TCRα homing endonuclease variants

Abstract

The present disclosure provides improved genome editing compositions and methods for editing TCRα genes.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 414,266, filed on October 28, 2016, and U.S. Provisional Application No. 62 / 406,689, filed on October 11, 2016, each of which is incorporated herein by reference in its entirety.

[0003] Statement regarding sequence listing

[0004] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is BLBD_077_02WO_ST25.txt. The text file is 114KB, created on October 11, 2017, and submitted electronically through EFS-Web, concurrently with the submission of the specification. Technical Field

[0005] The present disclosure relates to improved genome editing polypeptides and compositions. More specifically, the present disclosure relates to nuclease variants and compositions for editing the human T cell receptor alpha (TCR alpha) gene. Background Art

[0006] The number of people affected by cancer worldwide doubled between 1975 and 2000. Cancer is the second leading cause of morbidity and mortality worldwide, with approximately 14.1 million new cases and 8.2 million cancer-related deaths in 2012. The most common cancers are breast cancer, lung and bronchial cancer, prostate cancer, colon and rectal cancer, bladder cancer, melanoma of the skin, non-Hodgkin's lymphoma, thyroid cancer, kidney and renal pelvis cancer, endometrial cancer, leukemia, and pancreatic cancer. The number of new cancer cases is expected to increase to 22 million in the next two decades.

[0007] The immune system plays a key role in detecting and fighting human cancer. Most transformed cells are quickly detected by immune sentinels and destroyed by activating antigen-specific T cells via cloned expressed T cell receptors (TCRs). Therefore, cancer can be considered an immune disorder, and the immune system cannot exert the necessary anti-tumor response to permanently suppress and eliminate the disease. In order to more effectively fight cancer, some immunotherapy interventions developed in the past few decades have focused on enhancing T cell immunity. These treatments have only produced sporadic cases of disease remission and have not achieved substantial overall success.

[0008] Recently, adoptive cellular immunotherapy strategies based on the isolation, modification, expansion, and re-infusion of T cells have been studied and tested in early clinical trials. T cells are often the effector cells of choice for cancer immunotherapy due to their selective recognition and potent effector mechanisms. These treatments have shown variable success rates, but a small number of patients have experienced durable remissions, highlighting the unrealized potential of T cell-based immunotherapy.

[0009] Therefore, more effective, targeted, safer and durable therapies are needed to treat various forms of cancer and other immune disorders. In addition, methods and compositions that can accurately and reproducibly destroy endogenous TCR genes with high efficiency are needed. Today's standard of care for most cancers is unsatisfactory in some or all of these standards. Summary of the invention

[0010] The present disclosure relates generally, in part, to homing endonuclease (HE) variants and megaTALs that cleave a target site in exon 1 of the human TCRα gene, compositions comprising HE variants and megaTALs, and methods of using the same.

[0011] In various embodiments, the present disclosure contemplates, in part, a polypeptide comprising an I-OnuI homing endonuclease (HE) variant that cleaves a target site in a human T cell receptor alpha (TCRα) gene, wherein the variant comprises an amino acid substitution: SEQ ID L26I, R28D, N32R, K34N, S35E, V37N, G38R, S40R, E42S, G44R, V68K, A70T, G73S, N75R, S78M, K80R, L138M, T143N, S159P, S176A, C180H, F182G, I186K, S188V, S190G, K191T, L192A, G193K, Q195Y, Q197G, V199R, S201A, T203S, K207R, Y223S, K225R, S233R, D236E and V238E of any one of NO: 1-5 or a biologically active fragment thereof.

[0012] In various embodiments, the present disclosure contemplates, in part, a polypeptide comprising an I-OnuI homing endonuclease (HE) variant that cleaves a target site in a human T cell receptor alpha (TCRα) gene, wherein the variant comprises an amino acid substitution: SEQ ID L26I, R28D, N32R, K34N, S35E, V37N, G38R, S40R, E42S, G44R, V68K, A70T, G73S, N75R, S78M, K80R, L138M, T143N, S159P, S176A, E178D, C180H, F182G, I186K, S188V, S190G, K191T, L192A, G193K, Q195Y, Q197G, V199R, S201A, T203S, K207R, Y223S, K225R, S233R, D236E and V238E of any one of NO: 1-5 or a biologically active fragment thereof.

[0013] In certain embodiments, the biologically active fragment lacks 1, 2, 3, 4, 5, 6, 7 or 8 N-terminal amino acids compared to the corresponding wild-type I-OnuI HE.

[0014] In certain embodiments, the biologically active fragment lacks the 4 N-terminal amino acids compared to the corresponding wild-type I-OnuI HE.

[0015] In further embodiments, the biologically active fragment lacks the 8 N-terminal amino acids compared to the corresponding wild-type I-OnuI HE.

[0016] In some embodiments, the biologically active fragment lacks 1, 2, 3, 4, or 5 C-terminal amino acids compared to the corresponding wild-type I-OnuI HE.

[0017] In further embodiments, the biologically active fragment lacks the C-terminal amino acid compared to the corresponding wild-type I-OnuI HE.

[0018] In certain embodiments, the biologically active fragment lacks the two C-terminal amino acids compared to the corresponding wild-type I-OnuI HE.

[0019] In certain embodiments, the I-OnuI HE variant comprises an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 7-8, or a biologically active fragment thereof.

[0020] In another embodiment, the HE variant comprises the amino acid sequence shown in SEQ ID NO: 7 or a biologically active fragment thereof.

[0021] In certain embodiments, the HE variant comprises the amino acid sequence shown in SEQ ID NO: 8 or a biologically active fragment thereof.

[0022] In some embodiments, the polypeptide binds to the polynucleotide sequence shown in SEQ ID NO:17.

[0023] In certain embodiments, the polypeptide further comprises a DNA binding domain.

[0024] In further embodiments, the DNA binding domain is selected from the group consisting of: a TALE DNA binding domain and a zinc finger DNA binding domain.

[0025] In additional embodiments, the TALE DNA binding domain comprises about 9.5 TALE repeat units, about 10.5 TALE repeat units, about 11.5 TALE repeat units, about 12.5 TALE repeat units, about 13.5 TALE repeat units, about 14.5 TALE repeat units, or about 15.5 TALE repeat units.

[0026] In some embodiments, the TALE DNA binding domain comprises 11.5 TALE repeat units and binds to the polynucleotide sequence shown in SEQ ID NO:19.

[0027] In a specific embodiment, the TALE DNA binding domain comprises 10.5 TALE repeat units and binds to the polynucleotide sequence shown in SEQ ID NO:18.

[0028] In certain embodiments, the polypeptide binds to and cleaves the polynucleotide sequence shown in SEQ ID NO:20.

[0029] In specific embodiments, the zinc finger DNA binding domain comprises 2, 3, 4, 5, 6, 7 or 8 zinc finger motifs.

[0030] In additional embodiments, the polypeptide further comprises a peptide linker and a terminal processing enzyme or a biologically active fragment thereof.

[0031] In certain embodiments, the polypeptide further comprises a viral self-cleaving 2A peptide and a terminal processing enzyme or a biologically active fragment thereof.

[0032] In some embodiments, the end processing enzyme or biologically active fragment thereof has 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease, 5' flap endonuclease, helicase, or template-independent DNA polymerase activity.

[0033] In further embodiments, the terminal processing enzyme comprises Trex2 or a biologically active fragment thereof.

[0034] In certain embodiments, the polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 10 to 12 or a biologically active fragment thereof.

[0035] In certain embodiments, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 10 or a biologically active fragment thereof.

[0036] In another embodiment, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 11 or a biologically active fragment thereof.

[0037] In certain embodiments, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 12 or a biologically active fragment thereof.

[0038] In various embodiments, a polynucleotide encoding a polypeptide contemplated herein is provided.

[0039] In certain embodiments, the present disclosure contemplates, in part, an mRNA encoding a polypeptide contemplated herein.

[0040] In specific embodiments, the mRNA comprises the sequence set forth in any one of SEQ ID NOs: 16-19.

[0041] In some embodiments, a cDNA encoding a polypeptide contemplated herein is provided.

[0042] In certain embodiments, a vector comprising a polynucleotide encoding a polypeptide contemplated herein is provided.

[0043] In certain embodiments, a cell comprising a polypeptide contemplated herein is provided.

[0044] In further embodiments, a cell comprising a polynucleotide encoding a polypeptide contemplated herein is provided.

[0045] In further embodiments, a cell comprising a vector contemplated herein is provided.

[0046] In certain embodiments, a cell edited by a polypeptide contemplated herein is provided.

[0047] In certain embodiments, the cell is a hematopoietic cell.

[0048] In some embodiments, the cell is a T cell.

[0049] In certain embodiments, the cells are CD3+, CD4+ and / or CD8+ cells.

[0050] In certain embodiments, the cell is an immune effector cell.

[0051] In further embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

[0052] In certain embodiments, the cell is a Natural Killer (NK) cell or a Natural Killer T (NKT) cell.

[0053] In specific embodiments, the source of the cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Shown is a comparison of on-target and off-target sites (SEQ ID NOs: 17 and 21) of the I-OnuI HE variant designed to target a site in exon 1 of the constant region of the TCRα gene.

[0055] Figure 2 A schematic diagram of the strategy of transplanting a set of amino acids obtained from modular HE variants with high selectivity for "CCG" trinucleotide sequences (SEQ ID NOs: 17 and 67) into the first generation TCRα HE variant (SEQ ID NO: 6) to generate the second generation TCRα HE variant (TCRα2.0). The TCRα2.0 HE variant retains high selectivity for "CCG" at the desired substrate position.

[0056] Figure 3 shows the enhanced selectivity profiles of the first generation TCRα HE variant and TCRα 2.0 HE variant at substrate position p456. Panel (A) shows the yeast surface displayed cleavage maps of 64 possible nucleotide combinations and panel (B) shows a heat map of these activities.

[0057] Figure 4 Representative data for gene editing efficiency at the TCRα locus using CD3 surface expression as a readout are shown. The first generation TCRα megaTAL (A) (SEQ ID NO: 6) showed improved gene editing efficiency in human primary T cells compared to T cells treated with TCRα 2.0 megaTAL (SEQ ID NO: 7).

[0058] Figure 5 It is shown that the introduction of a targeted mutation (E178D) into TCRα2.0 megaTAL (B) to generate TCRα2.1 megaTAL (C) (SEQ ID NO: 8) restored the on-target cleavage activity of the first-generation TCRα megaTAL (A) while retaining the on-target selectivity of TCRα2.0 megaTAL.

[0059] Figure 6Yeast surface display profiling using bioinformatically derived putative off-target sites showed increased selectivity of the TCRα2.1 megaTAL.

[0060] Figure 7 Deep sequencing of amplicons generated from genomic DNA of primary T cells treated with megaTALs showed an increase in selectivity of TCRα2.1 megaTALs.

[0061] Figure 8 shows that appending additional TALE RVDs to the TCRα2.1 megaTAL to increase the TALE repeats from 10.5 to 11.5 (A) further increased the on-target activity of the TCRα2.1 megaTAL in primary T cells (SEQ ID NOs: 68 and 69) (B).

[0062] Fig. 9 It was shown that the addition of rationally designed minimal N-terminal TAL domains derived from different Xanthomonas members resulted in similar activity or in some cases modest increases in activity compared to the first generation TCRα megaTAL containing the X. oryzae N-terminal TAL domain.

[0063] A brief description of sequence identifiers

[0064] SEQ ID NO: 1 is the amino acid sequence of wild-type I-OnuI LAGLIDADG homing endonuclease (LHE).

[0065] SEQ ID NO: 2 is the amino acid sequence of wild-type I-OnuI LHE.

[0066] SEQ ID NO: 3 is the amino acid sequence of a biologically active fragment of wild-type I-OnuI LHE.

[0067] SEQ ID NO: 4 is the amino acid sequence of a biologically active fragment of wild-type I-OnuI LHE.

[0068] SEQ ID NO: 5 is the amino acid sequence of a biologically active fragment of wild-type I-OnuI LHE.

[0069] SEQ ID NO: 6 is the amino acid sequence of the I-OnuI LHE variant reprogrammed to bind to and cleave a target site in exon 1 of the constant region of the human TCRα gene.

[0070] SEQ ID NO: 7 is the amino acid sequence of the I-OnuI LHE variant reprogrammed to bind to and cleave a target site in exon 1 of the constant region of the human TCRα gene.

[0071] SEQ ID NO: 8 is the amino acid sequence of the I-OnuI LHE variant reprogrammed to bind to and cleave a target site in exon 1 of the constant region of the human TCRα gene.

[0072] SEQ ID NO: 9 is the amino acid sequence of a megaTAL that binds to and cleaves a target site in exon 1 of the constant region of the human TCRα gene.

[0073] SEQ ID NO: 10 is the amino acid sequence of a megaTAL that binds to and cleaves a target site in exon 1 of the constant region of the human TCRα gene.

[0074] SEQ ID NO: 11 is the amino acid sequence of a megaTAL that binds to and cleaves a target site in exon 1 of the constant region of the human TCRα gene.

[0075] SEQ ID NO: 12 is the amino acid sequence of a megaTAL that binds to and cleaves a target site in exon 1 of the constant region of the human TCRα gene.

[0076] SEQ ID NO: 13 is the mRNA sequence encoding a megaTAL that binds to and cleaves a target site in exon 1 of the constant region of the human TCRα gene.

[0077] SEQ ID NO: 14 is the mRNA sequence encoding a megaTAL that binds to and cleaves a target site in exon 1 of the constant region of the human TCRα gene.

[0078] SEQ ID NO: 15 is the mRNA sequence encoding a megaTAL that binds to and cleaves a target site in exon 1 of the constant region of the human TCRα gene.

[0079] SEQ ID NO: 16 is the mRNA sequence encoding a megaTAL that binds to and cleaves a target site in exon 1 of the constant region of the human TCRα gene.

[0080] SEQ ID NO: 17 is the I-OnuI LHE variant target site in exon 1 of the constant region of the human TCRα gene.

[0081] SEQ ID NO: 18 is the 10.5RVD TALE DNA binding domain target site in exon 1 of the constant region of the human TCRα gene.

[0082] SEQ ID NO: 19 is the 11.5RVD TALE DNA binding domain target site in exon 1 of the constant region of the human TCRα gene.

[0083] SEQ ID NO:20 is the megaTAL target site in exon 1 of the constant region of the human TCRα gene.

[0084] SEQ ID NO:21 is an off-target site in the human KAT2B gene for the TCRα nuclease targeting SEQ ID NO:17.

[0085] SEQ ID NOs:22-24 are the amino acid sequences encoding NTD variants megaTAL.

[0086] SEQ ID NOs:25-27 are mRNA sequences encoding NTD variants megaTAL.

[0087] SEQ ID NO:28 is the amino acid sequence encoding mouse Trex2.

[0088] SEQ ID NO:29 is the mRNA sequence encoding mouse Trex2.

[0089] SEQ ID NOs: 30-40 list the amino acid sequences of various linkers.

[0090] SEQ ID NOs: 41-65 list the amino acid sequences of the protease cleavage site and the self-cleaving polypeptide cleavage site.

[0091] In the foregoing sequences, X, if present, refers to any amino acid or absence of an amino acid. DETAILED DESCRIPTION

[0092] A. Overview

[0093] The present disclosure generally relates in part to improved genome editing compositions and methods of use thereof. Genome-edited immune effector cells provide many advantages over existing cell-based immunotherapies, including but not limited to improved safety due to reduced risk of undesired autoimmune responses, precision targeted therapy with more predictable therapeutic gene expression, improved durability and improved efficacy in the tumor microenvironment. Without wishing to be bound by any particular theory, the genome editing compositions contemplated in the various embodiments include homing endonucleases and megaTALs targeting the T cell receptor alpha (TCRα) gene, which are engineered to improve safety and increase target site specificity, selectivity, and catalytic activity.

[0094] In certain embodiments, modification of one or more TCR alpha alleles eliminates or substantially eliminates expression of the TCR alpha allele, reduces expression of the TCR alpha allele, and / or impairs, substantially impairs or eliminates one or more functions of the TCR alpha allele or renders the TCR alpha allele non-functional. In certain embodiments, TCR alpha functions include but are not limited to recruitment of CD3 to the cell surface, MHC-dependent recognition and binding of antigens, activation of TCR alpha beta signaling.

[0095] The genome editing compositions and methods considered in various embodiments include nuclease variants, which are designed to bind and cut the target site in the human T cell receptor alpha (TCRα) gene. The nuclease variants considered in a specific embodiment can be used to introduce double-strand breaks into a target polynucleotide sequence, which can be repaired by non-homologous end joining (NHEJ) in the absence of a polynucleotide template (e.g., a donor repair template), or by homology-directed repair (HDR) (i.e., homologous recombination) in the presence of a donor repair template. The nuclease variants considered in certain embodiments can also be designed to be a nickase, which generates single-stranded DNA breaks, which can be repaired using a base excision repair (BER) mechanism or homologous recombination of cells in the presence of a donor repair template. NHEJ is an error-prone process that often forms small insertions and deletions, which can destroy gene function. Homologous recombination requires homologous DNA as a template for repair, and homologous recombination can be used to produce an unlimited number of modifications, which are specified by introducing a donor DNA containing a desired sequence at the target site into a region flanked by the target site, and the target site is located on either side of the sequence with homology.

[0096] In a preferred embodiment, the genome editing compositions contemplated herein include a homing endonuclease variant or megaTAL targeted to the human TCRα gene.

[0097] In various embodiments, the TCRα gene targeting homing endonuclease variants or megaTALs contemplated herein have increased binding site selectivity or specificity for a target site compared to existing homing endonucleases or megaTALs targeting the TCRα gene.

[0098] In various embodiments, the TCRα gene targeting homing endonuclease variants or megaTALs contemplated herein have improved binding site selectivity or specificity for the target site while retaining high catalytic activity compared to existing homing endonucleases or megaTALs targeting the TCRα gene.

[0099] In a preferred embodiment, the genome editing compositions contemplated herein include a homing endonuclease variant or megaTAL and an end-processing enzyme, such as Trex2.

[0100] Unless specifically indicated to the contrary, the practice of specific embodiments will employ conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology within the skill of the art. For illustrative purposes, many of these conventional methods are described below. These techniques are fully described in the literature. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Method. Approach, Volumes I and II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes (Academic Press, New York, 1992); Transcription and Translation (B. Hames and S.Higgins, ed., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Current Protocols in Immunology (eds. QE Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, 1991); Annual Review of Immunology; and monographs in journals such as Advances in Immunology.

[0101] B. Definition

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of specific embodiments, preferred embodiments of compositions, methods and materials are described herein. For purposes of this disclosure, the following terms are defined as follows.

[0103] The articles "a," "an," and "the" are used herein to refer to one or more than one (ie, at least one, or one or more) of the grammatical object of the article. For example, "an element" means one element or one or more elements.

[0104] The use of an alternative conjunction (eg, "or") should be understood to mean one, both, or any combination of the alternatives.

[0105] The term "and / or" should be understood to mean one or both of the options.

[0106] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0107] In one embodiment, a range, such as 1 to 5, about 1 to 5, or about 1 to about 5, refers to every value encompassed within the range. For example, in one non-limiting and merely illustrative embodiment, the range “1 to 5” is equivalent to expressing 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0108] As used herein, the term "substantially" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, "substantially the same" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that produces approximately the same effect (e.g., a physiological effect) as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0109] Throughout this specification, unless the context requires otherwise, the words "comprise / comprises / comprising" will be understood to imply the inclusion of the stated one step or element or group of steps or elements but not the exclusion of any other step or element or any other group of steps or elements. "Consisting of" means including and limited to whatever follows the phrase "consisting of". Thus, the phrase "consisting of" means that the listed elements are required or mandatory, and that no other elements are present. "Consisting essentially of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified for the listed elements in the present disclosure. Thus, the phrase "consisting essentially of" means that the listed elements are required or mandatory, but that no other elements are present that substantially affect the activity or action of the listed elements.

[0110] References throughout this specification to "one embodiment / an embodiment", "a specific embodiment", "a related embodiment", "an embodiment", "an additional embodiment / a further embodiment", or combinations thereof, indicate that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment. Therefore, the aforementioned phrases appearing throughout this specification do not necessarily all refer to the same embodiment. In addition, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It should also be understood that the affirmative recitation of a feature in one embodiment serves as a basis for excluding the feature in a specific embodiment.

[0111] The term "ex vivo" generally refers to an activity occurring outside an organism, such as an experiment or measurement performed in or on a living tissue in an artificial environment outside the organism, preferably with minimal natural condition changes. In a specific embodiment, an "ex vivo" procedure relates to living cells or living tissues, which are taken from an organism and cultured or regulated in a laboratory device, usually under aseptic conditions, usually for a few hours or up to about 24 hours, but including up to 48 hours or 72 hours, depending on the environment. In certain embodiments, these tissues or cells can be collected and frozen, and then thawed for ex vivo processing. Using living cells or living tissues to continue tissue culture experiments or procedures longer than a few days is generally considered to be "in vitro", but in certain embodiments, this term can be used interchangeably with ex vivo.

[0112] The term "in vivo" generally refers to activities that occur within an organism. In one embodiment, the cell genome is transformed, edited or modified in vivo.

[0113] "Enhance" or "promote" or "increase" or "amplification" or "synergism" generally refers to the ability of the nuclease variants, genome editing compositions or genome-edited cells considered herein to produce, initiate or cause a greater response (i.e., physiological response) than the response caused by the vehicle or control. A measurable response may include an increase in the expression of a modified TCR or CAR, an increase in HR or HDR efficiency, an increase in binding site selectivity, an increase in binding site specificity, an increase in on-target binding, an increase in immune effector cell expansion, activation, persistence, and / or an increase in cancer cell death and killing ability, especially as apparent from the knowledge of the art and the description herein. An "increased" or "enhanced" amount is typically a "statistically significant" amount and can include an increase of 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points greater than 1 therebetween, e.g., 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, etc.) over the response produced by a vehicle or control.

[0114] "Reducing" or "reducing" or "reducing" or "reducing" or "mitigating" or "eliminating" or "inhibiting" or "reducing" generally refers to the ability of the nuclease variants, genome editing compositions, or genome-edited cells contemplated herein to produce, elicit, or cause a response (i.e., a physiological response) that is less than that elicited by a vehicle or control. A measurable response may include a reduction in endogenous TCR expression or function, a reduction in off-target binding, a reduction in the expression of biomarkers associated with immune effector cell exhaustion, and the like. A "reduced" or "decreased" amount is typically a "statistically significant" amount and can include a decrease of 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points greater than 1 therebetween, e.g., 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, etc.) compared to the response produced by the vehicle or control (reference response).

[0115] "Maintain" or "retain" or "no change" or "no substantial change" or "no substantial reduction" generally refers to the ability of a nuclease variant, genome editing composition, or genome-edited cell contemplated herein to produce, elicit, or cause a physiological response (i.e., downstream effect) that is substantially similar or comparable to that elicited by a vehicle or control. A comparable response is one that is not significantly different or measurably different from a reference response.

