOs: 11-33, 36-43, 46-67, 70-89, 92-1 18, 121-135, 138-156, 159-183, 186-199, 202-2 19, 222-243, 246-247, 250-266, 269-291, 294-3 13, 316-325, 328-330, 333-340, 343-357, 360-3 89, 392-399, 402-433, 436-462, 465-495, 498-5 Available in 01 and 504-529.

[1090] In certain embodiments, the engineered meganucleases of the present invention are homodimeric or heterodimeric. Each of the two subunits of the dimer is a dimer of SEQ ID NO: 1 (i.e., I -CreI). The engineered meganucleases disclosed herein are derived from certain Increased activity of engineered meganucleases against recognition sequences containing central sequences (e.g., cleavage) a modification (e.g., substitution) in a single subunit that confers increased cleavage activity; or It may contain modifications in both subunits.

[1091] In some instances, the first or second subunit of an I-CreI derived meganuclease The two subunits are at least 70% identical to wild-type I-CreI (SEQ ID NO: 1), 75%, at least 80%, at least 81%, at least 82%, at least 83% , at least 84%, at least 85%, at least 86%, at least 87%, less At least 88%, at least 89%, at least 90%, at least 91%, at least 92% %, at least 93%, at least 94%, at least 96%, at least 97%, at least may have at least 98%, at least 99%, or at least 99.5% sequence identity. In some embodiments, the first of any of the disclosed engineered meganucleases The subunit and / or the second subunit may be selected from the group consisting of positions 48, 50, 71, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104 excluding amino acid substitutions at one or more positions corresponding to positions 71, 72, 73, and 74. and a sequence similar to SEQ ID NO: 1 by at least 75%, at least 80%, at least 85%, or at least 88%, at least 90%, at least 92%, at least 94%, at least 96% , may have at least 97%, at least 98%, or at le...

Claims

1. ACAA, ACAG, ACAT, ACGA, ACGC, ACGG, ACGT, ATAA , ATAG, ATAT, ATGA, ATGG, TTGG, GCAA, GCAT, GCGA a recognition sequence comprising a central sequence selected from the group consisting of GCAG, TCAA, or TTAA; An engineered I-CreI-derived meganuclease that binds to and cleaves the first sequence. a first subunit and a second subunit, Each of the units comprises an amino acid sequence derived from SEQ ID NO: 1, and the first subunit and the second subunit is a sequence corresponding to positions 48, 50, 71, and 72 of SEQ ID NO: 1, respectively. , 73, 73B, and 74. I-CreI-derived meganuclease.

2. 2. The enzyme of claim 1, wherein the first subunit comprises one or more of the following residues: The meganucleases developed: (a) A, C, D, G, H, I, K, L, N, Q at the position corresponding to position 48 of SEQ ID NO: 1 , R, S, or T residues; (b) A, C, D, E, G, I, K, L, N, Q at the position corresponding to position 50 of SEQ ID NO: 1 , R, S, T, V, or W residues; (c) A, C, G, H, I, K, N, P, R, S at the position corresponding to position 71 of SEQ ID NO: 1 , or a T residue; (d) A, D, G, H, K, L, M, N, P, Q at the position corresponding to position 72 of SEQ ID NO: 1 , R, S, T, or V residues; (e) an A, C, G, I, S, T, or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an A, C, T, or S residue at a position corresponding to position 74 of SEQ ID NO:

1.

3. 10. The method of claim 1 or claim 9, wherein the second subunit comprises one or more of the following residues: Item 2. The engineered meganuclease (a) A, C, G, H, I, K, L, N, Q, R at the position corresponding to position 48 of SEQ ID NO: 1 , S, or T residues; (b) A, C, E, G, H, I, K, N, P, Q at the position corresponding to position 50 of SEQ ID NO: 1 , R, S, T, or V residues; (c) A, D, E, G, H, I, K, N, P, Q at the position corresponding to position 71 of SEQ ID NO: 1 , R, S, T, or Y residues; (d) A, C, E, G, H, I, K, M, N, P at a position corresponding to position 72 of SEQ ID NO: 1 , Q, R, S, T, V, or Y residues; (e) A, C, G, H, I, R, S, T, or a residue at the position corresponding to position 73 of SEQ ID NO:

1. V residue; and (f) an A, C, S, or T residue at a position corresponding to position 74 of SEQ ID NO:

1.

4. The central sequence is ACAA, ACAG, ACAT, ACGC, ACGG, or ACG T, wherein the first subunit comprises one or more of the following residues: The engineered meganuclease according to any one of claims 1 to 3. (a) A, C, G, H, I, K, L, N, Q, or or S residue; (b) A, C, K, Q, R, S, T, V, or a residue at the position corresponding to position 50 of SEQ ID NO: 1 W residue; (c) an A, G, P, or R residue at a position corresponding to position 71 of SEQ ID NO:1; (d) an H, K, P, Q, R, or T residue at a position corresponding to position 72 of SEQ ID NO:1; (e) an A, C, G, or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an S residue at a position corresponding to position 74 of SEQ ID NO:

1.

5. the central sequence consists of ATAA, ATAG, ATAT, ATGA, and ATGG, 4. The method of claim 1, wherein one subunit comprises one or more of the following residues: The engineered meganucleases described: (a) A, C, D, G, H, K, L, N, Q, S at the position corresponding to position 48 of SEQ ID NO: 1 , or a T residue; (b) C, D, E, G, I, K, N, R, S, T at the position corresponding to position 50 of SEQ ID NO: 1 , or V residue; (c) a G, H, I, K, N, R, or S residue at a position corresponding to position 71 of SEQ ID NO:1; (d) A, G, H, K, L, N, P, Q, R, S at the position corresponding to position 72 of SEQ ID NO: 1 , or a T residue; (e) an A, C, S, or T residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an A, C, or S residue at a position corresponding to position 74 of SEQ ID NO:

1.

