Mitochondrial DNA base editor with improved base editing efficiency

Abstract

The present invention relates to base editing of mitochondrial DNA. More specifically, the present invention relates to: a method for editing mitochondrial DNA bases by using a mitochondrial targeting sequence (MTS); and a base editing system used in the method. The base editing system comprises a DNA binding protein and a deaminase, or polynucleotides encoding these, and does not include an affinity tag or a polynucleotide encoding same. The present invention is useful for editing mitochondrial DNA bases of animal cells or plant cells.

Description

Mitochondrial DNA base editor with improved base editing efficiency

[0001] The present invention relates to base correction of mitochondrial DNA. More specifically, it relates to a method for base correction of mitochondrial DNA using a mitochondrial targeting sequence (MTS) and a base correction system used in such a method. The base correction system comprises a DNA binding protein and a deaminase, or a polynucleotide encoding them, and is characterized in that it does not include an affinity tag or a polynucleotide encoding them. The present invention is useful for base correction of mitochondrial DNA in animal or plant cells.

[0002] Fusion proteins that link DNA binding proteins and deaminase enzymes enable the induction of DNA mutations, such as single nucleotide conversions in a targeted manner to replace nucleotides or correct bases in the genome without generating DNA double-strand breaks (DSBs), to correct point mutations that cause genetic disorders, or to introduce desired single nucleotide mutations in prokaryotes and eukaryotic cells such as humans.

[0003] Programmable genome editing tools, such as zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeat (CRISPR) systems, and base editors composed of CRISPR-associated protein 9 (Cas9) variants and base deaminase proteins, have the potential to treat genetic diseases and improve crop traits through base sequence changes. However, these conventional genome editing tools are not suitable for correcting DNA bases in organelles such as mitochondria, particularly because they cannot deliver the guide RNA required to activate the most widely used CRISPR system to the organelles. Mitochondria encode several essential genes required for cellular respiration. Methods or tools for correcting genes in these organelles are crucial for studying the function of these genes and for treating mitochondrial genetic diseases.

[0004] Mitochondrial genetic diseases are caused by mutations in mitochondrial DNA itself or in the nuclear genes that encode mitochondrial proteins. Because mitochondrial DNA is constantly exposed to reactive oxygen species (ROS) generated during oxidative phosphorylation, it is more susceptible to mutation than nuclear DNA. Approximately 1 in 5,000 individuals carry a clinically confirmed pathogenic mitochondrial DNA mutation, which can be either homoplasmic or heteroplasmic. Many of these mutations are G>A or T>C substitutions, which can be corrected using adenine or cytosine base editors, respectively.

[0005] Double-stranded DNA-specific cytosine deaminase is an enzyme that can directly deaminate cytosine bases on double-stranded DNA, providing a new platform that can expand the scope of existing base editing systems (see Mok, BY et al. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature 583, 631-637 (2020), WO 2022 / 060185, etc.). Typically, double-stranded DNA-specific cytosine deaminase is split into two to reduce cytotoxicity, each fused to a DNA-binding protein, and its activity is induced through dimerization on the target DNA. This enables C-to-T base editing with high specificity and efficiency even in double-stranded DNA. This C-to-T base editor, based on double-stranded DNA-specific cytosine deaminase, is called DdCBE.

[0006] Meanwhile, adenine base correction can be achieved by linking a double-stranded DNA-specific cytosine deaminase to a DNA binding protein and an adenine deaminase capable of correcting adenine to guanine (A-to-G). A base editor (TALED, TALE-linked deaminase) that uses a TALE protein as a DNA binding protein and links a cytosine deaminase and an adenine deaminase to it can introduce a wide spectrum of base mutations, unlike a cytosine base editor that only causes C-to-T correction (see Cho, SI et al. Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases. Cell 185, 1764-1776.e12 (2022), WO 2022 / 060185, etc.). This A-to-G base editor is called TALED.

[0007] In addition, a mitochondrial DNA base editing system (so-called mitoABE or mitoCBE) has been developed that enables A-to-G or C-to-T base editing by selectively inducing a nick in one strand around a target base pair in mitochondrial DNA and then inducing deamination of adenine or cytosine in the formed single-stranded DNA region.

[0008] Most mitochondrial DNA base editing systems reported to date, including the above-described DdCBE and TALED, are known to be designed to include not only an MTS for mitochondrial localization, but also a tag sequence (e.g., HA or FLAG) useful for solubilizing, purifying, or detecting the base editing protein (e.g., fusion protein). However, the effect of the presence of a tag sequence on the base editing efficiency of a mitochondrial DNA base editor has not yet been specifically reported, and there are no specifically described cases of a base editor that does not include a tag sequence or a mitochondrial DNA base editor designed by intentionally removing or excluding a tag sequence.

[0009] The inventors of the present invention have made repeated efforts to improve the base editing efficiency of a mitochondrial DNA base editor including MTS along with a DNA binding protein and a deaminase, and have discovered for the first time that the base editing efficiency is improved by removing a tag sequence that has been used as a part of the base editing protein, such as HA or FLAG.

[0010] In addition, the inventors of the present invention screened for MTS sequences suitable for use in a mitochondrial DNA base editor and confirmed that the mitochondrial DNA base editing efficiency obtained when using various MTSs differed from the mitochondrial localization tendency of MTSs known through experiments using model proteins such as GFP and ATP6. Therefore, they attempted to further improve the base editing performance of a mitochondrial DNA base editor by using an MTS that had not been used before.

[0011] The present invention provides a DNA base correction system comprising a DNA binding protein and a deaminase, a base correction system excluding an affinity tag from a conventional base correction system known to be suitable for mitochondrial DNA base correction, and a base correction method using the same.

[0012] One aspect of the present invention provides a mitochondrial DNA base editing system comprising (1) a DNA binding protein, (2) at least one deaminase selected from adenine deaminase and cytosine deaminase, and (3) a base editor or a polynucleotide encoding the same, wherein the base editor comprises a mitochondrial targeting sequence (MTS) and does not comprise an affinity tag.

[0013] Another aspect of the present invention provides a method for base correction of mitochondrial DNA, comprising introducing the mitochondrial DNA base correction system as described above into a cell containing target DNA for base correction, or expressing the mitochondrial DNA base correction system within a cell containing target DNA for base correction.

[0014] Another aspect of the present invention provides a method for improving mitochondrial DNA base correction efficiency, comprising: measuring the mitochondrial DNA base correction efficiency of a base editor including an affinity tag; and then providing a base correction system including a tag-free base editor having the same amino acid sequence as the base editor but not including an affinity tag, or a polynucleotide encoding the same.

[0015] In various embodiments of the various aspects of the present invention, mitochondrial DNA base editing can occur in vitro or in vivo.

[0016] In various embodiments of the various aspects of the present invention, the base correction system may use MTSs that are not conventionally used for mitochondrial DNA base correction.

[0017] The mitochondrial DNA base editing system according to the present invention can provide significantly improved base editing efficiency compared to existing systems by removing affinity tags (e.g., HA, FLAG) from the base editor configuration. Furthermore, it was confirmed that the efficiency was further increased when a specific MTS, such as SDHD or SDHA, was used and the design did not include a tag sequence. Furthermore, this effect is commonly applicable to various base editing systems, such as DdCBE, TALED, and nickase-based base editing systems, and was consistently observed in various target genes and cell types. Therefore, the base editing system of the present invention can be utilized as a mitochondrial genome editing platform with high accuracy and efficiency, and can be usefully applied to research and development of therapeutics for mitochondrial diseases.

[0018] Figure 1 shows the results of a comparative experiment in HEK293T cells for the mitochondrial DNA base correction efficiency when using and not using tag sequences (FLAG, HA), targeting the human mitochondrial ND1 gene. COX8A and SOD2 are the MTSs used, L is ND1 Left TALE, R is ND1 Right TALE, 1397N and 1397C are DddAtox splitters, and 8e (TadA8e) and V28R are adenine deaminase. Proteins listed in [ ] are meant to constitute a single fusion protein. For example, [COX8A-3xFLAG-L-1397C-8e] represents a fusion protein (linker omitted) in which COX8A, 3xFLAG, L (Left TALE), 1397C (DddAtox splitter), and 8e (TadA8e) are linked. The outlined rectangles represent the DNA sequences recognized by each of the two DNA-binding proteins (TALE proteins). The above descriptions also apply to subsequent drawings. It should be noted that base editing efficiency increased when Tad8e or V28R was used as the adenine deaminase compared to when no tag sequence was used or when a tag sequence was used.

[0019] Figure 2 shows the results of a comparative experiment on the mitochondrial DNA base correction efficiency using and not using a tag sequence (FLAG, HA) in UDC cells, targeting the human mitochondrial ND1 gene. The TALE protein used is the same as that used in the results of Figure 1. Similar to the results of Figure 1, it should be noted that the base correction efficiency increased when the tag sequence was not used compared to when the tag sequence was used.

[0020] Figure 3 shows the results of measuring the base correction efficiency in the mitochondrial ND1 gene in UDC cells, divided into cases where the tag sequence (FLAG, HA) was used in both fusion proteins, deleted from only one of the two fusion proteins, and deleted from both fusion proteins. The TALE proteins used were the same as those used in the results of Figures 1 and 2. It should be noted that the base correction efficiency was highest when the tag sequence was not used in any of the fusion proteins used.

[0021] Figure 4 shows the results of measuring the base correction efficiency for the human mitochondrial ND1 gene in UDC cells, divided into cases where the COX8A MTS and SOD2 MTS sequences were used as MTS, when a tag sequence (FLAG, HA) was used (experimental results in the 2nd and 5th lines), when no tag sequence was used (experimental results in the 3rd and 6th lines), and when the SDHD MTS sequence was used as MTS without using a tag sequence (experimental results in the 4th and 7th lines). The base editor that provided the experimental results in the 2nd to 4th lines and the base editor that provided the experimental results in the 5th to 7th lines differ only in whether adenine deaminase (TadA8e) was used in conjunction with 1397C to the right TALE or to the left TALE. The TALE proteins used are the same as those used in the results in Figures 1 to 3. It should be noted that when the same MTS was used, the base correction efficiency was higher when the tag sequence was not used than when the tag sequence was used, and when the SDHD MTS was used as the MTS when the tag sequence was not used, the base correction efficiency was significantly higher.

[0022] Figure 5 shows the results of measuring the base correction efficiency for the human mitochondrial ND4 gene in UDC cells using new MTS sequences instead of the conventionally used MTS sequences (COX8A MTS, SOD MTS). The TALE proteins used here are ND4 Left TALE 90 and ND4 Right TALE 120. "90N" is the connection between ND4 Left TALE 90 and 1397N, and "120C" is the connection between ND4 Right TALE 120 and 1397C. In addition, adenine deaminase (TadA8e) was connected to 1397C. When SOD2 MTS and COX8A MTS were used, it was confirmed again that the base correction efficiency was significantly increased when the tag sequence was not used compared to when the tag sequence was used. All MTSs other than SOD2 MTS and COX8A MTS were used without the tag sequence.

[0023] Figure 6 shows the results of measuring the base correction efficiency in UDC cells targeting the human mitochondrial ND4 gene when the same or different MTSs were used for each of the two fusion proteins. The TALE proteins used were the same as those used in Figure 5. It should be noted that when the SDHD MTS sequence was used, a significantly improved base correction efficiency was obtained in all tested combinations, and among them, the base correction efficiency was highest when both fusion proteins used the SDHD MTS sequence.

[0024] Figures 7 and 8 demonstrate that mitochondrial base editing efficiency significantly increased when the tag sequence (FLAG or HA) was removed, even when using a nickase-based base editor. When targeting the G11778A mutation in the mitochondrial gene in UDC cells, in all cases tested, the base editing efficiency (A-to-G) was significantly improved in the absence of the tag sequence (Figure 8) compared to when the tag sequence was present (Figure 7).

[0025] Figures 9 and 10 show the results of a comparative analysis of the change in mitochondrial DNA base correction efficiency according to the removal of the tag sequence, targeting the G3460A mutation in the mitochondrial gene. A double-stranded DNA-specific cytosine deaminase based on DddAtox was used as the cytosine deaminase, and various MTSs (SOD2, COX8A, SDHD) were used as the MTS. In all experimental cases, the base correction efficiency (A-to-G) was significantly improved when the tag sequence was absent (Figure 10) compared to when the tag sequence was present (Figure 9). In addition, among the test groups in which the tag was removed, the base correction efficiency was the same or significantly higher when SDHD was used as the MTS than when SOD2 was used as the MTS.

[0026] Figures 11 to 13 show the results of comparing the base correction efficiency when SDHD, SDHA, and SDHDcut were used as MTSs targeting the G11778A mutation in the mitochondrial gene. Figures 11 and 12 show that the base correction efficiency equivalent to that of SDHD was obtained when SDHA and SDHDcut were used. SDHDcut is a shortened version of SDHD by removing 20 amino acids from the C-terminal side. Figure 13 shows the results of measuring the change in base correction efficiency of the mitochondrial base editor applied with SDHD MTS or SDHDcut MTS depending on the presence or absence of NES (nuclear export signal) in UDC cells, and shows that the base correction efficiency that is greatly improved by the absence of the tag sequence does not significantly vary depending on the presence or absence of NES.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the terms used herein are those well known and commonly used in the art.

[0028] The embodiments described in this specification and the configurations depicted in the drawings are only one embodiment of how the present invention is realized and do not fully represent the technical idea of ​​the present invention. Therefore, it should be understood that there may be various equivalents, modifications, and applicable examples that can replace them at the time of this application. In addition, the various aspects and embodiments described in this specification can be applied to other aspects and embodiments, and all combinations of the various elements described in the present invention fall within the scope of the present invention, and the scope of the present invention cannot be considered limited by the specific description described below.

[0029] In this specification, the use of the singular includes the plural unless specifically stated otherwise. As used herein, it should be noted that the singular form includes plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless otherwise stated.

[0030] The term “comprising” as used herein, unless otherwise specified, is understood to be an open-ended expression that essentially includes the described components, ingredients, steps, etc., but does not exclude the presence of other components, ingredients, steps, etc. Accordingly, the term “comprising” is interpreted to include the more limited meaning of “consisting of” or “consisting essentially of.”

[0031] The expression “for” as used in this specification and claims not only means that the composition or material is designed to be used for a particular use, but also includes the meaning that it has functions or characteristics that are suitable or useful for that use even if it is not actually used for that use.

[0032] The terms "correction," "editing," and "editing" as used herein are used interchangeably and refer to a method of altering a nucleic acid sequence by selectively modifying a specific genomic target. Such specific genomic target includes, but is not limited to, a gene, a promoter, an open reading frame, or any nucleic acid sequence.

[0033] The terms "base editing system" and "base correction system" as used herein are interchangeable and refer to a substance having an activity of changing a nucleic acid sequence by selective mutation of a genomic target, and include a combination of one or more different base editors. The terms "base editing system" or "base correction system" as used herein may be in the form of a polypeptide (which may be a fusion protein) or a polynucleotide, or a combination thereof, depending on the context, and may be a composition comprising one or more polypeptides (which may be fusion proteins) or polynucleotides, or a combination thereof. Accordingly, the terms "base correction system" or "base correction composition" as used herein may include one base editor or a combination of two or more different base editors, wherein the different base editors may be used simultaneously or separately.

[0034] The term “base editor” as used herein refers to an artificial gene editing protein that can selectively correct a specific base of a target DNA sequence to another base.

[0035] As used herein, the term "conservative amino acid substitution" refers to the replacement of some amino acids with amino acids of different properties while maintaining structural or functional similarity within a protein. Specifically, it refers to substitutions between amino acids with similar physicochemical properties (e.g., charge, size, hydrophobicity, polarity, etc.), and includes substitutions that substantially maintain the structural stability or biological function of the protein.

[0036] For example, substitutions within the following amino acid groups may be conservative substitutions:

[0037] Hydrophobic amino acid group: Ala, Val, Leu, Ile, Met

[0038] Polar uncharged amino acid group: Ser, Thr, Gln, Asn

[0039] Acidic amino acid group: Asp, Glu

[0040] Basic amino acid group: Lys, Arg, His

[0041] Aromatic amino acid group: Phe, Tyr, Trp

[0042] Such determination of substitution can be performed based on the standard amino acid classification that takes into account the charge, polarity, hydrophobicity, structural similarity, etc. of the amino acid, and a person skilled in the art can objectively determine whether the substitution is conservative by utilizing sequence alignment tools such as BLAST and Clustal Omega and conservation matrices (BLOSUM, PAM, etc.). When a specific amino acid sequence is described in this specification, it is interpreted that a variant in which one or more amino acids in the sequence are substituted with another amino acid corresponding to the conservative substitution is also included in the technical scope of the present invention.

[0043] The term "anionic amino acid" as used herein refers to an amino acid having a side chain that is negatively charged under physiological conditions (pH approximately 7.4), and includes glutamic acid (Glu, E) and aspartic acid (Asp, D). On the other hand, the term "cationic amino acid" refers to an amino acid having a side chain that is positively charged under physiological conditions (pH approximately 7.4), and includes lysine (Lys, K), arginine (Arg, R), histidine (His, H), and the like.

[0044] The term “sequence” in this specification may be interpreted as a nucleic acid (or polynucleotide) molecule or a protein (or polypeptide) molecule having a given sequence, depending on the context.

[0045] The term "other amino acid" as used herein means an amino acid selected from among alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, aspartic acid, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartic acid, glutamic acid, arginine, histidine, lysine, and all known variants of the above amino acids, excluding the amino acid that the wild-type protein originally has at the mutation position.

[0046] The term “sequence identity” or “sequence homology” as used herein means the number of residues present at the same position when two amino acid sequences or nucleic acid sequences are aligned, expressed as a percentage of the total length of the sequences. When it is said herein that a specific sequence “has at least X% sequence identity,” X can be, for example, 85%, 90%, 95%, 98%, or 99%. Sequence identity is typically calculated using BLAST (Basic Local Alignment Search Tool), ClustalW, EMBOSS, or other known sequence alignment algorithms, and is based on default parameters. For example, when aligning amino acid sequences using BLASTP, the identity value calculated using the BLOSUM62 matrix and gap penalty as default values ​​can be used as a basis. In addition, in this specification, “sequence identity” or “sequence homology” may include a value calculated according to an optimized global alignment or local alignment that takes into account insertions, deletions, substitutions, etc. during sequence alignment, and is also used as a standard for explaining the scope of functional equivalents that can maintain the technical effect of the invention.