[0116] The terms "specific binding affinity" or "specifically binds" or "specifically bound" or "specific targeting" describe the binding of one molecule to another molecule with a binding affinity greater than background binding, such as the binding of a DNA binding domain of a polypeptide to DNA. a (i.e., the equilibrium association constant for a specific binding interaction, in units of 1 / M) (e.g., the affinity or K a Greater than or equal to about 10 5 M -1 ) binds to or associates with a target site, then the binding domain "specifically binds" to the target site. In certain embodiments, the binding domain binds to or associates with a target site at a rate greater than or equal to about 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 or 10 13 M -1 K a A "high affinity" binding domain is one that has at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 or larger K a Those binding domains.

[0117] Alternatively, affinity can be defined as the equilibrium dissociation constant (K) for a specific binding interaction in units of M. d )(For example, 10 -5 M to 10 -13M, or less). The affinity of nuclease variants comprising one or more DNA binding domains for a DNA target site contemplated in a particular embodiment can be readily determined using conventional techniques (e.g., yeast cell surface display), or by binding association, or by displacement assays using labeled ligands.

[0118] In one embodiment, the affinity of specific binding is about 2 times greater than background binding, about 5 times greater than background binding, about 10 times greater than background binding, about 20 times greater than background binding, about 50 times greater than background binding, about 100 times greater than background binding, or about 1000 times greater than background binding or more.

[0119] The terms "selectively binds / selectively bound / selectively binding" or "selectively targeting" describe the preferential binding of a molecule to a target molecule in the presence of multiple off-target molecules (on-target binding). In certain embodiments, the HE or megaTAL selectively binds to an on-target DNA binding site about 5, 10, 15, 20, 25, 50, 100, or 1000 times or more frequently than the HE or megaTAL binds to an off-target DNA target binding site.

[0120] "On target" refers to the target site sequence.

[0121] "Off-target" refers to sequences that are similar but not identical to the target site sequence.

[0122] A "target site" or "target sequence" is a chromosomal or extrachromosomal nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind and / or cleave if sufficient binding and / or cleavage conditions exist. When reference is made to a polynucleotide sequence or SEQ ID NO. of only one strand of a reference target site or target sequence, it is understood that the target site or target sequence bound and / or cleaved by a nuclease variant is double-stranded and includes the reference sequence and its complementary sequence. In a preferred embodiment, the target site is a sequence in a human TCRα gene.

[0123] "Gene" refers to a DNA region encoding a gene product, as well as all DNA regions that regulate the production of a gene product, whether or not such regulatory sequences are adjacent to the coding and / or transcribed sequences. A gene includes, but is not limited to, promoter sequences, enhancers, silencers, insulators, boundary elements, terminators, polyadenylation sequences, post-transcriptional response elements, translational regulatory sequences (e.g., ribosome binding sites and internal ribosome entry sites), origins of replication, matrix attachment sites, and locus control regions.

[0124] As used herein, the term "genetically engineered" or "genetically modified" refers to the total genetic material added to a cell chromosomally or extrachromosomally in the form of DNA or RNA. The genetic modification can be targeted or non-targeted to a specific site in the cell genome. In one embodiment, the genetic modification is site-specific. In one embodiment, the genetic modification is not site-specific.

[0125] As used herein, the term "genome editing" refers to the replacement, deletion and / or introduction of genetic material at a target site in the genome of a cell that restores, corrects, disrupts and / or modifies the expression of a gene or gene product. Genome editing contemplated in particular embodiments includes the introduction of one or more nuclease variants into a cell to generate DNA damage at or adjacent to a target site in the genome of the cell, optionally in the presence of a donor repair template.

[0126] As used herein, the term "amount" refers to "an amount effective" or "an effective amount" of a nuclease variant, genome editing composition, or genome-edited cell sufficient to achieve beneficial or desired prophylactic or therapeutic results, including clinical results.

[0127] A "prophylactically effective amount" refers to an amount of a nuclease variant, a genome editing composition, or a genome edited cell sufficient to achieve the desired prophylactic result. Typically, but not necessarily, a prophylactic effective amount is less than a therapeutically effective amount because a prophylactic dose is used in a subject before or at an early stage of a disease.

[0128] A "therapeutically effective amount" of a nuclease variant, a genome editing composition, or a genome-edited cell may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the beneficial effects of treatment outweigh any toxic or deleterious effects. The term "therapeutically effective amount" includes an amount that effectively "treats" a subject (e.g., a patient). When a therapeutic amount is indicated, the doctor can determine the exact amount of the composition to be administered in a particular embodiment according to the instructions and taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and patient (subject) symptoms.

[0129] C. Nuclease variants

[0130] The nuclease variants considered in the specific embodiments herein are suitable for target sites in genome editing TCRα genes, and include one or more DNA binding domains and one or more DNA cleavage domains (e.g., one or more endonucleases and / or exonucleases domains), and optionally one or more linkers considered herein. The terms "reprogrammed nuclease", "remodeled nuclease" or "nuclease variant" are used interchangeably and refer to a nuclease including one or more DNA binding domains and one or more DNA cleavage domains, wherein the nuclease has been designed and / or modified from a parent nuclease or a naturally occurring nuclease to bind to and cleave a double-stranded DNA target sequence in exon 1 of a constant region of a human TCRα gene.

[0131] In a specific embodiment, the nuclease variant binds to and cleaves a target sequence in exon 1 of the constant region of a human TCRα gene, preferably at SEQ ID NO: 17 in exon 1 of the constant region of a human TCRα gene, and more preferably at the sequence "ATTC" in SEQ ID NO: 17 in exon 1 of the constant region of a human TCRα gene.

[0132] Nuclease variants can be designed and / or modified from naturally occurring nucleases or from previous nuclease variants. The nuclease variants considered in a particular embodiment can further include one or more additional functional domains, for example, an end processing enzyme domain showing an end processing enzyme of a 5'-3' exonuclease, a 5'-3' alkaline exonuclease, a 3'-5' exonuclease (e.g., Trex2), a 5' flap endonuclease, a helicase, a template-dependent DNA polymerase, or a template-independent DNA polymerase activity.

[0133] Illustrative examples of nuclease variants that bind and cleave a target sequence in the TCRα gene include, but are not limited to, homing endonuclease variants (meganuclease variants) and megaTALs.

[0134] 1. Homing endonucleases (meganucleases) variants

[0135] In various embodiments, a homing endonuclease or a meganuclease is reprogrammed to introduce a double-strand break (DSB) into a target site in a TCRα gene. In a specific embodiment, a homing endonuclease variant introduces a double-strand break into exon 1 of a human TCRα gene constant region, preferably SEQ ID NO: 17 in exon 1 of a human TCRα gene constant region, more preferably SEQ ID NO: 17 in exon 1 of a human TCRα gene constant region "ATTC" in sequence. "Homing endonuclease" and "meganuclease" are used interchangeably and refer to naturally occurring nucleases that recognize 12-45 base pair cleavage sites, and are generally divided into five families based on sequence and structural motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK.

[0136] A "reference homing endonuclease" or "reference meganuclease" refers to a wild-type homing endonuclease or a homing endonuclease found in nature. In one embodiment, a "reference homing endonuclease" refers to a wild-type homing endonuclease that has been modified to increase basal activity.

[0137] "Remodeled homing endonuclease", "reprogrammed homing endonuclease", "homing endonuclease variant", "remodeled meganuclease", "reprogrammed meganuclease" or "meganuclease variant" refers to a homing endonuclease comprising one or more DNA binding domains and one or more DNA cleavage domains, wherein the homing endonuclease has been designed and / or modified from a parent homing endonuclease or a naturally occurring homing endonuclease to bind to and cleave a DNA target sequence in a TCRα gene. A homing endonuclease variant can be designed and / or modified from a naturally occurring homing endonuclease or from another homing endonuclease variant. The homing endonuclease variants contemplated in particular embodiments may further include one or more additional functional domains, for example, an end processing enzyme domain of an end processing enzyme that exhibits 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template-dependent DNA polymerase, or template-independent DNA polymerase activity.

[0138] In certain embodiments, the homing endonuclease variants contemplated herein include increased selectivity for a target site compared to existing homing endonucleases, e.g., SEQ ID NO: 6. In certain embodiments, the homing endonucleases contemplated herein include increased selectivity for a target site compared to existing homing endonucleases while retaining catalytic activity, e.g., SEQ ID NO: 6.

[0139] Homing endonuclease (HE) variants do not exist in nature and can be obtained by recombinant DNA technology or by random mutagenesis. HE variants can be obtained by making one or more amino acid changes (e.g., mutations, substitutions, additions or deletions of one or more amino acids) in naturally occurring HE or HE variants. In certain embodiments, HE variants include one or more amino acid changes in the DNA recognition interface.

[0140] HE variants contemplated in certain embodiments may further include one or more linkers and / or additional functional domains, for example, an end-processing enzyme domain of an end-processing enzyme that exhibits 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template-dependent DNA polymerase, or template-independent DNA polymerase activity. In certain embodiments, the HE variant is introduced into a T cell having an end-processing enzyme that exhibits 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template-dependent DNA polymerase, or template-independent DNA polymerase activity. The HE variant and the 3' processing enzyme may be introduced separately, for example, in different vectors or separate mRNAs, or together, for example, as a fusion protein, or in a polycistronic construct separated by a viral self-cleaving peptide or IRES element.

[0141] "DNA recognition interface" refers to the HE amino acid residues that interact with the nucleic acid target base and those residues that are adjacent. For each HE, the DNA recognition interface includes an extensive network of side chain to side chain and side chain to DNA contacts, most of which must be unique to the recognition of a specific nucleic acid target sequence. Thus, the amino acid sequence of the DNA recognition interface corresponding to a specific nucleic acid sequence varies significantly and is characteristic of any natural HE or HE variant. As a non-limiting example, the HE variants contemplated in a particular embodiment can be derived by constructing a library of HE variants in which one or more amino acid residues located in the DNA recognition interface of a natural HE (or previously generated HE variant) are different. The library can be screened for target cleavage activity relative to each predicted TCRα target site using a cleavage assay (see, e.g., Jarjour et al., 2009, Nuc. Acids Res., 37(20):6871-6880).

[0142] LAGLIDADG homing endonucleases (LHEs) are the most intensively studied family of homing endonucleases, encoded primarily in organellar DNA in archaea and green algae and fungi, and display the highest overall DNA recognition specificity. LHEs include one or two LAGLIDADG catalytic motifs per protein chain and function as homodimers or single-chain monomers, respectively. Structural studies of LAGLIDADG proteins have identified a highly conserved core structure (Stoddard, 2005) characterized by an αββαββα fold, in which the LAGLIDADG motif belongs to the first helix of this fold. The efficient and specific cleavage of LHEs represents a protein scaffold to derive new highly specific and selective endonucleases. However, engineering LHEs to bind and cleave non-native or non-canonical target sites requires the selection of appropriate LHE scaffolds at up to two-thirds of the base pair positions in the target site, examination of the target locus, selection of putative target sites, and extensive alteration of the LHEs to alter their DNA contact points and cleavage specificity.

[0143] Illustrative examples of LHE include, but are not limited to, I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-Cp aMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-Hje MI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-Os oMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI and I-Vdi141I.

[0144] In one embodiment, the reprogrammed LHE or LHE variant is an I-OnuI variant. See, e.g., SEQ ID NO: 7-8.

[0145] In one embodiment, the reprogrammed I-OnuI LHE or I-OnuI variant targeting the TCRα gene is generated by natural I-OnuI or its biologically active fragment (SEQ ID NO: 1-5). In a preferred embodiment, the reprogrammed I-OnuI LHE or I-OnuI variant targeting the human TCRα gene is generated by an existing I-OnuI variant.

[0146] In one embodiment, the I-OnuI LHE that binds to and cleaves the human TCRα gene comprises at least 99% sequence identity to the DNA recognition interface of the I-OnuI LHE variants shown in SEQ ID NO: 7 or SEQ ID NO: 8 and biologically active fragments thereof.

[0147] In a specific embodiment, the I-OnuI LHE variant that binds to and cleaves the human TCRα gene comprises one or more amino acid substitutions or modifications in the DNA recognition interface of I-OnuI, a biologically active fragment thereof, and / or another variant thereof as shown in SEQ ID NO:7 or SEQ ID NO:8.

[0148] In certain embodiments, the HE variant cleaves the TCRα exon 1 constant region target site and comprises the following amino acid substitutions: SEQ ID L26I, R28D, N32R, K34N, S35E, V37N, G38R, S40R, E42S, G44R, V68K, A70T, G73S, N75R, S78M, K80R, L138M, T143N, S159P, S176A, C180H, F182G, I186K, S188V, S190G, K191T, L192A, G193K, Q195Y, Q197G, V199R, S201A, T203S, K207R, Y223S, K225R, S233R, D236E and V238E of any one of NO: 1-5 or a biologically active fragment thereof.

[0149] In certain embodiments, the HE variant cleaves the TCRα exon 1 constant region target site and comprises the following amino acid substitutions: SEQ ID L26I, R28D, N32R, K34N, S35E, V37N, G38R, S40R, E42S, G44R, V68K, A70T, G73S, N75R, S78M, K80R, L138M, T143N, S159P, S176A, E178D, C180H, F182G, I186K, S188V, S190G, K191T, L192A, G193K, Q195Y, Q197G, V199R, S201A, T203S, K207R, Y223S, K225R, S233R, D236E and V238E of any one of NO: 1-5 or a biologically active fragment thereof.

[0150] In certain embodiments, the I-OnuI LHE variant that binds to and cleaves the human TCRα gene comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8, or a biologically active fragment thereof.

[0151] In certain embodiments, the I-OnuI LHE variant comprises the amino acid sequence shown in any one of SEQ ID NOs: 7-8 or a biologically active fragment thereof.

[0152] In certain embodiments, the I-OnuI LHE variant comprises the amino acid sequence shown in SEQ ID NO: 7 or a biologically active fragment thereof.

[0153] In certain embodiments, the I-OnuI LHE variant comprises the amino acid sequence shown in SEQ ID NO: 8 or a biologically active fragment thereof.

[0154] 2. MegaTAL

[0155] In various embodiments, megaTALs including homing endonuclease variants are reprogrammed to introduce double-strand breaks (DSBs) into target sites in exon 1 of the constant region of a human TCRα gene. In specific embodiments, megaTALs introduce DSBs into exon 1 of the constant region of a human TCRα gene, preferably at SEQ ID NO: 17 in exon 1 of the constant region of a human TCRα gene, more preferably at the sequence "ATTC" in SEQ ID NO: 17 in exon 1 of the constant region of a human TCRα gene. "megaTAL" refers to a polypeptide including a TALE DNA binding domain and a homing endonuclease variant that binds to and cuts a DNA target sequence in a TCRα gene, and optionally includes one or more linkers and / or additional functional domains, for example, an end processing enzyme domain of an end processing enzyme that exhibits 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, or template-independent DNA polymerase activity.

[0156] In certain embodiments, megaTALs can be introduced into cells together with an end-processing enzyme that exhibits 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2), 5' flap endonuclease, helicase, template-dependent DNA polymerase, or template-independent DNA polymerase activity. The megaTAL and 3' processing enzyme can be introduced separately, e.g., in different vectors or separate mRNAs, or together, e.g., as a fusion protein, or in a polycistronic construct separated by a viral self-cleaving peptide or IRES element.

[0157] A "TALE DNA binding domain" is the DNA binding portion of a transcription activator-like effector (TALE or TAL effector) that mimics plant transcriptional activators to manipulate the plant transcriptome (see, e.g., Kay et al., 2007, Science, 318:648-651). TALE DNA binding domains contemplated in particular embodiments are engineered de novo or from naturally occurring TALEs, such as AvrBs3 from Xanthomonas campestris pv. vesicatoria, Xanthomonas gardneri, Xanthomonas translucens, Xanthomonas axonopodis, Xanthomonas perforans, Xanthomonas alfalfa, Xanthomonas citri, Xanthomonase uvesicatoria, and Xanthomonas oryzae, and brg11 and hpx17 from Ralstonia solanacearum. Illustrative examples of TALE proteins used to derive and design DNA binding domains are disclosed in U.S. Pat. No. 9,017,967 and references cited therein, all of which are incorporated herein by reference in their entirety.

[0158] In certain embodiments, a megaTAL comprises a TALE DNA binding domain comprising one or more repeat units that participate in the binding of the TALE DNA binding domain to its corresponding target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length. Each TALE DNA binding domain repeat unit comprises 1 or 2 DNA binding residues that constitute a repeat variable diresidue (RVD), typically at position 12 and / or 13 of the repeat. The natural (canonical) code for DNA recognition of these TALE DNA binding domains has been determined such that HD sequences at positions 12 and 13 bind to cytosine (C), NG binds to T, NI binds to A, NN binds to G or A, and NG binds to T. In certain embodiments, non-canonical (atypical) RVDs are contemplated.

[0159] Illustrative examples of non-canonical RVDs suitable for use with specific megaTALs contemplated in particular embodiments include, but are not limited to, HH, KH, NH, NK, NQ, RH, RN, SS, NN, SN, KN for recognizing guanine (G); NI, KI, RI, HI, SI for recognizing adenine (A); NG, HG, KG, RG for recognizing thymine (T); RD, SD, HD, ND, KD, YG for recognizing cytosine (C); NV, HN for recognizing A or G; and H*, HA, KA, N*, NA, NC, NS, RA, S* for recognizing A or T or G or C, where (*) indicates the absence of the amino acid at position 13. Additional illustrative examples of RVDs suitable for use with specific megaTALs contemplated in particular embodiments further include those disclosed in U.S. Pat. No. 8,614,092, which is incorporated herein by reference in its entirety.

[0160] In specific embodiments, megaTALs contemplated herein include TALE DNA binding domains that include 3 to 30 repeat units. In certain embodiments, megaTALs include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 TALE DNA binding domain repeat units. In a preferred embodiment, megaTALs contemplated herein include TALE DNA binding domains that include 5-15 repeat units, more preferably 7-15 repeat units, more preferably 9-15 repeat units, more preferably 9, 10, 11, 12, 13, 14, or 15 repeat units.

[0161] In specific embodiments, the megaTALs contemplated herein include a TALE DNA binding domain and an additional single truncated TALE repeat unit, wherein the TALE DNA binding domain includes 3 to 30 repeat units, and the additional single truncated TALE repeat unit includes 20 amino acids located at the C-terminus of a set of TALE repeat units, i.e., it is an additional C-terminal half TALE DNA binding domain repeat unit (amino acids -20 to -1 of the C-cap disclosed elsewhere herein, see below). Thus, in specific embodiments, the megaTALs contemplated herein include a TALE DNA binding domain that includes 3.5 to 30.5 repeat units. In certain embodiments, the megaTAL comprises 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, 15.5, 16.5, 17.5, 18.5, 19.5, 20.5, 21.5, 22.5, 23.5, 24.5, 25.5, 26.5, 27.5, 28.5, 29.5 or 30.5 TALE DNA binding domain repeat units. In a preferred embodiment, the megaTAL contemplated herein comprises a TALE DNA binding domain comprising 5.5-15.5 repeat units, more preferably 7.5-15.5 repeat units, more preferably 9.5-15.5 repeat units, more preferably 9.5, 10.5, 11.5, 12.5, 13.5, 14.5 or 15.5 repeat units.

[0162] In certain embodiments, megaTALs include a TAL effector construct that includes an "N-terminal domain (NTD)" polypeptide, one or more TALE repeat domains / units, a "C-terminal domain (CTD)" polypeptide, and a homing endonuclease variant. In some embodiments, the NTD, TALE repeats, and / or CTD domains are from the same species. In other embodiments, one or more of the NTD, TALE repeats, and / or CTD domains are from different species.

[0163] As used herein, the term "N-terminal domain (NTD)" polypeptide refers to the sequence flanking the N-terminal portion or fragment of a naturally occurring TALE DNA binding domain. If present, the NTD sequence can be of any length, as long as the TALE DNA binding domain repeat unit retains the ability to bind DNA. In a specific embodiment, the NTD polypeptide includes at least 120 to at least 140 or more amino acids at the N-terminus of the TALE DNA binding domain (0 is the amino acid 1 of the most N-terminal repeat unit). In a specific embodiment, the NTD polypeptide includes at least about 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 or at least 140 amino acids at the N-terminus of the TALE DNA binding domain. In one embodiment, a megaTAL contemplated herein comprises an NTD polypeptide that is at least about amino acid +1 to +122 to at least about +1 to +137 (0 is amino acid 1 of the most N-terminal repeat unit) of a Xanthomonas TALE protein. In specific embodiments, the NTD polypeptide comprises at least about 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or 137 amino acids N-terminal to the TALE DNA binding domain of a Xanthomonas TALE protein. In one embodiment, a megaTAL contemplated herein comprises an NTD polypeptide that is at least amino acid +1 to +121 (0 is amino acid 1 of the most N-terminal repeat unit) of a Ralstonia TALE protein. In particular embodiments, the NTD polypeptide comprises at least about 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136 or 137 amino acids at the N-terminus of the TALE DNA binding domain of the Ralstonia TALE protein.

[0164] As used herein, the term "C-terminal domain (CTD)" polypeptide refers to the sequence flanking the C-terminal portion or fragment of a naturally occurring TALE DNA binding domain. If present, the CTD sequence can be of any length as long as the TALE DNA binding domain repeat unit retains the ability to bind DNA. In specific embodiments, the CTD polypeptide includes at least 20 to at least 85 or more amino acids C-terminal to the last full repeat of the TALE DNA binding domain (the first 20 amino acids are half-repeat units C-terminal to the last C-terminal full repeat unit). In specific embodiments, the CTD polypeptide includes at least 20 to at least 85 or more amino acids C-terminal to the last full repeat of the TALE DNA binding domain (the first 20 amino acids are half-repeat units C-terminal to the last C-terminal full repeat unit). at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 443, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or at least 85 amino acids. In one embodiment, the megaTALs contemplated herein include a CTD polypeptide that is at least about amino acids -20 to -1 of a Xanthomonas TALE protein (-20 being amino acid 1 of the half-repeat unit C-terminal to the last C-terminal full repeat unit). In specific embodiments, the CTD polypeptide comprises at least about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids C-terminal to the last full repeat of the TALE DNA binding domain of a Xanthomonas TALE protein. In one embodiment, a megaTAL contemplated herein comprises a CTD polypeptide that is at least about amino acids -20 to -1 of a Ralstonia TALE protein (-20 being amino acid 1 of the half-repeat unit C-terminal to the last C-terminal full repeat unit). In particular embodiments, the CTD polypeptide comprises at least about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids C-terminal to the last complete repeat of the TALE DNA binding domain of the Ralstonia TALE protein.

[0165] In particular embodiments, megaTALs contemplated herein include fusion polypeptides comprising a TALE DNA binding domain engineered to bind to a target sequence, a homing endonuclease reprogrammed to bind and cleave a target sequence, and an optional NTD and / or CTD polypeptide, the TALE DNA binding domain, the homing endonuclease, and the optional NTD and / or CTD polypeptides being optionally connected to one another via one or more linker polypeptides contemplated elsewhere herein. Without wishing to be bound by any particular theory, it is contemplated that a megaTAL comprising a TALE DNA binding domain and an optional NTD and / or CTD polypeptide is fused to a linker polypeptide, which is further fused to a homing endonuclease variant. Thus, the TALE DNA binding domain binds to a DNA target sequence within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides of a target sequence bound by the DNA binding domain of a homing endonuclease variant. In this way, the megaTALs considered herein increase the specificity, selectivity, and efficiency of genome editing.