6. the central sequence consists of GCAA, GCAT, GCGA, or GCAG, and the first 4. The method of claim 1, wherein the subunit comprises one or more of the following residues: Engineered meganucleases: (a) an A, H, K, or R residue at a position corresponding to position 48 of SEQ ID NO:1; (b) a C, K, L, Q, R, S, T, or V residue at a position corresponding to position 50 of SEQ ID NO: 1; Base; (c) an A, G, H, N, R, S, or T residue at a position corresponding to position 71 of SEQ ID NO:1; (d) A, G, H, M, N, P, Q, R, S, T at the position corresponding to position 72 of SEQ ID NO: 1 , or V residue; (e) an A, C, I, T, or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an A or S residue at a position corresponding to position 74 of SEQ ID NO:

1.

7. the central sequence consists of TTGG or TTAA, and the first subunit has the following residues: The engineered meganucleic acid of any one of claims 1 to 3, comprising one or more of the following groups: Aze: (a) a K, N, R, or S residue at a position corresponding to position 48 of SEQ ID NO:1; (b) a C, E, K, R, S, T, or V residue at a position corresponding to position 50 of SEQ ID NO:1; (c) an A, G, K, N, R, or S residue at a position corresponding to position 71 of SEQ ID NO:1; (d) A, D, H, K, N, Q, R, S, or a residue at the position corresponding to position 72 of SEQ ID NO:

1. T residue; (e) an I or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an A, S, or T residue at a position corresponding to position 74 of SEQ ID NO:

1.

8. the central sequence consists of TCAA and the first subunit consists of one of the following residues:

4. The engineered meganuclease of any one of claims 1 to 3, comprising: (a) an A, G, H, K, N, Q, R, or S residue at a position corresponding to position 48 of SEQ ID NO: 1; Base; (b) a C, R, S, or T residue at a position corresponding to position 50 of SEQ ID NO:1; (c) a G, R, S, or T residue at a position corresponding to position 71 of SEQ ID NO:1; (d) a G, H, P, R, S, or T residue at a position corresponding to position 72 of SEQ ID NO:1; (e) an I or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an A or S residue at a position corresponding to position 74 of SEQ ID NO:

1.

9. the central sequence is ACAA, ACAG, ACAT, ACGC, ACGG, or ACGT wherein the second subunit comprises one or more of the following residues: The engineered meganuclease of any one of claims 1 to 4, (a) A, C, G, H, K, L, N, Q, R, S at the position corresponding to position 48 of SEQ ID NO: 1 , or a T residue; (b) A, C, G, H, K, L, N, Q, R, S at the position corresponding to position 50 of SEQ ID NO: 1 , or a T residue; (c) A, D, E, G, H, K, N, P, R, S at the position corresponding to position 71 of SEQ ID NO: 1 , or a T residue; (d) A, G, H, K, M, N, P, P, Q, R at the position corresponding to position 72 of SEQ ID NO: 1 , S, or T residues; (e) A, C, G, H, I, R, S, T, or a residue at the position corresponding to position 73 of SEQ ID NO:

1. V residue; (f) optionally, an R residue at position (73B) immediately following the position corresponding to position 73 of SEQ ID NO:1; groups; and (g) an A, C, S, or T residue at a position corresponding to position 74 of SEQ ID NO:

1.

10. the central sequence consists of ATAA, ATAG, ATAT, ATGA, or ATGG; The second subunit comprises one or more of the following residues:

6. The engineered meganuclease of any one of clauses 5: (a) A, C, G, H, K, N, Q, R, S, or or T residue; (b) A, C, E, I, K, N, Q, R, S, or or T residue; (c) A, C, E, I, K, N, Q, R, S, or or T residue; (d) A, G, H, K, N, Q, R, S, T, V at the position corresponding to position 72 of SEQ ID NO: 1 , or Y residue; (e) an A, C, G, H, I, R, S, or V residue at a position corresponding to position 73 of SEQ ID NO: 1; Base; (f) optionally, an R residue at position (73B) immediately following the position corresponding to position 73 of SEQ ID NO:1; groups; and (g) an A, C, S, or T residue at a position corresponding to position 74 of SEQ ID NO:

1.

11. the central sequence consists of GCAA, GCAT, GCGA, or GCAG, and the second 10. The method of claim 1, wherein the subunits comprise one or more of the following residues: The engineered meganuclease of any one of claims 1 to 4, (a) A, C, G, H, I, K, L, N, Q, R at the position corresponding to position 48 of SEQ ID NO: 1 , S, or T residues; (b) C, E, H, K, Q, R, S, T, or a residue at a position corresponding to position 50 of SEQ ID NO: 1 V residue; (c) an A, G, H, K, R, S, T, or Y residue at a position corresponding to position 71 of SEQ ID NO: 1; Base; (d) A, C, E, G, H, K, N, Q, R, S at the position corresponding to position 72 of SEQ ID NO: 1 , T, or Y residues; (e) an A, C, G, H, I, R, S, or V residue at a position corresponding to position 73 of SEQ ID NO: 1; groups; and (f) an A, S, or T residue at a position corresponding to position 74 of SEQ ID NO:

1.