[0047] The term "homolog" as used herein refers to a protein or nucleic acid that has homology or similarity to a specific protein or gene sequence and exhibits functionally similar biological activity. Homologs may perform the same or similar function, but may be of different species or may have some variation in the amino acid or base sequence. Homologs may be naturally occurring or artificially modified, and are considered to provide the same technical effect within the scope of the present invention.

[0048] The term "ortholog" as used herein refers to a gene or protein derived from two or more species that share a common ancestor, and thus corresponds to a corresponding gene or protein found in different species but with the same evolutionary origin. Generally, orthologs have a high degree of sequence homology between different species and are known to perform similar biological functions. As used herein, an ortholog of a specific protein may include a variant that performs essentially the same function as the original protein, even if a portion of the sequence contains amino acid substitutions, insertions, or deletions.

[0049] The term "functional variant" as used herein refers to a protein that substantially maintains the basic biological function of a polypeptide (e.g., a protein) having a specific amino acid sequence, or has an activity essentially similar to that of the protein, despite having one or more amino acids in the entire sequence conservatively or non-conservatively substituted, or modified, such as insertion, deletion, or substitution. For example, some differences in the sequence may be considered functional variants of the protein described herein, as long as the protein performs the effective function intended in the present invention, such as substrate recognition, catalytic activity, binding ability, or specificity for a target molecule. Such functional variants may be generated by spontaneous mutation, evolutionary modification, induced mutation, or genetic engineering methods.

[0050] As used herein, the terms "target" or "target site" refer to a pre-identified nucleic acid sequence of any composition and / or length. Such target sites include, but are not limited to, genes, promoters, or any nucleic acid sequence.

[0051] The term "fusion protein" as used herein refers to a protein in which two or more different protein (polypeptide) sequences or functional domains are combined into a single continuous polypeptide chain. Such a fusion protein may retain the original biological function of each component or be endowed with new functional properties, and may include a linker sequence between the components. When designating components of a fusion protein herein, unless otherwise specified, the left-to-right direction refers to the N-terminus to the C-terminus, respectively. Additionally, the linker sequence used may not be explicitly indicated.

[0052] The term “expression” as used herein refers to the process by which a polynucleotide (e.g., DNA or mRNA) encoding a DNA base editor or a component thereof is introduced into a cell, and the base editor or a component protein thereof is produced through the cell’s transcription and / or translation mechanisms. The expression may be transient, or stable when integrated into the genome of a target cell. The expression product may be a single protein, or may be a fusion protein in which multiple functional domains are fused. In addition, the expression may be performed in the cytoplasm or organelles (e.g., chloroplasts, mitochondria, nuclei, etc.), and for this purpose, the expression product may additionally include an organelle targeting sequence (MTS, CTS, etc.), a nuclear export signal (NLS), or an extranuclear export signal (NES). The expression level and location may vary depending on the sequence of the polynucleotide being introduced, the promoter, codon optimization, target cell type, introduction method, etc., and a person skilled in the art can appropriately adjust it according to the purpose.

[0053] The term "monomeric base editor" as used herein means a base editor that exists in the form of a single fusion protein.

[0054] As used herein, the term "dimeric base editor" refers to a base editor that exists in the form of two fusion proteins. Among the two fusion proteins, the fusion protein that binds to a DNA sequence located 5' upstream of the spacer region may be referred to as the "first fusion protein" or the "left fusion protein," and the fusion protein that binds to a DNA sequence located 3' downstream of the spacer region may be referred to as the "second fusion protein" or the "right fusion protein." Similarly, the DNA binding protein included in the first fusion protein may be expressed by the modifier "first" or "left," and the DNA binding protein included in the second fusion protein may be expressed by the modifier "second" or "right." Unless otherwise indicated, when the name of a DNA binding protein includes the suffix "N," it refers to a protein that binds to the N-terminal region of DddAtox or an ortholog thereof. Also, unless otherwise indicated, the fusion protein that binds to the Left TALE is specified first, followed by + to indicate the fusion protein that binds to the Right TALE. Also, unless otherwise indicated, when the name of the DNA binding protein includes the suffix "C", it means a protein that binds the C-term portion of DddAtox or its ortholog, and in the adenine base corrector used in this case, TadA8e is bound after the C-term of DddAtox or its ortholog. For example, unless otherwise indicated, [MTS-90N] + [MTS-120C] refers to proteins such as "MTS-Left TALE 90-DddAtox N-term" and "MTS-RightTALE 120-DddAtox C-term-TadA8e".

[0055] In some embodiments, the base editor may be a monomeric base editor, and in other embodiments, it may consist of a dimeric base editor that exists in the form of two fusion proteins.

[0056] In this specification, binding of a fusion protein to a given nucleotide sequence means that the DNA binding protein included in the fusion protein recognizes and binds to the nucleotide sequence.

[0057] The term “CRISPR-associated nuclease” as used herein, also called Cas, generally refers to a protein that is a component of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, binds to a guide RNA, specifically binds to a target nucleotide sequence, and has the activity of cleaving or regulating the sequence. In this specification, “CRISPR-associated nuclease” and “Cas” are used interchangeably.

[0058] The term "organelle DNA" as used herein refers to the genetic material present in organelles other than the nucleus within a eukaryotic cell, including mitochondrial DNA or plastid DNA.

[0059] 1. MTS

[0060] The present invention relates to a mitochondrial base correction system and a base correction method, using MTS.

[0061] The above "MTS (mitochondrial targeting sequence)" refers to an amino acid sequence that induces the transport of a protein to the mitochondria after translation. MTS is generally located at the N-terminus and forms a unique α-helical structure with a repeating arrangement of basic and hydrophobic amino acids, which interacts with the mitochondrial inner membrane transport complex to achieve transport. The MTS that can be used in some embodiments of the present invention is not limited to a specific sequence, length, structure, or origin, and is understood to include any functional sequence that can effectively direct a protein to the mitochondria.

[0062] Information on MTS has been identified from various biological proteins, and relevant sequences are widely available through public databases such as UniProt, NCBI, and MitoCarta. Using this publicly available sequence information, skilled artisans can select or combine MTS sequences appropriate for the desired protein to design the protein.

[0063] The MTS included in the DNA base editor of the present invention can be derived from various mitochondrial proteins existing in nature, and a sequence artificially designed to have a specific mitochondrial transport function can also be utilized. For example, MTS derived from human SOD2 (superoxide dismutase 2) protein (e.g., MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD), MTS derived from COX8A (cytochrome c oxidase subunit 8A) (e.g., MASVLTPLLLRGLTGSARRLPVPRAKIHSL), or MTS derived from human mitochondrial ATP synthase F1β subunit (e.g., MLGFVGRVAAAPASGALRRLTPSASLPPAQLLLRAAPTAVHPVRDYAAQ) are known as widely used representative sequences, and similarly, various mutants having a protein transport function to mitochondria are also well recognized by those skilled in the art.

[0064] The MTS that can be used in the present invention can be any signal sequence having the ability to translocate into mitochondria, and can be a natural MTS present at the N-terminus of various mitochondrial proteins, or an artificially synthesized MTS can also be used. For example, it may be an MTS sequence of a protein selected from the group consisting of COX8A, SOD2, COX10, RPM2, NDUS7, Sirt5, QCR2, ATP5G2, LACTB, COX2, spilv1, SDHA, SDHB, SDHC, SDHD, ATP9, OPA1, ATP6, ADCK3, ATP9, GHITM, P0644B06.24-2, NDUFAB1, ATP5G3, LOC (also known as LOC 100282174), BSC1L, COX5B, NUDFC2 and PDK4, or a truncated form thereof, or a conservative amino acid substitution thereof.

[0065] The base correction system and base correction method according to the present invention include an MTS, thereby enabling the base editor to efficiently move and be delivered to mitochondria.

[0066] For example, the MTS sequences usable in the present invention may include, but are not limited to, the following MTS or truncated forms thereof (e.g., truncated forms in which some amino acids at the C-terminal side are removed).

[0067] SOD2_MTS:

[0068] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD

[0069] COX8A_MTS:

[0070] MASVLTPLLLRGLTGSARRRLPVPRAKIHSL

[0071] crATP6 MTS:

[0072] MALQQAAPRVFGLLGRAPVALGQSGILTGSSGFKNQGFNGSLQSVENHVYAQAFSTSSQEEQAAPSIQGASGMKLPGMAGSMLLGKSRSGLRTGSMVPFAAQQAMNM

[0073] hsADCK3 MTS:

[0074] MAAILGDTIMVAKGLVKLTQAAVETHLQHLGIGGELIMAARALQSTAVEQIGMFLGKVQGQDKHEEYFAENFGGPEGEFHFSVPHAAGASTDFSSASAPDQSAPPSLGHAHSEGPAPAYVASGPFREAGFPGQASSPLGRANGRLFANPRDFSAMGFQRRF

[0075] hsATP5G3 MTS:

[0076] MFACAKLACTPSLIRAGSRVAYRPISASVLSRPEASRTGEGGSTVFNGAQNGVSQLIQREFQTSAISR

[0077] ncATP9 MTS:

[0078] MASTRVLASRLASQMAASAKVARPAVRVAQVSKRTIQTGSPLQTLKRTQMTSIVNATTRQAFQKRA

[0079] zmLOC:

[0080] MALLRAAVSELRRRGRGALTPLPALSSLLSSLSPRSPSASTRPEPNNPHADRRHVIALRRCPPLPASAVLAPELLHARGLLPRHWSHASPLSTSSSSSRPADKAQLTWVDKWIPEAARPY

[0081] hsATP5G2 MTS:

[0082] MPELILYVAITLSVAERLVGPGHACAEPSFRSSRCSAPLCLLCSGSSSPATAPHPLKMFACSKFVSTPSLVKSTSQLLSRPLSAVVLKRPEILTDESLSSLAVSCPLTSLVSSRSFQTSAISRIDTA

[0083] hsGHITM MTS:

[0084] MLAARLVCLRTLPSRVFHPAFTKASPVVKNSITKNQWLLTPSRE

[0085] hsSDHD MTS:

[0086] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH

[0087] hsNDUFAB1 MTS:

[0088] MASRVLSAYVSRLPAAFAPLPRVRMLAVARPLSTALCSAGTQTRLGTLQPALVLAQVPGRVTQLCRQY

[0089] hsOPA1 MTS:

[0090] MWRLRRAAVACEVCQSLVKHSSGIKGSLPLQKHLVSRSIYHSHHPTLKLQRPQLRTSFQQFSSLTNLPLRKLKFSPIKYGYQPRRN

[0091] hsSDHA MTS:

[0092] MSGVRGLSRLLSARRLALAKAWPTVLQTGTRGFHFTV

[0093] In some embodiments, MTS designed with sequences optimized for targeting efficiency by combining or partially modifying the above sequences may be used.

[0094] When MTS is used in the base correction system according to the present invention or the base correction method using the same, its position may vary, but it may preferably be directly or indirectly linked to the N-terminus of the DNA binding protein (e.g., via a linker and / or other protein component).

[0095] The present invention is characterized in that it does not include an affinity tag between the MTS and the DNA binding protein.

[0096] In some embodiments of the present invention, the MTS is directly linked to the N-terminus of the DNA binding protein.

[0097] In some embodiments of the present invention, the MTS may be linked to the N-terminus of the DNA binding protein via a linker. The linker used may have a sequence appropriately optimized so as not to affect the function of the fusion protein comprising the DNA binding protein.

[0098] In some embodiments of the present invention, when the MTS is linked to the N-terminus of the DNA binding protein via a linker or other protein component, the linker or other protein component may comprise less than 5, less than 4, less than 3, less than 2, or less than 1 anionic amino acid.

[0099] Through experiments, the inventors of the present invention have confirmed that specific combinations of MTS sequences are effective in increasing base-editing activity or reducing off-target effects. Accordingly, the base-editing system of the present invention can be provided in a form with enhanced intracellular target delivery efficiency and functional stability through the selection of an appropriate MTS.

[0100] According to some embodiments, certain MTSs can selectively enhance base correction efficiency for certain target genes, and this effect is confirmed through experimental data of the examples described herein.

[0101] The present invention, which improves DNA base editing efficiency by not including a tag sequence, can be applied even when any MTS is used. In the present invention, the MTS is preferably COX8A, SOD2, SDHA, SDHD, ATP6, ADCK3, ATP5G3, ATP9, LOC, ATP5G2, GHITM, NDUFAB, or OPA1, or an ortholog thereof, or a truncated form thereof (e.g., a truncated form in which some amino acid residues at the C-terminus are removed), or a functional variant thereof. In the present invention, the MTS is preferably SDHA or an ortholog thereof, or a truncated form thereof (e.g., a truncated form in which some amino acid residues at the C-terminus are removed), or a functional variant thereof. In the present invention, the MTS is preferably SDHD or an ortholog thereof, or a truncated form thereof (e.g., a truncated form in which some amino acid residues at the C-terminus are removed), or a functional variant thereof.

[0102] When the base correction system according to the present invention or the base correction method using the same uses two fusion proteins (or polynucleotide(s) encoding the same), at least one of the two fusion proteins has an MTS. Preferably, both of the two fusion proteins can have an MTS, and in this case, the two fusion proteins can include the same MTS or different MTS. In addition, one or more MTS sequences can be repeatedly arranged in one fusion protein, and such repeatedly arranged MTS sequences can be composed of the same MTS sequences or composed of different MTS sequences.

[0103] 2. Tag-free

[0104] The present invention is a mitochondrial base correction system and base correction method that does not use an affinity tag.

[0105] The terms "affinity tag", "tag", and "tag" as used herein refer to tags commonly used in the biotechnology field to facilitate the solubilization, purification, or detection of a base-editing protein (which may be a fusion protein). Such tags include, but are not limited to, biotin carboxylase carrier protein (BCCP) tag, myc tag, calmodulin tag, FLAG tag, HA (hemagglutinin) tag, polyhistidine tag (also called His-tag), maltose binding protein (MBP) tag, nus tag, glutathione-S-transferase (GST) tag, green fluorescent protein (GFP) tag, thioredoxin tag, S-tag, Softag (e.g., Softag 1, Softag 3), strep tag, biotin ligase tag, FlAsH tag, V5 tag, and SBP tag. Mitochondrial base editing editor proteins known to date have been described primarily as containing HA tags or FLAG tags.

[0106] In preferred embodiments, the affinity tag of the present invention is a tag relatively rich in anionic amino acids.

[0107] As used herein, the expression “tag-free” or “tag-free” refers to a state in which a protein does not contain a tag as defined above. That is, it does not contain an affinity tag, a functional tag, or a detection / tracking label sequence other than the target protein itself. Such tag-free proteins contribute to minimizing structural interference, non-specific interactions, or functional inhibition that may be caused by tags, and enable the functional proteins used in the base correction system of the present invention to more accurately and efficiently exert their intended biological activity within cells.

[0108] In some embodiments of the base correction system and base correction method of the present invention that do not include an affinity tag, the MTS can be directly linked to a DNA binding protein.

[0109] In some embodiments of the base correction system and base correction method of the present invention that do not include an affinity tag, the MTS may be linked indirectly (e.g., via a linker and / or other protein component) to the DNA binding protein.

[0110] In some embodiments of the present invention, when the MTS is linked to the N-terminus of the DNA binding protein via a linker or other protein component, the linker or other protein component comprises less than 5, less than 4, less than 3, less than 2, or less than 1 anionic amino acid.

[0111] The base correction system and base correction method of the present invention improve base correction efficiency by removing these tags. Furthermore, tag removal reduces the possibility of inducing an immune response to foreign proteins and minimizes protein stability, structural interference, and functional inhibition within mitochondria. Consequently, correction efficiency in mitochondrial DNA is improved and the possibility of off-target effects is reduced.

[0112] 3. DNA base editor

[0113] One aspect of the present invention is

[0114] (1) DNA binding protein,

[0115] (2) one or more deaminase selected from adenine deaminase and cytosine deaminase, and

[0116] (3) Including MTS,

[0117] This is about base editors that do not include affinity tags.

[0118] In some embodiments, the base editor may further comprise a nickase.

[0119] These DNA base editors have the ability to selectively correct specific DNA bases, and are particularly useful for inducing the conversion of cytosine (C) base to thymine (T) (C→T) or adenine (A) base to guanine (G) (A→G).

[0120] The DNA base editor of the present invention may exist in the form of one or more fusion proteins, and may optionally be in a dimeric form consisting of two fusion proteins. Such fusion proteins may include a linker sequence between the protein components.

[0121] A. DNA binding protein

[0122] A DNA base editor according to the present invention comprises one or more DNA binding proteins.

[0123] The "DNA binding protein" used in the DNA base editor according to the present invention means a protein that can recognize a specific base sequence and selectively bind to the sequence, and the binding specificity has a programmable characteristic according to the target sequence. In that sense, the DNA binding protein included in the DNA base editor according to the present invention is any programmable DNA binding protein suitable for use in DNA base editing. Those skilled in the art are well aware of the types of programmable DNA binding proteins suitable for use in DNA base editing. Examples of such DNA-binding proteins include zinc finger proteins and TALE (transcription activator-like effector, TALE) proteins, which can be designed to recognize specific base sequences through modular repeat sequences, dead Cas proteins (e.g., dCas9, dCas12a) that are targeted by guide RNA, and other artificially designed DNA recognition domains. These DNA-binding proteins can be fused to enzymatic proteins (e.g., nickases, cytosine deaminase, or adenine deaminase) to induce a desired biochemical reaction at a specific location in the genome.

[0124] The DNA binding protein used in the DNA base editor according to the present invention is not limited to a specific amino acid sequence or structure, and generally includes a programmable protein or functional variant thereof that has the ability to recognize a specific base sequence and selectively bind to that sequence. The DNA binding protein may be naturally occurring, a variant thereof, or an artificially designed protein. The DNA binding protein that can be used in the DNA base editor according to the present invention is understood to encompass all proteins capable of selectively binding to target DNA to achieve the purpose of the present invention, regardless of the specific sequence or origin.