[0166] In one embodiment, the megaTAL comprises a homing endonuclease variant and a TALE DNA binding domain that binds to a nucleotide sequence within about 4, 5, or 6 nucleotides (preferably 5 or 6 nucleotides) upstream of the binding site of the reprogrammed homing endonuclease.

[0167] In one embodiment, the megaTAL comprises a homing endonuclease variant and a TALE DNA binding domain that binds to the nucleotide sequence set forth in SEQ ID NO: 18, which is 6 nucleotides upstream of the nucleotide sequence bound and cleaved by the homing endonuclease variant (SEQ ID NO: 17). In a preferred embodiment, the megaTAL target sequence is SEQ ID NO: 18.

[0168] In one embodiment, the megaTAL comprises a homing endonuclease variant and a TALE DNA binding domain that binds to the nucleotide sequence set forth in SEQ ID NO: 19, which is 5 nucleotides upstream of the nucleotide sequence bound and cleaved by the homing endonuclease variant (SEQ ID NO: 17). In a preferred embodiment, the megaTAL target sequence is SEQ ID NO: 19.

[0169] In certain embodiments, megaTALs contemplated herein include an NTD of about 122 amino acids to 137 amino acids, about 9.5, about 10.5, about 11.5, about 12.5, about 13.5, about 14.5, or about 15.5 binding repeat units, a CTD of about 20 amino acids to about 85 amino acids, and an I-OnuI LHE variant. In certain embodiments, any one, two, or all of the NTD, DNA binding domain, and CTD can be designed from the same species or different species in any suitable combination.

[0170] In specific embodiments, a megaTAL contemplated herein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 10-12.

[0171] In particular embodiments, the megaTAL contemplated herein comprises the amino acid sequence set forth in SEQ ID NO:10.

[0172] In particular embodiments, a megaTAL contemplated herein comprises the amino acid sequence set forth in SEQ ID NO:11.

[0173] In particular embodiments, the megaTAL contemplated herein comprises the amino acid sequence set forth in SEQ ID NO:12.

[0174] In certain embodiments, the megaTAL includes a TALE DNA binding domain, and the I-OnuI LHE variant binds to and cleaves the nucleotide sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 20.

[0175] 3. Terminal processing enzyme

[0176] The genome editing compositions and methods considered in a particular embodiment include editing the cell genome using one or more copies of a nuclease variant and an end processing enzyme. In a particular embodiment, a single polynucleotide encodes a homing endonuclease variant and an end processing enzyme, which is separated by a linker, a self-cleaving peptide sequence (e.g., a 2A sequence), or separated by an IRES sequence. In a particular embodiment, the genome editing composition includes a polynucleotide encoding a nuclease variant and a polynucleotide encoding a separate end processing enzyme. In a particular embodiment, in addition to the tandem copies of the end processing enzyme separated by the self-cleaving peptide, the genome editing composition includes a polynucleotide encoding a homing endonuclease variant end processing enzyme single polypeptide fusion.

[0177] The term "end processing enzyme" refers to an enzyme that modifies the exposed ends of a polynucleotide chain. The polynucleotide can be double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA, double-stranded hybrids of DNA and RNA, and synthetic DNA (e.g., containing bases other than A, C, G, and T). The end processing enzyme can modify the exposed ends of the polynucleotide chain by adding one or more nucleotides, removing one or more nucleotides, removing or modifying phosphate groups, and / or removing or modifying hydroxyls. The end processing enzyme can modify the ends at the endonuclease cleavage site or the ends generated by other chemical or mechanical means, such as shearing (e.g., by fine-gauge needles, heating, ultrasonic treatment, mini-bead rolling and atomization), ionizing radiation, ultraviolet radiation, oxygen free radicals, chemical hydrolysis, and chemotherapeutic agents.

[0178] In certain embodiments, genome editing compositions and methods contemplated in certain embodiments comprise editing the genome of a cell using a homing endonuclease variant or a megaTAL and a DNA end processing enzyme.

[0179] The term "DNA end processing enzyme" refers to an enzyme that modifies the exposed ends of DNA. DNA end processing enzymes can modify flat ends or staggered ends (ends with 5' or 3' overhangs). DNA end processing enzymes can modify single-stranded or double-stranded DNA. DNA end processing enzymes can modify the ends at the endonuclease cleavage site or the ends generated by other chemical or mechanical means, such as shearing (e.g., by fine-gauge needles, heating, ultrasonic treatment, mini-bead rolling and atomization), ionizing radiation, ultraviolet radiation, oxygen free radicals, chemical hydrolysis and chemotherapeutic agents. DNA end processing enzymes can modify the exposed DNA ends by adding one or more nucleotides, removing one or more nucleotides, removing or modifying phosphate groups and / or removing or modifying hydroxyls.

[0180] Illustrative examples of DNA end processing enzymes suitable for use with particular embodiments contemplated herein include, but are not limited to, 5'-3' exonucleases, 5'-3' alkaline exonucleases, 3'-5' exonucleases, 5' flap endonucleases, helicases, phosphatases, hydrolases, and template-independent DNA polymerases.

[0181] Additional illustrative examples of DNA end processing enzymes suitable for use with the specific embodiments contemplated herein include, but are not limited to, Trex2, Trex1, Trex1 without a transmembrane domain, Apollo, Artemis, DNA2, Exo1, ExoT, ExoIII, Fen1, Fan1, MreII, Rad2, Rad9, TdT (terminal deoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, lambda exonuclease, Sox, vaccinia DNA polymerase, exonuclease I, exonuclease III, exonuclease VII, NDK1, NDK5, NDK7, NDK8, WRN, T7-exonuclease gene 6, avian myeloblastosis virus integrin (IN), Bloom, thermosensitive phosphatase, alkaline phosphatase, polynucleotide kinase (PNK), ApeI, mung bean nuclease, Hex1, TTRAP (TDP2), Sgs1, Sae2, CUP, Polμ, Polλ, MUS81, EME1, EME2, SLX1, SLX4 and UL-12.

[0182] In certain embodiments, the genome editing compositions and methods for editing the genome of cells contemplated herein include polypeptides comprising a homing endonuclease variant or a megaTAL and an exonuclease. The term "exonuclease" refers to an enzyme or domain that cleaves a phosphodiester bond at the end of a polynucleotide chain by a hydrolytic reaction that breaks the phosphodiester bond at the 3' or 5' end.

[0183] Illustrative examples of exonucleases suitable for use with specific embodiments contemplated herein include, but are not limited to, hExoI, yeast ExoI, E. coli ExoI, hTREX2, mouse TREX2, rat TREX2, hTREX1, mouse TREX1, rat TREX1, and rat TREX1.

[0184] In a specific embodiment, the DNA end processing enzyme is a 3' or 5' exonuclease, preferably Trex1 or Trex2, more preferably Trex2, even more preferably human or mouse Trex2.

[0185] D. Peptide

[0186] Various polypeptides are contemplated herein, including but not limited to homing endonuclease variants, megaTALs, and fusion polypeptides. In preferred embodiments, the polypeptides include the amino acid sequences shown in SEQ ID NOs: 1-12 and 22-24. Unless otherwise specified to the contrary, "polypeptide," "polypeptide fragment," "peptide," and "protein" are used interchangeably and are conventionally used as sequences of amino acids. In one embodiment, "polypeptide" includes fusion polypeptides and other variants. Polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides are not limited to a specific length, for example, they can include full-length protein sequences, full-length protein fragments, or fusion proteins, and can include post-translational modifications of the polypeptide (e.g., glycosylation, acetylation, phosphorylation, etc.) and other modifications known in the art (including naturally occurring and non-naturally occurring).

[0187] As used herein, "isolated protein," "isolated peptide," or "isolated polypeptide," etc. refers to a peptide or polypeptide molecule that is synthesized, separated and / or purified in vitro from the cellular environment and from association with other components of the cell, i.e., it has no significant association with in vivo substances.

[0188] Illustrative examples of polypeptides contemplated in particular embodiments include, but are not limited to, homing endonuclease variants, megaTALs, end-processing nucleases, fusion polypeptides, and variants thereof.

[0189] Polypeptides include "polypeptide variants". Polypeptide variants may differ from naturally occurring polypeptides by one or more amino acid substitutions, deletions, additions and / or insertions. These variants may be naturally occurring or may be generated synthetically, for example, by modifying one or more amino acids of the above polypeptide sequence. For example, in certain embodiments, it may be desirable to improve the biological properties of a homing endonuclease, megaTAL, etc. that binds to and cleaves a target site in a human TCRα gene by introducing one or more substitutions, deletions, additions and / or insertions into the polypeptide. In specific embodiments, the polypeptide comprises a polypeptide having at least about 65%, 70%, 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% or 99% amino acid identity to any of the reference sequences contemplated herein, typically wherein the variant retains at least one biological activity of the reference sequence.

[0190] Polypeptide variants include biologically active "polypeptide fragments". Illustrative examples of biologically active polypeptide fragments include DNA binding domains, nuclease domains, etc. As used herein, the term "biologically active fragment" or "minimum biologically active fragment" refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10% or at least 5% of the activity of a naturally occurring polypeptide. In a preferred embodiment, the biological activity is binding affinity and / or cleavage activity for a target sequence. In certain embodiments, a polypeptide fragment may include an amino acid chain of at least 5 to about 1700 amino acids in length. It will be appreciated that in certain embodiments, the length of the fragments is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 In some embodiments, the polypeptides include 9, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more amino acids. In a specific embodiment, the polypeptide includes a biologically active fragment of a homing endonuclease variant. In a specific embodiment, the polypeptides described herein can include one or more amino acids represented as "X". If present in an amino acid SEQ ID NO, "X" represents any amino acid. One or more "X" residues may be present at the N-terminus and C-terminus of the amino acid sequence set forth in a particular SEQ ID NO contemplated herein. If an "X" amino acid is absent, the remaining amino acid sequence set forth in the SEQ ID NO may be considered a biologically active fragment.

[0191] In a specific embodiment, the polypeptide includes a biologically active fragment of a homing endonuclease variant, such as SEQ ID NO: 6-7 or megaTAL (SEQ ID NO: 10-12). The biologically active fragment may include an N-terminal truncation and / or a C-terminal truncation. In a specific embodiment, the biologically active fragment lacks 1, 2, 3, 4, 5, 6, 7 or 8 N-terminal amino acids of the homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence or includes a deletion thereof, more preferably including a deletion of 4 N-terminal amino acids of the homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence. In a specific embodiment, the biologically active fragment lacks 1, 2, 3, 4 or 5 C-terminal amino acids of the homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence or includes a deletion thereof, more preferably including a deletion of 2 C-terminal amino acids of the homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence. In a particularly preferred embodiment, the biologically active fragment lacks or includes a deletion of the 4 N-terminal amino acids and 2 C-terminal amino acids of the homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence.

[0192] In a specific embodiment, the I-OnuI variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7 or 8 of the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and / or a deletion of 1, 2, 3, 4 or 5 of the following C-terminal amino acids: R, G, S, F, V.

[0193] In a specific embodiment, the I-OnuI variant comprises a deletion or substitution of 1, 2, 3, 4, 5, 6, 7 or 8 of the following N-terminal amino acids: M, A, Y, M, S, R, R, E; a deletion or substitution of 1, 2, 3, 4 or 5 of the following C-terminal amino acids: R, G, S, F, V.

[0194] In a specific embodiment, the I-OnuI variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7 or 8 of the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and / or a deletion of 1 or 2 of the following C-terminal amino acids: F, V.

[0195] In a specific embodiment, the I-OnuI variant comprises a deletion or substitution of 1, 2, 3, 4, 5, 6, 7 or 8 of the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and / or a deletion or substitution of 1 or 2 of the following C-terminal amino acids: F, V.

[0196] As described above, polypeptides can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in DNA. Methods for mutagenesis and nucleotide sequence changes are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA., 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367-382), U.S. Pat. No. 4,873,192, Watson, JD et al., (Molecular Biology of the Gene, 4th edition, Benjamin / Cummings, Menlo Park, California, 1987) and references cited therein. Guidance as to appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (National Biomedical Research Foundation, Washington, DC).

[0197] In certain embodiments, variant will contain one or more conservative substitutions. "Conservative substitution" is that one of the amino acid substitutions has another amino acid of similar properties, so that the technical staff of the peptide chemistry field will expect the secondary structure and the hydrophobic nature (hydropathic nature) of the polypeptide to be substantially unchanged. The polynucleotide and polypeptide structure that can be considered in a specific embodiment are modified, and the polypeptide comprises a polypeptide with at least about and still obtains a functional molecule encoding a variant or a derivative polypeptide with a desired characteristic. When it is necessary to change the amino acid sequence of a polypeptide to produce an equivalent or even improved variant polypeptide, those skilled in the art can, for example, change one or more codons of the encoding DNA sequence, for example, according to Table 1.

[0198] Table 1 - Amino acid codons

[0199]

[0200] Computer programs well known in the art, such as DNASTAR, DNA Strider, Geneious, MacVector or Vector NTI software, can be used to find guidance for determining which amino acid residues can be replaced, inserted or deleted without eliminating biological activity. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., replacements of similar charged or uncharged amino acids. Conservative amino acid changes involve replacement of one of the amino acid families associated in its side chain. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan and tyrosine are sometimes primarily classified as aromatic amino acids. In peptides or proteins, suitable conservative amino acid replacements are known to those skilled in the art, and can generally be carried out without changing the biological activity of the resulting molecule. Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter the biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th ed., 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0201] In one embodiment, when expression of two or more polypeptides is desired, the polynucleotide sequences encoding them may be separated by an IRES sequence, as disclosed elsewhere herein.

[0202] The polypeptides contemplated in certain embodiments include fusion polypeptides. In certain embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to polypeptides having at least two, three, four, five, six, seven, eight, nine or ten polypeptide fragments.

[0203] In another embodiment, two or more polypeptides can be expressed as a fusion protein including one or more self-cleaving polypeptide sequences disclosed elsewhere herein.

[0204] In one embodiment, the fusion proteins contemplated herein include one or more DNA binding domains and one or more nucleases, as well as one or more linkers and / or self-cleaving polypeptides.

[0205] In one embodiment, the fusion proteins contemplated herein include a nuclease variant; a linker or self-cleaving peptide; and a terminal processing enzyme, including but not limited to a 5'-3' exonuclease, a 5'-3' alkaline exonuclease, and a 3'-5' exonuclease (eg, Trex2).

[0206] Fusion polypeptides may include one or more polypeptide domains or fragments, including but not limited to signal peptides, cell penetrating peptide domains (CPP), DNA binding domains, nuclease domains, etc., epitope tags (e.g., maltose binding protein ("MBP"), glutathione S transferase (GST), HIS6, MYC, FLAG, V5, VSV-G and HA), polypeptide linkers and polypeptide cleavage signals. Fusion polypeptides are usually linked C-terminus to N-terminus, but they can also be linked C-terminus to C-terminus, N-terminus to N-terminus or N-terminus to C-terminus. In specific embodiments, the polypeptides of the fusion protein can be in any order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences and interspecies homologs, as long as the desired activity of the fusion polypeptide is retained. Fusion polypeptides can be produced by chemical synthesis methods or by chemical connection between two parts, or can generally be prepared using other standard techniques. The connecting DNA sequence comprising the fusion polypeptide is operably linked to suitable transcriptional or translational control elements, as disclosed elsewhere herein.

[0207] Fusion polypeptides may optionally include a linker that can be used to connect one or more polypeptides or domains within a polypeptide. Peptide linker sequences can be used to separate any two or more polypeptide components by a sufficient distance to ensure that each polypeptide folds into its appropriate secondary and tertiary structures, thereby allowing the polypeptide domains to perform their desired functions. Such peptide linker sequences are incorporated into fusion polypeptides using standard techniques in the art. Suitable peptide linker sequences can be selected based on the following factors: (1) they are able to adopt a flexible extended conformation; (2) they cannot adopt a secondary structure that can interact with the functional epitopes on the first and second polypeptides; and (3) lack hydrophobic or charged residues that may react with the functional epitopes of the polypeptides. Preferred peptide linker sequences contain Gly, Asn, and Ser residues. Other near-neutral amino acids, such as Thr and Ala, can also be used in linker sequences. Amino acid sequences that can be used as linkers include those disclosed in Maratea et al., Gene, 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA, 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. When a specific fusion polypeptide fragment contains a non-essential N-terminal amino acid region that can be used to separate functional domains and prevent spatial interference, a linker sequence is not required. Preferred linkers are typically flexible amino acid subsequences that are synthesized as part of a recombinant fusion protein. The length of the linker polypeptide can be 1 to 200 amino acids, 1 to 100 amino acids, or 1 to 50 amino acids, including all integer values ​​therebetween.

[0208] Exemplary linkers include, but are not limited to, the following amino acid sequences: Glycine polymer (G) n ; Glycine-serine polymer (G 1-5 S 1-5 ) n , wherein n is an integer of at least one, two, three, four or five; glycine-alanine polymer; alanine-serine polymer; GGG (SEQ ID NO: 30); DGGGS (SEQ ID NO: 31); TGEKP (SEQ ID NO: 32) (see, e.g., Liu et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS), 5525-5530 (1997)); GGRR (SEQ ID NO: 33) (Pomerantz et al., 1995, supra); (GGGGS) n, wherein n=1, 2, 3, 4 or 5 (SEQ ID NO:34) (Kim et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS)), 93, 1156-1160 (1996); EGKSSGSGSESKVD (SEQ ID NO:35) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA, 87:1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO:36) (Bird et al., 1988, Science, 242:423-426), GGRRGGGS (SEQ ID NO:37); LRQRDGERP (SEQ ID NO:38); LRQKDGGGSERP (SEQ ID NO:39); LRQKD(GGGS)2ERP (SEQ ID NO:40). NO:40). Alternatively, flexible linkers can be rationally designed using computer programs capable of modeling the DNA binding site and the peptide itself (Desjarlais and Berg, PNAS, 90:2256-2260 (1993), PNAS, 91:11099-11103 (1994)) or by phage display methods.

[0209] The fusion polypeptide may further include a polypeptide cleavage signal between each polypeptide domain described herein or between an endogenous open reading frame and a polypeptide encoded by a donor repair template. In addition, a polypeptide cleavage site may be placed in any linker peptide sequence. Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites, such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites) and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004, "Traffic", 5 (8); 616-26).

[0210] Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., 1997, J. Gener. Virol., 78, 699-722; Scymczak et al. (2004), Nature Biotech., 5, 589-594). Exemplary protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, barley mosaic virus (byovirus) NIa protease, protease encoded by barley mosaic virus RNA-2, aphthous stolonifer L protease, enterovirus 2A protease, rhinovirus 2A protease, picornavirus 3C protease, cowpea mosaic virus 24K protease, nematode-borne polyhedron virus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow spot virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to its high cleavage stringency, in one embodiment TEV (Tobacco Etch Virus) protease cleavage sites are preferred, such as EXXYXQ (G / S) (SEQ ID NO: 41), such as ENLYFQG (SEQ ID NO: 42) and ENLYFQS (SEQ ID NO: 43), where X represents any amino acid (cleavage caused by TEV occurs between Q and G or Q and S).

[0211] In certain embodiments, the self-cleavage polypeptide site comprises a 2A or 2A-like site, sequence or domain (Donnelly et al., 2001, J. Gen. Virol., 82: 1027-1041). In a specific embodiment, the viral 2A peptide is an aphthous stomatitis virus 2A peptide, a potato virus Y 2A peptide or a cardiovirus 2A peptide.

[0212] In one embodiment, the viral 2A peptide is selected from the group consisting of: foot-and-mouth disease virus (FMDV) 2A peptide, equine rhinitis A virus (ERAV) 2A peptide, β-tetrasomal virus (TaV) 2A peptide, porcine teschovirus-1 (PTV-1) 2A peptide, Theiler virus 2A peptide and encephalomyocarditis virus 2A peptide.

[0213] Illustrative examples of 2A sites are provided in Table 2.

[0214] Table 2: Exemplary 2A sites comprising the following sequence:

[0215] SEQ ID NO:44 GSGATNFSLLKQAGDVEENPGP SEQ ID NO:45 ATNFSLLKQAGDVEENPGP SEQ ID NO:46 LLKQAGDVEENPGP SEQ ID NO:47 GSGEGRGSLLTCGDVEENPGP SEQ ID NO:48 EGRGSLLTCGDVEENPGP SEQ ID NO:49 LLTCGDVEENPGP SEQ ID NO:50 GSGQCTNYALLKLAGDVESNPGP SEQ ID NO:51 QCTNYALLKLAGDVESNPGP SEQ ID NO:52 LLKLAGDVESNPGP SEQ ID NO:53 GSGVKQTLNFDLLKLAGDVESNPGP SEQ ID NO:54 VKQTLNFDLLKLAGDVESNPGP SEQ ID NO:55 LLKLAGDVESNPGP SEQ ID NO:56 LLNFDLLKLAGDVESNPGP SEQ ID NO:57 TLNFDLLKLAGDVESNPGP SEQ ID NO:58 LLKLAGDVESNPGP SEQ ID NO:59 NFDLLKLAGDVESNPGP SEQ ID NO:60 QLLNFDLLKLAGDVESNPGP SEQ ID NO:61 APVKQTLNFDLLKLAGDVESNPGP SEQ ID NO:62 VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT SEQ ID NO:63 LNFDLLKLAGDVESNPGP SEQ ID NO:64 LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP SEQ ID NO:65 EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP

[0216] E. Polynucleotide

[0217] In a particular embodiment, polynucleotides encoding one or more homing endonuclease variants, megaTALs, terminal processing enzymes, and fusion polypeptides contemplated herein are provided. As used herein, the term "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA / RNA hybrids. Polynucleotides can be single-stranded or double-stranded and are recombinant, synthetic, or isolated. Polynucleotides include, but are not limited to, pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, synthetic RNA, genomic RNA (gRNA), positive strand RNA (RNA (+)), negative strand RNA (RNA (-)), tracrRNA, crRNA, single guide RNA (sgRNA), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA. Polynucleotides refer to polymeric forms of nucleotides, either ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotides, of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000, or at least 15000 or more nucleotides in length, and all intermediate lengths. It will be readily appreciated that in this context, "intermediate lengths" means any length between the cited values, e.g., 6, 7, 8, 9 nucleotides, etc., 101, 102, 103 nucleotides, etc.; 151, 152, 153 nucleotides, etc.; 201, 202, 203 nucleotides, etc. In specific embodiments, the polynucleotide or variant has at least or about 50%, 55%, 60%, 65%, 70%, 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%, or 100% sequence identity to a reference sequence.