12. the central sequence consists of TTGG or TTAA, and the second subunit has the following residues: The engineered compound according to any one of claims 1 to 3 or claim 7, comprising one or more of the following groups: Selected meganucleases: (a) an A, K, S, or T residue at a position corresponding to position 48 of SEQ ID NO:1; (b) a C, E, K, R, or T residue at a position corresponding to position 50 of SEQ ID NO:1; (c) an A, D, G, K, Q, R, S, or T residue at a position corresponding to position 71 of SEQ ID NO: 1; Base; (d) a G, I, R, S, T, or V residue at a position corresponding to position 72 of SEQ ID NO:1; (e) an I, R, or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an A, S, or T residue at a position corresponding to position 74 of SEQ ID NO:

1.

13. the central sequence consists of TCAA and the second subunit consists of one of the following residues: The engineered meganucleic acid of any one of claims 1 to 3 or claim 8, comprising Aze: (a) a K or S residue at a position corresponding to position 48 of SEQ ID NO:1; (b) a C, K, R, or T residue at a position corresponding to position 50 of SEQ ID NO:1; (c) a G, R, or T residue at a position corresponding to position 71 of SEQ ID NO:1; (d) a G, P, R, S, or T residue at a position corresponding to position 72 of SEQ ID NO:1; (e) an I or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an A, S, or T residue at a position corresponding to position 74 of SEQ ID NO:

1.

14. 2. The engineered meganuclease of claim 1, wherein: (a) the central sequence is ACAA and the first subunit is selected from the group consisting of SEQ ID NOs: 11-33 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (b) the central sequence is ACAG and the first subunit is SEQ ID NO: 36-43 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (c) the central sequence is ACAT and the first subunit is SEQ ID NO: 46-67 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (d) the central sequence is ACGA and the first subunit is selected from the group consisting of SEQ ID NOs: 70-89 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (e) the central sequence is ACGC and the first subunit is SEQ ID NO: 92-11 8, containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of nothing, (f) the central sequence is ACGG and the first subunit is SEQ ID NO: 121-1 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 35 include, (g) the central sequence is ACGT and the first subunit is SEQ ID NO: 138-1 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 56 include, (h) the central sequence is ATAA and the first subunit is SEQ ID NO: 159-1 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 83 include, (i) the central sequence is ATAG and the first subunit is SEQ ID NO: 186-199 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (j) the central sequence is ATAT and the first subunit is SEQ ID NO: 202-203 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 19 include, (k) the central sequence is ATGA and the first subunit is SEQ ID NO: 222-222 Residues corresponding to any one of residues 48, 50, 71, 72, 73, and 74 of 43 include, (l) the central sequence is ATGG and the first subunit is SEQ ID NO: 246-247 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 47 include, (m) the central sequence is TTGG and the first subunit is SEQ ID NO: 250-252 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 66 include, (n) the central sequence is GCAA and the first subunit is SEQ ID NO: 269-2 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 91 include, (o) the central sequence is GCAT and the first subunit is SEQ ID NO: 294-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 13 include, (p) the central sequence is GCGA and the first subunit is SEQ ID NO: 316-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 25 include, (q) the central sequence is GCAG and the first subunit is SEQ ID NO: 328-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of the 30 include, (r) the central sequence is TCAA and the first subunit is SEQ ID NO: 333-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 40 Contains, or (s) the central sequence is TTAA and the first subunit is selected from the group consisting of SEQ ID NOs: 343 to 349; Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 357 Includes.

15. 15. The engineered meganuclease of any one of claims 1 to 14, wherein: (a) the central sequence is ACAA and the second subunit is a sequence selected from the group consisting of SEQ ID NOs: 11 to 33 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (b) the central sequence is ACAG and the second subunit is SEQ ID NO: 36-43 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (c) the central sequence is ACAT and the second subunit is SEQ ID NO: 46-67 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (d) the central sequence is ACGA and the second subunit is selected from the group consisting of SEQ ID NOs: 70-89 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (e) the central sequence is ACGC and the second subunit is SEQ ID NO: 92-11 8, containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of nothing, (f) the central sequence is ACGG and the second subunit is SEQ ID NO: 121-1 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 35 include, (g) the central sequence is ACGT and the second subunit is SEQ ID NO: 138-1 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 56 include, (h) the central sequence is ATAA and the second subunit is SEQ ID NO: 159-1 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 83 include, (i) the central sequence is ATAG and the second subunit is SEQ ID NO: 186-199 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (j) the central sequence is ATAT and the second subunit is SEQ ID NO: 202-203 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 19 include, (k) the central sequence is ATGA and the second subunit is SEQ ID NO: 222-222 Residues corresponding to any one of residues 48, 50, 71, 72, 73, and 74 of 43 include, (l) the central sequence is ATGG and the second subunit is SEQ ID NO: 246-247 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 47 include, (m) the central sequence is TTGG and the second subunit is SEQ ID NO: 250-252 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 66 include, (n) the central sequence is GCAA and the second subunit is SEQ ID NO: 269-2 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 91 include, (o) the central sequence is GCAT and the second subunit is SEQ ID NO: 294-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 13 include, (p) the central sequence is GCGA and the second subunit is SEQ ID NO: 316-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 25 include, (q) the central sequence is GCAG and the second subunit is SEQ ID NO: 328-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of the 30 include, (r) the central sequence is TCAA and the second subunit is SEQ ID NO: 333-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 40 Contains, or (s) the central sequence is TTAA and the second subunit is selected from the group consisting of SEQ ID NOs: 343 to 349; Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 357 Includes:

16. ACAA, ACAG, ACAT, ACGA, ACGC, ACGG, ACGT, ATAA , ATAG, ATAT, ATGA, ATGG, TTGG, GCAA, GCAT, GCGA A meganuclease recognition sequence containing a central sequence consisting of GCAG, TCAA, or TTAA. A method for cleaving double-stranded DNA at a target site comprising a sequence comprising: The double-stranded DNA is contacted with the engineered meganuclease of any one of claims 1 to 15. contacting the engineered meganuclease with the recognition sequence, wherein the engineered meganuclease binds to and cleaves the recognition sequence. method.