[0125] The DNA base editor according to the present invention comprises one or more DNA binding proteins. The DNA binding proteins are proteins capable of selectively binding to a specific DNA sequence, enabling specific recognition of the target site for base editing.

[0126] In some embodiments, the DNA binding protein may be selected from the group consisting of a zinc finger protein, a TALE protein, a CRISPR-associated nuclease, or a combination thereof. Reference may be made to prior disclosures regarding zinc finger proteins, TALE proteins, and CRISPR-associated nucleases, including WO 2022 / 060185 and WO2022 / 017745, which are incorporated by reference herein in their entirety.

[0127] The above "zinc finger protein (ZFP)" generally binds to zinc ions (Zn 2+) refers to a protein or protein domain that forms a stabilized structure through the combination of zinc finger domains and has the function of binding to a specific DNA sequence. Such zinc finger proteins contain one or more "zinc finger (ZF)" structures. Zinc finger proteins have sequence specificity that allows them to bind to a DNA sequence consisting of a specific 3-4 base pair, and by designing them by continuously combining multiple zinc finger domains, they can have high specificity and binding affinity for a long target DNA sequence.

[0128] Zinc finger proteins that can be used in some embodiments of the present invention include naturally occurring proteins or artificial recombinants, mutants, functional variants, or variants with improved specificity derived therefrom, and are not limited in origin or sequence composition, as long as they can specifically bind to a desired target DNA sequence. Those skilled in the art can design and produce a desired zinc finger protein using previously disclosed ZFP libraries, genetic engineering methods, and techniques for analyzing DNA-binding specificity.

[0129] Zinc finger proteins have a relatively small molecular weight and can bind to target sequences with only their pure protein structure without relying on an RNA guide sequence, so they have the advantage of being easy to apply even in delivery systems with vector size limitations or in environments where RNA is unstable.

[0130] The above "TALE protein" is generally based on a transcription activator-like factor derived from the plant pathogenic bacteria Xanthomonas genus, and refers to a DNA binding protein with sequence specificity that can bind to a specific DNA sequence. The TALE protein is composed of a series of repeat modules, each module consisting of about 34 amino acids, of which two amino acid residues at positions 12 and 13 (so-called RVD, repeat-variable diresidue) determine the binding specificity for a single DNA base. By designing a combination of these modules, a TALE protein with sequence specificity tailored to a desired target DNA sequence can be generated. As used herein, the TALE-repeat modules may be referred to as a "TALE array", a "TALE repeat sequence", etc., and the expression "TALE protein" means a configuration in which an N-terminal domain and a C-terminal domain (which may include a half domain) are included on both sides of the TALE array, respectively.

[0131] The term "N-terminal domain (NTD)" as used herein refers to a region located at the amino terminus of a TALE protein, which includes a sequence that contributes to the alignment of DNA binding sites or maintenance of protein stability. For example, in a TALE protein derived from Xanthomonas, the N-terminal domain may be composed of a sequence approximately between amino acids 1 and 150. However, such sequences are merely examples, and the "N-terminal domain" in the present specification also includes variants, homologs, or artificially designed sequences of the sequence as long as the sequence can perform the above function. A person skilled in the art can select or design a suitable N-terminal domain sequence based on the structure and function of a known TALE protein.

[0132] The term "C-terminal domain (CTD)" used herein refers to a region located at the carboxy terminus of a TALE protein, which includes a sequence that performs protein stability or other regulatory functions. For example, in Xanthomonas TALE, a sequence corresponding to amino acids 800 to 900 may be included. However, the "C-terminal domain" in the present specification is not limited to such sequence, and also includes other biological sequences or artificial sequences that can perform the same or similar functions. Such sequences can be easily selected or designed by a person skilled in the art based on publicly available TALE protein information.

[0133] TALE proteins that can be used in some embodiments of the present invention include naturally occurring TALE sequences or recombinants, mutants, functional variants, or forms with artificially controlled sequence specificity derived therefrom, and are not limited in sequence composition or origin, as long as they can bind to a desired DNA sequence. Those skilled in the art can utilize published TALE libraries and TALE design algorithms to create TALE proteins that specifically bind to various DNA target sequences. For example, Kim, Yongsub, et al. "A library of TAL effector nucleases spanning the human genome." Nature biotechnology 31.3 (2013): 251-258.

[0134] TALE proteins have the advantage of not requiring guide RNA, precisely recognizing target DNA sequences by directly assembling repeat modules at the protein level, and being free from constraints on PAM sequences. Therefore, TALE proteins are particularly advantageous in complex genomic environments or situations requiring flexible targeting.

[0135] The above "CRISPR-associated nuclease" is also called "Cas protein" and generally refers to a protein having nuclease activity capable of cleaving DNA or RNA derived from the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system, which is an acquired immune system of bacteria or archaea. These Cas proteins generally form a complex with a guide RNA, recognize a target nucleic acid sequence complementary to the base sequence of the guide RNA, and then induce cleavage (nicking or double-strand break) at the corresponding site. Representative examples include Cas9 (e.g., Streptococcus pyogenesCas9), Cas12a (Cpf1), Cas12b, Cas13, and Cas14.

[0136] CRISPR-associated nucleases that may be used in some embodiments of the present invention may include naturally occurring proteins, functional variants, conservative amino acid substitutions, truncated forms, or variants in which the enzymatic activity is altered or eliminated, and also include inactive forms (dead Cas or dCas), nickase forms (nCas), or forms that include fusions with various functional domains (e.g., deaminase, transcription factor, etc.).

[0137] When the base editor according to the present invention has two fusion proteins, the two fusion proteins each comprise a DNA binding protein, which may be identical or different. That is, one of the two fusion proteins may be, for example, a zinc finger protein, a TALE protein, or a CRISPR-associated nuclease, and the other may independently be, for example, a zinc finger protein, a TALE protein, or a CRISPR-associated nuclease.

[0138] B. Deaminase

[0139] The DNA base editor according to the present invention comprises a deaminase.

[0140] The "deaminase" included in the DNA base editor according to the present invention refers to a biological catalyst protein that changes the chemical structure of a base by removing the amino group (-NH2) present in a specific nucleotide base. Through the deamination reaction, cytosine (C) is converted to uracil (U), and adenine (A) is converted to inosine (I). Since inosine is recognized as guanine (G) during DNA replication or transcription, the conversion of A·T → G·C becomes possible.

[0141] The DNA base editor according to the present invention includes an adenine deaminase or a cytosine deaminase, and these may be included together.

[0142] B1. Cytosine deaminase

[0143] In some embodiments, the deaminase included in the DNA base editor according to the present invention is a cytosine deaminase.

[0144] The "cytosine deaminase" included in the DNA base editor according to the present invention generally refers to an enzyme that catalyzes a deamination reaction that converts the cytosine base in DNA to uracil. This enzyme converts cytosine to uracil by removing the amino group (-NH2) of cytosine, thereby inducing a C:G → T:A conversion in the corresponding base pair.

[0145] The cytosine deaminase that can be used in some embodiments of the present invention is not limited to a specific amino acid sequence, structural characteristics, biological species of origin, or name, and includes all wild-type proteins, artificial or evolutionary modifications, functional variants, orthologs, conservative amino acid substitutions, etc., as long as the enzyme has a biological activity that can act on a cytosine base on DNA to induce a deamination reaction. Those skilled in the art can easily access numerous literatures and public databases (e.g., GenBank, UniProt, REBASE, etc.) that are already known regarding the sequence, structure, and function of such enzymes, and can implement a cytosine deaminase for implementing selective DNA editing according to a specific purpose.

[0146] In some embodiments, the cytosine deaminase can be an apolipoprotein B editing complex (APOBEC), an activation-induced deaminase (AID), a double strand DNA-specific cytosine deaminase, or a tRNA-specific adenosine deaminase (TadA) with C-to-T proofreading activity.

[0147] (a) double-stranded DNA-specific cytosine deaminase

[0148] In some embodiments, the cytosine deaminase is a double-stranded DNA-specific cytosine deaminase.

[0149] As used herein, the term "double-stranded DNA-specific cytosine deaminase" refers to an enzyme that deamines the cytosine base in double-stranded DNA, converting it to uracil. Unlike typical cytosine deaminases, this enzyme has the characteristic of directly recognizing and reacting with cytosine within the normal double-stranded DNA structure, rather than single-stranded DNA, as its substrate.

[0150] An example of such a double-stranded DNA-specific deaminase is DddA (DddAtox), a toxic protein derived from Burkholderia cenocepacia, which is known to selectively recognize and convert cytosine in double-stranded DNA to uracil. Because this enzyme can generally induce cytotoxicity, it may be desirable to split it into two parts and fuse them with a DNA-binding protein for use, if necessary.

[0151] The double-stranded DNA-specific cytosine deaminase used in the present invention is not limited to the above examples, and is understood to include all proteins or functional variants thereof that have the activity of deaminating cytosine using double-stranded DNA as a substrate, regardless of its origin, amino acid sequence, structure, or name. Such double-stranded DNA-specific cytosine deaminase and variants thereof have already been described in various documents. For example, WO 2022 / 060185, WO 2022 / 221337, WO 2022 / 155265, WO 2023 / 081855, WO 2023 / 097226, WO 2024 / 112441, WO 2024 / 107263, etc., which are incorporated by reference in their entirety by this application, and contents already known prior to the present application may be cited.

[0152] In some embodiments, the cytosine deaminase is DddAtox, a cytosine deaminase from Burkholderia cenocepacia, or a variant thereof. The amino acid sequence of DddAtox is as follows.

[0153] wild-type DddAtox:

[0154] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNNSNSPKSPTKGGC (SEQ ID NO: 1)

[0155] As variants of DddAtox, various variants are also known that are characterized by the context of the cytosine (C) base to be base-edited, the editing efficiency, the improved specificity, etc., and these include DddA2, DddA3, DddA4, DddA5, DddA6, DddA7, DddA8, DddA9, DddA10, DddA11, etc. With regard to these variants, reference may be made to the contents that were already known prior to the present application, including, for example, WO 2022 / 221337, which is incorporated by reference in its entirety by this application.

[0156] In some embodiments, the present invention can utilize full-length or split forms (e.g., 1333N / 1333C or 1397N / 1397C) of DddAtox as a cytosine deaminase, wherein the split forms are each comprised of separate fusion proteins and can function cooperatively. As used herein, "cooperatively functioning" with respect to split forms of cytosine deaminase means that although existing as separate proteins, they exert cytosine deaminase activity through dimerization.

[0157] Although DddAtox can be used alone, due to its high toxicity and strong enzymatic activity, it is preferably used divided into N-terminal and C-terminal fragments (in the form of split DddAtox) for safer and more efficient DNA base editing. In this case, DddAtox exists as two fragments, each of which is incorporated into or linked to an independent fusion protein to function. When the cytosine deaminase used in the present invention is used in the form of a first fragment and a second fragment, the first fragment and the second fragment do not have deamination activity, and the deaminization activity is exhibited only when the two fragments are adjacent to each other. In other words, in order for the cytosine deaminase to be used in the form of two fragments, the sequences of the two fragments must be combined to form the full-length sequence of the cytosine deaminase, and those skilled in the art of base editing using cytosine deaminase are well aware of this.

[0158] When DddAtox exists in the form of two fragments, one of the two fragments may include a sequence from the N-terminus to the 33rd, 44th, 54th, 68th, 82nd, 98th or 108th amino acid of the amino acid sequence of SEQ ID NO: 1, and the other of the two fragments may include a sequence from the 34th, 45th, 55th, 69th, 83rd, 99th or 109th amino acid of the amino acid sequence of SEQ ID NO: 1 to the C-terminus.

[0159] In some embodiments, when cytosine deaminase is used in the form of two fragments, it may be in the form of 1333N and 1333C having the following amino acid sequences.

[0160] 1333N:

[0161] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGG

[0162] 1333C:

[0163] PTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNNSNSPKSPTKGGC

[0164] In some embodiments, when cytosine deaminase is used in the form of two fragments, it may be in the form of 1397N and 1397C having the following amino acid sequences.

[0165] 1397N:

[0166] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG

[0167] 1397C:

[0168] AIPVKRGATGETKVFTGNNSNSPKSPTKGGC

[0169] In some embodiments, when cytosine deaminase is used in the form of two fragments, it may be in the form of DddA11 1333N and DddA11 1333C having the following amino acid sequences.

[0170] DddA11 1333N:

[0171] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFISGG

[0172] DddA11 1333C:

[0173] PTPYPNYVSAGHVEGQSALFMRDNGISEGLVFHNNPKGTCGFCVNMIETLLPENAKMTVVPPEGAIPVKRGATGETKVFIGNNSNSPKSPTKGGC

[0174] In some embodiments, when cytosine deaminase is used in the form of two fragments, it may be in the form of DddA11 1397N and DddA11 1397C having the following amino acid sequences.

[0175] DddA11 1397N:

[0176] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFISGGPTPYPNYVSAGHVEGQSALFMRDNGISEGLVFHNNPKGTCGFCVNMIETLLPENAKMTVVPPEG

[0177] DddA11 1397C:

[0178] AIPVKRGATGETKVFIGNNSNSPKSPTKGGC

[0179] In some embodiments, when DddAtox or a variant thereof is present in the form of a fragment of 1333N and 1333C, one or more amino acids selected from the group consisting of positions 3, 5, 10, 11, 13, 14, 15, 16, 17, 18, 19, 28, 30 and 31 of 1333N or one or more amino acids selected from the group consisting of positions 13, 16, 17, 20, 21, 28, 29, 30, 31, 32, 33, 56, 57, 58 and 60 of 1333C may be substituted with another amino acid. When DddAtox or its variant exists in the form of fragments 1397N and 1397C, one or more amino acids selected from the group consisting of positions 87, 88, 91, 92, 95, 100, 101, 102 and 103 of 1397N or one or more amino acids selected from the group consisting of positions 13, 14, 15 and 16 of 1397C may be substituted with another amino acid. In some embodiments, the other amino acid is alanine.

[0180] In some embodiments, when DddAtox or a variant thereof exists in the form of fragments 1333N and 1333C, the amino acid at positions 56, 57 or 58 of 1333C may be substituted with another amino acid. When DddAtox or a variant thereof exists in the form of fragments 1397N and 1397C, the amino acid at positions 100, 101 or 102 of 1397N may be substituted with another amino acid. In some embodiments, the other amino acid is alanine.

[0181] The use of these variants allows for highly efficient and precise C-to-T editing without causing undesirable off-target C-to-T editing, as the two pairs of cleavage fragments derived from DddAtox or its variants, each linked to a DNA-binding protein, fail to function properly when they fail to bind DNA. Reference may be made to prior disclosures, including, for example, WO 2022 / 060185, which is incorporated herein by reference in its entirety.

[0182] In some embodiments, the cytosine deaminase is a variant of a double-stranded DNA-specific cytosine deaminase derived from Ruminococcus, having the following amino acid sequence, and referred to herein as WC03.

[0183] WC03:

[0184] NANCNQEKPVLPKYDGKTTEGVMVTPDGKQISFKSGNSSTPSGSYPQYKAQSASHVEGKAALYMRENGINEATVFHNNPNGTCGFCDRQVPALLPKGAKLTVVPPSNSVANNVRAIPVPKTYIGNSTVPKIK

[0185] The above WC03 can be used as a full-length protein, or, if necessary, can be provided in a split form, with each split fused to a DNA-binding protein. In this case, the two splits act cooperatively to selectively convert cytosine in double-stranded DNA to uracil.

[0186] In some embodiments, WC03 is used in the following split form:

[0187] WC03_S257N:

[0188] NANCNQEKPVLPKYDGKTTEGVMVTPDGKQISFKSGNSSTPSGSYPQYKAQSASHVEGKAALYMRENGINEATVFHNNPNGTCGFCDRQVPALLPKGAKLTVVPPSNS

[0189] WC03_S257C:

[0190] VANNVRAIPVPKTYIGNSTVPKIK

[0191] In some embodiments, the cytosine deaminase used in the present invention may be used in a full-length form, and the full-length cytosine deaminase used in this case (e.g., DddA) tox ) are amino acid sequences that have been modified to have no or only low toxicity.

[0192] DddA tox The C-terminus of DNA has a specific concentration of positively charged amino acids. Since DNA is negatively charged, it binds to positively charged amino acids in proteins. By replacing these positively charged amino acids, DddA is formed. tox By weakening the binding force of DddA to DNA, intracellular toxicity can be reduced or eliminated. In other words, if a positively charged amino acid is substituted to make it non-toxic, cloning using E. coli is possible, resulting in full-length DddA. toxcan be secured. Based on this, the non-toxic full-length cytosine deaminase is DddA of sequence number 1. tox It can be provided by replacing one or more, two or more, three or more, four or more, or five or more amino acids in the amino acid sequence with another amino acid (e.g., alanine), and in this regard, reference may be made to contents already known prior to the present application, including, for example, WO 2022 / 060185, which is incorporated by reference in its entirety by this application.

[0193] In some embodiments, when a full-length cytosine deaminase is used as the cytosine deaminase, such cytosine deaminase may have one or more amino acid substitutions selected from the group consisting of a substitution of S at position 37 with G, a substitution of G at position 59 with S, a substitution of A at position 109 with V, and a substitution of S at position 129 with G in the amino acid sequence of SEQ ID NO: 1.

[0194] A cytosine deaminase mutant having all of the following substitutions: S to G at position 37, G to S at position 59, A to V at position 109, and S to G at position 129 in the amino acid sequence of SEQ ID NO: 1 is commonly referred to as “GSVG.”

[0195] GSVG:

[0196] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLEGKVFSSGGTPPYPNYANAGHVESQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGVIPVKRGATGETKVFTGNSNGPKSPTKGGC

[0197] In addition, the non-toxic full-length DddAtox may comprise an amino acid sequence selected from the group consisting of the following amino acid sequences.