[0218] In a particular embodiment, the polynucleotide can be codon optimized. As used herein, the term "codon optimized" refers to replacing codons in a polynucleotide encoding a polypeptide to increase the expression, stability and / or activity of the polypeptide. Factors affecting codon optimization include, but are not limited to, one or more of the following: (i) changes in codon preferences between preference tables of two or more organisms or genes or synthetic constructions, (ii) changes in codon preference within an organism, gene or genome, (iii) systematic changes in codons (including the environment), (iv) changes in codons according to which tRNA is decoded, (v) changes in codons according to GC%, whether overall or at a position in a triplet, (vi) changes in the degree of similarity to a reference sequence (e.g., a naturally occurring sequence), (vii) changes in codon frequency cutoffs, (viii) structural properties of mRNA transcribed from a DNA sequence, (ix) prior knowledge of the function of the DNA sequence on which the design of the codon substitution set is based, (x) systematic changes in the codon set for each amino acid, and / or (xi) separation and removal of false translation start sites.

[0219] As used herein, the term "nucleotide" refers to a heterocyclic nitrogenous base connected to a phosphorylated sugar by an N-glycosidic bond. Nucleotide should be understood to include natural bases and a variety of modified bases recognized in the art. These bases are generally located at the 1' position of the nucleotide sugar portion. Nucleotide generally includes a base, a sugar and a phosphate group. In ribonucleic acid (RNA), the sugar is ribose, and in deoxyribonucleic acid (DNA), the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group present in ribose. Exemplary natural nitrogenous bases include purine, adenosine (A) and guanidine (G), as well as pyrimidine, cytidine (C) and thymidine (T) (or in the context of RNA, uracil (U)). The C-1 atom of deoxyribose is bonded to the N-1 of pyrimidine or the N-9 of purine. Nucleotide is generally a monophosphate, a diphosphate or a triphosphate. Nucleotides may or may not be modified at the sugar, phosphate and / or base moieties (also interchangeably referred to as nucleotide analogs, nucleotide derivatives, modified nucleotides, non-natural nucleotides and non-standard nucleotides; see, e.g., WO 92 / 07065 and WO 93 / 15187). Examples of modified nucleic acid bases are summarized by Limbach et al. (1994, Nucleic Acids Res., 22, 2183-2196).

[0220] Nucleotide can also be considered as the phosphate ester of nucleoside, wherein esterification occurs on the hydroxyl connected to the C-5 of sugar. As used herein, the term "nucleoside" refers to that a heterocyclic nitrogenous base is connected to sugar by an N-glycosidic bond. Nucleoside is considered to include natural bases in the art, and also includes well-known modified bases. These bases are generally located at the 1' position of the nucleoside sugar moiety. Nucleoside generally includes a base and a sugar group. Nucleoside can be or not modified (also interchangeably referred to as nucleoside analogs, nucleoside derivatives, modified nucleosides, non-natural nucleosides or non-standard nucleosides) at sugar and / or base moieties. Also as mentioned above, Limbach et al. (1994, " Nucleic Acids Res. ", 22,2183-2196) summarizes the examples of modified nucleic acid bases.

[0221] Illustrative examples of polynucleotides include, but are not limited to, polynucleotides encoding SEQ ID NOs: 1-12 and 22-24 and polynucleotide sequences set forth in SEQ ID NOs: 13-16 and 25-27.

[0222] In various illustrative embodiments, polynucleotides contemplated herein include, but are not limited to, polynucleotides encoding homing endonucleases variants, megaTALs, end-processing enzymes, fusion polypeptides, and expression vectors, viral vectors, and transfer plasmids comprising the polynucleotides contemplated herein.

[0223] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to polynucleotides that exhibit substantial sequence identity to a reference polynucleotide sequence or that hybridize to a reference sequence under stringent conditions as defined below. These terms also encompass polynucleotides that differ from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Thus, the terms "polynucleotide variant" and "variant" encompass polynucleotides in which one or more nucleotides have been added or deleted or modified or in which one or more nucleotides have been substituted with a different nucleotide. At this point, it is well understood in the art that certain changes, including mutations, additions, deletions, and substitutions, may be made to a reference polynucleotide such that the altered polynucleotide retains the biological function or activity of the reference polynucleotide.

[0224] In one embodiment, polynucleotides include nucleotide sequences that hybridize with target nucleic acid sequences under stringent conditions. Hybridization under "stringent conditions" describes a hybridization scheme in which nucleotide sequences with at least 60% identity to each other remain hybridized. Typically, stringent conditions are selected to be approximately 5 ° C lower than the thermal melting point (Tm) of a specific sequence under a determined ionic strength and pH. Tm is a temperature (under a determined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize with the target sequence when balanced. Since the target sequence is usually present in excess, 50% of the probes are occupied when balanced under Tm.

[0225] As used herein, the description "sequence identity" or, for example, including "a sequence having 50% identity to..." refers to the degree to which a sequence has identity on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis over a comparison window. Therefore, the "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) are present in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Nucleotides and polypeptides comprising at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein, typically wherein the polypeptide variant retains at least one biological activity of the reference polypeptide.

[0226] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" is at least 12 monomer units in length, but typically 15 to 18 monomer units, and in most cases at least 25 monomer units, comprising nucleotides and amino acid residues. Because two polynucleotides can each include (1) a sequence that is similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence), and (2) a sequence that is different between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 consecutive positions, typically about 50 to about 100 consecutive positions, and more typically about 100 to about 150 consecutive positions, in which a sequence is compared to a reference sequence of the same number of consecutive positions, and the two sequences are then optimally aligned. For the best comparison of two sequences, the comparison window can include about 20% or less addition or deletion (that is, gap) compared with the reference sequence (which does not include addition or deletion). The best comparison for comparing the sequence of the comparison window can be carried out by the computerization of the algorithm (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package 7.0 version, Genetics Computer Group, 575 Science Drive Madison, WI, USA), or by the inspection and best comparison (that is, causing the highest homology percentage on the comparison window) generated by any one of the selected methods. It is also possible to refer to the BLAST program family, such as Altschul et al., 1997, " Nucleic Acids Research (Nucl.Acids Res.) ", 25:3389. The detailed discussion of sequence analysis can be found in Ausubel et al., " Modern Molecular Biology Experiment Guide (Current Protocolsin Molecular Biology) ", John Wiley & Sons Inc., 1994-1998, the unit 19.3 of the 15th chapter.

[0227] As used herein, "isolated polynucleotide" refers to a polynucleotide purified from sequences flanking its naturally occurring state, e.g., a DNA fragment that has been removed from sequences normally adjacent to the fragment. In specific embodiments, "isolated polynucleotide" refers to complementary DNA (cDNA), recombinant polynucleotides, synthetic polynucleotides, or other polynucleotides that do not exist in nature and have been artificially produced.

[0228] In various embodiments, the polynucleotides include mRNA encoding polypeptides contemplated herein, including but not limited to homing endonuclease variants, megaTALs, and end-processing enzymes. In certain embodiments, the mRNA includes a cap, one or more nucleotides, and a poly(A) tail.

[0229] As used herein, the term "5' cap" or "5' cap structure" or "5' cap moiety" refers to a chemical modification that has been incorporated into the 5' end of an mRNA. The 5' cap is involved in nuclear export, mRNA stability and translation.

[0230] In a specific embodiment, the mRNA contemplated herein includes a 5' cap comprising a 5'-ppp-5'-triphosphate bond between the terminal guanosine cap residue of the mRNA molecule and the 5'-terminal transcribed sense nucleotide. This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue.

[0231] Illustrative examples of 5' caps suitable for use with specific embodiments of mRNA polynucleotides contemplated herein include, but are not limited to: unmethylated 5' cap analogs, such as G(5')ppp(5')G, G(5')ppp(5')C, G(5')ppp(5')A; methylated 5' cap analogs, such as m 7 G(5′)ppp(5′)G、m 7 G(5′)ppp(5′)C and m 7 G(5′)ppp(5′)A; dimethylated 5′ cap analogs, such as m 2,7 G(5′)ppp(5′)G、m 2,7 G(5′)ppp(5′)C and m 2,7 G(5′)ppp(5′)A; trimethylated 5′ cap analogs, such as m 2,2,7 G(5′)ppp(5′)G、m 2,2,7 G(5′)ppp(5′)C and m 2,2,7 G(5′)ppp(5′)A; dimethylated symmetrical 5′ cap analogs, such as m 7 G(5′)pppm 7 (5′)G, m 7 G(5′)pppm 7 (5′)C and m 7 G(5′)pppm 7 (5′)A; and anti-reverse 5′ cap analogs, such as anti-reverse cap analog (ARCA) caps, designated 3′O-Me-m 7 G(5′)ppp(5′)G、2′O-Me-m 7 G(5′)ppp(5′)G、2′O-Me-m 7G(5′)ppp(5′)C, 2′O-Me-m 7 G(5′)ppp(5′)A、m 7 2′d(5′)ppp(5′)G、m 7 2′d(5′)ppp(5′)C、m 7 2′d(5′)ppp(5′)A, 3′O-Me-m 7 G(5′)ppp(5′)C, 3′O-Me-m 7 G(5′)ppp(5′)A、m 7 3′d(5′)ppp(5′)G、m 7 3′d(5′)ppp(5′)C、m 7 3'd(5')ppp(5')A and its tetraphosphate derivatives) (see, e.g., Jemielity et al., Ribonucleic Acid (RNA), 9: 1108-1122 (2003)).

[0232] In certain embodiments, the mRNA includes a 5' cap, which is a 7-methylguanylate ("m7G"), linked to the 5'-end of the first transcribed nucleotide via a triphosphate bridge, resulting in m 7 G(5')ppp(5')N, wherein N is any nucleoside.

[0233] In some embodiments, the mRNA includes a 5' cap, wherein the cap is a Cap0 structure (the Cap0 structure lacks the 2'-O-methyl residue of the ribose linked to bases 1 and 2), a Cap1 structure (the Cap1 structure has a 2'-O-methyl residue at base 2), or a Cap2 structure (the Cap2 structure has a 2'-O-methyl residue linked to bases 2 and 3).

[0234] In one embodiment, the mRNA comprises m 7 G(5′)ppp(5′)G cap.

[0235] In one embodiment, the mRNA includes an ARCA cap.

[0236] In particular embodiments, the mRNAs contemplated herein include one or more modified nucleosides.

[0237] In one embodiment, the mRNA includes one or more modified nucleosides selected from the group consisting of pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinemethyluridine, 1-taurinemethyl-pseudouridine, 5-taurinemethyl-2-thio-uridine, 1-taurinemethyl-4-thio-uridine, 5-methyl-uridine, 1- methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyridine pyrrole-cytidine, pyrrole-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine , 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wyosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine and N2,N2-dimethyl-6-thio-guanosine.

[0238] In one embodiment, the mRNA comprises one or more modified nucleosides selected from the group consisting of pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinemethyluridine, 1-taurinemethyl-pseudouridine, 5-taurinemethyl-2-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinemethyl-2-methyluridine, 5-taurinemethyl-2-methyluridine, 5-carboxy ... -thio-uridine, 1-taurinemethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine and 4-methoxy-2-thio-pseudouridine.

[0239] In one embodiment, the mRNA comprises one or more modified nucleosides selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrole-cytidine, pyrrole-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4- Thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zeblin, 5-aza-zeblin, 5-methyl-zeblin, 5-aza-2-thio-zeblin, 2-thio-zeblin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine and 4-methoxy-1-methyl-pseudoisocytidine.

[0240] In one embodiment, the mRNA comprises one or more modified nucleosides selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine , N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylaminoformyladenosine, N6-threonylaminoformyladenosine, 2-methylthio-N6-threonylaminoformyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine and 2-methoxy-adenine.

[0241] In one embodiment, the mRNA includes one or more modified nucleosides selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wyobutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine and N2,N2-dimethyl-6-thio-guanosine.

[0242] In one embodiment, the mRNA includes one or more pseudouridines, one or more 5-methyl-cytosines, and / or one or more 5-methyl-cytidines.

[0243] In one embodiment, the mRNA includes one or more pseudouridines.

[0244] In one embodiment, the mRNA includes one or more 5-methyl-cytidines.

[0245] In one embodiment, the mRNA includes one or more 5-methyl-cytosines.

[0246] In specific embodiments, the mRNA contemplated herein includes a poly(A) tail to help protect the mRNA from exonuclease degradation, stabilize the mRNA and promote translation. In certain embodiments, the mRNA includes a 3' poly(A) tail structure.

[0247] In particular embodiments, the poly(A) tail is at least about 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or at least about 500 or more adenine nucleotides in length, or any intermediate number of adenine nucleotides. In certain embodiments, the poly(A) tail is at least about 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175 60, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 9, 200, 201, 202, 203, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319,

[0248] In certain embodiments, the length of the poly(A) tail is about 10 to about 500 adenine nucleotides, about 50 to about 500 adenine nucleotides, about 100 to about 500 adenine nucleotides, about 150 to about 500 adenine nucleotides, about 200 to about 500 adenine nucleotides, about 250 to about 500 adenine nucleotides, about 300 to about 500 adenine nucleotides, about 50 to about 450 adenine nucleotides, or about 500 to about 500 adenine nucleotides. adenine nucleotides, about 50 to about 400 adenine nucleotides, about 50 to about 350 adenine nucleotides, about 100 to about 500 adenine nucleotides, about 100 to about 450 adenine nucleotides, about 100 to about 400 adenine nucleotides, about 100 to about 350 adenine nucleotides, about 100 to about 300 adenine nucleotides, about 150 to about 500 adenine nucleotides, about 150 to about 450 adenine nucleotides, about 150 to about 400 adenine nucleotides, about 150 to about 350 adenine nucleotides, about 150 to about 300 adenine nucleotides, about 150 to about 250 adenine nucleotides, about 150 to about 200 adenine nucleotides, about 200 to about 500 adenine nucleotides, about 200 to about 450 adenine nucleotides, about 200 to about 40 In some embodiments, the present invention provides adenine nucleotides in an amount of about 200 to about 350 adenine nucleotides, about 200 to about 300 adenine nucleotides, about 250 to about 500 adenine nucleotides, about 250 to about 450 adenine nucleotides, about 250 to about 400 adenine nucleotides, about 250 to about 350 adenine nucleotides, or about 250 to about 300 adenine nucleotides or any intermediate range of adenine nucleotides.

[0249] Terms describing the direction of a polynucleotide include: 5' (usually the end of a polynucleotide with a free phosphate group) and 3' (usually the end of a polynucleotide with a free hydroxyl (OH) group). A polynucleotide sequence can be annotated in a 5' to 3' direction or a 3' to 5' direction. For DNA and mRNA, the 5' to 3' strand is referred to as the "justice," "positive," or "coding" strand because its sequence is identical to that of the pre-messenger (pre-mRNA) [except for uracil (U) in RNA, rather than thymine (T) in DNA]. For DNA and mRNA, the complementary 3' to 5' strand, which is the strand transcribed by RNA polymerase, is referred to as the "template," "antisense," "negative," or "non-coding" strand. As used herein, the term "reverse" refers to a 5' to 3' sequence written in a 3' to 5' direction or a 3' to 5' sequence written in a 5' to 3' direction.

[0250] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., nucleotide sequences) that are related by the base pairing rules. For example, the complementary strand of the DNA sequence 5'AGTCATG 3' is 3'TCAGTAC 5'. The latter sequence is usually written as a reverse complement, with the 5' end on the left and the 3' end on the right, i.e., 5'CATGACT 3'. A sequence that is equal to its reverse complement is called a palindrome. Complementarity can be "partial", in which only some nucleic acid bases are matched according to the base pairing rules. Alternatively, there can be "complete" or "full" complementarity between nucleic acids.

[0251] Polynucleotides can be prepared, manipulated, expressed and / or delivered using any of the various mature techniques known and available in the art. In order to express the desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into a suitable vector. The desired polypeptide can also be expressed by delivering the mRNA encoding the polypeptide into a cell.

[0252] Illustrative examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements, such as SleepingBeauty, PiggyBac.

[0253] Additional illustrative examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), bacteriophages (e.g., lambda phage or M13 phage), and animal viruses.

[0254] Illustrative examples of viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses and integration-defective lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40).

[0255] Illustrative examples of expression vectors include, but are not limited to, the pClneo vector (Promega) for expression in mammalian cells; the pLenti4 / V5-DEST vector (Promega) for lentivirus-mediated gene transfer and expression in mammalian cells; TM 、pLenti6 / V5-DEST TM and pLenti6.2 / V5-GW / lacZ (Invitrogen). In certain embodiments, the coding sequence of the polypeptide disclosed herein can be linked to these expression vectors for expressing the polypeptide in mammalian cells.

[0256] In a specific embodiment, carrier is an additional carrier or a carrier maintained outside the chromosome. As used herein, term "additional" refers to a carrier that can replicate and is not integrated into the chromosomal DNA of the host and can not gradually lose from the host cell of division, which also represents that the carrier replicates outside the chromosome or additionally.

[0257] In certain embodiments, to achieve efficient translation of each of the plurality of polypeptides, the polynucleotide sequences may be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides.

[0258] As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry into the start codon (e.g., ATG) of a cistron (protein coding region), resulting in cap-independent translation of the gene. See, e.g., Jackson et al., 1990, Trends Biochem Sci, 15(12):477-83) and Jackson and Kaminski, 1995, RNA, 1(10):985-1000. Examples of IRES commonly used by those skilled in the art include those described in U.S. Pat. No. 6,692,736. Additional examples of "IRES" known in the art include, but are not limited to, IRES obtainable from picornaviruses (Jackson et al., 1990) and IRES obtainable from viral or cellular mRNA sources, such as, for example, immunoglobulin heavy chain binding protein (BiP), vascular endothelial growth factor (VEGF) (Huez et al., 1998, Mol. Cell. Biol., 18(11):6178-6190), fibroblast growth factor 2 (FGF-2) and insulin-like growth factor (IGFII), translation initiation factor eIF4G and yeast transcription factors TFIID and HAP4, encephalomyocarditis virus (EMCV) commercially available from Novagen (Duke et al., 1992, J. Virol., 66(3):1602-9) and VEGF IRES (Huez et al., 1998, Mol Cell Biol., 18(11):6178-90). IRES have also been reported in viral genomes of Picornaviridae, Dicistroviridae, and Flaviviridae species, as well as in HCV, Friedreich's murine leukemia virus (FrMLV), and Moloney's murine leukemia virus (MoMLV).

[0259] In one embodiment, the IRES used in the polynucleotides contemplated herein is the EMCV IRES.

[0260] In a specific embodiment, the polynucleotide includes a polynucleotide having a consensus Kozak sequence and encoding a desired polypeptide. As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly promotes the initial binding of mRNA to the small subunit of the ribosome and increases translation. The consensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO: 66), where R is a purine (A or G) (Kozak, 1986, Cell, 44 (2): 283-92, and Kozak, 1987, Nucleic Acids Res., 15 (20): 8125-48).

[0261] Elements that direct efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression.

[0262] In a specific embodiment, the vector includes a polyadenylation sequence 3' of a polynucleotide encoding a polypeptide to be expressed. The term "poly (A) site" or "poly (A) sequence" used herein refers to a DNA sequence that instructs the termination and polyadenylation of a nascent RNA transcript by RNA polymerase II. The polyadenylation sequence can promote mRNA stability by adding a poly (A) tail to the 3' end of the coding sequence, thus contributing to improving translation efficiency. Cutting and polyadenylation are guided by the poly (A) sequence in RNA. The core poly (A) sequence of mammalian pre-mRNA has two recognition elements flanking the cutting-polyadenylation site. Typically, the almost invariant AAUAAA hexamer is located 20-50 nucleotides upstream of a more variable element rich in U or GU residues. The cutting of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5' cleavage product. In a specific embodiment, the core poly (A) sequence is a synthetic poly (A) sequence (e.g., AATAAA, ATTAAA, AGTAAA). Illustrative examples of poly (A) sequences include, but are not limited to, the SV40 poly (A) sequence, the bovine growth hormone poly (A) sequence (BGHpA), the rabbit β-globin poly (A) sequence (rβgpA), or another suitable heterologous or endogenous poly (A) sequence known in the art.

[0263] In certain embodiments, polynucleotides encoding one or more nuclease variants, megaTALs, terminal processing enzymes, or fusion polypeptides can be introduced into hematopoietic cells, such as T cells, by non-viral and viral methods. In certain embodiments, delivery of one or more polynucleotides encoding nucleases and / or donor repair templates can be provided by the same method or by different methods, and / or by the same vector or by different vectors.

[0264] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is usually connected to a carrier nucleic acid molecule, for example, inserted into a carrier nucleic acid molecule. The vector may contain sequences that direct autonomous replication in the cell, or may contain sequences sufficient to allow integration into the host cell DNA. In a specific embodiment, a non-viral vector is used to deliver one or more polynucleotides contemplated herein to a T cell.

[0265] Illustrative examples of non-viral vectors include, but are not limited to, plasmids (eg, DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0266] Illustrative methods of non-viral delivery of polynucleotides contemplated in particular embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran mediated transfer, biolistics, and heat shock.

[0267] Illustrative examples of polynucleotide delivery systems suitable for use in particular embodiments contemplated in particular embodiments include, but are not limited to, those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipid transfection reagents are commercially available (e.g., Transfectam TM and Lipofectin TM ). Cationic and neutral lipids suitable for effective receptor recognition lipofection of polynucleotides have been described in the literature. See, for example, Liu et al. (2003), Gene Therapy, 10:180–187; and Balazs et al. (2011), Journal of Drug Delivery, 2011:1-12. Antibody-targeted, bacteria-derived, non-biological nanocell-based delivery is also contemplated in certain embodiments.

[0268] F. Cells

[0269] The genome-edited cells prepared by the methods contemplated in specific embodiments include one or more gene edits in the TCRα gene, and provide improved cell-based therapeutics for preventing, treating or ameliorating at least one symptom of cancer, GVHD, infectious diseases, autoimmune diseases, immunodeficiency, or conditions associated therewith. Without wishing to be bound by any particular theory, it is believed that the genome-edited immune effector cells prepared by the methods contemplated herein have excellent properties, including enhanced and improved in vivo safety, efficacy, and durability.

[0270] The genome-edited cells contemplated in specific embodiments can be autologous / autogeneic ("one's own") or non-autologous ("non-one's own", e.g., allogeneic, homologous, or xenogeneic). As used herein, "autologous" refers to cells from the same subject. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the comparison cells. As used herein, "homologous" refers to cells of different subjects that are genetically identical to the comparison cells. As used herein, "xenogeneic" refers to cells of a different species from the comparison cells. In a preferred embodiment, the cells are obtained from a mammalian subject. In a more preferred embodiment, the cells are obtained from a primate subject, optionally from a non-human primate. In a most preferred embodiment, the cells are obtained from a human subject.

[0271] "Isolated cells" refer to non-naturally occurring cells, such as cells not found in nature, modified cells, engineered cells, etc., which have been obtained from an in vivo tissue or organ and are substantially free of extracellular matrix.

[0272] As used herein, the term "cell population" refers to a plurality of cells, which may be composed of any number and / or combination of homogeneous or heterogeneous cell types, as described elsewhere herein. For example, in order to transduce T cells, a cell population may be isolated or obtained from peripheral blood. The cell population may account for about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the target cell type to be edited. In certain embodiments, T cells may be isolated or purified from a heterogeneous cell population using methods known in the art.

[0273] Illustrative examples of cell types whose genomes may be edited using the compositions and methods contemplated herein include, but are not limited to, cell lines, primary cells, stem cells, progenitor cells, and differentiated cells, and mixtures thereof.