17. ACAA, ACAG, ACAT, ACGA, ACGC, ACGG, ACGT, ATAA , ATAG, ATAT, ATGA, ATGG, TTGG, GCAA, GCAT, GCGA , GCAG, TCAA, or TTAA. The present invention relates to a method for producing a phospholipase C10-containing ... and the second subunit each comprise an engineered amino acid sequence derived from SEQ ID NO:

1. A method for enhancing the cleavage activity of a meganuclease, comprising: The second subunits are located at positions 48, 50, 71, 72, and 73 of SEQ ID NO: 1, respectively. 3, and one or more positions corresponding to positions 74, and the modified nuclease is It has enhanced cleavage activity compared to the control engineered megaknuclease. Including, a method.

18. the step of modifying the first subunit to include one or more of the following residues:

18. The method of claim 17, comprising modifying the (a) A, C, D, G, H, I, K, L, N, Q at the position corresponding to position 48 of SEQ ID NO: 1 , R, S, or T residues; (b) A, C, D, E, G, I, K, L, N, Q at the position corresponding to position 50 of SEQ ID NO: 1 , R, S, T, V, or W residues; (c) A, C, G, H, I, K, N, P, R, S at the position corresponding to position 71 of SEQ ID NO: 1 , or a T residue; (d) A, D, G, H, K, L, M, N, P, Q at the position corresponding to position 72 of SEQ ID NO: 1 , R, S, T, or V residues; (e) an A, C, G, I, S, T, or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an A, C, T, or S residue at a position corresponding to position 74 of SEQ ID NO:

1.

19. the step of modifying the second subunit to include one or more of the following residues: The method of claim 17 or 18, comprising modifying the (a) A, C, G, H, I, K, L, N, Q, R at the position corresponding to position 48 of SEQ ID NO: 1 , S, or T residues; (b) A, C, E, G, H, I, K, N, P, Q at the position corresponding to position 50 of SEQ ID NO: 1 , R, S, T, or V residues; (c) A, D, E, G, H, I, K, N, P, Q at the position corresponding to position 71 of SEQ ID NO: 1 , R, S, T, or Y residues; (d) A, C, E, G, H, I, K, M, N, P at a position corresponding to position 72 of SEQ ID NO: 1 , Q, R, S, T, V, or Y residues; (e) A, C, G, H, I, R, S, T, or a residue at the position corresponding to position 73 of SEQ ID NO:

1. V residue; and (f) an A, C, S, or T residue at a position corresponding to position 74 of SEQ ID NO:

1.

20. The central sequence is ACAA, ACAG, ACAT, ACGC, ACGG, or ACG T, and said modifying step modifies said first subunit to one of the following residues: The method of any one of claims 17 to 19, modified to include: (a) A, C, G, H, I, K, L, N, Q, or or S residue; (b) A, C, K, Q, R, S, T, V, or a residue at the position corresponding to position 50 of SEQ ID NO: 1 W residue; (c) an A, G, P, or R residue at a position corresponding to position 71 of SEQ ID NO:1; (d) an H, K, P, Q, R, or T residue at a position corresponding to position 72 of SEQ ID NO:1; (e) an A, C, G, or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an S residue at a position corresponding to position 74 of SEQ ID NO:

1.

21. the central sequence consists of ATAA, ATAG, ATAT, ATGA, or ATGG; wherein said modifying step modifies said first subunit to include one or more of the following residues: The method according to any one of claims 17 to 19, comprising modifying the (a) A, C, D, G, H, K, L, N, Q, S at the position corresponding to position 48 of SEQ ID NO: 1 , or a T residue; (b) C, D, E, G, I, K, N, R, S, T at the position corresponding to position 50 of SEQ ID NO: 1 , or V residue; (c) a G, H, I, K, N, R, or S residue at a position corresponding to position 71 of SEQ ID NO:1; (d) A, G, H, K, L, N, P, Q, R, S at the position corresponding to position 72 of SEQ ID NO: 1 , or a T residue; (e) an A, C, S, or T residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an A, C, or S residue at a position corresponding to position 74 of SEQ ID NO:

1.

22. the central sequence consists of GCAA, GCAT, GCGA, or GCAG, and modifying the first subunit to include one or more of the following residues: The method according to any one of claims 17 to 19, comprising: (a) an A, H, K, or R residue at a position corresponding to position 48 of SEQ ID NO:1; (b) a C, K, L, Q, R, S, T, or V residue at a position corresponding to position 50 of SEQ ID NO: 1; Base; (c) an A, G, H, N, R, S, or T residue at a position corresponding to position 71 of SEQ ID NO:1; (d) A, G, H, M, N, P, Q, R, S, T at the position corresponding to position 72 of SEQ ID NO: 1 , or V residue; (e) an A, C, I, T, or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an A or S residue at a position corresponding to position 74 of SEQ ID NO:

1.

23. the central sequence consists of TTGG or TTAA, and the modifying step comprises The claim includes modifying the subunit to include one or more of the following residues:

20. The method according to any one of claims 17 to 19, (a) a K, N, R, or S residue at a position corresponding to position 48 of SEQ ID NO:1; (b) a C, E, K, R, S, T, or V residue at a position corresponding to position 50 of SEQ ID NO:1; (c) an A, G, K, N, R, or S residue at a position corresponding to position 71 of SEQ ID NO:1; (d) A, D, H, K, N, Q, R, S, or a residue at the position corresponding to position 72 of SEQ ID NO:

1. T residue; (e) an I or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an A, S, or T residue at a position corresponding to position 74 of SEQ ID NO:

1.