[0198] A1341D KRKKA variant:

[0199] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYDNAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTAGGC

[0200] AAAAA 변이체:

[0201] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVAAGATGETAVFTGNSNSPASPTAGGC

[0202] AAAAK 변이체:

[0203] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVAAGATGETAVFTGNSNSPASPTKGGC

[0204] AAKAA 변이체:

[0205] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVAAGATGETKVFTGNSNSPASPTAGGC

[0206] AAKAK 변이체:

[0207] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVAAGATGETKVFTGNSNSPASPTKGGC

[0208] KAAAA 변이체:

[0209] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKAGATGETAVFTGNSNSPASPTAGGC

[0210] E1347A 변이체:

[0211] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVAGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC

[0212] SSVG 변이체:

[0213] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVESQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGVIPVKRGATGETKVFTGNSNGPKSPTKGGC

[0214] GSAG 변이체:

[0215] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLEGKVFSSGGPTPYPNYANAGHVESQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNGPKSPTKGGC

[0216] GSVS 변이체:

[0217] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLEGKVFSSGGPTPYPNYANAGHVESQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGVIPVKRGATGETKVFTGNSNSPKSPTKGGC

[0218] (b) 기타 시토신 탈아미노효소

[0219] In some embodiments, the cytosine deaminase that can be used in the present invention is APOBEC1. This enzyme typically acts on single-stranded DNA, but can be useful for base editing when fused with an enzyme domain having nickase function.

[0220] The above “APOBEC1” is an abbreviation for Apolipoprotein B mRNA Editing Catalytic Polypeptide 1, and refers to an enzyme that has the activity of deaminating cytosine to uracil. APOBEC1 is originally known as an enzyme involved in editing apolipoprotein B mRNA in mammalian hepatocytes, etc., but this enzyme can exhibit the activity of deaminating cytosine bases within a specific sequence of DNA or RNA. The APOBEC1 that can be used in the present invention is not necessarily limited to the naturally occurring wild type, and also includes functional mutants, orthologs, or artificially improved proteins that exhibit similar activity.

[0221] In some embodiments, the cytosine deaminase is APOBEC1 or an ortholog thereof having the following amino acid sequence:

[0222] APOBEC1:

[0223] MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFI YIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK

[0224] In some embodiments, the cytosine deaminase that can be used in the present invention is AID. This enzyme typically acts on single-stranded DNA, but can be useful for base editing when fused with an enzyme domain having nickase function.

[0225] The above “AID” ​​stands for Activation-Induced Cytidine Deaminase, which refers to a cytosine deaminase that plays an essential role in the generation of antibody diversity in the immune system. The AID that can be used in some embodiments of the present invention is not necessarily limited to the naturally occurring wild type, but also includes functional variants thereof, orthologs, or artificially improved proteins that exhibit similar activity.

[0226] In some embodiments, the cytosine deaminase is AID or an ortholog thereof having the amino acid sequence:

[0227] AID:

[0228] MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL

[0229] In some embodiments, the cytosine deaminase that can be used in the present invention is a variant of TadA8e that has been mutated to have cytosine deaminase activity.

[0230] TadA8e:

[0231] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN (SEQ ID NO: 2)

[0232] These variants may include, for example, one or more amino acid residues 6, 26, 27, 28, 46, 48, 49, 61, 74, 76, 77, 82, 96, 107, 108, 112, 114, 115, 119, 122, 127, 142, 143, 151, 154 and 158 of the amino acid sequence of SEQ ID NO: 2 (TadA8e) being mutated to other amino acids. With respect to the composition of such cytosine deaminases, reference may be made to known matters prior to the present application, including WO 2022 / 060185, WO 2023 / 086953, which are incorporated by reference in their entirety herein.

[0233] Thus, the cytosine deaminase used in the present invention is not limited to the name or origin of the enzyme, and includes any protein, a homologue thereof, or a variant thereof having biological activity capable of inducing base correction by selectively converting a cytosine base on a target DNA to uracil.

[0234] B2. Adenine deaminase

[0235] In some embodiments, the deaminase included in the DNA base editor according to the present invention is adenine deaminase. As used herein, "adenine deaminase" is also referred to as "AD."

[0236] The above "adenine deaminase" generally refers to an enzyme that catalyzes the deamination reaction that converts the adenine base in DNA to inosine. Inosine acts similarly to guanine during DNA replication or transcription, resulting in an A:T → G:C conversion in the corresponding base pair.

[0237] The adenine deaminase that can be used in some embodiments of the present invention is not limited to a specific amino acid sequence, structural characteristics, biological species or name, and includes all wild-type proteins, artificial or evolutionary modifications, functional variants, orthologs, conservative amino acid substitutions, etc., as long as the enzyme has biological activity capable of inducing a deamination reaction by acting on an adenine base on DNA. Those skilled in the art can implement an adenine deaminase suitable for a specific purpose by referring to various literature and public databases (e.g., GenBank, UniProt, etc.) regarding the sequence, structure, and function of such enzymes.

[0238] For example, TadA (tRNA-specific adenosine deaminase) from Escherichia coli is a well-known representative adenine deaminase that originally has the activity of deaminating adenosine in tRNA, and has been improved to acquire the activity that can act on DNA through specific mutations or protein engineering.

[0239] In some embodiments, the adenine deaminase has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to TadA having the amino acid sequence of SEQ ID NO: 2 or an ortholog thereof, or a functional variant thereof, or a conservative amino acid substitution thereof.

[0240] In some embodiments, the adenine deaminase has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to TadA8e having the amino acid sequence of SEQ ID NO: 2, or a functional variant thereof, or a conservative amino acid substitution thereof. Such variants may include, for example, one or more amino acids selected from the group consisting of positions 28, 30, 46, 48, 49, 82, 84, 106, 108, 110, and 111 of the amino acid sequence of TadA8e (SEQ ID NO: 2) in which one or more amino acids are mutated to another amino acid or a conservative amino acid substitution thereof. For example, the amino acid variant may include one or more amino acid substitutions selected from the group consisting of V28Q, V28R, A48W, F84M, V106A, K110S, K110T, K110V, R111F, R111Q, R111S, R111T, and R111Y. V28Q means that the 28th valine (V) is mutated to glutamine (Q), and a person skilled in the art who is familiar with amino acid symbols can easily understand the meaning of the mutant notations. With regard to the composition of an adenine deaminase that can be used in the present invention, contents already known prior to the present application, including WO 2022 / 060185, WO 2023 / 086953, etc., which are incorporated by reference in their entirety by this application, may be cited.

[0241] In some embodiments, the adenine deaminase is a variant of TadA8e having the amino acid sequence: V28R.

[0242] V28R:

[0243] SEVEFSHEYWMRHALTLAKRARDERERPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN

[0244] C. MTS

[0245] The DNA base editor according to the present invention comprises MTS.

[0246] With regard to MTS included in the base editor of the present invention, the contents described under the “1. MTS” section of this specification are quoted as is.

[0247] D. Nikkaze

[0248] In some embodiments, a DNA base editor according to the present invention may comprise a nickase.

[0249] A nickase is any enzyme that has the activity of producing a single-stranded break (also known as a "nick") in double-stranded DNA, i.e., cleaving one strand of the DNA double helix but not the other strand.

[0250] The nickase may be selected from the group consisting of MutH, BspD6I, FokI (including homodimers or heterodimers), BsaI, BsmBI, BsmAI, BsrDI, CviPII, BspQI, AlwI and I-TevI, fragments thereof, and mutants thereof, or may be conservative amino acid substitutions thereof.

[0251] The above nickase may be used as a single fusion protein linked to a DNA binding protein and / or another protein, or may be expressed as a separate protein. When multiple fusion proteins are used in the base correction composition according to the present invention, the nickase may be included in only one of the multiple fusion proteins, or the same or different nickases may be included in each of the multiple fusion proteins. When used in the form of a fusion protein, the location of the nickase may vary.

[0252] In some embodiments, when the DNA base editor according to the present invention comprises a nickase, the DNA base editor may comprise a cytosine deaminase or an adenine deaminase. When comprising a cytosine deaminase, it is preferred that the cytosine deaminase used exhibits a deamination activity specific to single-stranded DNA.

[0253] In some embodiments, the nickase is MutH* having the following amino acid sequence:

[0254] MutH*:

[0255] SQPRPLLSPPETEEQLLAQAQQLSGYTLGELAALAGLVTPENLKRDKGWIGVLLEIWLGASAGSKPEQDFAALGVELKTIPVDSLGRPLATTAVCVAPLTGNSGVTWETSHVRH KLKRVLWIPVEGERSIPLAKRRVGSPLLWSPNEEEDRQLREDWEELMMIVLGQIERITARHGEYLQIRPKAANAKALTEAIGARGERILTLPRGFYLKKNFTSALLARHFLIQ

[0256] In some embodiments, the nickase is BspD6I having the following amino acid sequence:

[0257] BspD6I:

[0258] RQLEEVIDLLEVYHEKKNVIEEKIKARFIANKNTVFEWLTWNGFIILGNALEYKNNFVIDEELQPVTHAAGNQPDMEIIYEDFIVLGEVTTSKGATQFKMESEPVTRHYLN KKKELEKQGVEKELYCLFIAPEINKNTFEEFMKYNIVQNTRIIPLSLKQFNMLLMVQKKLIEKGRRLSSYDIKNLMVSLYRTTIECERKYTQIKAGLEETLNNWVVDKEVRF

[0259] E. Additional polypeptide elements (linker, NES, UGI, UDG, etc.)

[0260] The DNA base editor according to the present invention may further comprise additional polypeptide or protein components in addition to (i) a DNA binding protein, (ii) a deaminase selected from adenine deaminase and cytosine deaminase, and (iii) MTS. These additional components are used for the purpose of improving base editing efficiency or controlling intracellular delivery to the target sequence and localization within organelles.

[0261] In some embodiments, the DNA base editor of the present invention may comprise uracil DNA glycosylase (UDG).

[0262] The above "UDG" refers to a DNA repair enzyme that recognizes and excises uracil bases within DNA. UDG typically plays an early role in the base excision repair (BER) pathway, recognizing and excising uracil, which has been aberrantly inserted into DNA due to cytosine deamination. This enzyme is conserved across diverse organisms, including bacteria, eukaryotes, archaea, and viruses, and performs a crucial enzymatic function in targeted gene editing technology.

[0263] The UDG used in the present invention is not limited to a specific amino acid sequence, structure, origin, or biological species, and is generally understood as a concept encompassing all proteins having the activity of recognizing and removing uracil bases in DNA, their truncated forms, or functional variants thereof. UDG is also called UNG (uracil-N-glycosylase).

[0264] When UDG is used in the base correction system according to the present invention, it may be included as part of a fusion protein comprising a DNA binding protein and a deaminase, or it may be expressed (or used) as a separate polypeptide.

[0265] In some embodiments, the DNA base editor of the present invention may comprise a uracil-DNA glycosylase inhibitor (UGI).

[0266] The above "UGI" refers to a protein or polypeptide that inhibits the activity of UDG, an enzyme that typically removes uracil from DNA. This UGI is used to induce stable base substitutions by preventing uracil from being removed by UDG after cytosine is deaminated to uracil during the DNA base proofreading process.

[0267] The UGI that can be used in some embodiments of the present invention is not limited to a specific amino acid sequence, origin or structural characteristic, and is understood as a concept that includes any protein or functional variant thereof that has the function of inhibiting the activity of UDG.

[0268] When UGI is used in the base correction system according to the present invention, it may be included as part of a fusion protein comprising a DNA binding protein and a deaminase, or it may be expressed (or used) as a separate polypeptide.

[0269] In some embodiments, the DNA base editor of the present invention may comprise a NES.

[0270] The above "NES (nuclear export signal)" refers to a peptide sequence or a functional variant thereof capable of inducing protein movement into the cytoplasm. NES generally has the function of binding to a nuclear export receptor (exportin) to transport a protein from the nucleus to the cytoplasm, and typically has a structural characteristic in which 4 to 5 hydrophobic amino acids are arranged at specific intervals. The NES used in some embodiments of the present invention is not limited to a specific sequence, structure, or origin, and may include various forms of nuclear export sequences as long as the functional characteristics are maintained.

[0271] The NES used in some embodiments of the present invention may be derived from various proteins existing in nature, and artificially designed sequences may also be utilized. For example, the NES derived from the HIV-1 Rev protein (e.g., LQLPPLERLTL), the NES derived from the PKI protein (e.g., LALKLAGLDI), or the NES derived from the NS2 protein of the mouse minivirus (MVM) (e.g., VDEMTKKFGTLTIHDTEK) are widely known as representative sequences, and various variants having similar nuclear export functions are also well known to those skilled in the art.

[0272] These signal sequences are generally preferably located at the N-terminus of the fusion protein comprising the DNA binding protein and the deaminase, and can be appropriately designed depending on the cellular organelle in which the proofreading system is to function.

[0273] F. fusion protein

[0274] All or part of the components constituting the DNA base editor according to the present invention may be present in the form of a fusion protein.

[0275] When the DNA base editor according to the present invention comprises a double-stranded DNA-specific cytosine deaminase as a cytosine deaminase, the cytosine deaminase exists in one full-length form or in two split forms.

[0276] When the double-stranded DNA-specific cytosine deaminase is used in its full-length form, the DNA base editor according to the present invention comprises a single fusion protein, wherein the fusion protein comprises MTS, a DNA binding protein, and the double-stranded DNA-specific cytosine deaminase, and optionally may comprise an adenine deaminase. In this case, the double-stranded DNA-specific cytosine deaminase used in its full-length form is a non-toxic variant (e.g., GSVG).

[0277] When a double-stranded DNA-specific cytosine deaminase is used in the form of two fragments, the DNA base editor according to the present invention comprises two fusion proteins. The two fusion proteins each independently comprise a DNA binding protein, and the cytosine deaminase exists in the form of two fragments, one fragment being included in each fusion protein. The adenine deaminase may optionally be included, and when the adenine deaminase is included, it may be included in either or both of the two fusion proteins. MTS may also be included in either or both of the two fusion proteins, but is preferably included in both fusion proteins.

[0278] Even when the DNA base editor according to the present invention comprises a nickase, the DNA base editor according to the present invention may comprise one or two fusion proteins.

[0279] When the DNA base editor according to the present invention comprises a nickase and comprises one fusion protein, the fusion protein comprises MTS, a DNA binding protein, and a nickase, and comprises an adenine deaminase or a cytosine deaminase.

[0280] When the DNA base editor according to the present invention comprises a nickase and comprises two fusion proteins, the two fusion proteins each independently comprise one DNA binding protein, and one of the two fusion proteins comprises a nickase and the other fusion protein comprises an adenine deaminase or a cytosine deaminase.

[0281] In all embodiments, the fusion protein comprised in the DNA base editor according to the present invention does not comprise an affinity tag.

[0282] F-1. Unit fusion protein A

[0283] In some embodiments, the DNA base editor according to the present invention may comprise (i) a DNA binding protein, (ii) a cytosine deaminase (derived from a double-stranded DNA-specific cytosine deaminase) in the form of a single fusion protein.

[0284] The sequence of (i) DNA binding protein and (ii) cytosine deaminase included in the fusion protein may vary.

[0285] In some preferred embodiments, the sequence of the components within the fusion protein is as follows. The sequence below is intended to indicate the relative positions of the components shown, and other components that may be present, including linkers, are omitted.

[0286] (N-terminal) [DNA binding protein] - [cytosine deaminase] (C-terminal)

[0287] The above-mentioned polypeptide components may be directly linked to each other or linked via one or more linkers.

[0288] The above-described unit fusion protein preferably comprises an MTS at the N-terminus. In some embodiments, the MTS is directly linked to the DNA binding protein, or when linked via a linker or other polypeptide component, the linker or other polypeptide component comprises fewer than five anionic amino acids.

[0289] Various additional polypeptide components described in the "E. Additional Polypeptide Components" section above may be added to the arrangement described above. These may also be directly linked to other polypeptide components or linked via one or more linkers.

[0290] F-2. Unit fusion protein B

[0291] In some embodiments, a DNA base editor according to the present invention may comprise (i) a DNA binding protein, (ii) a cytosine deaminase (derived from a double-stranded DNA-specific cytosine deaminase), and (iii) an adenine deaminase in the form of a single fusion protein.

[0292] The sequence of (i) DNA binding protein, (ii) cytosine deaminase, and (iii) adenine deaminase included in the fusion protein may vary.

[0293] In some preferred embodiments, the sequence of the components within the fusion protein is as follows. The sequence below is intended to indicate the relative positions of the components shown, and other components that may be present, including linkers, are omitted.

[0294] (N-terminal) [DNA binding protein] - [cytosine deaminase] - [adenine deaminase] (C-terminal)

[0295] (N-terminal) [DNA binding protein] - [adenine deaminase] - [cytosine deaminase] (C-terminal)

[0296] The above-mentioned polypeptide components may be directly linked to each other or linked via one or more linkers.

[0297] The above-described unit fusion protein preferably comprises an MTS at the N-terminus. In some embodiments, the MTS is directly linked to the DNA binding protein, or when linked via a linker or other polypeptide component, the linker or other polypeptide component comprises fewer than five anionic amino acids.

[0298] Various additional polypeptide elements described in the above “E. Additional Polypeptide Elements” section may be added to the arrangement described above. These may also be directly linked to other polypeptide elements or linked via one or more linkers.

[0299] F-3. Unit fusion protein C

[0300] In some embodiments, the DNA base editor according to the present invention may comprise (i) a DNA binding protein, (ii) a nickase, and (iii) a cytosine deaminase (a single-stranded DNA-specific cytosine deaminase) in the form of a single fusion protein.

[0301] The sequence of (i) DNA binding protein, (ii) nickase, and (iii) cytosine deaminase included in the fusion protein can vary.

[0302] In some preferred embodiments, the sequence of the components within the fusion protein is as follows. The sequence below is intended to indicate the relative positions of the components shown, and other components that may be present, including linkers, are omitted.

[0303] (N-terminal) [DNA binding protein] - [nickase] - [cytosine deaminase] (C-terminal)

[0304] (N-terminal) [DNA binding protein] - [cytosine deaminase] - [nickase] (C-terminal)

[0305] The above-mentioned polypeptide components may be directly linked to each other or linked via one or more linkers.

[0306] The above-described unit fusion protein preferably comprises an MTS at the N-terminus. In some embodiments, the MTS is directly linked to the DNA binding protein, or when linked via a linker or other polypeptide component, the linker or other polypeptide component comprises fewer than five anionic amino acids.