[0274] In a preferred embodiment, the genome editing compositions and methods are used to edit hematopoietic cells, more preferably immune cells, and even more preferably T cells.

[0275] The term "T cell" or "T lymphocyte" is recognized in the art and is intended to include thymocytes, immune effector cells, regulatory T cells, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cell can be a T helper (Th) cell, such as a T helper 1 (Th1) or T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD4 + T cells)CD4 + T cells, cytotoxic T cells (CTL; ​​CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - T cells or any other T cell subsets. In one embodiment, the T cells are immune effector cells. In one embodiment, the T cells are NKT cells. Other illustrative T cell populations suitable for specific embodiments include naive T cells and memory T cells.

[0276] In various embodiments, the genome-edited cells include immune effector cells that include TCRα genes edited by the compositions and methods contemplated herein. An "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). Exemplary immune effector cells contemplated in specific embodiments are T lymphocytes, particularly cytotoxic T cells (CTLs; CD8 + T cells), TILs, and helper T cells (HTL; CD4 + In one embodiment, the immune effector cells comprise natural killer (NK) cells. In one embodiment, the immune effector cells comprise natural killer T (NKT) cells.

[0277] "Effective T cells" and "immature T cells" are used interchangeably in certain embodiments and refer to a T cell phenotype in which the T cells are able to proliferate with a decrease in differentiation. In certain embodiments, immature T cells have the phenotype of "immature T cells". In certain embodiments, immature T cells include one or more or all of the following biological markers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, and CD38. In one embodiment, immature T cells include one or more or all of the following biological markers: CD62L, CD127, CD197, and CD38. In one embodiment, immature T cells lack expression of CD57, CD244, CD160, PD-1, CTLA4, and LAG3.

[0278] T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or tumors.

[0279] In particular embodiments, the population of cells includes immune effector cells or T cells that include a homing endonuclease variant or megaTAL that targets exon 1 of the constant region of a TCR alpha gene as contemplated herein.

[0280] In particular embodiments, the population of cells includes immune effector cells or T cells that include a homing endonuclease variant polypeptide or a megaTAL polypeptide that targets exon 1 of a constant region of a TCRα gene as contemplated herein.

[0281] In particular embodiments, the cell population includes immune effector cells or T cells, which include a vector encoding a homing endonuclease variant or a megaTAL that targets exon 1 of the constant region of a TCRα gene as contemplated herein.

[0282] In particular embodiments, the cell population includes immune effector cells or T cells that include mRNA encoding a homing endonuclease variant or megaTAL that targets exon 1 of the constant region of a TCRα gene as contemplated herein.

[0283] G. Compositions and Formulations

[0284] The composition considered in a particular embodiment may include one or more polypeptides, polynucleotides, vectors including them, and genome editing compositions and genome editing cell compositions, as considered herein. The genome editing compositions and methods considered in a particular embodiment can be used to edit target sites in human T cell receptor alpha (TCR alpha) genes in cells or cell populations. In a preferred embodiment, the genome editing composition is used to edit the TCR alpha gene in hematopoietic cells (e.g., T cells or immune effector cells).

[0285] In various embodiments, the compositions contemplated herein include nuclease variants, and optional terminal processing enzymes, such as 3'-5' exonucleases (Trex2). Nuclease variants can be in the form of mRNA, which is introduced into cells by polynucleotide delivery methods disclosed above (e.g., electroporation, lipid nanoparticles, etc.). In one embodiment, a composition comprising mRNA encoding a homing endonuclease variant or megaTAL and optional 3'-5' exonucleases is introduced into cells by polynucleotide delivery methods disclosed above. The composition can be used to generate genome-edited cells or genome-edited cell populations by error-prone NHEJ.

[0286] In various embodiments, the compositions contemplated herein include a donor repair template. The composition can be delivered to a cell expressing or expressing a nuclease variant and an optional end processing enzyme. In one embodiment, the composition can be delivered to a cell expressing or expressing a homing endonuclease variant or megaTAL and an optional 3'-5' exonuclease. Expression of a gene editing enzyme in the presence of a donor repair template can be used to generate a genome-edited cell or a genome-edited cell population by HDR.

[0287] Compositions include but are not limited to pharmaceutical compositions. "Pharmaceutical composition" refers to a composition prepared in a pharmaceutically acceptable or physiologically acceptable solution that is administered to a cell or animal alone or in combination with one or more other treatment modalities. It should also be understood that the composition may also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies or other various pharmaceutically active agents, if desired. There is virtually no restriction on other components that may also be included in the composition, provided that the additional agents do not adversely affect the composition.

[0288] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without causing excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0289] The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or vehicle used together with the therapeutic cells. Illustrative examples of pharmaceutical carriers can be sterile liquids, such as cell culture media, water and oils, the oils comprising those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. In a particular embodiment, suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. Unless any conventional culture medium or reagent is incompatible with the active ingredient, its use in the therapeutic composition is considered. The supplemented active ingredient can also be incorporated into the composition.

[0290] In one embodiment, the composition comprising a pharmaceutically acceptable carrier is suitable for administration to a subject. In a specific embodiment, the composition comprising a carrier is suitable for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration. In a specific embodiment, the composition comprising a pharmaceutically acceptable carrier is suitable for intraventricular, intraspinal or intrathecal administration. Pharmaceutically acceptable carriers include sterile aqueous solutions, cell culture media or dispersions. The use of these culture media and reagents for pharmaceutically active substances is well known in the art. Unless any conventional culture media or reagents are incompatible with the transduced cells, their use in pharmaceutical compositions is considered.

[0291] In certain embodiments, the compositions contemplated herein include genetically modified T cells and a pharmaceutically acceptable carrier. Compositions including cell-based compositions contemplated herein can be administered alone or in combination with other suitable compounds by enteral or parenteral administration methods to achieve the desired therapeutic goal.

[0292] Pharmaceutically acceptable carriers must have sufficiently high purity and sufficiently low toxicity to make them suitable for use to human subjects to be treated. It should also maintain or increase the stability of the composition. Pharmaceutically acceptable carriers can be liquid or solid, and in view of the planned mode of administration, when combined with other components of the composition, pharmaceutically acceptable carriers are selected to provide required volume, consistency, etc. For example, pharmaceutically acceptable carriers can be but are not limited to adhesives (e.g., pregelatinized corn starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose, etc.), fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate, calcium hydrogen phosphate, etc.), lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.), disintegrants (e.g., starch, sodium starch glycolate, etc.), or wetting agents (e.g., sodium lauryl sulfate, etc.). Other suitable pharmaceutically acceptable carriers for the compositions contemplated herein include, but are not limited to, water, saline solutions, alcohol, polyethylene glycol, gelatin, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like.

[0293] These carrier solutions may also contain buffers, diluents, and other suitable additives. The term "buffer" as used herein refers to a solution or liquid whose chemical composition neutralizes an acid or base without significant pH change. Examples of buffers contemplated herein include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), normal / physiological saline (0.9% NaCl).

[0294] The pharmaceutically acceptable carrier may be present in an amount sufficient to maintain the pH of the composition at about 7. Alternatively, the pH of the composition is in the range of about 6.8 to about 7.4, for example, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3 and 7.4. In yet another embodiment, the pH of the composition is about 7.4.

[0295] Compositions contemplated herein may include non-toxic pharmaceutically acceptable culture media. The composition may be a suspension. The term "suspension" as used herein refers to non-adherent conditions in which cells are not attached to a solid support. For example, cells maintained as a suspension may be agitated or stirred and not adhered to a support, such as a culture dish.

[0296] In certain embodiments, the compositions contemplated herein are formulated in suspension, wherein the genome-edited T cells are dispersed in an acceptable liquid culture medium or solution (e.g., saline or serum-free culture medium), an intravenous (IV) bag, etc. Acceptable diluents include, but are not limited to, water, Promali, Ringer's solution, isotonic sodium chloride (saline) solution, serum-free cell culture medium, and culture medium suitable for cryogenic storage (e.g., culture medium).

[0297] In certain embodiments, the pharmaceutically acceptable carrier is substantially free of natural proteins of human or animal origin and is suitable for storing compositions comprising genome-edited T cell populations. The therapeutic composition is intended for administration to human patients and is therefore substantially free of cell culture components, such as bovine serum albumin, horse serum, and fetal bovine serum.

[0298] In some embodiments, the composition is formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects. In specific embodiments, the pharmaceutically acceptable cell culture medium is a serum-free medium.

[0299] Compared to serum-containing culture media, serum-free culture media have several advantages, including simplifying and better defining the composition, reducing the degree of contamination, eliminating potential sources of infectious agents and reducing costs. In various embodiments, serum-free culture media are animal-free and may optionally be protein-free. Optionally, the culture media may contain biopharmaceutically acceptable recombinant proteins. "Animal-free" culture media refers to culture media in which components are derived from non-animal sources. Recombinant proteins replace natural animal proteins in animal-free culture media, and nutrients are obtained from synthetic, plant or microbial sources. In contrast, "protein-free" culture media are defined as being substantially protein-free.

[0300] Illustrative examples of serum-free culture media for use in particular compositions include, but are not limited to, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

[0301] In a preferred embodiment, the composition comprising genome-edited T cells is formulated in Viagra.

[0302] In various embodiments, the composition comprising the genome-edited T cells is formulated in a cryopreservation medium. For example, a cryopreservation medium with a cryopreservative can be used to maintain high cell viability results after thawing. Illustrative examples of cryopreservation medium for specific compositions include, but are not limited to, CryoStor CS10, CryoStor CS5, and CryoStor CS2.

[0303] In one embodiment, the composition is formulated in a solution comprising 50:50 of CryoStor A and CryoStor CS10.

[0304] In a particular embodiment, the composition is substantially free of mycoplasma, endotoxin and microbial contamination. With respect to endotoxin, "substantially free" means that the endotoxin content per cell dose is less than the content allowed by the FDA for biological agents, which is a total endotoxin of 5EU / kg body weight per day, i.e., for an average of 70 kg of people, each total cell dose is 350EU. In a particular embodiment, the composition comprising hematopoietic stem cells or progenitor cells transduced with a retroviral vector contemplated herein contains about 0.5EU / mL to about 5.0EU / mL, or about 0.5EU / mL, 1.0EU / mL, 1.5EU / mL, 2.0EU / mL, 2.5EU / mL, 3.0EU / mL, 3.5EU / mL, 4.0EU / mL, 4.5EU / mL or 5.0EU / mL.

[0305] In certain embodiments, compositions and formulations suitable for the delivery of polynucleotides, including but not limited to one or more mRNA encoding one or more reprogramming nucleases and optionally, end-processing enzymes, are contemplated.

[0306] Exemplary formulations for ex vivo delivery may also include the use of various transfection agents known in the art, such as calcium phosphate, electroporation, heat shock, and various liposome preparations (i.e., lipid-mediated transfection). As described in more detail below, liposomes are lipid bilayers that encapsulate a portion of an aqueous fluid. DNA spontaneously associates to the outer surface of cationic liposomes (by virtue of their charge), and these liposomes will interact with the cell membrane.

[0307] In certain embodiments, the formulation of pharmaceutically acceptable carrier solutions is well known to those skilled in the art, such as developing appropriate dosing and treatment regimens to use the specific compositions described herein in various treatment regimens, including, for example, enteral and parenteral, such as intravascular, intravenous, intraarterial, intraosseous, intraventricular, intracerebral, intracranial, intraspinal, intrathecal, and intramedullary administration and formulation. Those skilled in the art will appreciate that certain embodiments contemplated herein may include other formulations, such as those well known in the pharmaceutical art, which are described, for example, in Remington: The Science and Practice of Pharmacy, Vol. I and Vol. II, 22nd Edition, Loyd V. Allen Jr., ed., Philadelphia, Pennsylvania: Pharmaceutical Press; 2012, the entire contents of which are incorporated herein by reference.

[0308] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0309] Although the foregoing embodiments have been described in detail by way of illustration and example for purposes of clear understanding, it will be readily apparent to those of ordinary skill in the art, based on the teachings contemplated herein, that certain changes and modifications may be made without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only, and not as limitations. Those skilled in the art will readily recognize various non-critical parameters that may be changed or modified to produce substantially similar results.

[0310] Examples

[0311] Example 1

[0312] Improved selectivity of I-OnuI variants targeting the human TCRα gene

[0313] Suboptimal selectivity regions were identified in the DNA recognition interface region of the I-OnuI variant (SEQ ID NO:6) targeting exon 1 in the constant region of the human TCRα gene ("original TCRαI-OnuI variant"). Positions p4, p5, and p6 in the target site were found to be relatively tolerant to base pair substitutions. Nucleotides were found in a frequent off-target site in the TCRα on-target site (CCG at p456) (SEQ ID NO:17) and in intron 6 of the KAT2B gene (ACA at p456, see Osborne et al., 2015) ( Figure 1, SEQ ID NO: 21). I-OnuI variants including domains with enhanced selectivity at these positions were developed to improve the overall selectivity properties and specific reduction of activity at KAT2B off-target sites (SEQ ID NO: 21).

[0314] Examination of the crystal structure of the wild-type I-OnuI protein (e.g., SEQ ID NOs: 1-5) in complex with its natural target site identified a subset of 8 amino acid residues that may contact or approach position p456. Local analysis of another I-OnuI variant targeting the human TCRα gene identified amino acids with significantly higher CCG selectivity compared to the original TCRα targeting I-OnuI variant. The amino acids responsible for high CCG triplet selectivity in the TCRα targeting I-OnuI variant were inserted into the original TCRα I-OnuI variant to generate the TCRα2.0 I-OnuI variant (SEQ ID NO: 7) ( Figure 2 ). TCRα2.0 I-OnuI variant enzymatic activity was evaluated for all 64 nucleotide combinations at the p456 substrate position using yeast surface display (Figure 3). The TCRα2.0 I-OnuI variant exhibited significantly better substrate discrimination, as marked by cleavage of less substrate than the original TCRαI-OnuI variant.

[0315] Example 2

[0316] Improving the catalytic activity of TCRα 2.0 megaTAL

[0317] Disruption of the TCRα locus prevents trafficking of CD3 to the cell surface. CD3 expression can be measured using fluorescently labeled anti-CD3 antibodies and flow cytometry. Gene editing efficiency is inversely proportional to CD3 fluorescence.

[0318] Primary human T cells were activated with CD3 and CD28 and electroporated with in vitro transcribed mRNA encoding either the original TCRα HE variant (e.g., SEQ ID NO: 13) or the TCRα2.0 HE variant (e.g., SEQ ID NO: 14) in the form of a megaTAL. Despite exhibiting significantly enhanced selectivity at the p456 substrate position, the TCRα2.0 megaTAL exhibited reduced catalytic activity, as indicated by increased CD3 expression compared to the original TCRα megaTAL ( Figure 4 ). Genomic DNA was subsequently isolated from megaTAL-treated cells and reduced catalytic activity was confirmed by using PCR in combination with Tracking Indels at Target Sites (TIDE, see Brinkman et al., 2014).

[0319] A single amino acid substitution E178D that increases catalytic activity in another I-OnuI variant was inserted into the active site of TCRα2.0 megaTAL to generate TCRα2.1 megaTAL (SEQ ID NO: 8). In vitro transcribed mRNA encoding TCRα2.1 megaTAL (e.g., SEQ ID NO: 15) was electroporated into primary human T cells. TCRα2.1 megaTAL increased CD3 knockdown by 10-fold compared to TCRα2.0 megaTAL. Figure 5 .

[0320] Example 3

[0321] TCRα2.1 I-OnuI variants and TCRα2.1 megaTALs exhibit enhanced on-target-off-target discrimination

[0322] Therapeutic genome editing carries a degree of risk due to the extremely low frequency of off-target editing events. Genome editing enzymes can be deeply characterized for off-target editing events using existing methods (see Tsai et al., 2016).

[0323] Potential off-target sites were identified using a bioinformatics algorithm that scanned the human genome for 22-bp sequences similar to the TCRα target sequence (SEQ ID NO: 17). The first 764 22-bp sequences returned from the bioinformatics search were assayed using yeast surface display to determine cleavage of the original TCRα HE variant and the TCRα2.1 HE variant. Figure 6 ), the TCRα2.1 HE variant exhibited increased selectivity for the TCRα target sequence, consistent with the results obtained in Example 1.

[0324] A second bioinformatics search was performed using sequence information of the top 50 cleaved substrates from the 764 substrate set to further inform the search. These were assayed to determine cleavage of the original TCRα and TCRα2.1 HE. The refined TCRα2.1 HE variants exhibited an enhanced selectivity profile compared to the original TCRα HE variants.

[0325] A second assay using an integrase-deficient lentiviral vector (IDLV, see Gabriel, 2011) was used to identify additional off-target sites generated in vivo. In short, IDLV was captured at double-strand break sites within enzyme-treated cells and blank-treated cells. The frequency and location of IDLV integration can then be plotted and compared between the two populations. An increase in the frequency of IDLV uptake at specific genomic sites in enzyme-treated samples compared to blank-treated samples indicates enzymatic cleavage. Using this method, the primary off-target site of the original TCRα mega TAL was identified in intron six of the KAT2B gene (SEQ ID NO:21, Osborne et al., 2015).

[0326] The identified putative off-target sites were evaluated by deep sequencing of amplicons generated from megaTAL-treated donor-derived primary T cells ( Figure 7 ). The TCRα2.1 megaTAL eliminated off-target editing at the KAT2B locus and exhibited enhanced selectivity for on-target sites, with reduced activity at potential off-target sites compared to the original TCRα megaTAL.

[0327] Example 4

[0328] Expansion of the TAL DNA-binding domain increases TCRα genome editing

[0329] MegaTALs comprise a TALE DNA binding domain fused to a meganuclease construct to increase binding affinity or addressability to a desired target sequence (see Boissel et al., 2013). To further increase on-target editing efficiency at the TCRα locus, an additional TALE RVD is appended to the C-terminus of the TALE DNA binding domain to increase the total length of the TALE DNA binding domain array from 10.5 to 11.5 RVD repeats and generate the 11.5RVD TCRα2.1 megaTAL (e.g., SEQ ID NO: 12). The 11.5RVD TALE DNA binding domain recognizes the original TALE binding site (SEQ ID NO: 18) with an additional nucleotide at the 3' end (SEQ ID NO: 19).

[0330] Human primary T cells were electroporated with in vitro transcribed mRNA encoding 10.5 RVD and 11.5 RVD TCRα2.1 megaTALs. Compared to TCRα editing using 10.5 RVD TCRα2.1 megaTALs, 11.5 RVD TCRα2.1 megaTALs increased TCRα editing by 30-50% as measured by CD3 knockdown using flow cytometry (Figure 8).

[0331] Example 5

[0332] Variant N-terminal TALE constructs

[0333] MegaTAL is a hybrid nuclease comprising a homing endonuclease fused to a TAL effector (TALE) construct comprising an N-terminal domain, a DNA binding domain, and a C-terminal domain. Some TALE constructs include a minimal N-terminal region (Δ-154') of approximately 136 amino acids, a variable number of repeats containing a 34 amino acid RVD (terminating with a truncated 20 amino acid repeat), and a minimal C-terminal region of approximately 63 amino acids.

[0334] The TALE constructs fused to the homing endonucleases described in the previous examples comprise sequences derived from the Xanthomonas oryzae TALE protein. In addition to the Xanthomonas oryzae TALE domain sequences, the minimal N-terminal TALE domain (NTD) region can be derived from other members of the Xanthomonas genus, such as Xanthomonas translucent and Xanthomonas citri. Multiple sequence alignments of the N-terminal TALE domains show that Xanthomonas oryzae has 85% and 87% sequence similarity with Xanthomonas translucent and Xanthomonas citri, respectively. To evaluate whether alternative species NTDs could be used to further enhance TAL activity, hybrid NTDs were generated by rationally substituting and combining the following amino acid positions in endogenous X. translucensis and X. citrinum sequences: V152G, D153K, D165E, E166K, I167L, P169L, A173P, T174I, Q180E, M183I, H191Q, V205I, V207A, I224V, V227I, A236V, G249S, G257S, V271I (e.g., SEQ ID NOs. NO:21); V152G, D153K, T170K, V171A, V183I, A200P, A236T, A239V, A244V, E245Q, G257S, L260V, V271I (e.g., SEQ ID NO:24); T170K, A200P, Q230H, W231C, S232G, A236T, A239V, A244V, E245Q, G257S (e.g., SEQ ID NO:23). The hybrid sequence (e.g., SEQ ID NO:22, 23, 24) showed 80% similarity to the N-terminal TAL domain of Xanthomonas oryzae. Each of these minimal TALE domains was individually grafted onto the N-terminus of the TALE DNA binding domain of the original TCRα I-OnuI variant megaTAL. In vitro transcribed mRNA encoding variant megaTALs including either TALE N-terminal domain (NTD) variants or the X. oryzae NTD was electroporated into human primary T cells to assess the editing rate of the TCRα locus. Flow cytometric analysis of CD3 expression showed that TCRα megaTALs including the NTD variant domain exhibited similar activity, and in some cases slightly higher activity ( Fig. 9 ).