24. the central sequence consists of TCAA, and the modifying step modifies the first subunit to: Any of claims 17 to 19, including modifications to include one or more of the following residues: The method according to any one of claims 1 to 4, (a) an A, G, H, K, N, Q, R, or S residue at a position corresponding to position 48 of SEQ ID NO: 1; Base; (b) a C, R, S, or T residue at a position corresponding to position 50 of SEQ ID NO:1; (c) a G, R, S, or T residue at a position corresponding to position 71 of SEQ ID NO:1; (d) a G, H, P, R, S, or T residue at a position corresponding to position 72 of SEQ ID NO:1; (e) an I or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an A or S residue at a position corresponding to position 74 of SEQ ID NO:

1.

25. The central sequence is ACAA, ACAG, ACAT, ACGC, ACGG, or ACG T, and said modifying step modifies said second subunit to one of the following residues: The method of any one of claims 17 to 20, modified to include: (a) A, C, G, H, K, L, N, Q, R, S at the position corresponding to position 48 of SEQ ID NO: 1 , or a T residue; (b) A, C, G, H, K, L, N, Q, R, S at the position corresponding to position 50 of SEQ ID NO: 1 , or a T residue; (c) A, D, E, G, H, K, N, P, R, S at the position corresponding to position 71 of SEQ ID NO: 1 , or a T residue; (d) A, G, H, K, M, N, P, P, Q, R at the position corresponding to position 72 of SEQ ID NO: 1 , S, or T residues; (e) A, C, G, H, I, R, S, T, or a residue at the position corresponding to position 73 of SEQ ID NO:

1. V residue; (f) optionally, an R residue at position (73B) immediately following the position corresponding to position 73 of SEQ ID NO:1; groups; and (g) an A, C, S, or T residue at a position corresponding to position 74 of SEQ ID NO:

1.

26. the central sequence consists of ATAA, ATAG, ATAT, ATGA, or ATGG; wherein said modifying step modifies said second subunit to include one or more of the following residues: The method according to any one of claims 17 to 19 or claim 21, comprising modifying the method as follows: method: (a) A, C, G, H, K, N, Q, R, S, or or T residue; (b) A, C, E, I, K, N, Q, R, S, or or T residue; (c) A, C, E, I, K, N, Q, R, S, or or T residue; (d) A, G, H, K, N, Q, R, S, T, V at the position corresponding to position 72 of SEQ ID NO: 1 , or Y residue; (e) an A, C, G, H, I, R, S, or V residue at a position corresponding to position 73 of SEQ ID NO: 1; Base; (f) optionally, an R residue at position (73B) immediately following the position corresponding to position 73 of SEQ ID NO:1; groups; and (g) an A, C, S, or T residue at a position corresponding to position 74 of SEQ ID NO:

1.

27. the central sequence consists of GCAA, GCAT, GCGA, or GCAG, and modifying the second subunit to include one or more of the following residues: The method of any one of claims 17 to 19 or claim 22, comprising: (a) A, C, G, H, I, K, L, N, Q, R at the position corresponding to position 48 of SEQ ID NO: 1 , S, or T residues; (b) C, E, H, K, Q, R, S, T, or a residue at a position corresponding to position 50 of SEQ ID NO: 1 V residue; (c) an A, G, H, K, R, S, T, or Y residue at a position corresponding to position 71 of SEQ ID NO: 1; Base; (d) A, C, E, G, H, K, N, Q, R, S at the position corresponding to position 72 of SEQ ID NO: 1 , T, or Y residues; (e) an A, C, G, H, I, R, S, or V residue at a position corresponding to position 73 of SEQ ID NO: 1; groups; and (f) an A, S, or T residue at a position corresponding to position 74 of SEQ ID NO:

1.

28. the central sequence consists of TTGG or TTAA, and the modifying step is 1, comprising modifying the hydroxyl group of the hydroxyl group to include one or more of the following residues: The method of any one of claims 7 to 19 or 23, (a) an A, K, S, or T residue at a position corresponding to position 48 of SEQ ID NO:1; (b) a C, E, K, R, or T residue at a position corresponding to position 50 of SEQ ID NO:1; (c) an A, D, G, K, Q, R, S, or T residue at a position corresponding to position 71 of SEQ ID NO: 1; Base; (d) a G, I, R, S, T, or V residue at a position corresponding to position 72 of SEQ ID NO:1; (e) an I, R, or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an A, S, or T residue at a position corresponding to position 74 of SEQ ID NO:

1.

29. the central sequence consists of TCAA, and the modifying step modifies the second subunit to:

19. The method of claim 17, wherein the amino acid sequence is modified to include one or more of the following residues:

25. The method of any one of claims 24 to 24, (a) a K or S residue at a position corresponding to position 48 of SEQ ID NO:1; (b) a C, K, R, or T residue at a position corresponding to position 50 of SEQ ID NO:1; (c) a G, R, or T residue at a position corresponding to position 71 of SEQ ID NO:1; (d) a G, P, R, S, or T residue at a position corresponding to position 72 of SEQ ID NO:1; (e) an I or V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an A, S, or T residue at a position corresponding to position 74 of SEQ ID NO:

1.