[0307] Various additional polypeptide elements described in the above “E. Additional Polypeptide Elements” section may be added to the arrangement described above. These may also be directly linked to other polypeptide elements or linked via one or more linkers.

[0308] F-4. Unit fusion protein D

[0309] In some embodiments, the DNA base editor according to the present invention may comprise (i) a DNA binding protein, (ii) a nickase, and (iii) an adenine deaminase in the form of a single fusion protein.

[0310] The sequence of (i) DNA binding protein, (ii) nickase, and (iii) adenine deaminase included in the fusion protein can vary.

[0311] In some preferred embodiments, the sequence of the components within the fusion protein is as follows. The sequence below is intended to indicate the relative positions of the components shown, and other components that may be present, including linkers, are omitted.

[0312] (N-terminal) [DNA binding protein] - [nickase] - [adenine deaminase] (C-terminal)

[0313] (N-terminal) [DNA binding protein] - [adenine deaminase] - [nickase] (C-terminal)

[0314] The above-mentioned polypeptide components may be directly linked to each other or linked via one or more linkers.

[0315] The above-described unit fusion protein preferably comprises an MTS at the N-terminus. In some embodiments, the MTS is directly linked to the DNA binding protein, or when linked via a linker or other polypeptide component, the linker or other polypeptide component comprises fewer than five anionic amino acids.

[0316] Various additional polypeptide components described in the "E. Additional Polypeptide Components" section above may be added to the arrangement described above. These may also be directly linked to other polypeptide components or linked via one or more linkers.

[0317] F-5. Unit fusion protein E

[0318] In some embodiments, the DNA base editor according to the present invention may comprise (i) a DNA binding protein and (ii) a nickase in the form of a single fusion protein.

[0319] The sequence of (i) DNA binding protein and (ii) nickase included in the fusion protein may vary.

[0320] In some preferred embodiments, the sequence of the components within the fusion protein is as follows. The sequence below is intended to indicate the relative positions of the components shown, and other components that may be present, including linkers, are omitted.

[0321] (N-terminal) [DNA binding protein] - [nickase] (C-terminal)

[0322] The above-mentioned polypeptide components may be directly linked to each other or linked via one or more linkers.

[0323] The above unit fusion protein is preferably used together with a separate unit fusion protein comprising adenine deaminase or cytosine deaminase.

[0324] The above-described unit fusion protein preferably comprises an MTS at the N-terminus. In some embodiments, the MTS is directly linked to the DNA binding protein, or when linked via a linker or other polypeptide component, the linker or other polypeptide component comprises fewer than five anionic amino acids.

[0325] Various additional polypeptide elements described in the above “E. Additional Polypeptide Elements” section may be added to the arrangement described above. These may also be directly linked to other polypeptide elements or linked via one or more linkers.

[0326] F-6. Unit fusion protein F

[0327] In some embodiments, the DNA base editor according to the present invention may comprise (i) a DNA binding protein, (ii) a cytosine deaminase (a single-stranded DNA-specific cytosine deaminase) in the form of a single fusion protein.

[0328] The sequence of (i) DNA binding protein and (ii) cytosine deaminase included in the fusion protein may vary.

[0329] In some preferred embodiments, the sequence of the components within the fusion protein is as follows. The sequence below is intended to indicate the relative positions of the components shown, and other components that may be present, including linkers, are omitted.

[0330] (N-terminal) [DNA binding protein] - [cytosine deaminase] (C-terminal)

[0331] The above-mentioned polypeptide components may be directly linked to each other or linked via one or more linkers.

[0332] The above unit fusion protein is preferably used together with a separate unit fusion protein containing a nickase.

[0333] The above-described unit fusion protein preferably comprises an MTS at the N-terminus. In some embodiments, the MTS is directly linked to the DNA binding protein, or when linked via a linker or other polypeptide component, the linker or other polypeptide component comprises fewer than five anionic amino acids.

[0334] Various additional polypeptide components described in the "E. Additional Polypeptide Components" section above may be added to the arrangement described above. These may also be directly linked to other polypeptide components or linked via one or more linkers.

[0335] F-7. Unit fusion protein G

[0336] In some embodiments, a DNA base editor according to the present invention may comprise (i) a DNA binding protein and (ii) an adenine deaminase in the form of a single fusion protein.

[0337] The sequence of (i) DNA binding protein and (ii) adenine deaminase included in the fusion protein may vary.

[0338] In some preferred embodiments, the sequence of the components within the fusion protein is as follows. The sequence below is intended to indicate the relative positions of the components shown, and other components that may be present, including linkers, are omitted.

[0339] (N-terminal) [DNA binding protein] - [adenine deaminase] (C-terminal)

[0340] The above unit fusion protein is preferably used in conjunction with a separate unit fusion protein comprising a nickase or a double-stranded DNA-specific cytosine deaminase.

[0341] The above-mentioned polypeptide components may be directly linked to each other or linked via one or more linkers.

[0342] The above-described unit fusion protein preferably comprises an MTS at the N-terminus. In some embodiments, the MTS is directly linked to the DNA binding protein, or when linked via a linker or other polypeptide component, the linker or other polypeptide component comprises fewer than five anionic amino acids.

[0343] Various additional polypeptide elements described in the above “E. Additional Polypeptide Elements” section may be added to the arrangement described above. These may also be directly linked to other polypeptide elements or linked via one or more linkers.

[0344] F-8. Linker

[0345] The term "linker" as used herein refers to an amino acid linker, which is an amino acid sequence that covalently connects two or more functional protein domains, peptides, or other biological molecular elements. Such linkers serve to provide sufficient flexibility, length, or spatial separation so that each connected component can maintain its own structural or functional activity, and may sometimes be designed to include a specific secondary structure (e.g., an α-helix) or recognition sequence. For example, a repeating sequence based on glycine (G) and serine (S) (e.g., GGGGS)n is known as a representative example that confers high flexibility and water solubility.

[0346] The linker that can be used in the DNA editing editor according to the present invention is not limited to a specific amino acid sequence, length, or structure. It can be a naturally occurring sequence or an artificially designed sequence, and any amino acid sequence with various lengths, sequence combinations, or structural characteristics is included, as long as the function of each component connected via the linker is substantially maintained. Those skilled in the art can select or design an appropriate linker based on the characteristics of the target domain and the intended application. Selecting an appropriate linker is crucial for reducing interference between each domain and improving overall base editing efficiency.

[0347] In some embodiments, the DNA editing editor according to the present invention may comprise one or more linkers selected from the following linkers:

[0348] 2a.a. Linker: GS

[0349] 5a.a. Linker: TGEKQ

[0350] 10a.a. Linker: SGAQGSTLDF

[0351] 13a.a. Linker: AAEFGIRIPGEKP

[0352] 14a.a. Linker: AAEFGIHGVPAAMG

[0353] 16a.a. Linker: SGSETPGTSESATPES

[0354] 24a.a. Linker: SGTPHEVGVYTLSGTPHEVGVYTL

[0355] 32a.a. Linker: GSGGSSGGSSGSETPGTSESATPESSGGSSGGS

[0356] F-9. Monomeric Base Editor

[0357] According to one embodiment of the present invention, the base editor may be a monomeric form composed of a single fusion protein. The protein includes a mitochondrial targeting sequence (MTS) for translocation into mitochondria, a DNA binding protein capable of binding to a specific target sequence, and an adenine deaminase or cytosine deaminase for base editing.

[0358] Monomeric base editors offer the advantages of minimizing vector size due to their single protein structure and facilitating expression and assembly. Furthermore, their single transport pathway into mitochondria allows for more consistent enzymatic activity.

[0359] In some embodiments where the base editor according to the present invention is a monomeric base editor, the unit fusion protein may be any one of unit fusion proteins A to D.

[0360] In the case of a monomeric base editor comprising a single fusion protein A or B, the cytosine deaminase is preferably a non-toxic variant (e.g., GSVG) of a double-stranded DNA-specific cytosine deaminase.

[0361] A monomeric base editor containing a unit fusion protein A is suitable for C-to-T proofreading.

[0362] A monomeric base editor containing a unit fusion protein B is suitable for A-to-G proofreading.

[0363] In the case of a monomeric base editor comprising a unit fusion protein C or D, the cytosine deaminase is preferably a cytosine deaminase exhibiting deamination activity toward single-stranded DNA (e.g., APOBEC1 or AID).

[0364] A monomeric base editor containing a unit fusion protein C is suitable for C-to-T proofreading.

[0365] A monomeric base editor containing a unit fusion protein D is suitable for A-to-G proofreading.

[0366] F-10. Dimeric Base Editor

[0367] In some embodiments, the base editor of the present invention may be a dimeric form comprising two fusion proteins, each of which comprises a DNA binding protein capable of binding to a specific target sequence. The DNA binding proteins contained in each of the two fusion proteins may be of the same or different types.

[0368] In some embodiments where the base editor of the present invention is in a dimeric form, at least one of the two fusion proteins comprises MTS, and it is preferred that both fusion proteins each comprise MTS.

[0369] In some embodiments where the base editor of the present invention is in a dimeric form, at least one of an adenine deaminase or a cytosine deaminase is included in at least one of the two fusion proteins.

[0370] The dimeric form provides structural flexibility, allowing each fusion protein to bind to adjacent target sites, leading to synergistic or specific activation between the two domains. Specifically, when the cytosine deaminase is split into two halves, each incorporated into a left / right fusion protein, this can reduce nonspecific activity at off-target sites and enable efficient editing at the target site.

[0371] Furthermore, when only one fusion protein contains adenine deaminase, selective or simultaneous correction of adenine and cytosine is possible, depending on the combination. This structure offers the advantage of flexibility in complex base editing situations.

[0372] In some embodiments where the base editor according to the present invention is a monomeric base editor, the unit fusion protein may be any one of the unit fusion proteins A, B, E, F and G, and the combination of two fusion proteins may be as follows.

[0373] [Combination 1] First fusion protein: unit fusion protein A; Second fusion protein: unit fusion protein A

[0374] [Combination 2] First fusion protein: unit fusion protein A; Second fusion protein: unit fusion protein B

[0375] [Combination 3] First fusion protein: Unit fusion protein B; Second fusion protein: Unit fusion protein A

[0376] [Combination 4] First fusion protein: unit fusion protein E; Second fusion protein: unit fusion protein F

[0377] [Combination 5] First fusion protein: unit fusion protein E; Second fusion protein: unit fusion protein G

[0378] [Combination 6] First fusion protein: unit fusion protein F; Second fusion protein: unit fusion protein E

[0379] [Combination 7] First fusion protein: unit fusion protein G; Second fusion protein: unit fusion protein E

[0380] The above combinations 1, 4 and 6 are suitable for C-to-T correction.

[0381] The above combinations 2, 3, 5 and 7 are suitable for A-to-T correction.

[0382] In some embodiments where the base editor according to the present invention is a dimeric base editor, DNA editing occurs in the region between the DNA sequences to which the two DNA binding proteins included in the two fusion proteins each bind, said region being referred to as a "spacer."

[0383] G. Base correction efficacy

[0384] The base editor according to the present invention has optimized various structural elements to enhance the base editing efficiency of mitochondrial DNA. In particular, the introduction of the following components was effective in improving editing efficiency:

[0385] (A) Optimization of the mitochondrial targeting sequence (MTS)

[0386] Instead of MTS, which is commonly used for mitochondrial DNA base correction, such as COX8A, the present invention screens various new MTS candidates to secure MTS with high mitochondrial DNA base correction efficiency.

[0387] (B) Remove tag sequence

[0388] By removing affinity tag sequences such as FLAG and HA, the negative impact on intracellular protein expression and movement was minimized, thereby increasing base editing efficiency.

[0389] (C) Domain configuration combination

[0390] By evaluating various combinations of cytosine deaminase in full-length or split form, presence and location of adenine deaminase, and fusion protein arrangement in monomeric or dimer form, it was confirmed that certain combinations exhibited high correction efficiency.

[0391] This combination of optimized elements enables effective base editing within mitochondria, which can be used to treat genetic diseases by correcting specific genetic mutations.

[0392] In some embodiments, the base editor according to the present invention is suitable for correcting (A-to-G) the G11778A mutation in a mitochondrial gene. The G11778A mutation in a mitochondrial gene is a genetic mutation that occurs in patients with LHON, a type of optic neuropathy of unknown cause that causes loss of vision in both eyes. When LOC, SDHD, SDHA, or GHITM are used as MTS, the A-to-G correction efficiency for the G11778A mutation is high, and the correction efficiency is particularly excellent when SDHD or SDHA is used, and it was confirmed that the same effect is obtained even when SDHD is truncated at the C-terminus.

[0393] In some embodiments, the base editor according to the present invention is suitable for correcting (A-to-G) the G3460A mutation in a mitochondrial gene. The G3460A mutation in a mitochondrial gene is also a genetic mutation found in patients with LHON. It has been confirmed that the use of SDHD as an MTS results in a high A-to-G correction efficiency for the G3460A mutation.

[0394] 4. Polynucleotide

[0395] One aspect of the present invention relates to a polynucleotide encoding the DNA base editor of the present invention. With respect to the "DNA base editor of the present invention," the content described in the "3. DNA base editor" section of this specification is incorporated herein by reference.

[0396] The polynucleotide according to the present invention is a polynucleotide encoding the DNA base editor of the present invention as described above, and the polynucleotide may be DNA or RNA. The DNA or RNA includes a sequence contained in mRNA, cDNA, synthetic DNA, plasmid DNA, linear DNA, or a viral vector.

[0397] The polynucleotide according to the present invention may be a single polynucleotide encoding the monomeric base editor as described above, or may be separate polynucleotides each encoding two fusion proteins constituting the dimeric base editor, and may be produced in the form of a single polynucleotide using the Golden Gate technique.

[0398] A person skilled in the art can easily obtain the amino acid sequence of each constituent protein by referring to the contents disclosed in the present application specification and the published protein sequences registered in databases such as NCBI GenBank and UniProt, and produce a polynucleotide according to the present invention. Based on the obtained amino acid sequences, a nucleotide sequence can be designed considering the codon usage frequency suitable for the host organism, and further, it is obvious to a person skilled in the art or within the scope of routine experimental techniques to produce a polynucleotide containing the sequence using commercial services for custom production of synthetic genes (e.g., IDT, GenScript, etc.) and vector cloning, PCR amplification, and DNA assembly technologies (Gibson assembly, Golden Gate, etc.).

[0399] The polynucleotide(s) may include expression control sequences (e.g., promoter) in addition to the coding region so as to sufficiently express the function of the encoded protein(s).

[0400] In some embodiments of the present invention, the polynucleotide may be codon optimized to suit the type of expression system (e.g., bacteria, plants, mammalian cells, etc.). Codon optimization is a general technique for increasing gene expression efficiency, and can increase protein expression by adjusting the nucleotide sequence according to the tRNA utilization frequency of the target organism.

[0401] 5. Base correction system

[0402] One aspect of the present invention relates to a mitochondrial DNA base editing system comprising the base editor of the present invention or a polynucleotide encoding the same. With respect to the "base editor of the present invention" and the "polynucleotide" encoding the same, the contents described in the "3. DNA base editor" and "4. polynucleotide" sections of this specification are incorporated herein by reference.

[0403] The base correction system according to the present invention may comprise a single base editor or polynucleotide molecule, or may comprise two or more base editors or polynucleotides. Such configurations are as described throughout this specification.

[0404] 6. Base correction composition

[0405] One aspect of the present invention relates to a mitochondrial DNA base editing composition comprising the DNA base editor of the present invention or a polynucleotide encoding the same. With respect to the "DNA base editor of the present invention" and the "polynucleotide" encoding the same, the contents described in the "3. DNA base editor" section of this specification are incorporated herein by reference.

[0406] The base correction composition according to the present invention is for correcting bases in nuclear or mitochondrial DNA.

[0407] The base correction composition according to the present invention may comprise the DNA base editor of the present invention or a polynucleotide encoding the same as described above and a biocompatible carrier.

[0408] The above "biocompatible carrier" refers to a material that can be included in the composition according to the present invention, and which does not significantly inhibit physiological functions when in contact with a biological system—e.g., cells, tissues, organs, or entire organisms of humans, animals, or plants—and does not induce toxic or immune reactions, etc. Such a carrier can be selected in various ways depending on pharmaceutical, biological, or agricultural use, and can play a role in improving the stability, permeability, and delivery efficiency of a base correction enzyme or a protein, nucleic acid, or auxiliary molecule related thereto.

[0409] The biocompatible carrier that can be used in the base correction composition according to the present invention is not limited to a specific chemical structure, physical form or origin, and may include, for example, a buffer solution, a surfactant, a liposome, a nanoparticle, a hydrogel, a polymeric material (e.g., PEG, PVA, PLA, PLGA), a natural or synthetic polysaccharide (e.g., dextran, hyaluronic acid, chitosan), a plant- or microbial-derived polymer, a liposome, a micelle, a biopolymer, or a combination thereof. In addition, the biocompatible carrier may be appropriately selected or combined depending on a specific delivery route or application target (e.g., human tissue, animal tissue, plant tissue, etc.), and may also include a biologically acceptable solvent, preservative, stabilizer, buffer, surfactant, reducing agent, etc.

[0410] A person skilled in the art can easily select and combine a carrier suitable for a given application purpose, delivery route, or target organism based on information already widely known through various literature and public databases regarding the types, properties, and application methods of the biocompatible carriers.

[0411] The base correction composition according to the present invention may be provided in various physical forms. The physical form may be selected based on the intended application, route of administration, stability, storage conditions, or manufacturing process, and falls within the general pharmaceutical design criteria for enhancing the efficacy and ease of use of the composition.

[0412] In some embodiments, the base correction composition according to the present invention may be in the form of a liquid, suspension, gel, powder, lyophilisate, tablet, capsule, or injectable composition. It may also be formulated as a liposome, nanoparticle, lipid nanoparticle (LNP), or other delivery particle.

[0413] The base correction composition aspect of the present invention is not limited to the above physical form, and all formulation modifications that can be appropriately selected and manufactured by a person skilled in the art according to the purpose are included in the scope of the present invention.