[0335] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Therefore, the claims are not limited by this disclosure. Sequence Listing <110> bluebird bio, Inc. Jarjour, Jordan Pogson, Mark <120> TCRα homing endonuclease variants <130> BLBD-075 / 00US <140> PCT / US2017 / 056178 <141> 2017-10-11 <150> US 62 / 406,689 <151> 2016-10-11 <150> US 62 / 414,266 <151> 2016-10-28 <160> 69 <170> SIPOSequenceListing 1.0 <210> 1 <211> 303 <212> PRT <213> Ophiostoma novo-ulmi <400> 1 Met Ala Tyr Met Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr 1 5 10 15 Gly Phe Ala Asp Ala Glu Gly Ser Phe Leu Leu Arg Ile Arg Asn Asn 20 25 30 Asn Lys Ser Ser Val Gly Tyr Ser Thr Glu Leu Gly Phe Gln Ile Thr 35 40 45 Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp 50 55 60 Lys Val Gly Val Ile Ala Asn Ser Gly Asp Asn Ala Val Ser Leu Lys 65 70 75 80 Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys 85 90 95 Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Met Leu Phe Lys Gln 100 105 110 Ala Phe Cys Val Met Glu Asn Lys Glu His Leu Lys Ile Asn Gly Ile 115 120 125 Lys Glu Leu Val Arg Ile Lys Ala Lys Leu Asn Trp Gly Leu Thr Asp 130 135 140 Glu Leu Lys Lys Ala Phe Pro Glu Ile Ile Ser Lys Glu Arg Ser Leu 145 150 155 160 Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser 165 170 175 Gly Glu Gly Cys Phe Phe Val Asn Leu Ile Lys Ser Lys Ser Lys Leu 180 185 190 Gly Val Gln Val Gln Leu Val Phe Ser Ile Thr Gln His Ile Lys Asp 195 200 205 Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile 210 215 220 Lys Glu Lys Asn Lys Ser Glu Phe Ser Trp Leu Asp Phe Val Val Thr 225 230 235 240 Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn 245 250 255 Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val 260 265 270 Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp 275 280 285 Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe 290 295 300 <210> 2 <211> 303 <212> PRT <213> Ophiostoma novo-ulmi <400> 2 Met Ala Tyr Met Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr 1 5 10 15 Gly Phe Ala Asp Ala Glu Gly Ser Phe Leu Leu Arg Ile Arg Asn Asn 20 25 30 Asn Lys Ser Ser Val Gly Tyr Ser Thr Glu Leu Gly Phe Gln Ile Thr 35 40 45 Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp 50 55 60 Lys Val Gly Val Ile Ala Asn Ser Gly Asp Asn Ala Val Ser Leu Lys 65 70 75 80 Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys 85 90 95 Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln 100 105 110 Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile 115 120 125 Lys Glu Leu Val Arg Ile Lys Ala Lys Leu Asn Trp Gly Leu Thr Asp 130 135 140 Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Ser Leu 145 150 155 160 Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser 165 170 175 Gly Glu Gly Cys Phe Phe Val Asn Leu Ile Lys Ser Lys Ser Lys Leu 180 185 190 Gly Val Gln Val Gln Leu Val Phe Ser Ile Thr Gln His Ile Lys Asp 195 200 205 Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile 210 215 220 Lys Glu Lys Asn Lys Ser Glu Phe Ser Trp Leu Asp Phe Val Val Thr 225 230 235 240 Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn 245 250 255 Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val 260 265 270 Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp 275 280 285 Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe 290 295 300 <210> 3 <211> 303 <212> PRT <213> Ophiostoma novo-ulmi <220> <221> MOD_RES <222> (1)..(3) <223> Any amino acid or no <400> 3 Xaa Xaa Xaa Met Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr 1 5 10 15 Gly Phe Ala Asp Ala Glu Gly Ser Phe Leu Leu Arg Ile Arg Asn Asn 20 25 30 Asn Lys Ser Ser Val Gly Tyr Ser Thr Glu Leu Gly Phe Gln Ile Thr 35 40 45 Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp 50 55 60 Lys Val Gly Val Ile Ala Asn Ser Gly Asp Asn Ala Val Ser Leu Lys 65 70 75 80 Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys 85 90 95 Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln 100 105 110 Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile 115 120 125 Lys Glu Leu Val Arg Ile Lys Ala Lys Leu Asn Trp Gly Leu Thr Asp 130 135 140 Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Ser Leu 145 150 155 160 Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser 165 170 175 Gly Glu Gly Cys Phe Phe Val Asn Leu Ile Lys Ser Lys Ser Lys Leu 180 185 190 Gly Val Gln Val Gln Leu Val Phe Ser Ile Thr Gln His Ile Lys Asp 195 200 205 Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile 210 215 220 Lys Glu Lys Asn Lys Ser Glu Phe Ser Trp Leu Asp Phe Val Val Thr 225 230 235 240 Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn 245 250 255 Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val 260 265 270 Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp 275 280 285 Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe 290 295 300 <210> 4 <211> 303 <212> PRT <213> Ophiostoma novo-ulmi <220> <221> MOD_RES <222> (1)..(4) <223> Any amino acid or no <220> <221> MOD_RES <222> (302)..(303) <223> Any amino acid or no <400> 4 Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr 1 5 10 15 Gly Phe Ala Asp Ala Glu Gly Ser Phe Leu Leu Arg Ile Arg Asn Asn 20 25 30 Asn Lys Ser Ser Val Gly Tyr Ser Thr Glu Leu Gly Phe Gln Ile Thr 35 40 45 Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp 50 55 60 Lys Val Gly Val Ile Ala Asn Ser Gly Asp Asn Ala Val Ser Leu Lys 65 70 75 80 Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys 85 90 95 Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln 100 105 110 Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile 115 120 125 Lys Glu Leu Val Arg Ile Lys Ala Lys Leu Asn Trp Gly Leu Thr Asp 130 135 140 Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Ser Leu 145 150 155 160 Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser 165 170 175 Gly Glu Gly Cys Phe Phe Val Asn Leu Ile Lys Ser Lys Ser Lys Leu 180 185 190 Gly Val Gln Val Gln Leu Val Phe Ser Ile Thr Gln His Ile Lys Asp 195 200 205 Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile 210 215 220 Lys Glu Lys Asn Lys Ser Glu Phe Ser Trp Leu Asp Phe Val Val Thr 225 230 235 240 Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn 245 250 255 Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val 260 265 270 Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp 275 280 285 Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa 290 295 300 <210> 5 <211> 303 <212> PRT <213> Ophiostoma novo-ulmi <220> <221> MOD_RES <222> (1)..(8) <223> Any amino acid or no <220> <221> MOD_RES <222> (302)..(303) <223> Any amino acid or no <400> 5 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ile Asn Pro Trp Ile Leu Thr 1 5 10 15 Gly Phe Ala Asp Ala Glu Gly Ser Phe Leu Leu Arg Ile Arg Asn Asn 20 25 30 Asn Lys Ser Ser Val Gly Tyr Ser Thr Glu Leu Gly Phe Gln Ile Thr 35 40 45 Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp 50 55 60 Lys Val Gly Val Ile Ala Asn Ser Gly Asp Asn Ala Val Ser Leu Lys 65 70 75 80 Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys 85 90 95 Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln 100 105 110 Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile 115 120 125 Lys Glu Leu Val Arg Ile Lys Ala Lys Leu Asn Trp Gly Leu Thr Asp 130 135 140 Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Ser Leu 145 150 155 160 Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser 165 170 175 Gly Glu Gly Cys Phe Phe Val Asn Leu Ile Lys Ser Lys Ser Lys Leu 180 185 190 Gly Val Gln Val Gln Leu Val Phe Ser Ile Thr Gln His Ile Lys Asp 195 200 205 Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile 210 215 220 Lys Glu Lys Asn Lys Ser Glu Phe Ser Trp Leu Asp Phe Val Val Thr 225 230 235 240 Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn 245 250 255 Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val 260 265 270 Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp 275 280 285 Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa 290 295 300 <210> 6 <211> 303 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Synthetic I-OnuI Variant <400> 6 Met Ala Tyr Met Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr 1 5 10 15 Gly Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg 20 25 30 Asn Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr 35 40 45 Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp 50 55 60 Lys Val Gly Lys Ile Thr Asn Ser Gly Asp Arg Ala Val Met Leu Arg 65 70 75 80 Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys 85 90 95 Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln 100 105 110 Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile 115 120 125 Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Thr Asp 130 135 140 Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu 145 150 155 160 Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser 165 170 175 Gly Asp Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Asn Ala 180 185 190 Lys Val Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp 195 200 205 Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile 210 215 220 Trp Glu Lys Asn Lys Ser Glu Phe Ser Trp Leu Glu Phe Val Val Thr 225 230 235 240 Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn 245 250 255 Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val 260 265 270 Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp 275 280 285 Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe 290 295 300 <210> 7 <211> 303 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Synthetic I-OnuI LHE variants <220> <221> MOD_RES <222> (1)..(4) <223> Any amino acid or no <220> <221> MOD_RES <222> (302)..(303) <223> Any amino acid or no <400> 7 Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr 1 5 10 15 Gly Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg 20 25 30 Asn Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr 35 40 45 Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp 50 55 60 Lys Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg 65 70 75 80 Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys 85 90 95 Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln 100 105 110 Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile 115 120 125 Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp 130 135 140 Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu 145 150 155 160 Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala 165 170 175 Gly Glu Gly His Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala 180 185 190 Lys Val Tyr Val Gly Leu Arg Phe Ala Ile Ser Gln His Ile Arg Asp 195 200 205 Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile 210 215 220 Arg Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Glu Val Thr 225 230 235 240 Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn 245 250 255 Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val 260 265 270 Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp 275 280 285 Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa 290 295 300 <210> 8 <211> 303 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Synthetic I-OnuI LHE variants <220> <221> MOD_RES <222> (1)..(4) <223> Any amino acid or no <220> <221> MOD_RES <222> (302)..(303) <223> Any amino acid or none <400> 8 Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr 1 5 10 15 Gly Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg 20 25 30 Asn Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr 35 40 45 Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp 50 55 60 Lys Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg 65 70 75 80 Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys 85 90 95 Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln 100 105 110 Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile 115 120 125 Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp 130 135 140 Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu 145 150 155 160 Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala 165 170 175 Gly Asp Gly His Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala 180 185 190 Lys Val Tyr Val Gly Leu Arg Phe Ala Ile Ser Gln His Ile Arg Asp 195 200 205 Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile 210 215 220 Arg Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Glu Val Thr 225 230 235 240 Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn 245 250 255 Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val 260 265 270 Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp 275 280 285 Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa 290 295 300 <210> 9 <211> 877 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Synthetic I-OnuI Variant megaTAL Construct <400> 9 Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu Arg 1 5 10 15 Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val 20 25 30 Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe 35 40 45 Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly 50 55 60 Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu Ala 65 70 75 80 Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala Arg 85 90 95 Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro Pro 100 105 110 Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly Gly 115 120 125 Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly 130 135 140 Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn 145 150 155 160 Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 165 170 175 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 180 185 190 Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 195 200 205 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 210 215 220 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 225 230 235 240 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 245 250 255 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val 260 265 270 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 275 280 285 Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu 290 295 300 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 305 310 315 320 Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 325 330 335 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 340 345 350 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 355 360 365 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 370 375 380 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly 385 390 395 400 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 405 410 415 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn 420 425 430 Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 435 440 445 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 450 455 460 Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 465 470 475 480 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 485 490 495 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala 500 505 510 Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His 515 520 525 Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala Val 530 535 540 Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn Arg 545 550 555 560 Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val Gly 565 570 575 Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly Phe 580 585 590 Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn Asn 595 600 605 Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu His 610 615 620 Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys Val 625 630 635 640 Gly Lys Ile Thr Asn Ser Gly Asp Arg Ala Val Met Leu Arg Val Thr 645 650 655 Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr Pro 660 665 670 Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala Phe 675 680 685 Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys Glu 690 695 700 Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Thr Asp Glu Leu 705 710 715 720 Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile Asn 725 730 735 Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser Gly Asp 740 745 750 Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Asn Ala Lys Val 755 760 765 Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys Asn 770 775 780 Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp Glu 785 790 795 800 Lys Asn Lys Ser Glu Phe Ser Trp Leu Glu Phe Val Val Thr Lys Phe 805 810 815 Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr Leu 820 825 830 Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala Lys 835 840 845 Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu Ile 850 855 860 Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe 865 870 875 <210> 10 <211> 877 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Synthetic megaTAL construct <400> 10 Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu Arg 1 5 10 15 Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val 20 25 30 Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe 35 40 45 Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly 50 55 60 Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu Ala 65 70 75 80 Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala Arg 85 90 95 Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro Pro 100 105 110 Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly Gly 115 120 125 Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly 130 135 140 Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn 145 150 155 160 Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 165 170 175 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 180 185 190 Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 195 200 205 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 210 215 220 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 225 230 235 240 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 245 250 255 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val 260 265 270 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 275 280 285 Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu 290 295 300 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 305 310 315 320 Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 325 330 335 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 340 345 350 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 355 360 365 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 370 375 380 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly 385 390 395 400 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 405 410 415 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn 420 425 430 Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 435 440 445 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 450 455 460 Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 465 470 475 480 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 485 490 495 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala 500 505 510 Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His 515 520 525 Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala Val 530 535 540 Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn Arg 545 550 555 560 Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val Gly 565 570 575 Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly Phe 580 585 590 Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn Asn 595 600 605 Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu His 610 615 620 Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys Val 625 630 635 640 Gly Lys Ile Thr Asn Ser Gly Asp Arg Ala Val Met Leu Arg Val Thr 645 650 655 Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr Pro 660 665 670 Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala Phe 675 680 685 Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys Glu 690 695 700 Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Thr Asp Glu Leu 705 710 715 720 Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile Asn 725 730 735 Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser Gly Asp 740 745 750 Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Asn Ala Lys Val 755 760 765 Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys Asn 770 775 780 Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp Glu 785 790 795 800 Lys Asn Lys Ser Glu Phe Ser Trp Leu Glu Phe Val Val Thr Lys Phe 805 810 815 Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr Leu 820 825 830 Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala Lys 835 840 845 Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu Ile 850 855 860 Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe 865 870 875 <210> 11 <211> 875 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Synthetic megaTAL construct <400> 11 Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu Arg 1 5 10 15 Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val 20 25 30 Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe 35 40 45 Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly 50 55 60 Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu Ala 65 70 75 80 Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala Arg 85 90 95 Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro Pro 100 105 110 Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly Gly 115 120 125 Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly 130 135 140 Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn 145 150 155 160 Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 165 170 175 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 180 185 190 Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 195 200 205 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 210 215 220 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 225 230 235 240 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 245 250 255 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val 260 265 270 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 275 280 285 Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu 290 295 300 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 305 310 315 320 Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 325 330 335 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 340 345 350 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 355 360 365 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 370 375 380 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly 385 390 395 400 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 405 410 415 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn 420 425 430 Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 435 440 445 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 450 455 460 Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 465 470 475 480 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 485 490 495 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala 500 505 510 Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His 515 520 525 Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala Val 530 535 540 Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn Arg 545 550 555 560 Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val Gly 565 570 575 Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly Phe 580 585 590 Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn Asn 595 600 605 Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu His 610 615 620 Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys Val 625 630 635 640 Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg Val Thr 645 650 655 Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr Pro 660 665 670 Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala Phe 675 680 685 Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys Glu 690 695 700 Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu Leu 705 710 715 720 Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile Asn 725 730 735 Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala Gly Asp 740 745 750 Gly His Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala Lys Val 755 760 765 Tyr Val Gly Leu Arg Phe Ala Ile Ser Gln His Ile Arg Asp Lys Asn 770 775 780 Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Arg Glu 785 790 795 800 Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Glu Val Thr Lys Phe 805 810 815 Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr Leu 820 825 830 Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala Lys 835 840 845 Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu Ile 850 855 860 Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg 865 870 875 <210> 12 <211> 909 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Synthetic megaTAL construct <400> 12 Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu Arg 1 5 10 15 Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val 20 25 30 Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe 35 40 45 Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly 50 55 60 Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu Ala 65 70 75 80 Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala Arg 85 90 95 Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro Pro 100 105 110 Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly Gly 115 120 125 Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly 130 135 140 Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn 145 150 155 160 Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 165 170 175 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 180 185 190 Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 195 200 205 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 210 215 220 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 225 230 235 240 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 245 250 255 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val 260 265 270 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 275 280 285 Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu 290 295 300 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 305 310 315 320 Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 325 330 335 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 340 345 350 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 355 360 365 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 370 375 380 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly 385 390 395 400 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 405 410 415 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn 420 425 430 Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 435 440 445 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 450 455 460 Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 465 470 475 480 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 485 490 495 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 500 505 510 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 515 520 525 Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Ser Ile 530 535 540 Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn 545 550 555 560 Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp 565 570 575 Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val 580 585 590 Asn Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg 595 600 605 Val Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr 610 615 620 Gly Phe Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg 625 630 635 640 Asn Asn Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr 645 650 655 Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp 660 665 670 Lys Val Gly Lys Ile Thr Asn Ser Ser Asp Arg Ala Val Met Leu Arg 675 680 685 Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys 690 695 700 Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln 705 710 715 720 Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile 725 730 735 Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp 740 745 750 Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu 755 760 765 Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ala 770 775 780 Gly Asp Gly His Phe Gly Val Asn Leu Lys Lys Val Lys Gly Thr Ala 785 790 795 800 Lys Val Tyr Val Gly Leu Arg Phe Ala Ile Ser Gln His Ile Arg Asp 805 810 815 Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile 820 825 830 Arg Glu Lys Asn Lys Ser Glu Phe Arg Trp Leu Glu Phe Glu Val Thr 835 840 845 Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn 850 855 860 Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val 865 870 875 880 Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp 885 890 895 Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg 900 905 <210> 13 <211> 2634 <212> RNA <213> Artificial Sequence <220> <221> gene <222> ()..() <223> Synthetic I-OnuI Variant megaTAL Construct <400> 13 augggauccg cgccaccuaa gaagaaacgc aaagucgugg aucuacgcac gcucggcuac 60 agucagcagc agcaagagaa gaucaaaccg aaggugcguu cgacaguggc gcagcaccac 120 gaggcacugg ugggccaugg guuuacacac gcgcacaucg uugcgcucag ccaacacccg 180 gcagcguuag ggaccgucgc ugucacguau cagcacauaa ucacggcguu gccagaggcg acacacgaag acaucguugg cgucggcaaa cagugguccg gcgcacgcgc ccuggaggcc uugcucacgg augcggggga ccgccguuac covered aggccacuu gugaagauug caaaacgugg cggcgugacc gcaauggagg cagugcaugc aucgcgcaau 480. gcacugacgg gugccccccu gaaccugacc ccggaccaag ugguggcuau cgccagcaac auuggcggca agcaagcgcu cgaaacggug cagcggcugu ugccggugcu gugccaggac 540 cauggccuga ccccggacca agugguggcu aucgccagca acauuggcgg caagcaagcg 660. cucgaaacgg ugcagcggcu guugccggug cugugccagg accauggccu gaccccggac 720. 720. 720. 720. 720. 720. 720. 720. 720. 720. 720. 720 780. cuguugccgg ugcugugcca ggaccauggc cugaccccgg accaagugg ggcuaucgcc agcaacggug gcggcaagca agcgcucgaa acggugcagc ggcuguugcc ggugcugugc caggaccaug gccugacucc ggaccaagug guggcuaucg ccagccacga uggcggcaag caagcgcucg aaacggugca gcggcuguug ccggugcugu gccaggacca uggccugacu 960 ccggaccaag ugguggcuau cgccagccac gauggcggca agcaagcgcu cgaaacggug 1020 cagcggcugu ugccggugcu gugccaggac cauggcuga ccccggacca agugguggcu 1080 aucgccagca acauuggcgg caagcaagcg cucgaaacgg ugcagcggcu guugccggug 1140 cugugccagg accauggccu gaccccggac caguuggugg cuaucgccag caacauggc 1200 ggcaagcaag cgcucgaaac ggugcagcgg cuguugccgg ugcugugcca ggaccauggc 1260 cugaccccgg accaaguggu ggcuaucgcc agcaacggug gcggcaagca agcgcucgaa 1320 acggugcagc ggcuguugcc ggugcugugc caggaccaug gccugacccc ggaccaagug 1380 guggcuaucg ccagcaacaa uggcggcaag caagcgcucg aaacggugca gcggcuguug 1440 ccggugcugu gccaggacca uggccugacc ccggaccaag ugguggcuau cgccagcaac 1500 auuggcggca agcaagcgcu cgaaagcauu guggcccagc ugagccggcc ugaucccggcg 1560 1620 auggaugcag ugaaaaaggg auugccgcac gcgccggaau ugaucagaag agucaaucgc 1680 cguauuggcg aacgcacguc ccaucgcguu gcgauaucua gagugggagg aagcucgcgc 1740 agagagucca ucaacccaug gauucugacu gguuucgcug augccgaagg aucauucaua 1800 cuagacaucc gcaaccgaaa caacgaaagc aacagauacc gaacuucgcu gagauuccag 1860 aucaccguc acaacaagga caaaucgauu cuggagaaua uccagucgac uuggaagguc 1920 ggcaagauca caaacagcgg cgacagagcc gucaugcuga gggucacccg uuucgaagau 1980 uugaaaguga uuaucgacca cuucgagaaa uauccgcuga uuacccagaa auugggcgau 2040 uacaaguugu uuaaacaggc auucagcguc auggagaaca aagaacaucu uaaggagaau 2100 gggauuaagg agcucguacg aaucaaagcu aagaugaauu ggggucucac ugacgaauug 2160 aaaaaagcau uuccagagaa cauuagcaaa gagcgccccc uuaucaauaa gaacauuccg 2220 aauuucaaau ggcuggcugg auucacaucu ggugauggcu acuucggcgu gaaucuaaaa 2280 aagguaaagg gcaacgcaaa gguauaccug ggacugagau ucucaaucuc acaggacauc 2340 agagacaaga accugaugaa uucauugaua acauaccuag gcugugguuc caucugggag 2400 aagaacaagu cugaguucag uuggcucgag uucgucguaa ccaaauucag cgauaucaac 2460 gacaagauca uuccgguauu ccaggaaaau acucugauug gcgucaaacu cgaggacuuu 2520 gaagauuggu gcaagguugc caaauugauc gaagagaaga aacaccugac cgaauccggu 2580 uuggaugaga uuaagaaaau caagcugaac augaacaaag gucgugucuu cuga 2634 <210> 14 <211> 2634 <212> RNA <213> Artificial Sequence <220> <221> gene <222> ()..() <223> Synthetic megaTAL construct <400> 14 augggauccg cgccaccuaa gaagaaacgc aaagucgugg aucuacgcac gcucggcuac 60 agucagcagc agcaagagaa gaucaaaccg aaggugcguu cgacaguggc gcagcaccac 120 gaggcacugg ugggccaugg guuuacacac gcgcacaucg uugcgcucag ccaacacccg 180 gcagcguuag ggaccgucgc ugucacguau cagcacauaa ucacggcguu gccagaggcg 240 acacacgaag acaucguugg cgucggcaaa cagugguccg gcgcacgcgc ccuggaggcc uugcucacgg augcggggga ccgccguuac covered aggccacuu gugaagauug caaaacgugg cggcgugacc gcaauggagg cagugcaugc aucgcgcaau 480. gcacugacgg gugccccccu gaaccugacc ccggaccaag ugguggcuau cgccagcaac auuggcggca agcaagcgcu cgaaacggug cagcggcugu ugccggugcu gugccaggac 540 cauggccuga ccccggacca agugguggcu aucgccagca acauuggcgg caagcaagcg 660. cucgaaacgg ugcagcggcu guugccggug cugugccagg accauggccu gaccccggac 720. 720. 720. 720. 720. 720. 720. 720. 720. 720. 720. 720 780. cuguugccgg ugcugugcca ggaccauggc cugaccccgg accaagugg ggcuaucgcc agcaacggug gcggcaagca agcgcucgaa acggugcagc ggcuguugcc ggugcugugc caggaccaug gccugacucc ggaccaagug guggcuaucg ccagccacga uggcggcaag caagcgcucg aaacggugca gcggcuguug ccggugcugu gccaggacca uggccugacu ccggaccaag ugguggcuau cgccagccac gauggcggca agcaagcgcu cgaaacggug 1020 cagcggcugu ugccggugcu gugccaggac cauggcuga ccccggacca agugguggcu 1080 aucgccagca acauuggcgg caagcaagcg cucgaaacgg ugcagcggcu guugccggug 1140 cugugccagg accauggccu gaccccggac caguuggugg cuaucgccag caacauggc 1200 ggcaagcaag cgcucgaaac ggugcagcgg cuguugccgg ugcugugcca ggaccauggc 1260 cugaccccgg accaaguggu ggcuaucgcc agcaacggug gcggcaagca agcgcucgaa 1320 acggugcagc ggcuguugcc ggugcugugc caggaccaug gccugacccc ggaccaagug 1380 guggcuaucg ccagcaacaa uggcggcaag caagcgcucg aaacggugca gcggcuguug 1440 ccggugcugu gccaggacca uggccugacc ccggaccaag ugguggcuau cgccagcaac 1500 auuggcggca agcaagcgcu cgaaagcauu guggcccagc ugagccggcc ugaucccggcg 1560 1620 auggaugcag ugaaaaaggg auugccgcac gcgccggaau ugaucagaag agucaucgc 1680 cguauuggcg aacgcacguc ccaucgcguu gcgauaucua gagugggagg aagcucgcgc 1740 agagagucca ucaacccaug gauucugacu gguuucgcug augccgaagg aucauucaua 1800 cuagacaucc gcaaccgaaa caacgaaagc aacagauacc gaacuucgcu gagauuccag 1860 aucaccguc acaacaagga caaaucgauu cuggagaaua uccaguccac uuggaagguc 1920 ggcaagauca caaacagcag ugacagagcc gucaugcuga gggucacccg uuucgaagau 1980 uugaaaguga uuaucgacca cuucgagaaa uauccgcuga uuacccagaa auugggcgau 2040 uacaaguugu uuaaacaggc auucagcguc auggagaaua aagaacaucu uaaggagaau 2100 gggauuaagg agcucguacg aaucaaagcu aagaugaauu ggggucucaa ugacgaauug 2160 aaaaaagcau uuccagagaa cauuagcaaa gagcgccccc uuaucaauaa gaacauuccg 2220 aauuucaaau ggcuggcugg auuccagcu ggugaaggcc auuucggcgu gaaucuaaaa 2280 aagguaaagg gcaccgcaaa gguauacgug ggacugagau ucgcuaucuc acaggacauc 2340 agagacaaga accugaugaa uucauugaua acauaccuag gcugugguuc caucagagag 2400 aagaacaagu cugaguucag auggcucgag uucgaaguaa ccaaauucag cgauaucaac 2460 gacaagauca uuccgguauu ccaggaaaau acucugauug gcgucaaacu cgaggacuuu 2520 gaagauuggu gcaagguugc caaauugauc gaagagaaga aacaccugac cgaauccggu 2580 uuggaugaga uuaagaaaau caagcugaac augaacaaag gucgugucuu cuga 2634 <210> 15 <211> 2628 <212> RNA <213> Artificial Sequence <220> <221> gene <222> ()..() <223> Synthetic megaTAL construct <400> 15 augggauccg cgccaccuaa gaagaaacgc aaagucgugg aucuacgcac gcucggcuac 60 agucagcagc agcaagagaa gaucaaaccg aaggugcguu cgacaguggc gcagcaccac 120 gaggcacugg ugggccaugg guuuacacac gcgcacaucg uugcgcucag ccaacacccg 180 gcagcguuag ggaccgucgc ugucacguau cagcacauaa ucacggcguu gccagaggcg 240 acacacgaag acaucguugg cgucggcaaa cagugguccg gcgcacgcgc ccuggaggcc 300 uugcucacgg augcggggga ccgccguuac covered aggccacuu gugaagauug caaaacgugg cggcgugacc gcaauggagg cagugcaugc aucgcgcaau 480. gcacugacgg gugccccccu gaaccugacc ccggaccaag ugguggcuau cgccagcaac auuggcggca agcaagcgcu cgaaacggug cagcggcugu ugccggugcu gugccaggac 540 cauggccuga ccccggacca agugguggcu aucgccagca acauuggcgg caagcaagcg 660. cucgaaacgg ugcagcggcu guugccggug cugugccagg accauggccu gaccccggac 720. 720. 720. 720. 720. 720. 720. 720. 720. 720. 720. 