30. The method according to any one of claims 17 to 29, (a) the central sequence is ACAA and the first subunit is selected from the group consisting of SEQ ID NOs: 11-33 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (b) the central sequence is ACAG and the first subunit is SEQ ID NO: 36-43 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (c) the central sequence is ACAT and the first subunit is SEQ ID NO: 46-67 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (d) the central sequence is ACGA and the first subunit is selected from the group consisting of SEQ ID NOs: 70-89 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (e) the central sequence is ACGC and the first subunit is SEQ ID NO: 92-11 8, containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of nothing, (f) the central sequence is ACGG and the first subunit is SEQ ID NO: 121-1 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 35 include, (g) the central sequence is ACGT and the first subunit is SEQ ID NO: 138-1 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 56 include, (h) the central sequence is ATAA and the first subunit is SEQ ID NO: 159-1 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 83 include, (i) the central sequence is ATAG and the first subunit is SEQ ID NO: 186-199 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (j) the central sequence is ATAT and the first subunit is SEQ ID NO: 202-203 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 19 include, (k) the central sequence is ATGA and the first subunit is SEQ ID NO: 222-222 Residues corresponding to any one of residues 48, 50, 71, 72, 73, and 74 of 43 include, (l) the central sequence is ATGG and the first subunit is SEQ ID NO: 246-247 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 47 include, (m) the central sequence is TTGG and the first subunit is SEQ ID NO: 250-252 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 66 include, (n) the central sequence is GCAA and the first subunit is SEQ ID NO: 269-2 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 91 include, (o) the central sequence is GCAT and the first subunit is SEQ ID NO: 294-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 13 include, (p) the central sequence is GCGA and the first subunit is SEQ ID NO: 316-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 25 include, (q) the central sequence is GCAG and the first subunit is SEQ ID NO: 328-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of the 30 include, (r) the central sequence is TCAA and the first subunit is SEQ ID NO: 333-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 40 Contains, or (s) the central sequence is TTAA and the first subunit is selected from the group consisting of SEQ ID NOs: 343 to 349; Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 357 Includes.

31. The method according to any one of claims 17 to 30, (a) the central sequence is ACAA and the second subunit is a sequence selected from the group consisting of SEQ ID NOs: 11 to 33 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (b) the central sequence is ACAG and the second subunit is SEQ ID NO: 36-43 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (c) the central sequence is ACAT and the second subunit is SEQ ID NO: 46-67 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (d) the central sequence is ACGA and the second subunit is selected from the group consisting of SEQ ID NOs: 70-89 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (e) the central sequence is ACGC and the second subunit is SEQ ID NO: 92-11 8, containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of nothing, (f) the central sequence is ACGG and the second subunit is SEQ ID NO: 121-1 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 35 include, (g) the central sequence is ACGT and the second subunit is SEQ ID NO: 138-1 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 56 include, (h) the central sequence is ATAA and the second subunit is SEQ ID NO: 159-1 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 83 include, (i) the central sequence is ATAG and the second subunit is SEQ ID NO: 186-199 containing residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 、 (j) the central sequence is ATAT and the second subunit is SEQ ID NO: 202-203 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 19 include, (k) the central sequence is ATGA and the second subunit is SEQ ID NO: 222-222 Residues corresponding to any one of residues 48, 50, 71, 72, 73, and 74 of 43 include, (l) the central sequence is ATGG and the second subunit is SEQ ID NO: 246-247 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 47 include, (m) the central sequence is TTGG and the second subunit is SEQ ID NO: 250-252 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 66 include, (n) the central sequence is GCAA and the second subunit is SEQ ID NO: 269-2 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 91 include, (o) the central sequence is GCAT and the second subunit is SEQ ID NO: 294-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 13 include, (p) the central sequence is GCGA and the second subunit is SEQ ID NO: 316-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 25 include, (q) the central sequence is GCAG and the second subunit is SEQ ID NO: 328-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of the 30 include, (r) the central sequence is TCAA and the second subunit is SEQ ID NO: 333-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 40 Contains, or (s) the central sequence is TTAA and the second subunit is selected from the group consisting of SEQ ID NOs: 343 to 349; Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 357 Includes.

32. From GTAA, GTAG, GTAT, GTGA, GTGC, GTGG, or GTGT an engineered meganuclease that binds to and cleaves a recognition sequence comprising a central sequence comprising: a first subunit and a second subunit, wherein the first subunit is 1, wherein the first subunit comprises an amino acid sequence derived from positions 48, 50, and 60 of SEQ ID NO:

1. 71, 72, 73, and 74, Engineered meganucleases.

33. 33. The method of claim 32, wherein the first subunit comprises one or more of the following residues: Engineered meganucleases: (a) A, C, G, H, K, L, M, N, Q, R at the position corresponding to position 48 of SEQ ID NO: 1 , S, T, or V residues; (b) A, C, E, G, I, K, L, Q, R, S at the position corresponding to position 50 of SEQ ID NO: 1 , T, or V residues; (c) A, D, E, F, G, H, I, K, L, N at the position corresponding to position 71 of SEQ ID NO: 1 , Q, R, S, T, V, or Y residues; (d) A, C, D, G, H, K, M, N, P, Q at the position corresponding to position 72 of SEQ ID NO: 1 , R, S, T, V, W, or Y residues; (e) A, C, I, L, N, R, S, T, or a residue at the position corresponding to position 73 of SEQ ID NO: 1 V residue; and (f) an A, C, G, S, or T residue at a position corresponding to position 74 of SEQ ID NO:

1.