[0414] The base correction composition according to the present invention can be applied in various ways to ensure effective delivery to the target cell, tissue, or organism. The application method may vary depending on the target species, cell type, delivery route, or formulation characteristics, and is selected based on the stability, efficacy, and biological compatibility of the composition.

[0415] The base correction composition of the present invention can be applied to plants or plant cells. Methods for applying to plants may include agroinfiltration, Agrobacterium-mediated delivery, gene gun delivery, electroporation, or direct intratissue injection. The composition to be applied may be prepared in the form of protein, DNA, mRNA, or ribonucleoprotein (RNP), and may be used with a delivery vehicle capable of penetrating plant cell walls (e.g., cell-penetrating peptide, non-targeting nanoparticle, etc.) depending on the purpose.

[0416] The base correction composition of the present invention may be applied to animals or animal cells. Methods for application to animal cells include liposomes, lipofectamine, polymer-based carriers (e.g., PEI), electroporation, microinjection, and gene delivery methods using viral vectors (e.g., AAV, lentivirus). For in vivo application, the composition may be administered via various routes, such as intramuscular, intravenous, intraperitoneal, intravitreal, or intracerebral. The composition may be formulated in the form of protein, mRNA, DNA, or ribonucleoprotein (RNP), and may be combined with lipid nanoparticles (LNPs) or membrane fusion peptides to increase delivery efficiency and target specificity. These delivery methods may be selected comprehensively considering the type of target cell or tissue, the therapeutic purpose, and the possibility of inducing an immune response.

[0417] The base correction composition of the present invention can be flexibly applied to various biological subjects, and any in vivo, ex vivo, or in vitro delivery method that can be selected by a person skilled in the art to achieve the desired effect is included in the scope of application of the present invention.

[0418] The base correction composition of the present invention can be used to directly manipulate cells in an extracellular environment (in vitro), and can also be used for the purpose of directly inducing base correction in an intracellular environment (in vivo).

[0419] The base correction composition according to the present invention can be usefully applied to base correction technology that enables precise manipulation of genetic sequences by selectively converting specific DNA bases in vivo or in vitro. In particular, the composition can be used to precisely correct a desired target base sequence.

[0420] Because this correction reaction occurs without DNA double-strand breaks, it has the advantage of being less mutagenic and enhancing genome stability compared to existing gene editing technologies. Therefore, the composition of the present invention can be widely utilized in various fields, including plant variety improvement, industrial microorganism improvement, and biotechnology research.

[0421] 7. Transmitter

[0422] One aspect of the present invention relates to a delivery system comprising the DNA base editor of the present invention or a polynucleotide encoding the same. With respect to the "DNA base editor of the present invention" and the "polynucleotide" encoding the same, the contents described in the "3. DNA base editor" and "4. polynucleotide" sections of this specification are incorporated herein by reference.

[0423] The delivery vehicle according to the present invention refers to a means for effectively delivering the DNA base editor of the present invention or one or more polynucleotides (e.g., DNA, mRNA, etc.) encoding the same to a target site in a cell or a living body. Such a delivery vehicle may include various components to enhance the cell penetration efficiency, intracellular stability, organelle targeting ability, or in vivo distribution characteristics of the editor component, and preferably has acceptable properties such as biocompatibility, biodegradability, and non-immunogenicity. The delivery vehicle of the present invention aims to increase the efficiency and specificity of gene correction, while minimizing cytotoxicity and reducing the possibility of affecting non-target tissues.

[0424] The term "vector" or "delivery vehicle" as used herein refers to a biological or non-biological means capable of effectively delivering the DNA base editor of the present invention or one or more polynucleotides encoding it into cells. These delivery vehicles can be categorized into various types based on their structure, origin, mechanism of action, etc., and can be appropriately selected depending on the intended application target (e.g., plant cells, bacteria, etc.) and administration method.

[0425] In some embodiments, the vector may be a viral vector, including but not limited to adeno-associated virus (AAV), lentivirus, adenovirus, retrovirus, bacteriophage-based vector, and the like.

[0426] In other embodiments, the carrier may be a non-viral carrier, including, for example, lipid nanoparticles (LNPs), polymeric nanoparticles, cationic liposomes, lipofectins, peptide-based carriers, electroporation, or nanoneedle-based systems.

[0427] Additionally, in some embodiments for application to plants, the carrier may comprise a physical delivery means implemented by Agrobacterium tumefaciens strains, plant virus-based vectors, protoplast delivery systems, or gene gun technology.

[0428] Additionally, in some embodiments applied to animal cells, the delivery vehicle may be selected considering the structural characteristics and physiological environment of the animal cell. In the case of viral delivery vehicles, adeno-associated virus (AAV), lentivirus, adenovirus, retrovirus, etc. can be widely used because of their excellent cell infection specificity and high gene expression efficiency. Non-viral delivery vehicles may include lipid nanoparticles (LNP), cationic liposomes, polymeric nanoparticles, cell-penetrating peptides, electroporation, microinjection, or water-soluble polymer conjugates. These delivery vehicles can be appropriately selected depending on the type of target cell, therapeutic purpose, possibility of inducing an immune response, route of administration, etc., and in vivo delivery is possible through various routes such as intravenous, intramuscular, intraperitoneal, or intravitreal. In some cases, the delivery vehicle may additionally include a tissue-specific targeting function (ligand-based targeting).

[0429] A person skilled in the art can select and combine appropriate carriers based on known techniques, depending on the characteristics of a specific base editor system, the delivery route, and the type of cell or organism to which it is applied.

[0430] The delivery system according to the present invention is applicable to various cells and organisms and can be selectively adjusted according to the purpose. Specifically, the delivery target includes a eukaryotic cell or a prokaryotic cell, and in some embodiments, the delivery target may be a plant tissue, cell, or embryo.

[0431] 8. Base correction method

[0432] One aspect of the present invention relates to a method for correcting a base in mitochondrial DNA, comprising introducing the DNA base correction system of the present invention as described above into a cell containing target DNA for base correction, or expressing the DNA base editor within a cell containing target DNA for base correction. With respect to the "DNA base correction system of the present invention," the contents described in the "5. Base correction system" section of this specification and related sections cited therefrom are incorporated herein by reference.

[0433] In some embodiments, the method comprises introducing a DNA base editor, a polynucleotide encoding the same, a base correction composition, and a carrier as described above into a cell comprising target DNA for base correction.

[0434] The above method is a method for correcting bases in mitochondrial DNA.

[0435] The above base correction method can be performed in vitro, ex vivo, or in vivo, and can be designed for various application purposes, such as research purposes and trait improvement purposes. In particular, the DNA base editor of the present invention can efficiently correct bases in target DNA, and thus has wide applicability, such as the introduction of agriculturally useful traits and the exploration of functional genes.

[0436] The components used in the base correction method of the present invention may include one or more of the DNA base editor described above, a polynucleotide encoding the same (e.g., mRNA or DNA), and a carrier (e.g., adeno-associated virus vector, lipid nanoparticle, etc.) containing the components. The components may be introduced into cells singly or in combination, and when present in the form of a fusion protein, stable expression and base correction activity can be provided through optimized binding between the constituent proteins. In addition, the components may additionally include NES, etc., as needed.

[0437] The base correction method of the present invention is characterized by selectively correcting a specific base on DNA to another base, and the target of correction may be various, such as a mutation causing a genetic disease, an abnormal expression control region, or an artificial mutation for imparting a specific trait.

[0438] The method for expressing the DNA base editor or its components for implementing the base correction method of the present invention is not particularly limited and can be performed using technical means widely known to those skilled in the art. For example, a polynucleotide encoding a desired protein can be cloned into an appropriate expression vector, then introduced into a cell to induce transcription and / or translation, thereby causing expression. The expression can include both transient or stable expression within the cell, and can be implemented in various ways depending on the type of expression vector (e.g., plasmid, viral vector, etc.), the selection of promoter, the cell type, the introduction method, etc. Those skilled in the art can select and apply an appropriate expression system and conditions considering the desired cell type and base correction efficiency.

[0439] In the base correction method of the present invention, the DNA base editor or a composition comprising the same can be introduced into cells through various physical or chemical methods. For example, lipofection, electroporation, microinjection, viral vector delivery, nanoparticle delivery, Agrobacterium-mediated delivery, etc. can be used, and an appropriate delivery method can be selected depending on the type of cell being introduced, the target gene location, the target organism species, etc. In addition, the introduction conditions (e.g., pH, temperature, incubation time, introduction amount, etc.) can be easily optimized by a person skilled in the art, taking into account the desired base correction efficiency and cell viability.

[0440] 9. Methods for improving base correction efficiency

[0441] One aspect of the present invention relates to a method for improving mitochondrial DNA base editing efficiency, characterized by removing an affinity tag from a mitochondrial base editing system.

[0442] In some embodiments, the method comprises:

[0443] (1) a step of measuring the base correction efficiency of a base editor comprising a DNA binding protein, (2) one or more deaminase selected from adenine deaminase and cytosine deaminase, (3) MTS, and (4) an affinity tag for target mitochondrial DNA, and

[0444] A step of providing a base correction system comprising a tag-free base editor having the same amino acid sequence as the base editor but not including an affinity tag, or a polynucleotide encoding the same.

[0445] The base editor including the affinity tag used in the above method and the tagless base editor are identical except for the presence of the tag. Regarding the possible configurations of the "base editor", including "DNA binding protein", "adenine deaminase", "cytosine deaminase", "nickase", "MTS", and "affinity tag" that may be components of these, the contents described in the sections "1. MTS", "2. Tagless", "3. DNA base editor" and related sections cited therefrom are cited as is.

[0446] Among the above methods, the base correction efficiency can be measured by introducing the base editor or a polynucleotide encoding the base editor into a cell containing target mitochondrial DNA, or by expressing the base editor or a polynucleotide encoding the base editor into a cell containing target mitochondrial DNA for base correction.

[0447] The above cells may be in vitro or in vivo environments.

[0448] Techniques for introducing or expressing such base editors or polynucleotides encoding them into cells are readily apparent to those skilled in the art, and may be appropriately selected depending on the type of target cell, culture conditions, delivery method, etc. For example, in vitro environments, electroporation, liposome-based delivery vehicles, microinjection, etc. may be used, while in vivo environments, viral vectors, lipid nanoparticles (LNPs), intramuscular or intravenous injection, etc. may be utilized depending on the intended purpose.

[0449] Base editing efficiency can be measured by isolating mitochondrial DNA from cells where editing has been performed, amplifying the target region via PCR, and analyzing the amplified product. Analytical methods include Sanger sequencing, next-generation sequencing (NGS), digital PCR (dPCR), or post-cloning sequencing. Depending on the purpose, the A-to-G or C-to-T editing ratio, off-target rate, and editing specificity can be quantitatively assessed.

[0450] Additionally, the measurement of base correction efficiency can be performed by contacting the base editor or a polynucleotide encoding the same with isolated DNA comprising a target mitochondrial DNA sequence in an extracellular environment. This method includes the step of mixing the isolated DNA comprising the target mitochondrial DNA sequence and the base correction system (e.g., a polynucleotide encoding the base editor) for which correction efficiency is to be evaluated in the form of reactants in a reaction tube. More specifically, with respect to the isolated DNA, DNA or mRNA (or a vector including the same) encoding the base editor can be added to the reaction system together, and the base editor produced through an in vitro transcription and / or translation process can be allowed to react with the target DNA.

[0451] These reactions may include factors and enzymes required for transcription and translation reactions, and other components required for DNA proofreading. For example, DNA polymerase, RNA polymerase, four types of NTPs (corresponding to adenine, cytosine, guanine, and uracil), cap analogs, ribonuclease inhibitors, protease inhibitors, a mixture of amino acids, tRNA, aminoacyl-tRNA synthetase, methionyl-tRNA transformylase, ribosomes containing rRNA, initiation factors, elongation factors, termination factors, pyrophosphatase, MgCl2, antioxidants, buffers, polyamines, and the like, some or all of which may be provided in the form of a cell lysate (e.g., reticulocyte lysate).

[0452] The base-corrected DNA generated from the reaction can be used directly for sequencing analysis without purification. Analytical methods, such as Sanger sequencing, NGS, dPCR, or post-cloning base sequence analysis, can be used, as previously described. This allows quantitative evaluation of A-to-G or C-to-T correction efficiency, off-target reaction rates, and specificity.

[0453] 10. Corrective organisms

[0454] The DNA base correction composition of the present invention can be applied to the genomes of various eukaryotic cells, including animal cells and plant cells.

[0455] A composition according to the present invention can be introduced into an embryo of a human or non-human animal, and the embryo can be transplanted into a surrogate mother and made pregnant to produce a gestational age-corrected animal.

[0456] The composition according to the present invention can be introduced into an animal's fertilized egg and cultured.

[0457] The fertilized eggs obtained above can be implanted into a surrogate mother and delivered. The non-human transgenic animal may further include a step of confirming whether the animal is transgenic after delivery. The non-human transgenic animals can be mated to produce offspring transgenic animals.

[0458] The above "offspring" refers to all viable offspring of transgenic animals produced by mating with the non-human transgenic animals, and more specifically, refers to the Fl generation produced by mating the transgenic animals with each other or with normal animals using the transgenic animals as parents, the F2 generation produced by mating animals of the Fl generation with normal animals, and subsequent generations, but is not limited thereto.

[0459] The above-mentioned mating may be characterized by mating with the transgenic animal or a normal animal. The present invention may include cells, tissues, and by-products isolated from the transgenic animal or the offspring transgenic animal. The by-products may refer to all materials derived from the transgenic rabbit, but may preferably be characterized by being selected from the group consisting of blood, serum, urine, feces, saliva, organs, and skin, but is not limited thereto.

[0460] The present invention may be as follows based on the above-described contents, but is not limited thereto.

[0461] 1. A mitochondrial DNA base correction composition comprising one or more proteins or one or more polynucleotides encoding the same,

[0462] The protein comprises a DNA binding protein, an enzyme protein and a mitochondrial targeting sequence (MTS), but does not contain an affinity tag.

[0463] A base correction composition, or a mitochondrial DNA base correction method using the same, wherein the enzyme protein is at least one protein selected from the group consisting of adenine deaminase and cytosine deaminase.

[0464] 2. A base correction composition according to the above-mentioned first paragraph, wherein the at least one protein is present in the form of at least one fusion protein, or a mitochondrial DNA base correction method using the same.

[0465] 3. A base correction composition according to the above-mentioned first or second clause, wherein the DNA binding protein is independently selected from the group consisting of a zinc finger protein, a TALE (transcription activator-like effector) protein, and a CRISPR-associated nuclease, or a mitochondrial DNA base correction method using the same.

[0466] 4. A base correction composition according to any one of the above-mentioned clauses 1 to 3, wherein the adenine deaminase is TadA (tRNA-specific adenosine deaminase) or a variant thereof, or a mitochondrial DNA base correction method using the same.

[0467] 5. A base correction composition according to the above-mentioned clause 4, wherein the adenine deaminase comprises the amino acid sequence of SEQ ID NO: 2 (TadA8e) or a conservative variant thereof, or a mitochondrial DNA base correction method using the same.

[0468] 6. A base correction composition according to the above-mentioned paragraph 4, wherein the adenine deaminase comprises a variant in which one or more amino acids selected from the group consisting of positions 28, 30, 46, 48, 49, 82, 84, 106, 108, 110, and 111 of the amino acid sequence of SEQ ID NO: 2 are substituted with another amino acid, or a conservative amino acid substitution thereof, or a mitochondrial DNA base correction method using the same.

[0469] 7. A base correction composition, or a mitochondrial DNA base correction method using the same, in the above-mentioned paragraph 6, wherein the adenine deaminase is a protein having the amino acid sequence of SEQ ID NO: 25 (V28R) or a conservative amino acid substitution thereof.

[0470] 8. A base correction composition according to any one of the above-mentioned clauses 1 to 7, wherein the cytosine deaminase is APOBEC (apolipoprotein B editing complex), AID (activation-induced deaminase) or DddA, or a mutant thereof, or a mutant of TadA (tRNA-specific adenosine deaminase), or a method for base correction of mitochondrial DNA using the same.

[0471] 9. A base correction composition, or a mitochondrial DNA base correction method using the same, in the above-mentioned paragraph 8, wherein the cytosine deaminase is DddAtox having the amino acid sequence of SEQ ID NO: 1 or a homolog thereof, or a conservative amino acid substitution thereof, and exists in the form of a full-length or two fragments.

[0472] 10. A base correction composition in the above-mentioned paragraph 8, wherein the cytosine deaminase exists in the form of two fragments, one of the two fragments includes a sequence from the N-terminus to the 33rd, 44th, 54th, 68th, 82nd, 98th or 108th amino acid in the amino acid sequence of SEQ ID NO: 1, and the other of the two fragments includes a sequence from the 34th, 45th, 55th, 69th, 99th or 109th amino acid in the amino acid sequence of SEQ ID NO: 1 to the C-terminus, or a mitochondrial DNA base correction method using the same.

[0473] 11. A base correction composition, or a mitochondrial DNA base correction method using the same, in the above-mentioned paragraph 10, wherein one of the two fragments comprises the amino acid sequence of 1333N or a conservative amino acid substitution thereof, and the other comprises the amino acid sequence of 1333C or a conservative amino acid substitution thereof; or one of the two fragments comprises the amino acid sequence of 1397N or a conservative amino acid substitution thereof, and the other comprises the amino acid sequence of 1397C or a conservative amino acid substitution thereof.

[0474] 12. In the above-mentioned paragraph 8, the cytosine deaminase exists in the form of two fragments, one of the two fragments is a mutant in which one or more amino acids selected from the group consisting of positions 3, 5, 10, 11, 13, 14, 15, 16, 17, 18, 19, 28, 30 and 31 of 1333N are substituted with another amino acid, or a conservative substitution thereof, and the other is a mutant in which one or more amino acids selected from the group consisting of positions 13, 16, 17, 20, 21, 28, 29, 30, 31, 32, 33, 56, 57, 58 and 60 of 1333C are substituted with another amino acid, or a conservative substitution thereof;

[0475] A base correction composition, or a mitochondrial DNA base correction method using the same, wherein one of the two fragments is a mutant in which one or more amino acids selected from the group consisting of positions 87, 88, 91, 92, 95, 100, 101, 102, and 103 of 1397N are substituted with another amino acid, or a conservative substitution thereof, and the other is a mutant in which one or more amino acids selected from the group consisting of positions 13, 14, 15, and 16 of 1397C are substituted with another amino acid, or a conservative substitution thereof.