720 780. cuguugccgg ugcugugcca ggaccauggc cugaccccgg accaagugg ggcuaucgcc agcaacggug gcggcaagca agcgcucgaa acggugcagc ggcuguugcc ggugcugugc caggaccaug gccugacucc ggaccaagug guggcuaucg ccagccacga uggcggcaag caagcgcucg aaacggugca gcggcuguug ccggugcugu gccaggacca uggccugacu ccggaccaag ugguggcuau cgccagccac gauggcggca agcaagcgcu cgaaacggug cagcggcugu ugccggugcu gugccaggac cauggcuga ccccggacca agugguggcu 1080 aucgccagca acauuggcgg caagcaagcg cucgaaacgg ugcagcggcu guugccggug 1140 cugugccagg accauggccu gaccccggac caguuggugg cuaucgccag caacauggc 1200 ggcaagcaag cgcucgaaac ggugcagcgg cuguugccgg ugcugugcca ggaccauggc 1260 cugaccccgg accaaguggu ggcuaucgcc agcaacggug gcggcaagca agcgcucgaa 1320 acggugcagc ggcuguugcc ggugcugugc caggaccaug gccugacccc ggaccaagug 1380 guggcuaucg ccagcaacaa uggcggcaag caagcgcucg aaacggugca gcggcuguug 1440 ccggugcugu gccaggacca uggccugacc ccggaccaag ugguggcuau cgccagcaac 1500 auuggcggca agcaagcgcu cgaaagcauu guggcccagc ugagccggcc ugaucccggcg 1560 1620 auggaugcag ugaaaaaggg auugccgcac gcgccggaau ugaucagaag agucaucgc 1680 cguauuggcg aacgcacguc ccaucgcguu gcgauaucua gagugggagg aagcucgcgc 1740 agagagucca ucaacccaug gauucugacu gguuucgcug augccgaagg aucauucaua 1800 cuagacaucc gcaaccgaaa caacgaaagc aacagauacc gaacuucgcu gagauuccag 1860 aucaccguc acaacaagga caaaucgauu cuggagaaua uccaguccac uuggaagguc 1920 ggcaagauca caaacagcag ugacagagcc gucaugcuga gggucacccg uuucgaagau 1980 uugaaaguga uuaucgacca cuucgagaaa uauccgcuga uuacccagaa auugggcgau 2040 uacaaguugu uuaaacaggc auucagcguc auggagaaua aagaacaucu uaaggagaau 2100 gggauuaagg agcucguacg aaucaaagcu aagaugaauu ggggucucaa ugacgaauug 2160 aaaaaagcau uuccagagaa cauuagcaaa gagcgccccc uuaucaauaa gaacauuccg 2220 aauuucaaau ggcuggcugg auuccagcu ggugauggcc auuucggcgu gaaucuaaaa 2280 aagguaaagg gcaccgcaaa gguauacgug ggacugagau ucgcuaucuc acaggacauc 2340 agagacaaga accugaugaa uucauugaua acauaccuag gcugugguuc caucagagag 2400 aagaacaagu cugaguucag auggcucgag uucgaaguaa ccaaauucag cgauaucaac 2460 gacaagauca uuccgguauu ccaggaaaau acucugauug gcgucaaacu cgaggacuuu 2520 gaagauuggu gcaagguugc caaauugauc gaagagaaga aacaccugac cgaauccggu 2580 uuggaugaga uuaagaaaau caagcugaac augaacaaag gucguuga 2628 <210> 16 <211> 2733 <212> RNA <213> Artificial Sequence <220> <221> gene <222> ()..() <223> Synthetic megaTAL construct <400> 16 augggauccg ccccuccgaa gaagaagcgg aaagucgugg accuccggac ccuggguuac 60 ucucagcagc agcaggagaa gaucaagccg aaggugcggu cgacuguggc ccagcaucac 120 gaggcccugg ugggacacgg cuucacccac gcccacauug uggcccugag ccagcacccg 180 gcagcgcugg gaaccguggc cgugaccuac cagcacauca uuacugcccu gccugaagcg 240 acccacgagg auaucguggg cgucggaaag cagugguccg gagccagagc cuuggaggcu 300 cugcugacug acgccggaga gcugcggggc ccgccccugc aacuggauac cggccagcuc 360 gugaaaaucg ccaagagagg aggagugacc gccauggaag ccgugcaugc aucccgcaau 420 gcacugacug gugcacccccu gaaccucacu ccugaccagg ucgucgcuau cgcaagcaac 480 aucggaggga aacaagcucu cgagacagug cagcgccucc ugccagugcu uugccaggac 540 cacggccuga cuccagacca gguggucgcu auugcgucga acauuggagg gaagcaagcc 600 cuugaaaccg ugcagaggcu gcucccggug cugugccaag accauggacu caccccggac 660 caaguggugg cuauugcuag caacauuggc gguaagcagg cgcuggagac aguccagcgg 720 cugcugccgg uguugugcca agauccggu cuuaccccag accaagucgu ggcgauugcc 780 uccaacggug gcggcaagca agcacucgaa acuguccaga gacugcuccc ugugcucugu 840 caagaccacg gguugacccc cgaccaagug guggccaucg ccucccauga uggagaaaag 900 caggcccucg agacugucca gcgacugcuc cccguguugu gucaggauca uggauugacg 960 cccgaucagg ucguggccau ugccucccac gacgguggaa agcaagcgcu ggaaacugug 1020 cagcgguugc ugccgguccu gugccaggac cacggacuga cuccggacca gguggucgcc 1080 aucgcaucca acauuggugg caagcaggcu cucgaaaccg uccaacgccu guugccggug 1140 1200 ggcaaacagg cccuggaac cgugcaacgc cugcugcccg uccucugcca ggaccacggu 1260 cugaccccug accaggucgu cgcgaucgcg ucaaaugggg ggggcaaaca ggcucuggaa 1320 acggugcagc ggcuccuucc gguguuaugc caggaccacg ggcugacucc cgaccaggug 1380 guggcgaucg ccucgaacaa cggaggcaaa caagcccugg agacugugca gagacucug 1440 cccgugcugu gccaagacca ugggcucacc ccugaucagg ugguggcaau cgccucaaac 1500 aucggcggga agcaggcacu ggaaacugug cagagacucc ugcccgugcu gugccaagac 1560 caugggcuga ccccggacca agugguggcu aucgccuccc acgacgggg caaacaggcc 1620 cuggaucca ucgucgcuca gcugagccgg ccugacccag cacucgccgc ccugaccaau 1680 gaccaucugg ucgcccuggc cugccuggga ggcagacccg cgauggacgc ggucaagaag 1740 ggucugccgc acgccccuga gcuuauucgg agagugaaca ggcgcaucgg ugaacgcacc 1800 ucccaucggg ucgcaaucuc uagagugggc ggaucguccc ggcgggaguc caucaauccu 1860 uggauccuga ccggcuucgc cgacgccgaa ggcuccuuca uccuggacau caggaacagg 1920 aacaacgagu caaacaggua ccgcaccucc cuucgguucc agauuacucu gcacaacaag gauaagucca uccucgagaa cauccaguca accuggaaag ugggcaagau cacuaacucc 2040 ucggaccgcg cagugaugcu ccgggugacc cgcuucgagg accugaaggu gaucauugac 2100 cacuucgaga aguacccucu cauaacccag aagcugggag auuacaagcu guuuaagcag 2160 gcguucuccg ugauggagaa caaagaacac cuuaaggaga augggauuaa ggaacugguc 2220 cgcauuaagg ccaagaugaa cuggggacug aacgacgagu ugaaaaggc auuuccugaa 2280 aacaucucca aggaacggcc gcucaucaac aagaacauc ccaauuucaa guggcuggcg 2340 ggguucacug ccggggacgg acacuucgga gugaaccuga agaaggugaa gggcaccgcc 2400 aagguguacg ugggccugcg guucgcgauc agccagcaca uccgggauaa gaaccugaug 2460 aacagccuca ucaccuaccu gggaugcgga agcauccggg agaagaacaa gucagaauuc 2520 cgauggcugg auuugaagu cauccacgug 2580 ooccaggaga accccuaoooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooderrrrrrrrrrhoooooooooooooooooooooooooooooooooooerder 2640 gccaagcuca ucgaagagaa gaagcaccug accgaagcg gccuggauga gauaagaag 2700 auuaagcuca acaugaaaa gggaagauag uag 2733 <210> 17 <211> 22 <212> DNA <213> Homo sapiens <400> 17 tgtctgccta is cleaning it 22 <210> 18 <211> 11 <212> DNA <213> Homo sapiens <400> 18 aaatccagtg and 11 <210> 19 <211> 12 <212> DNA <213> Homo sapiens <400> 19 aaatccagtg and 12 <210> 20 <211> 39 <212> DNA <213> Homo sapiens <400> 20 aaatccagtg acagtctgt ctgcctattc accgatttt 39 <210> 21 <211> 22 <212> DNA <213> Homo sapiens <400> 21 agtatgccta ttctacaaga aa 22 <210> 22 <211> 877 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Synthetic NTD variant megaTAL construct <400> 22 Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Gly Lys Leu Cys 1 5 10 15 Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Leu Lys Leu Lys Ala 20 25 30 Arg Pro Ile Val Ala Gln His His Glu Ala Leu Ile Gly His Gly Phe 35 40 45 Thr Arg Ala Gln Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly 50 55 60 Thr Ile Ala Ala Lys Tyr Gln Ala Met Ile Ala Ala Leu Pro Glu Ala 65 70 75 80 Thr His Glu Asp Val Val Gly Ile Gly Lys Gln Trp Ser Gly Ala Arg 85 90 95 Thr Leu Glu Val Leu Leu Thr Val Ser Gly Glu Leu Arg Ser Pro Pro 100 105 110 Leu Gln Leu Asp Thr Ser Gln Leu Leu Lys Ile Ala Lys Arg Gly Gly 115 120 125 Val Thr Ala Ile Glu Ala Val His Ala Trp Arg Asn Ala Leu Thr Gly 130 135 140 Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn 145 150 155 160 Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 165 170 175 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 180 185 190 Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 195 200 205 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 210 215 220 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 225 230 235 240 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 245 250 255 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val 260 265 270 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 275 280 285 Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu 290 295 300 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 305 310 315 320 Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 325 330 335 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 340 345 350 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 355 360 365 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 370 375 380 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly 385 390 395 400 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 405 410 415 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn 420 425 430 Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 435 440 445 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 450 455 460 Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 465 470 475 480 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 485 490 495 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala 500 505 510 Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His 515 520 525 Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala Val 530 535 540 Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn Arg 545 550 555 560 Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val Gly 565 570 575 Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly Phe 580 585 590 Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn Asn 595 600 605 Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu His 610 615 620 Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys Val 625 630 635 640 Gly Lys Ile Thr Asn Ser Gly Asp Arg Ala Val Met Leu Arg Val Thr 645 650 655 Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr Pro 660 665 670 Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala Phe 675 680 685 Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys Glu 690 695 700 Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Thr Asp Glu Leu 705 710 715 720 Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile Asn 725 730 735 Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser Gly Asp 740 745 750 Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Asn Ala Lys Val 755 760 765 Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys Asn 770 775 780 Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp Glu 785 790 795 800 Lys Asn Lys Ser Glu Phe Ser Trp Leu Glu Phe Val Val Thr Lys Phe 805 810 815 Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr Leu 820 825 830 Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala Lys 835 840 845 Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu Ile 850 855 860 Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe 865 870 875 <210> 23 <211> 877 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Synthetic NTD variant megaTAL construct <400> 23 Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu Ser 1 5 10 15 Thr Phe Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val 20 25 30 Arg Ser Thr Val Ala Gln His His Ala Ala Leu Val Gly His Gly Phe 35 40 45 Thr His Ala His Ile Val Glu Leu Ser Lys His Pro Pro Ala Leu Gly 50 55 60 Thr Ile Ala Ala Arg Tyr Ser Glu Met Ile Ala Ala Leu Pro Glu Ala 65 70 75 80 Thr His Glu Asp Ile Val Gly Val Gly Lys His Cys Ala Gly Ala Arg 85 90 95 Thr Leu Glu Val Leu Leu Met Val Val Gln Glu Leu Arg Ala Pro Pro 100 105 110 Leu Gln Leu Val Thr Ser Gln Leu Leu Lys Ile Ala Lys Arg Gly Gly 115 120 125 Val Thr Ala Val Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly 130 135 140 Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn 145 150 155 160 Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 165 170 175 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 180 185 190 Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 195 200 205 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 210 215 220 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 225 230 235 240 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 245 250 255 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val 260 265 270 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 275 280 285 Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu 290 295 300 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 305 310 315 320 Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 325 330 335 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 340 345 350 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 355 360 365 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 370 375 380 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly 385 390 395 400 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 405 410 415 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn 420 425 430 Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 435 440 445 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 450 455 460 Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 465 470 475 480 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 485 490 495 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala 500 505 510 Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His 515 520 525 Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala Val 530 535 540 Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn Arg 545 550 555 560 Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val Gly 565 570 575 Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly Phe 580 585 590 Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn Asn 595 600 605 Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu His 610 615 620 Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys Val 625 630 635 640 Gly Lys Ile Thr Asn Ser Gly Asp Arg Ala Val Met Leu Arg Val Thr 645 650 655 Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr Pro 660 665 670 Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala Phe 675 680 685 Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys Glu 690 695 700 Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Thr Asp Glu Leu 705 710 715 720 Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile Asn 725 730 735 Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser Gly Asp 740 745 750 Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Asn Ala Lys Val 755 760 765 Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys Asn 770 775 780 Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp Glu 785 790 795 800 Lys Asn Lys Ser Glu Phe Ser Trp Leu Glu Phe Val Val Thr Lys Phe 805 810 815 Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr Leu 820 825 830 Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala Lys 835 840 845 Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu Ile 850 855 860 Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe 865 870 875 <210> 24 <211> 877 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Synthetic NTD Variant megaTAL Construct <400> 24 Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Gly Lys Leu Ser 1 5 10 15 Thr Phe Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Ala 20 25 30 Arg Ser Thr Val Ala Gln His His Ala Ala Leu Ile Gly His Gly Phe 35 40 45 Thr His Ala His Ile Val Glu Leu Ser Lys His Pro Pro Ala Leu Gly 50 55 60 Thr Ile Ala Ala Arg Tyr Ser Glu Met Ile Ala Ala Leu Pro Glu Ala 65 70 75 80 Thr His Glu Asp Ile Val Gly Val Gly Lys Gln Trp Ser Gly Ala Arg 85 90 95 Thr Leu Glu Val Leu Leu Met Val Val Gln Glu Leu Arg Ala Pro Pro 100 105 110 Leu Gln Leu Val Thr Ser Gln Leu Val Lys Ile Ala Lys Arg Gly Gly 115 120 125 Val Thr Ala Ile Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly 130 135 140 Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn 145 150 155 160 Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 165 170 175 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 180 185 190 Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 195 200 205 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 210 215 220 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 225 230 235 240 Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val 245 250 255 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val 260 265 270 Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp 275 280 285 Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu 290 295 300 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr 305 310 315 320 Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 325 330 335 Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly 340 345 350 Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 355 360 365 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 370 375 380 His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly 385 390 395 400 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 405 410 415 Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn 420 425 430 Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val 435 440 445 Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala 450 455 460 Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 465 470 475 480 Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala 485 490 495 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala 500 505 510 Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His 515 520 525 Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala Val 530 535 540 Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn Arg 545 550 555 560 Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val Gly 565 570 575 Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly Phe 580 585 590 Ala Asp Ala Glu Gly Ser Phe Ile Leu Asp Ile Arg Asn Arg Asn Asn 595 600 605 Glu Ser Asn Arg Tyr Arg Thr Ser Leu Arg Phe Gln Ile Thr Leu His 610 615 620 Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys Val 625 630 635 640 Gly Lys Ile Thr Asn Ser Gly Asp Arg Ala Val Met Leu Arg Val Thr 645 650 655 Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr Pro 660 665 670 Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala Phe 675 680 685 Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys Glu 690 695 700 Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Thr Asp Glu Leu 705 710 715 720 Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile Asn 725 730 735 Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser Gly Asp 740 745 750 Gly Tyr Phe Gly Val Asn Leu Lys Lys Val Lys Gly Asn Ala Lys Val 755 760 765 Tyr Val Gly Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp Lys Asn 770 775 780 Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Ser Ile Trp Glu 785 790 795 800 Lys Asn Lys Ser Glu Phe Ser Trp Leu Glu Phe Val Val Thr Lys Phe 805 810 815 Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr Leu 820 825 830 Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala Lys 835 840 845 Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu Ile 850 855 860 Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe 865 870 875 <210> 25 <211> 2634 <212> RNA <213> Artificial Sequence <220> <221> gene <222> ()..() <223> Synthetic NTD Variant megaTAL Construct <400> 25 augggauccg cgccaccuaa gaagaaacgc aaagucggca agcugugcac ccugggcuac 60 agccagcagc agcaggagaa gcugaagcug aaggccagac ccaucguggc ccagcaccac 120 gaggcccuga ucggccacgg cuucaccaga gcccagaucg uggcccugag ccagcacccc 180 gccgccccugg gcaccaucgc cgccaaguac caggccauga ucgccgcccc gcccgaggcc 240 acccacgagg acgugguggg caucggcaag caguggagcg gcgccagaac ccuggaggug 300 360 degree rotation cugaagaucg ccaagagagg cggcgugacc gccaucgagg ccgugcacgc cuggcgcaau 420 gcacugacgg gugcccccccu gaaccugacc ccggaccaag ugguggcuau cgccagcaac 480 auuggcggca agcaagcgcu cgaaacggug cagcggugu ugccggugcu gugccaggac 540 cauggcuga ccccggacca agugguggcu aucgccagca acauuggcgg caagcaagcg 600 cucgaaacgg ugcagcggcu guugccggug cugugccagg accauggccu gaccccggac 660 caaguggugg cuaucgccag caacauuggc ggcaagcaag cgcucgaaac ggugcagcgg 720 cuguugccgg ugcugugcca ggaccauggc cugaccccgg accaagugggu ggcuaucgcc 780 agcaacggug gcggcaagca agcgcucgaa acggugcagc ggcuguugcc ggugcugugc 840 caggaccaug gccugacucc ggaccaagug guggcuaucg ccagccacga uggcggcaag 900 caagcgcucg aaacggugca gcggcuguug ccggugcugu gccaggacca uggccugacu 960 ccggaccaag ugguggcuau cgccagccac gauggcggca agcaagcgcu cgaaacggug 1020 cagcggcugu ugccggugcu gugccaggac cauggcuga ccccggacca agugguggcu 1080 aucgccagca acauuggcgg caagcaagcg cucgaaacgg ugcagcggcu guugccggug 1140 cugugccagg accauggccu gaccccggac caguuggugg cuaucgccag caacauggc 1200 ggcaagcaag cgcucgaaac ggugcagcgg cuguugccgg ugcugugcca ggaccauggc 1260 cugaccccgg accaaguggu ggcuaucgcc agcaacggug gcggcaagca agcgcucgaa 1320 acggugcagc ggcuguugcc ggugcugugc caggaccaug gccugacccc ggaccaagug 1380 guggcuaucg ccagcaacaa uggcggcaag caagcgcucg aaacggugca gcggcuguug 1440 ccggugcugu gccaggacca uggccugacc ccggaccaag ugguggcuau cgccagcaac 1500 auuggcggca agcaagcgcu cgaaagcauu guggcccagc ugagccggcc ugaucccggcg 1560 uuggccgcgu ugaccaacga ccaccucguc gccuuggccu gccucggcgg acguccugcc 1620 auggaugcag ugaaaaaggg auugccgcac gcgccggaau ugaucagaag agucaaucgc 1680 cguauuggcg aacgcacguc ccaucgcguu gcgauaucua gagugggagg aagcucgcgc 1740 agagagucca ucaacccaug gauucugacu gguuucgcug augccgaagg aucauucaua 1800 cuagacaucc gcaaccgaaa caacgaaagc aacagauacc gaacuucgcu gagauuccag 1860 aucaccguc acaacaagga caaaucgauu cuggagaaua uccagucgac uuggaagguc 1920 ggcaagauca caaacagcgg cgacagagcc gucaugcuga gggucacccg uuucgaagau 1980 uugaaaguga uuaucgacca cuucgagaaa uauccgcuga uuacccagaa auugggcgau 2040 uacaaguugu uuaaacaggc auucagcguc auggagaaca aagaacaucu uaaggagaau 2100 gggauuaagg agcucguacg aaucaaagcu aagaugaauu ggggucucac ugacgaauug 2160 aaaaaagcau uuccagagaa cauuagcaaa gagcgccccc uuaucaauaa gaacauuccg 2220 aauuucaaau ggcuggcugg auucacaucu ggugauggcu acuucggcgu gaaucuaaaa 2280 aagguaaagg gcaacgcaaa gguauacgug ggacugagau ucucaaucuc acagcacauc 2340 agagacaaga accugaugaa uucauugaua acauaccuag gcugugguuc caucugggag 2400 aagaacaagu cugaguucag uuggcucgag uucgucguaa ccaaauucag cgauaucaac 2460 gacaagauca uuccgguauu ccaggaaaau acucugauug gcgucaaacu cgaggacuuu 2520 gaagauuggu gcaagguugc caaauugauc gaagagaaga aacaccugac cgaauccggu 2580 uuggaugaga uuaagaaaau caagcugaac augaacaaag gucgugucuu cuga 2634 <210> 26 <211> 2634 <212> RNA <213> Artificial Sequence <220> <221> gene <222> ()..() <223> Synthetic NTD Variant megaTAL Construct <400> 26 augggauccg cgccaccuaa gaagaaacgc aaagucgugg accugagcac cuucggcuac 60 agccagcagc agcaggagaa gaucaagccc aaggugagaa gcaccguggc ccagcaccac 120 gccgcccugg ugggccacgg cuucacccac gcccacaucg uggagcugag caagcacccc 180 cccgcccugg gcaccaucgc cgccagauac agcgagauga ucgccgccccu gcccgaggcc 240 acccacgagg acaucguggg cgugggcaag cacugcgccg gcgccagaac ccuggaggug 300 360 cugaagaucg ccaagagagg cggcgugacc gccguggagg ccgugcacgc cagccgcaau 420 gcacugacgg gugcccccccu gaaccugacc ccggaccaag ugguggcuau cgccagcaac 480 auuggcggca agcaagcgcu cgaaacggug cagcggugu ugccggugcu gugccaggac 540 cauggcuga ccccggacca agugguggcu aucgccagca acauuggcgg caagcaagcg 600 cucgaaacgg ugcagcggcu guugccggug cugugccagg accauggccu gaccccggac 660 caaguggugg cuaucgccag caacauuggc ggcaagcaag cgcucgaaac ggugcagcgg 720 cuguugccgg ugcugugcca ggaccauggc cugaccccgg accaagugggu ggcuaucgcc 780 agcaacggug gcggcaagca agcgcucgaa acggugcagc ggcuguugcc ggugcugugc 840 caggaccaug gccugacucc ggaccaagug guggcuaucg ccagccacga uggcggcaag 900 caagcgcucg aaacggugca gcggcuguug ccggugcugu gccaggacca uggccugacu 960 ccggaccaag ugguggcuau cgccagccac gauggcggca agcaagcgcu cgaaacggug 1020 cagcggcugu ugccggugcu gugccaggac cauggcuga ccccggacca agugguggcu 1080 aucgccagca acauuggcgg caagcaagcg cucgaaacgg ugcagcggcu guugccggug 1140 cugugccagg accauggccu gaccccggac caguuggugg cuaucgccag caacauggc 1200 ggcaagcaag cgcucgaaac ggugcagcgg cuguugccgg ugcugugcca ggaccauggc 1260 cugaccccgg accaaguggu ggcuaucgcc agcaacggug gcggcaagca agcgcucgaa 1320 acggugcagc ggcuguugcc ggugcugugc caggaccaug gccugacccc ggaccaagug 1380 guggcuaucg ccagcaacaa uggcggcaag caagcgcucg aaacggugca gcggcuguug 1440 ccggugcugu gccaggacca uggccugacc ccggaccaag ugguggcuau cgccagcaac 1500 auuggcggca agcaagcgcu cgaaagcauu guggcccagc ugagccggcc ugaucccggcg 1560 1620 auggaugcag ugaaaaaggg auugccgcac gcgccggaau ugaucagaag agucaaucgc 1680 cguauuggcg aacgcacguc ccaucgcguu gcgauaucua gagugggagg aagcucgcgc 1740 agagagucca ucaacccaug gauucugacu gguuucgcug augccgaagg aucauucaua 1800 cuagacaucc gcaaccgaaa caacgaaagc aacagauacc gaacuucgcu gagauuccag 1860 aucaccguc acaacaagga caaaucgauu cuggagaaua uccagucgac uuggaagguc 1920 ggcaagauca caaacagcgg cgacagagcc gucaugcuga gggucacccg uuucgaagau 1980 uugaaaguga uuaucgacca cuucgagaaa uauccgcuga uuacccagaa auugggcgau 2040 uacaaguugu uuaaacaggc auucagcguc auggagaaca aagaacaucu uaaggagaau 2100 gggauuaagg agcucguacg aaucaaagcu aagaugaauu ggggucucac ugacgaauug 2160 aaaaaagcau uuccagagaa cauuagcaaa gagcgccccc uuaucaauaa gaacauuccg 2220 aauuucaaau ggcuggcugg auucacaucu ggugauggcu acuucggcgu gaaucuaaaa 2280 aagguaaagg gcaacgcaaa gguauaccug ggacugagau ucucaaucuc acaggacauc 2340 agagacaaga accugaugaa uucauugaua acauaccuag gcugugguuc caucugggag 2400 aagaacaagu cugaguucag uuggcucgag uucgucguaa ccaaauucag cgauaucaac 2460 gacaagauca uuccgguauu ccaggaaaau acucugauug gcgucaaacu cgaggacuuu 2520 gaagauuggu gcaagguugc caaauugauc gaagagaaga aacaccugac cgaauccggu 2580 uuggaugaga uuaagaaaau caagcugaac augaacaaag gucgugucuu cuga 2634 <210> 27 <211> 2634 <212> RNA <213> Artificial Sequence <220> <221> gene <222> ()..() <223> Synthetic NTD Variant megaTAL Construct <400> 27 augggauccg cgccaccuaa gaagaaacgc aaagucggca agcugagcac cuucggcuac 60 agccagcagc agcaggagaa gaucaagccc aaggccagaa gcaccguggc ccagcaccac 120 gccgcccuga ucggccacgg cuucacccac gcccacaucg uggagcugag caagcacccc 180 cccgcccugg gcaccaucgc cgccagauac agcgagauga ucgccgcccu gcccgaggcc 240 acccacgagg acaucguggg cgugggcaag caguggagcg gcgccagaac ccuggaggug 300 360 gugaagaucg ccaagagagg cggcgugacc gccaucgagg ccgugcacgc cagccgcaau 420 gcacugacgg gugcccccccu gaaccugacc ccggaccaag ugguggcuau cgccagcaac 480 auuggcggca agcaagcgcu cgaaacggug cagcggugu ugccggugcu gugccaggac 540 cauggcuga ccccggacca agugguggcu aucgccagca acauuggcgg caagcaagcg 600 cucgaaacgg ugcagcggcu guugccggug cugugccagg accauggccu gaccccggac 660 caaguggugg cuaucgccag caacauuggc ggcaagcaag cgcucgaaac ggugcagcgg 720 cuguugccgg ugcugugcca ggaccauggc cugaccccgg accaagugggu ggcuaucgcc 780 agcaacggug gcggcaagca agcgcucgaa acggugcagc ggcuguugcc ggugcugugc 840 caggaccaug gccugacucc ggaccaagug guggcuaucg ccagccacga uggcggcaag 900 caagcgcucg aaacggugca gcggcuguug ccggugcugu gccaggacca uggccugacu 960 ccggaccaag ugguggcuau cgccagccac gauggcggca agcaagcgcu cgaaacggug 1020 cagcggcugu ugccggugcu gugccaggac cauggcuga ccccggacca agugguggcu 1080 aucgccagca acauuggcgg caagcaagcg cucgaaacgg ugcagcggcu guugccggug 1140 cugugccagg accauggccu gaccccggac caguuggugg cuaucgccag caacauggc 1200 ggcaagcaag cgcucgaaac ggugcagcgg cuguugccgg ugcugugcca ggaccauggc 1260 cugaccccgg accaaguggu ggcuaucgcc agcaacggug gcggcaagca agcgcucgaa 1320 acggugcagc ggcuguugcc ggugcugugc caggaccaug gccugacccc ggaccaagug 1380 guggcuaucg ccagcaacaa uggcggcaag caagcgcucg aaacggugca gcggcuguug 1440 ccggugcugu gccaggacca uggccugacc ccggaccaag ugguggcuau cgccagcaac 1500 auuggcggca agcaagcgcu cgaaagcauu guggcccagc ugagccggcc ugaucccggcg 1560 1620 auggaugcag ugaaaaaggg auugccgcac gcgccggaau ugaucagaag agucaucgc 1680 cguauuggcg aacgcacguc ccaucgcguu gcgauaucua gagugggagg aagcucgcgc 1740 agagagucca ucaacccaug gauucugacu gguuucgcug augccgaagg aucauucaua 1800 cuagacaucc gcaaccgaaa caacgaaagc aacagauacc gaacuucgcu gagauuccag 1860 aucaccguc acaacaagga caaaucgauu cuggagaaua uccagucgac uuggaagguc 1920 ggcaagauca caaacagcgg cgacagagcc gucaugcuga gggucacccg uuucgaagau 1980 uugaaaguga uuaucgacca cuucgagaaa uauccgcuga uuacccagaa auugggcgau 2040 uacaaguugu uuaaacaggc auucagcguc auggagaaca aagaacaucu uaaggagaau 2100 gggauuaagg agcucguacg aaucaaagcu aagaugaauu ggggucucac ugacgaauug 2160 aaaaaagcau uuccagagaa cauuagcaaa gagcgccccc uuaucaauaa gaacauuccg 2220 aauuucaaau ggcuggcugg auucacaucu ggugauggcu acuucggcgu gaaucuaaaa 2280 aagguaaagg gcaacgcaaa gguauaccug ggacugagau ucucaaucuc acaggacauc 2340 agagacaaga accugaugaa uucauugaua acauaccuag gcugugguuc caucugggag 2400 aagaacaagu cugaguucag uuggcucgag uucgucguaa ccaaauucag cgauaucaac 2460 gacaagauca uuccgguauu ccaggaaaau acucugauug gcgucaaacu cgaggacuuu 2520 gaagauuggu gcaagguugc caaauugauc gaagagaaga aacaccugac cgaauccggu 2580 uuggaugaga uuaagaaaau caagcugaac augaacaaag gucgugucuu cuga 2634 <210> 28 <211> 711 <212> RNA <213> Mus musculus <400> 28 augucugagc caccucgggc ugagaccuuu guauuccugg accuagaagc cacugggcuc 60 ccaaacaugg acccugagau ugcagagaua ucccuuuuug cuguucaccg cucuucccug 120 gagaacccag aacgggauga uucugguucc uuggugcugc cccguguucu ggacaagcuc 180 acacugugca ugugcccgga gcgccccuuu acugccaagg ccagugagau uacugguuug 240 agcagcgaaa gccugaugca cugcgggaag gcugguuuca auggcgcugu gguaaggaca 300 cugcagggcu uccuaagccg ccaggagggc cccaucugcc uuguggccca caauggcuuc 360 gauuaugacu ucccacugcu gugcacggag cuacaacguc ugggugccca ucugccccaa 420 gacacugucu gccuggacac acugccugca uugcggggcc uggaccgugc ucacagccac 480 ggcaccaggg cucaaggccg caaaagcuac agccuggcca gucucuucca ccgcuacuuc 540 caggcugaac ccagugcugc ccauucagca gaaggugaug ugcacacccu gcuucugauc 600 uuccugcauc gugcuccuga gcugcucgcc ugggcagaug agcaggcccg cagcugggcu 660 cauauugagc ccauguacgu gccaccugau gguccaagcc ucgaagccug a 711 <210> 29 <211> 236 <212> PRT <213> Mus musculus <400> 29 Met Ser Glu Pro Pro Arg Ala Glu Thr Phe Val Phe Leu Asp Leu Glu 1 5 10 15 Ala Thr Gly Leu Pro Asn Met Asp Pro Glu Ile Ala Glu Ile Ser Leu 20 25 30 Phe Ala Val His Arg Ser Ser Leu Glu Asn Pro Glu Arg Asp Asp Ser 35 40 45 Gly Ser Leu Val Leu Pro Arg Val Leu Asp Lys Leu Thr Leu Cys Met 50 55 60 Cys Pro Glu Arg Pro Phe Thr Ala Lys Ala Ser Glu Ile Thr Gly Leu 65 70 75 80 Ser Ser Glu Ser Leu Met His Cys Gly Lys Ala Gly Phe Asn Gly Ala 85 90 95 Val Val Arg Thr Leu Gln Gly Phe Leu Ser Arg Gln Glu Gly Pro Ile 100 105 110 Cys Leu Val Ala His Asn Gly Phe Asp Tyr Asp Phe Pro Leu Leu Cys 115 120 125 Thr Glu Leu Gln Arg Leu Gly Ala His Leu Pro Gln Asp Thr Val Cys 130 135 140 Leu Asp Thr Leu Pro Ala Leu Arg Gly Leu Asp Arg Ala His Ser His 145 150 155 160 Gly Thr Arg Ala Gln Gly Arg Lys Ser Tyr Ser Leu Ala Ser Leu Phe 165 170 175 His Arg Tyr Phe Gln Ala Glu Pro Ser Ala Ala His Ser Ala Glu Gly 180 185 190 Asp Val His Thr Leu Leu Leu Ile Phe Leu His Arg Ala Pro Glu Leu 195 200 205 Leu Ala Trp Ala Asp Glu Gln Ala Arg Ser Trp Ala His Ile Glu Pro 210 215 220 Met Tyr Val Pro Pro Asp Gly Pro Ser Leu Glu Ala 225 230 235 <210> 30 <211> 3 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Exemplary Linker Sequences <400> 30 Gly Gly Gly 1 <210> 31 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Exemplary Linker Sequences <400> 31 Asp Gly Gly Gly Ser 1 5 <210> 32 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Exemplary Linker Sequences <400> 32 Thr Gly Glu Lys Pro 1 5 <210> 33 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Exemplary Linker Sequences <400> 33 Gly Gly Arg Arg 1 <210> 34 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Exemplary Linker Sequences <400> 34 Gly Gly Gly Gly Ser 1 5 <210> 35 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Exemplary Linker Sequences <400> 35 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Val Asp 1 5 10 <210> 36 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Exemplary Linker Sequences <400> 36 Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Exemplary Linker Sequences <400> 37 Gly Gly Arg Arg Gly Gly Gly Ser 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Exemplary Linker Sequences <400> 38 Leu Arg Gln Arg Asp Gly Glu Arg Pro 1 5 <210> 39 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Exemplary Linker Sequences <400> 39 Leu Arg Gln Lys Asp Gly Gly Gly Ser Glu Arg Pro 1 5 10 <210> 40 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Exemplary Linker Sequences <400> 40 Leu Arg Gln Lys Asp Gly Gly Gly Ser Gly Gly Gly Ser Glu Arg Pro 1 5 10 15 <210> 41 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> ()..() <223> Cleavage sequence of TEV protease <220> <221> SITE <222> (2)..(3) <223> Xaa is any amino acid <220> <221> SITE <222> (5) <223> Xaa is any amino acid <220> <221> SITE <222> (7) <223> Xaa = Gly or Ser <400> 41 Glu Xaa Xaa Tyr Xaa Gln Xaa 1 5 <210> 42 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Cleavage sequence of TEV protease <400> 42 Glu Asn Leu Tyr Phe Gln Gly 1 5 <210> 43 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Cleavage sequence of TEV protease <400> 43 Glu Asn Leu Tyr Phe Gln Ser 1 5 <210> 44 <211> twenty two <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 44 Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 <210> 45 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 45 Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 1 5 10 15 Pro Gly Pro <210> 46 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 46 Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 1 5 10 <210> 47 <211> twenty one <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 47 Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu 1 5 10 15 Glu Asn Pro Gly Pro 20 <210> 48 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 48 Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro 1 5 10 15 Gly Pro <210> 49 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 49 Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro 1 5 10 <210> 50 <211> twenty three <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 50 Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp 1 5 10 15 Val Glu Ser Asn Pro Gly Pro 20 <210> 51 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 51 Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15 Asn Pro Gly Pro 20 <210> 52 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 52 Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro 1 5 10 <210> 53 <211> 25 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 53 Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala 1 5 10 15 Gly Asp Val Glu Ser Asn Pro Gly Pro 20 25 <210> 54 <211> twenty two <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 54 Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 <210> 55 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 55 Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro 1 5 10 <210> 56 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 56 Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro <210> 57 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 57 Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 1 5 10 15 Pro Gly Pro <210> 58 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 58 Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro 1 5 10 <210> 59 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 59 Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly 1 5 10 15 Pro <210> 60 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 60 Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15 Asn Pro Gly Pro 20 <210> 61 <211> twenty four <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 61 Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly 1 5 10 15 Asp Val Glu Ser Asn Pro Gly Pro 20 <210> 62 <211> 40 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 62 Val Thr Glu Leu Leu Tyr Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro 1 5 10 15 Arg Pro Leu Leu Ala Ile His Pro Thr Glu Ala Arg His Lys Gln Lys 20 25 30 Ile Val Ala Pro Val Lys Gln Thr 35 40 <210> 63 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 63 Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro 1 5 10 15 Gly Pro <210> 64 <211> 40 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 64 Leu Leu Ala Ile His Pro Thr Glu Ala Arg His Lys Gln Lys Ile Val 1 5 10 15 Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly 20 25 30 Asp Val Glu Ser Asn Pro Gly Pro 35 40 <210> 65 <211> 33 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> ()..() <223> Self-cleaving polypeptide comprising a 2A site <400> 65 Glu Ala Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln Thr Leu 1 5 10 15 Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly 20 25 30 Pro <210> 66 <211> 10 <212> DNA <213> Artificial Sequence <220> <221> gene <222> ()..() <223> Consensus Kozak sequence <400> 66 gccrccatgg 10 <210> 67 <211> twenty two <212> DNA <213> Artificial Sequence <220> <221> gene <222> ()..() <223> Synthetic constructs <220> <221> misc_feature <222> (1)..(14) <223> n is a, c, g, or t <220> <221> misc_feature <222> (18)..(22) <223> n is a, c, g, or t <400> 67 nnnnnnnnnn nnnnccgnnn nn 22 <210> 68 <211> 47 <212> DNA <213> Homo sapiens <400> 68 actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgat 47 <210> 69 <211> 47 <212> DNA <213> Homo sapiens <400> 69 atcaaaatcg gtgaataggc agacagactt gtcactggat ttagagt 47