34. 32 or claim 33, wherein the second subunit comprises one or more of the following residues:

34. The engineered meganuclease of claim 33: (a) a K residue at a position corresponding to position 48 of SEQ ID NO:1; (b) a Q residue at a position corresponding to position 50 of SEQ ID NO:1; (c) a G residue at a position corresponding to position 71 of SEQ ID NO:1; (d) an S residue at a position corresponding to position 72 of SEQ ID NO:1; (e) a V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an S residue at a position corresponding to position 74 of SEQ ID NO:

1.

35. 35. The engineered meganuclease of any one of claims 32 to 34, wherein: (a) the central sequence is GTAA and the first subunit is SEQ ID NO: 360-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 89 include, (b) the central sequence is GTAG and the first subunit is SEQ ID NO: 392-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 99 include, (c) the central sequence is GTAT and the first subunit is SEQ ID NO: 402-404 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 33 include, (d) the central sequence is GTGA and the first subunit is selected from the group consisting of SEQ ID NOs: 436-437 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 62 include, (e) the central sequence is GTGC and the first subunit is SEQ ID NO: 465-4 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 95 include, (f) the central sequence is GTGG and the first subunit is SEQ ID NO: 498-5 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of .01 Contains, or (g) the central sequence is GTGT and the first subunit is selected from the group consisting of SEQ ID NOs: 504 to 508; Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 529 Includes.

36. From GTAA, GTAG, GTAT, GTGA, GTGC, GTGG, or GTGT and cleaving double-stranded DNA at a target site containing a meganuclease recognition sequence comprising a central sequence comprising The method for detecting a target site comprises the step of: and contacting the engineered meganuclease of claim 1 with the engineered meganuclease of claim 1, , binding to and cleaving said recognition sequence.

37. From GTAA, GTAG, GTAT, GTGA, GTGC, GTGG, or GTGT a first subunit and a second subunit that bind to and cleave a recognition sequence comprising a central sequence comprising a first subunit comprising an amino acid sequence derived from SEQ ID NO:1; A method for enhancing the cleavage activity of a meganuclease comprising: The fragments are those corresponding to positions 48, 50, 71, 72, 73, and 74 of SEQ ID NO:

1. and modifying the modified nuclease at one or more positions in the target engineered meganuclease. The method comprises: having enhanced cleavage activity compared to a cleavage enzyme.

38. the step of modifying the first subunit to include one or more of the following residues:

38. The method of claim 37, comprising modifying the (a) A, C, G, H, K, L, M, N, Q, R at the position corresponding to position 48 of SEQ ID NO: 1 , S, T, or V residues; (b) A, C, E, G, I, K, L, Q, R, S at the position corresponding to position 50 of SEQ ID NO: 1 , T, or V residues; (c) A, D, E, F, G, H, I, K, L, N at the position corresponding to position 71 of SEQ ID NO: 1 , Q, R, S, T, V, or Y residues; (d) A, C, D, G, H, K, M, N, P, Q at the position corresponding to position 72 of SEQ ID NO: 1 , R, S, T, V, W, or Y residues; (e) A, C, I, L, N, R, S, T, or a residue at the position corresponding to position 73 of SEQ ID NO: 1 V residue; and (f) an A, C, G, S, or T residue at a position corresponding to position 74 of SEQ ID NO:

1.

39. 37 or claim 36, wherein the second subunit comprises one or more of the following residues: The method according to claim 38, (a) a K residue at a position corresponding to position 48 of SEQ ID NO:1; (b) a Q residue at a position corresponding to position 50 of SEQ ID NO:1; (c) a G residue at a position corresponding to position 71 of SEQ ID NO:1; (d) an S residue at a position corresponding to position 72 of SEQ ID NO:1; (e) a V residue at a position corresponding to position 73 of SEQ ID NO:1; and (f) an S residue at a position corresponding to position 74 of SEQ ID NO:

1.

40. The method according to any one of claims 37 to 39, (a) the central sequence is GTAA and the first subunit is SEQ ID NO: 360-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 89 include, (b) the central sequence is GTAG and the first subunit is SEQ ID NO: 392-3 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 99 include, (c) the central sequence is GTAT and the first subunit is SEQ ID NO: 402-404 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 33 include, (d) the central sequence is GTGA and the first subunit is selected from the group consisting of SEQ ID NOs: 436-437 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 62 include, (e) the central sequence is GTGC and the first subunit is SEQ ID NO: 465-4 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 95 include, (f) the central sequence is GTGG and the first subunit is SEQ ID NO: 498-5 Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of .01 Contains, or (g) the central sequence is GTGT and the first subunit is selected from the group consisting of SEQ ID NOs: 504 to 508; Residues corresponding to residues 48, 50, 71, 72, 73, and 74 of any one of 529 Includes:

41. The engineered meganucleic acid of any one of claims 1 to 15 or claims 32 to 35. A polynucleotide comprising a nucleic acid sequence encoding an ase.

42. 42. The polynucleotide of claim 41, which is mRNA.

43. The engineered meganucleic acid of any one of claims 1 to 15 or claims 32 to 35. A recombinant DNA construct comprising a polynucleotide comprising a nucleic acid sequence encoding a .

44. 44. The recombinant virus of claim 43, wherein the recombinant virus comprises the polynucleotide. DNA constructs.

45. The recombinant virus may be a recombinant adenovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant adenovirus.

45. The recombinant vector of claim 44, which is a virus or a recombinant adeno-associated virus (AAV). DNA constructs.

46. The recombinant virus of claim 44 or claim 45, wherein the recombinant virus is a recombinant AAV. DNA constructs.

47. The engineered meganucleic acid of any one of claims 1 to 15 or claims 32 to 35. A recombinant virus comprising a polynucleotide comprising a nucleic acid sequence encoding a tase.