[0476] 13. A base correction composition according to the above-mentioned paragraph 8, wherein the cytosine deaminase comprises an amino acid sequence of SEQ ID NO: 1 and exists in a full-length form, and is a variant in which one or more amino acids selected from the group consisting of positions 37, 59, 109, and 129 of the amino acid sequence of SEQ ID NO: 1 are substituted with another amino acid, or a conservative amino acid substitution thereof, or a mitochondrial DNA base correction method using the same.

[0477] 14. A base correction composition according to the above-mentioned 13th paragraph, wherein the cytosine deaminase comprises an amino acid sequence of SEQ ID NO: 1, and is a variant having one or more amino acid substitutions selected from the group consisting of a substitution of S at position 37 with G, a substitution of G at position 59 with S, a substitution of A at position 109 with V, and a substitution of S at position 129 with G, or a conservative amino acid substitution thereof, or a method for base correction of mitochondrial DNA using the same.

[0478] 15. A base correction composition according to any one of the above-mentioned claims 1 to 14, wherein at least one enzyme protein further comprises a nickase, or a mitochondrial DNA base correction method using the same.

[0479] 16. A base correction composition according to any one of the above-mentioned claims 1 to 15, wherein the at least one protein comprises a nuclear export signal (NES), or a mitochondrial DNA base correction method using the same. A base correction composition, or a mitochondrial DNA base correction method using the same.

[0480] 17. A base correction composition according to any one of the above-mentioned claims 1 to 16, wherein at least one protein exists in the form of two fusion proteins, each of the two fusion proteins including MTS, and the MTS included in the two fusion proteins have the same amino acid sequence, or a mitochondrial DNA base correction method using the same.

[0481] 18.. A base correction composition according to any one of the above-mentioned claims 1 to 17, wherein at least one protein exists in the form of two fusion proteins, each of the two fusion proteins including MTS, and the MTS included in the two fusion proteins have different amino acid sequences, or a mitochondrial DNA base correction method using the same.

[0482] 19. In any one of the above-mentioned paragraphs 1 to 17, wherein one or more proteins are present in the form of two fusion proteins, and the cytosine deaminase is present in the form of two splits,

[0483] One of the two fusion proteins comprises MTS, a DNA binding protein, and one of two cytosine deaminase cleavages,

[0484] A base correction composition, or a method for base correction of mitochondrial DNA using the same, wherein the other of the two fusion proteins comprises MTS, a DNA binding protein, the other of two cytosine deaminase cleavages, and an adenine deaminase.

[0485] 20. In any one of the above-mentioned clauses 1 to 17, wherein one or more proteins are present in the form of one fusion protein, and the cytosine deaminase is present in the form of a full-length protein,

[0486] A base correction composition comprising the fusion protein MTS, a DNA binding protein, a full-length form of cytosine deaminase and adenine deaminase, or a mitochondrial DNA base correction method using the same.

[0487] 21. A base correction composition according to any one of the above-mentioned clauses 1 to 20, wherein the MTS is composed of one or more identical or different MTS sequences linked together, or a mitochondrial DNA base correction method using the same.

[0488] 22. A base correction composition according to any one of the above-mentioned items 1 to 21, wherein the MTS independently comprises an amino acid sequence of MTS of SDHA, SDHB, SDHC, SDHD, ATP6, LOC, GHITM, ADCK3, ATP5G2, ATP5G3, ATP9, OPA1, COX8A, and SOD2, or a conservative amino acid substitution thereof, or a mitochondrial DNA base correction method using the same.

[0489] 23. A base correction composition according to the above-mentioned paragraph 22, wherein MTS comprises an amino acid sequence of SDHD or a conservative amino acid substitution thereof, or a mitochondrial DNA base correction method using the same.

[0490] 24. A base correction composition having improved base correction efficiency compared to a corresponding base correction composition comprising an affinity tag or a polynucleotide encoding the same, according to any one of the above-mentioned claims 1 to 23, or a mitochondrial DNA base correction method using the same.

[0491] 25. A base correction composition according to the above-mentioned clause 24, wherein the base correction efficiency is improved by at least two times, or a mitochondrial DNA base correction method using the same.

[0492] 26. A base correction composition according to any one of the above-mentioned clauses 1 to 25, wherein the mitochondrial DNA is mitochondrial DNA of an animal cell, or a DNA base correction method using the same.

[0493] 27. A base correction composition according to the above-mentioned clause 26, wherein the animal cell is a human cell, or a DNA base correction method using the same.

[0494] 28. A base correction composition according to any one of the above-mentioned clauses 1 to 25, wherein the mitochondrial DNA is mitochondrial DNA of a plant cell, or a DNA base correction method using the same.

[0495] 29. A delivery composition comprising a base correction composition according to any one of claims 1 to 28 mentioned above, for delivering the base correction composition to a cell, tissue or organism containing target DNA for base correction.

[0496] 30. A delivery composition according to the above-mentioned paragraph 29, wherein the base correction composition is in the form of one or more polynucleotides, which is a plasmid vector, a viral vector, a lipid nanoparticle, or a polymeric nanoparticle.

[0497] 31. A delivery composition according to the above-mentioned clause 30, which is an adeno-associated viral vector.

[0498] In another aspect, the present invention may be as follows based on the above-described contents, but is not limited thereto.

[0499] 1. (1) DNA binding protein,

[0500] (2) one or more deaminase selected from adenine deaminase and cytosine deaminase, and

[0501] (3) Contains a mitochondrial targeting sequence (MTS),

[0502] Does not include affinity tags,

[0503] A mitochondrial DNA base editing system comprising a base editor or a polynucleotide encoding the same.

[0504] 2. In the above-mentioned paragraph 1,

[0505] The above base editor

[0506] A cytosine deaminase is included, and the cytosine deaminase is a double-stranded DNA-specific cytosine deaminase, which exists in one full-length form or in two split forms, and when it exists in two split forms, it exhibits cytosine deaminizing activity through their dimerization.

[0507] Adenine deaminase is optionally included,

[0508] Mitochondrial DNA base editing system.

[0509] 3. In the second paragraph mentioned above,

[0510] The above base editor

[0511] (1) DNA binding protein, (2) cytosine deaminase, and (3) MTS in the form of two fusion proteins,

[0512] The two fusion proteins each independently comprise one DNA binding protein,

[0513] The cytosine deaminase exists in the form of two splits, one of which is included in each of the fusion proteins,

[0514] In the case where adenine deaminase is included, it is included in either or both of the two fusion proteins.

[0515] Mitochondrial DNA base editing system.

[0516] 4. A mitochondrial DNA base editing system according to any one of the above-mentioned clauses 1 to 3, wherein the base editor further comprises (4) a nickase.

[0517] 5. In the above-mentioned paragraph 4, the base editor comprises (1) a DNA binding protein, (2) a deaminase, (3) MTS, and (4) a nickase in the form of two fusion proteins,

[0518] The two fusion proteins each independently comprise one DNA binding protein,

[0519] One of the two fusion proteins comprises a nickase, and the other fusion protein comprises an adenine deaminase or a cytosine deaminase.

[0520] Mitochondrial DNA base editing system.

[0521] 6. A mitochondrial DNA base correction system according to any one of the above-mentioned clauses 1 to 5, wherein the DNA binding protein included in the base editor is independently selected from the group consisting of a zinc finger protein, a TALE (transcription activator-like effector) protein, and a CRISPR-associated nuclease.

[0522] 7. A mitochondrial DNA base editing system according to any one of the above-mentioned clauses 1 to 6, wherein the MTS included in the base editor is directly linked to a DNA binding protein.

[0523] 8. A mitochondrial DNA base correction system according to any one of the above-mentioned clauses 1 to 6, wherein there are less than 5 cationic amino acids between the MTS and the DNA binding protein.

[0524] 9. A mitochondrial DNA base correction system according to the above-mentioned paragraph 8, wherein the cationic amino acid is glutamic acid or aspartic acid.

[0525] 10. A mitochondrial DNA base editing system according to any one of the above-mentioned claims 1 to 9, wherein the base editor comprises two fusion proteins, each of the two fusion proteins comprising MTS, and the MTS included in the two fusion proteins have the same amino acid sequence.

[0526] 11. A mitochondrial DNA base editing system according to any one of the above-mentioned clauses 1 to 10, wherein the base editor comprises two fusion proteins, each of the two fusion proteins comprising MTS, and the MTS included in the two fusion proteins have different amino acid sequences.

[0527] 12. A mitochondrial DNA base editing system according to any one of the above-mentioned clauses 1 to 11, wherein the MTS included in the base editor is one or more identical or different MTS sequences connected in series.

[0528] 13. A mitochondrial DNA base editing system according to any one of the above-mentioned clauses 1 to 12, wherein the MTS included in the base editor independently comprises an amino acid sequence of an MTS of SDHA, SDHB, SDHC, SDHD, ATP6, LOC, GHITM, ADCK3, ATP5G2, ATP5G3, ATP9, OPA1, COX8A or SOD2, or a truncated form thereof, or a conservative amino acid substitution thereof.

[0529] 14. A mitochondrial DNA base editing system according to the above-mentioned clause 13, wherein the MTS included in the base editor comprises an amino acid sequence of SDHD, or a truncated form thereof, or a conservative amino acid substitution thereof.

[0530] 15. A mitochondrial DNA base editing system according to the above-mentioned clause 13, wherein the MTS included in the base editor comprises an amino acid sequence of SDHA, or a truncated form thereof, or a conservative amino acid substitution thereof.

[0531] 16. A mitochondrial DNA base correction system having improved base correction efficiency compared to a corresponding base system comprising an affinity tag or a polynucleotide encoding the same, according to any one of claims 1 to 15 mentioned above.

[0532] 17. A mitochondrial DNA base correction system according to the above-mentioned clause 16, wherein the base correction efficiency is improved by at least two times.

[0533] 18. A mitochondrial DNA base editing system according to any one of the above-mentioned clauses 1 to 17, wherein the base editor further comprises a nuclear export signal (NES).

[0534] 19. A mitochondrial DNA base correction system for use in in vitro DNA base correction according to any one of claims 1 to 18 mentioned above.

[0535] 20. A mitochondrial DNA base correction system for use in in vivo DNA base correction according to any one of claims 1 to 18 mentioned above.

[0536] 21. A mitochondrial DNA base correction system suitable for correcting an adenine (A) base to a guanine (G) base according to any one of claims 1 to 20 mentioned above.

[0537] 22. A mitochondrial DNA base correction system suitable for correcting a cytosine (C) base to a thymine (T) base according to any one of the above-mentioned clauses 1 to 20.

[0538] 23. A mitochondrial DNA base correction system according to any one of the above-mentioned clauses 1 to 22, wherein the mitochondrial DNA is mitochondrial DNA of an animal cell.

[0539] 24. A mitochondrial DNA base correction system according to the above-mentioned clause 23, wherein the animal cell is a human cell.

[0540] 25. A mitochondrial DNA base correction system according to any one of the above-mentioned clauses 1 to 22, wherein the mitochondrial DNA is mitochondrial DNA of a plant cell.

[0541] 26. A polynucleotide encoding a base editor as described in any one of claims 1 to 25 mentioned above.

[0542] 27. A mitochondrial DNA base correction composition comprising a base correction system according to any one of claims 1 to 25 mentioned above.

[0543] 28. A delivery vehicle for delivering the base correction system to a cell, tissue or organism containing target DNA for mitochondrial DNA base correction, comprising the base correction system described in any one of claims 1 to 25 mentioned above.

[0544] 29. A carrier according to the above-mentioned paragraph 28, wherein the base correction system comprises a polynucleotide, which is a plasmid vector, a viral vector, a lipid nanoparticle, or a polymeric nanoparticle.

[0545] 30. A method for base correction of mitochondrial DNA, comprising introducing a base correction system according to any one of the above-mentioned claims 1 to 25, a base correction composition according to the above-mentioned claim 27, or a carrier according to the above-mentioned claim 28 or 29 into a cell containing target DNA for base correction.

[0546] 31. An organism in which the mitochondrial DNA base has been corrected by the base correction method described in Article 30 mentioned above.

[0547] In another aspect, the present invention may be as follows based on the above-described contents, but is not limited thereto.

[0548] 1. A method for base correction of mitochondrial DNA, comprising introducing a mitochondrial DNA base correction system into a cell containing target DNA for base correction or expressing the mitochondrial DNA base correction system in a cell containing target DNA for base correction,

[0549] The above mitochondrial base editing system comprises a base editor or a polynucleotide encoding the same,

[0550] The above base editor

[0551] (1) DNA binding protein,

[0552] (2) one or more deaminase selected from adenine deaminase and cytosine deaminase, and

[0553] (3) Contains a mitochondrial targeting sequence (MTS),

[0554] Not including affinity tags,

[0555] Mitochondrial DNA base correction method.

[0556] 2. In the above-mentioned paragraph 1,

[0557] The above base editor

[0558] A cytosine deaminase is included, and the cytosine deaminase is a double-stranded DNA-specific cytosine deaminase, which exists in one full-length form or in two split forms, and when it exists in two split forms, it exhibits cytosine deaminizing activity through their dimerization.

[0559] Adenine deaminase is optionally included

[0560] Mitochondrial DNA base correction method.

[0561] 3. In the second paragraph mentioned above,

[0562] The above base editor

[0563] (1) DNA binding protein, (2) cytosine deaminase, and (3) MTS in the form of two fusion proteins,

[0564] The two fusion proteins each independently comprise one DNA binding protein,

[0565] The cytosine deaminase exists in the form of two splits, one of which is included in each of the fusion proteins,

[0566] In the case where adenine deaminase is included, it is included in either or both of the two fusion proteins.

[0567] Mitochondrial DNA base correction method.

[0568] 4. A method for correcting mitochondrial DNA bases according to any one of the above-mentioned clauses 1 to 3, wherein the base editor further comprises (4) a nickase.

[0569] 5. In the above-mentioned paragraph 4,

[0570] The above base editor

[0571] (1) DNA binding protein, (2) deaminase, (3) MTS, and (4) nickase in the form of two fusion proteins,

[0572] The two fusion proteins each independently comprise one DNA binding protein,

[0573] One of the two fusion proteins comprises a nickase, and the other fusion protein comprises an adenine deaminase or a cytosine deaminase.

[0574] Mitochondrial DNA base correction method.

[0575] 6. A method for correcting mitochondrial DNA bases according to any one of the above-mentioned clauses 1 to 5, wherein the DNA binding proteins included in the base editor are each independently selected from the group consisting of a zinc finger protein, a TALE (transcription activator-like effector) protein, and a CRISPR-associated nuclease.

[0576] 7. A method for correcting mitochondrial DNA bases, wherein the MTS included in the base editor is directly linked to a DNA binding protein, according to any one of the above-mentioned clauses 1 to 6.

[0577] 8. A method for correcting mitochondrial DNA bases, wherein the number of cationic amino acids present between the MTS included in the base editor and the DNA binding protein is less than 5, according to any one of the above-mentioned clauses 1 to 6.

[0578] 9. A method for correcting mitochondrial DNA bases in the above-mentioned paragraph 8, wherein the cationic amino acid is glutamic acid or aspartic acid.

[0579] 10. A method for base editing a mitochondrial DNA according to any one of the above-mentioned claims 1 to 9, wherein the base editor comprises two fusion proteins, each of the two fusion proteins comprising MTS, and the MTS included in the two fusion proteins have the same amino acid sequence.

[0580] 11. A method for base editing a mitochondrial DNA according to any one of the above-mentioned claims 1 to 10, wherein the base editor comprises two fusion proteins, each of the two fusion proteins comprising MTS, and the MTS included in the two fusion proteins have different amino acid sequences.

[0581] 12. A method for correcting mitochondrial DNA bases, wherein the MTS included in the base editor is one or more identical or different MTS sequences connected in series according to any one of the above-mentioned clauses 1 to 11.

[0582] 13. A method for base editing a mitochondrial DNA according to any one of the above-mentioned clauses 1 to 12, wherein the MTS included in the base editor independently comprises an amino acid sequence of an MTS of SDHA, SDHB, SDHC, SDHD, ATP6, LOC, GHITM, ADCK3, ATP5G2, ATP5G3, ATP9, OPA1, COX8A or SOD2, or a truncated form thereof, or a conservative amino acid substitution thereof.

[0583] 14. A method for correcting mitochondrial DNA bases, wherein the MTS included in the base editor comprises an amino acid sequence of SDHD, a truncated form thereof, or a conservative amino acid substitution thereof, in the above-mentioned paragraph 13.

[0584] 15. A mitochondrial DNA base correction method according to the above-mentioned 13th paragraph, wherein the MTS included in the base editor comprises an amino acid sequence of SDHA, or a truncated form thereof, or a conservative amino acid substitution thereof.

[0585] 16. A mitochondrial DNA base correction method having improved base correction efficiency compared to a DNA base correction method using a corresponding base correction system comprising an affinity tag or a polynucleotide encoding the same, according to any one of the above-mentioned claims 1 to 15.

[0586] 17. A mitochondrial DNA base correction method according to the above-mentioned clause 16, wherein the base correction efficiency is improved by at least two times.

[0587] 18. A method for correcting a mitochondrial DNA base according to any one of the above-mentioned clauses 1 to 17, wherein the base editor additionally includes a nuclear export signal (NES).

[0588] 19. A mitochondrial DNA base correction method for use in in vitro DNA base correction according to any one of claims 1 to 18 mentioned above.

[0589] 20. A mitochondrial DNA base correction method for use in in vivo DNA base correction according to any one of claims 1 to 18 mentioned above.

[0590] 21. A mitochondrial DNA base correction method for correcting an adenine (A) base to a guanine (G) base according to any one of the above-mentioned clauses 1 to 20.

[0591] 22. A mitochondrial DNA base correction method for correcting a cytosine (C) base to a thymine (T) base according to any one of the above-mentioned clauses 1 to 20.