Claims

1. A polypeptide comprising an I-OnuI homing endonuclease (HE) variant, which cleaves a target site in a human T cell receptor alpha (TCRα) gene, wherein the amino acid sequence of the I-OnuI HE variant is as shown in residues 5-301 of SEQ ID NO: 7 or 8.

2. The polypeptide of claim 1, wherein the amino acid sequence of the I-OnuIHE variant is as shown in residues 5-301 of SEQ ID NO:

7.

3. The polypeptide of claim 1, wherein the amino acid sequence of the I-OnuIHE variant is as shown in residues 5-301 of SEQ ID NO:

8.

4. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide binds to the polynucleotide sequence shown in SEQ ID NO:

17.

5. The polypeptide according to any one of claims 1 to 3, further comprising a DNA binding domain.

6. The polypeptide of claim 5, wherein the DNA binding domain is selected from the group consisting of a TALE DNA binding domain and a zinc finger DNA binding domain.

7. The polypeptide of claim 6, wherein the TALE DNA binding domain comprises 9.5 TALE repeat units, 10.5 TALE repeat units, 11.5 TALE repeat units, 12.5 TALE repeat units, 13.5 TALE repeat units, 14.5 TALE repeat units, or 15.5 TALE repeat units.

8. The polypeptide of claim 6 or claim 7, wherein the TALE DNA binding domain comprises 11.5 TALE repeat units and binds to the polynucleotide sequence shown in SEQ ID NO:

19.

9. The polypeptide of claim 6 or claim 7, wherein the TALE DNA binding domain comprises 10.5 TALE repeat units and binds to the polynucleotide sequence shown in SEQ ID NO:

18.

10. The polypeptide according to claim 6 or claim 7, wherein the polypeptide binds to and cleaves the polynucleotide sequence shown in SEQ ID NO:

20.

11. The polypeptide of claim 6, wherein the zinc finger DNA binding domain comprises 2, 3, 4, 5, 6, 7 or 8 zinc finger motifs.

12. The polypeptide according to any one of claims 1 to 3, further comprising a peptide linker and a terminal processing enzyme.

13. The polypeptide according to any one of claims 1 to 3, further comprising a viral self-cleaving 2A peptide and a terminal processing enzyme.

14. The polypeptide of claim 12, wherein the end processing enzyme has 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease, 5' flap endonuclease, helicase or template-independent DNA polymerase activity.

15. The polypeptide of claim 13, wherein the end processing enzyme has 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease, 5' flap endonuclease, helicase or template-independent DNA polymerase activity.

16. The polypeptide of claim 12, wherein the terminal processing enzyme comprises Trex2.

17. The polypeptide of claim 13, wherein the terminal processing enzyme comprises Trex2.

18. A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

11.

19. A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

12.

20. The polypeptide according to claim 18 or 19, further comprising a peptide linker and a terminal processing enzyme.

21. The polypeptide according to claim 18 or 19, further comprising a viral self-cleaving 2A peptide and a terminal processing enzyme.

22. The polypeptide of claim 20, wherein the end processing enzyme has 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease, 5' flap endonuclease, helicase or template-independent DNA polymerase activity.

23. The polypeptide of claim 21, wherein the end processing enzyme has 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease, 5' flap endonuclease, helicase or template-independent DNA polymerase activity.

24. The polypeptide of claim 20, wherein the terminal processing enzyme comprises Trex2.

25. The polypeptide of claim 21, wherein the terminal processing enzyme comprises Trex2.

26. A polynucleotide encoding the polypeptide according to any one of claims 1 to 25.

27. An mRNA encoding the polypeptide according to any one of claims 1 to 25.

28. A cDNA encoding the polypeptide according to any one of claims 1 to 25.

29. A vector comprising a polynucleotide encoding the polypeptide according to any one of claims 1 to 25.

30. A cell comprising a polypeptide according to any one of claims 1 to 25, wherein the cell is incapable of developing into an animal or a plant.

31. A cell comprising a polynucleotide encoding a polypeptide according to any one of claims 1 to 25, wherein the cell is incapable of developing into an animal or a plant.

32. A cell comprising the vector of claim 29, wherein the cell is incapable of developing into an animal or a plant.

33. A cell edited by the polypeptide according to any one of claims 1 to 25, wherein the cell is incapable of developing into an animal or a plant.

34. The cell of any one of claims 30 to 33, wherein the cell is a hematopoietic cell, a T cell, and / or an immune effector cell.

35. The cell according to any one of claims 30 to 33, wherein the cell is a CD3 + cells, CD4 + Cells, CD8 + cells, cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), helper T cells, natural killer (NK) cells and / or natural killer T (NKT) cells.

36. The cell of any one of claims 30 to 33, wherein the source of the cell is peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue or a tumor.

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