48. recombinant adenovirus, recombinant lentivirus, recombinant retrovirus, or recombinant 48. The recombinant virus of claim 47, which is an AAV.

49. 49. The recombinant virus of claim 48, which is a recombinant AAV.

50. Genetically modified eukaryotic cells having a target sequence disrupted in the chromosome of the genetically modified eukaryotic cell 1. A method for making a 1 to 15 or 32 to 35, which is expressed in a eukaryotic cell. A polynucleotide comprising a nucleic acid sequence encoding the engineered meganuclease of any one of claims 1 to 4. introducing a nucleotide, The engineered meganuclease generates a cleavage site in the chromosome at the recognition sequence, and the target wherein the target sequence is destroyed by non-homologous end joining at the cleavage site.

51. The nucleic acid is introduced into the eukaryotic cell by mRNA or a recombinant virus. Item 50. The method according to item 50.

52. 52. The method of claim 50 or claim 51, wherein the eukaryotic cell is a mammalian cell.

53. 53. The method of any one of claims 50 to 52, wherein the eukaryotic cell is a human cell.

54. 52. The method of claim 50 or claim 51, wherein the eukaryotic cell is a plant cell.

55. Genetically modified eukaryotic cells having a target sequence disrupted in the chromosome of the genetically modified eukaryotic cell 1. A method for making a A eukaryotic cell is cultured using an engineered vector according to any one of claims 1 to 15 or claims 32 to 35. introducing a meganuclease The engineered meganuclease generates a cleavage site in the chromosome at the recognition sequence, and the target wherein the target sequence is destroyed by non-homologous end joining at the cleavage site.

56. 56. The method of claim 55, wherein the eukaryotic cell is a mammalian cell.

57. 57. The method of claim 55 or claim 56, wherein the eukaryotic cell is a human cell.

58. 56. The method of claim 55, wherein the eukaryotic cell is a plant cell.

59. Genetically modified eukaryotic cells comprising an exogenous sequence of interest inserted into the chromosome of the genetically modified eukaryotic cell 1. A method for producing a gene encoding a gene encoding a gene for a eukaryotic cell, comprising: (a) any one of claims 1 to 15 or claims 32 to 35, which is expressed in said eukaryotic cell a first nucleic acid sequence encoding the engineered meganuclease of any one of claims 1 to 4; and (b) a second nucleic acid sequence comprising the sequence of interest; introducing one or more polynucleotides comprising the engineered meganuclease generates a cleavage site in the chromosome at a recognition sequence; The method wherein the sequence of interest is inserted into the chromosome at the cleavage site.

60. the second nucleic acid sequence further comprises a sequence homologous to a sequence adjacent to the cleavage site, 60. The method of claim 59, wherein the sequence of interest is inserted into the cleavage site by homologous recombination. 。

61. The first nucleic acid sequence is introduced into the eukaryotic cell by mRNA or a recombinant virus.

61. The method of claim 59 or claim 60,

62. 59 to 60, wherein the second nucleic acid is introduced into the eukaryotic cell by a recombinant virus.

61. The method of any one of claims 61 to 61.

63. 63. The method of any one of claims 59 to 62, wherein the eukaryotic cell is a mammalian cell.

64. 64. The method of any one of claims 59 to 63, wherein the eukaryotic cell is a human cell.

65. 63. The method of any one of claims 59 to 62, wherein the eukaryotic cell is a plant cell.

66. Genetically modified eukaryotic cells comprising an exogenous sequence of interest inserted into the chromosome of the genetically modified eukaryotic cell 1. A method for making a (a) an engineered megakaryon according to any one of claims 1 to 15 or claims 32 to 35; introducing the nuclease into a eukaryotic cell; and (b) introducing into said eukaryotic cell a polynucleotide comprising a nucleic acid sequence comprising said sequence of interest; and the engineered meganuclease generates a cleavage site in the chromosome at a recognition sequence, and Beauty The method wherein the sequence of interest is inserted into the chromosome at the cleavage site.

67. the polynucleotide further comprises a sequence homologous to a sequence adjacent to the cleavage site, 67. The method of claim 66, wherein the sequence of interest is inserted into the cleavage site by homologous recombination. Law.

68. 10. The method of claim 9, wherein the polynucleotide is introduced into the eukaryotic cell by a recombinant virus. 66 or 67. The method of claim 67.

69. 69. The method of any one of claims 66 to 68, wherein the eukaryotic cell is a mammalian cell.

70. 70. The method of any one of claims 66 to 69, wherein the eukaryotic cell is a human cell.

71. 71. The method of any one of claims 66 to 70, wherein the eukaryotic cell is a plant cell.

72. A genetically modified eukaryotic cell prepared by the method of any one of claims 50 to 71. 。

73. A pharmaceutical composition according to any one of claims 1 to 15 or claims 32 to 35, comprising a pharmaceutically acceptable carrier. The engineered meganuclease described herein or a nucleic acid encoding the engineered meganuclease. and a polynucleotide comprising the nucleic acid sequence.

74. 74. The pharmaceutical composition of claim 73, wherein the polynucleotide is mRNA.

75. 75. The pharmaceutical composition of claim 74, wherein the mRNA is encapsulated in a lipid nanoparticle. thing.

76. Any of claims 73 to 75, including a recombinant DNA construct comprising said polynucleotide. The pharmaceutical composition described in claim 1.

77. Any one of claims 73 to 76, comprising a recombinant virus comprising said polynucleotide. The pharmaceutical composition described in

78. 78. The pharmaceutical composition of claim 77, wherein the recombinant virus is a recombinant AAV.

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