[0592] 23. A method for correcting mitochondrial DNA bases according to any one of the above-mentioned clauses 1 to 22, wherein the mitochondrial DNA is mitochondrial DNA of an animal cell.

[0593] 24. A method for correcting mitochondrial DNA bases in the above-mentioned 23rd paragraph, wherein the animal cell is a human cell.

[0594] 25. A method for correcting mitochondrial DNA bases according to any one of the above-mentioned clauses 1 to 22, wherein the mitochondrial DNA is mitochondrial DNA of a plant cell.

[0595] 26. An organism in which a mitochondrial DNA base has been corrected by the base correction method described in any one of the above-mentioned clauses 1 to 25.

[0596] In another aspect, the present invention may be as follows based on the above-described contents, but is not limited thereto.

[0597] 1. Measuring the base correction efficiency of a base editor comprising (1) a DNA binding protein, (2) one or more deaminase selected from adenine deaminase and cytosine deaminase, (3) MTS, and (4) an affinity tag for target mitochondrial DNA,

[0598] Providing a base correction system comprising a tag-free base editor having the same amino acid sequence as the base editor but not including an affinity tag, or a polynucleotide encoding the same.

[0599] A method for improving the efficiency of mitochondrial DNA base editing.

[0600] 2. A method according to the above-mentioned first paragraph, wherein the base correction efficiency is measured by introducing the base editor into a cell containing target mitochondrial DNA or expressing the base editor in a cell containing target mitochondrial DNA for base correction.

[0601] 3. A method according to the above-mentioned first paragraph, wherein the base correction efficiency is measured by contacting the base editor with an isolated DNA containing a target mitochondrial DNA sequence in an extracellular environment.

[0602] 4. In any one of the above-mentioned clauses 1 to 3,

[0603] The base editor and tagless DNA base editor including the above affinity tag are

[0604] A cytosine deaminase is included, and the cytosine deaminase is a double-stranded DNA-specific cytosine deaminase, which exists in one full-length form or in two split forms, and when it exists in two split forms, it exhibits cytosine deaminizing activity through their dimerization.

[0605] Adenine deaminase is optionally included,

[0606] method.

[0607] 5. In any one of the above-mentioned clauses 1 to 4,

[0608] The base editor and tagless DNA base editor including the above affinity tag are

[0609] (1) DNA binding protein, (2) cytosine deaminase, and (3) MTS in the form of two fusion proteins,

[0610] The two fusion proteins each independently comprise one DNA binding protein,

[0611] The cytosine deaminase exists in the form of two splits, one of which is included in each of the fusion proteins,

[0612] In the case where adenine deaminase is included, it is included in either or both of the two fusion proteins.

[0613] method.

[0614] 6. A method according to any one of the above-mentioned clauses 1 to 5, wherein the base editor and tagless DNA base editor including the affinity tag further comprise (4) a nickase.

[0615] 7. In the above-mentioned paragraph 6,

[0616] The base editor and tagless DNA base editor including the above affinity tag are

[0617] (1) DNA binding protein, (2) deaminase, (3) MTS, and (4) nickase in the form of two fusion proteins,

[0618] The two fusion proteins each independently comprise one DNA binding protein,

[0619] One of the two fusion proteins comprises a nickase, and the other fusion protein comprises an adenine deaminase or a cytosine deaminase.

[0620] method.

[0621] 8. A method according to any one of the above-mentioned claims 1 to 7, wherein the DNA binding protein included in the base editor and tagless DNA base editor including the affinity tag is independently selected from the group consisting of a zinc finger protein, a TALE (transcription activator-like effector) protein, and a CRISPR-associated nuclease.

[0622] 9. A method according to any one of the above-mentioned clauses 1 to 8, wherein the MTS included in the base editor and tagless DNA base editor including the affinity tag is directly linked to a DNA binding protein.

[0623] 10. A method according to any one of the above-mentioned claims 1 to 8, wherein the number of cationic amino acids is less than 5 between the MTS and the DNA binding protein included in the base editor and tagless DNA base editor including the affinity tag.

[0624] 11. A method according to the above-mentioned clause 10, wherein the cationic amino acid is glutamic acid or aspartic acid.

[0625] 12. A method according to any one of the above-mentioned claims 1 to 11, wherein the base editor including the affinity tag and the tagless DNA base editor comprise two fusion proteins, each of the two fusion proteins comprising MTS, and the MTS included in the two fusion proteins have the same amino acid sequence.

[0626] 13. A method according to any one of the above-mentioned claims 1 to 11, wherein the base editor including the affinity tag and the tagless DNA base editor comprise two fusion proteins, each of the two fusion proteins comprising MTS, and the MTS included in the two fusion proteins have different amino acid sequences.

[0627] 14. A method according to any one of the above-mentioned clauses 1 to 13, wherein the MTS included in the base editor and tagless DNA base editor including the affinity tag is one or more identical or different MTS sequences connected in series.

[0628] 15. A method according to any one of the above-mentioned claims 1 to 14, wherein the MTS included in the base editor and tagless DNA base editor including the affinity tag each independently includes an amino acid sequence of an MTS of SDHA, SDHB, SDHC, SDHD, ATP6, LOC, GHITM, ADCK3, ATP5G2, ATP5G3, ATP9, OPA1, COX8A or SOD2, or a truncated form thereof, or a conservative amino acid substitution thereof.

[0629] 16. A method according to the above-mentioned paragraph 15, wherein the MTS included in the base editor and tagless DNA base editor including the affinity tag includes an amino acid sequence of SDHD, or a truncated form thereof, or a conservative amino acid substitution thereof.

[0630] 17. A method according to the above-mentioned paragraph 15, wherein the MTS included in the base editor and tagless DNA base editor including the affinity tag includes an amino acid sequence of SDHA, or a truncated form thereof, or a conservative amino acid substitution thereof.

[0631] 18. A method according to any one of the above-mentioned claims 1 to 17, wherein the tagless DNA base editor has at least a two-fold improvement in base correction efficiency compared to a base editor including the affinity tag.

[0632] 19. A method according to any one of the above-mentioned claims 1 to 18, wherein the base editor and tagless DNA base editor including the affinity tag further include a nuclear export signal (NES).

[0633] 20. A method according to any one of claims 1 to 19 mentioned above, wherein the base correction system comprising a tagless base editor or a polynucleotide encoding the same is suitable for in vitro DNA base correction.

[0634] 21. A method according to any one of claims 1 to 19 mentioned above, wherein the base correction system comprising a tagless base editor or a polynucleotide encoding the same is suitable for in vivo DNA base correction.

[0635] 22. A method according to any one of the above-mentioned claims 1 to 21, wherein the base correction system comprising a tagless base editor or a polynucleotide encoding the same is suitable for correcting an adenine (A) base to a guanine (G) base.

[0636] 23. A method according to any one of the above-mentioned claims 1 to 21, wherein the base correction system comprising a tagless base editor or a polynucleotide encoding the same is suitable for correcting a cytosine (C) base to a thymine (T) base.

[0637] 24. A method according to any one of the above-mentioned clauses 1 to 23, wherein the mitochondrial DNA is mitochondrial DNA of an animal cell.

[0638] 25. A method according to claim 24, wherein the animal cell is a human cell.

[0639] 26. A method according to any one of the above-mentioned claims 1 to 23, wherein the mitochondrial DNA is mitochondrial DNA of a plant cell.

[0640] 27. A mitochondrial DNA base correction system provided by the method described in any one of the above-mentioned items 1 to 26.

[0641] 28. A method for base correction of mitochondrial DNA, comprising introducing the mitochondrial DNA base correction system described in the above-mentioned paragraph 27 into a cell containing target DNA for base correction.

[0642] 29. An organism in which the mitochondrial DNA base has been corrected by the base correction method described in the above-mentioned paragraph 28.

[0643] Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are provided only to illustrate the present invention and the present invention is not limited thereto.

[0644] Example 1: Production of sTALEDs targeting the mtND1 and mtND4 genes

[0645] The first and second fusion proteins were constructed, which comprise split TALED (sTALED), an adenine base editor targeting the ND1 and ND4 genes of mitochondrial DNA. DNA sequences encoding the fusion proteins (including those with the FLAG or HA tag encoding sequence removed) were cloned using the Gibson assembly system using expression plasmids containing CMV and T7 promoters, SOD2 MTS or COX8A MTS, FLAG or HA tags, DddAtox 1397N or 1397C, TadA8e and / or TadA8e_V28R (also referred to as V28R) as templates. After obtaining the MTS sequences other than SOD2 MTS and COX8A MTS, the WC03 sequence which is a cytosine deaminase, the MutH* and BspD6I nickases, and the NES (nuclear export signal) amino acid sequences, codon optimization for expression in humans was performed, and the double-stranded DNA sequences were synthesized by IDT (Integrated DNA Technologies), and the synthesized double-stranded DNA fragments were included in the DNA sequences encoding the fusion proteins. Primers for each template were custom-made, and the DNA fragments required for Gibson assembly were amplified using PrimeSTAR® GXL DNA Polymerase (TAKARA), purified using a PCR SV mini kit (GeneAll), and these purified DNA fragments were assembled using a HiFi DNA assembly kit (NEB). The reassembled DNA was transformed into competent DH5α (enzynomics) E. coli cells by heat shock (42°C), and a single colony selectively grown on LB solid medium containing antibiotics was cultured in LB liquid medium (containing antibiotics) by shaking incubation overnight at 37°C.Afterwards, plasmid DNA was purified using a mini-prep using the Plasmid SV mini kit (GeneAll) according to the manufacturer's protocol. The purified plasmid DNA, i.e., the newly constructed expression plasmids, was sequenced using Sanger sequencing (Macrogen) to confirm proper cloning. The constructed DNA sequences are located between the CMV promoter and the T7 promoter and terminator.

[0646] To construct base editor fusion proteins containing TALE proteins, the TALE plasmid and the expression plasmids cloned above (including those with the DNA sequence encoding the tag sequence removed; and those with the DNA sequences encoding new MTS sequences) were constructed using the Golden-Gate assembly system.

[0647] Specifically, plasmids corresponding to target DNAs were selected from a set of TALE subarray plasmids consisting of a total of 424 (6 × 64 tripartite plasmids + 2 × 16 bipartite plasmids + 2 × 4 monopartite plasmids). These subarray plasmids encode repeat units required for TALE proteins to recognize specific DNA sequences and were designed to contain a BsaI restriction enzyme recognition site for Golden Gate assembly. Each repeat unit has specificity for a specific nucleotide (e.g., NI for A, HD for C, NN for G, NG for T) (Kim, Y. et al., 2013). The selected TALE subarray plasmids were cleaved with Bsa I restriction enzyme and then linked to form complementary sticky ends. Through this process, the assembled gene sequence was used to synthesize a TALE array between the TALE N-terminal domain (NTD) and TALE C-terminal domain (CTD) of the previously prepared vector, which was used to generate a base editor plasmid targeting a specific sequence.

[0648] Finally, the base sequences were verified to ensure that the base editors produced using Competent Sanger sequencing (Macrogen) were properly cloned.

[0649] The composition of the produced fusion proteins is as follows.

[0650] MTS - TALE protein - 2aa linker - DddAtox 1397N

[0651] MTS - 3x HA - TALE protein - 2aa linker - DddAtox 1397N

[0652] MTS - TALE protein - 2aa linker - DddAtox 1397C - 16aa linker - AD

[0653] MTS - 3X FLAG - TALE protein - 2aa linker - DddAtox 1397C - 16aa linker - AD

[0654] MTS - 3x HA - TALE protein - 2aa linker - MutH*

[0655] MTS - 3x HA - TALE protein - 2aa linker - BspD6I

[0656] MTS - 3x FLAG - TALE protein - 16aa linker - AD

[0657] MTS - TALE protein - 2aa linker - MutH*

[0658] MTS - TALE protein - 2aa linker - BspD6I

[0659] MTS - TALE protein - 16aa linker - AD

[0660] MTS - TALE protein - 2aa linker - WC03_S257N

[0661] MTS - TALE protein - 2aa linker - WC03_S257C - 16aa linker - AD

[0662] MTS - TALE protein - 2aa linker - WC03_S257N - NES

[0663] MTS - TALE protein - 2aa linker - WC03_S257C - 16aa linker - AD - NES

[0664]

[0665] Example 2: Transfection of HEK293T cells and UDC cells for ND1 gene correction.

[0666] HEK293T cells were cultured in 12-well Clear TC-Treated Multiple Well Plates (Corning) in DMEM (Welgene1) supplemented with 10% FBS and 1% penicillin antibiotics at 37°C in a 5% CO2 atmosphere. UDC cells were seeded at a density of 0.75 x 105 cells per well in 48-well Clear TC-Treated Multiple Well Plates (Corning). After 20-24 hours, 500 ng each of the plasmids encoding the first and second fusion proteins (1 μg in total) were mixed with Lipofectamine 2000 (Invitrogen) and Opti-MEM (gibco) and added to the cells pre-seeded in the 48-well plates for transfection. The transfected cells were cultured at 37°C in a 5% CO2 atmosphere while replacing the medium. After 3 days, the cells were harvested, the culture medium was removed, and 100 μL of cell lysis buffer (50 mM Tris-HCl pH 7.4 (Welgene), 1 mM EDTA pH 8.0 (Welgene), 0.05% sodium dodecyl sulfate (Welgene), 5 μL Proteinase K (Qiagen)) was added to each well, and incubated in a PCR machine at 50°C for 1 hour and 80°C for 20 minutes.

[0667]

[0668] Example 3: UDC cell transfection for gene correction of G3460A and G11778A mutations.

[0669] Patient-derived UDC cells containing G3460A and G11778A mutations, respectively, were cultured in Renal Epithelial cell Growth Media Bullet kit (REGM, Lonza) medium in 12-well Clear TC-Treated Multiple Well Plates (Corning) coated with 0.1% gelatin (Welgene) at 37°C in 5% CO2. The plasmids encoding the first and second fusion proteins (1 μg each, a total of 2 μg) were transfected into UDC cells (1.0 x 104 cells) by electroporation (1350 V, 30 ms, 1 pulse) using a NEON 10 μL device. The electroporated UDC cells were plated onto 8-well Clear TC-Treated Multiple Well Plates (Corning) coated with 0.1% gelatin and pre-incubated with culture medium. Transfected UDC cells were cultured at 37°C in 5% CO2 with replacement of the medium. After 6 days, cells were harvested, the medium was removed, and cell lysis was performed, as described in Example 2.

[0670]

[0671] Example 4: Sequencing and Base Correction Efficiency Analysis for Base Correction Verification

[0672] The reactants obtained in Example 2 or 3 were used as templates without purification, and sequences were analyzed by targeted deep sequencing to analyze the base correction ratio of the target region. To construct a deep sequencing library, nested first PCR and second PCR were performed using PrimeSTAR® GXL DNA Polymerase (TAKARA) using the first PCR as a template, and a third PCR was performed using an index-containing primer to add the final index sequence. The third PCR reaction product with the added index sequence was purified using a PCR SV mini kit (GeneAll), and paired-end sequencing was performed using a MiniSeq Mid Output Kit (illumina) using a MiniSeq system (illumina).

[0673] The correction efficiency of the first and second fusion protein combinations targeting the mitochondrial ND1 gene was compared and analyzed in HEK293T and UDC cells and presented as a heatmap (Figs. 1 to 4). In addition, the correction efficiency obtained by targeting the mitochondrial ND4 gene was compared and analyzed in UDC cells and presented as a heatmap (Figs. 5 and 6). In addition, the gene correction efficiency by fusion proteins that applied a nickase other than cytosine deaminase was analyzed in UDC cells using SOD2 and COX8A MTSs in which HA and FLAG tags were included in the base editor, and the correction efficiency of SDHD MTSs in which HA and FLAG tags were not included in the base editor, and the correction efficiency was presented in a graph (Figs. 7 and 8).

[0674] The correction efficiency in the first and second fusion protein combinations targeting the G3460A mutation was compared with the correction efficiency in UDC by tagged MTSs such as SOD2-3XHA and COX8A-3XFLAG, untagged SOD2, COX8A MTS, and untagged SDHD MTS (Figs. 9 and 10).

[0675] The correction efficiency was compared by applying WC03 instead of DddAtox, SDHD without tag, SDHDcut with the length of SDHD shortened, and SDHA MTS, and it was also confirmed that the improved base correction efficiency was applied as is in the base editor with additional NES (Figs. 11 to 13).

[0676] Experimental results showed that when the composition of the base editors was the same but the presence or absence of the tag sequence was different, the base correction efficiency was significantly increased when the tag sequence was not used compared to when the tag sequence was used. Experimental results obtained in UDC cells showed that when the tag sequence was not used, the A-to-G correction efficiency increased by at least two times compared to when the tag sequence was used, and by about three times when the adenine deaminase V28R was applied (see Fig. 2).

[0677] In the sTALED system, when only one side contained a FLAG or HA tag, the proofreading efficiency was analyzed compared to a base editor containing tags on both sides and a base editor containing no tags at all. As a result, it was confirmed that it is preferable for the base editor to not include FLAG or HA tags (see Fig. 3).

[0678] This increase in proofreading efficiency in base editors without such tags was also observed when switching to 1397N and 1397C-8e as the Left and Right TALE components, respectively, and was further improved when SDHD MTS was used instead of COX8A and SOD2 MTS (see Figures 4 and 5).

[0679] Furthermore, when we experimented with introducing various MTS sequences without using tags, we confirmed that the correction efficiency differed depending on the type of MTS used. As shown in Fig. 5, ATP6, LOC, SDHD, and NDUFAB1 MTSs showed correction efficiencies similar to or superior to the previously used COX8A or SOD2 MTSs, and in particular, SDHD MTS showed the highest correction efficiency. For example, in the ND4 gene correction results, when the tag sequence was used together with the SOD or COX8A MTS, the correction efficiency was 4.47% (based on the highest value at the adenine (A) base in the spacer region shown in Fig. 5), whereas when the MTS was changed to SDHD and the tag was removed, the correction efficiency increased to 29.74%, showing an efficiency improvement of more than 6 times.

[0680] According to a report by Chin et al. (Chin et al., 2018, Cell Reports 22, 2818-2826), SDHD MTS is known to have a relatively low mitochondrial localization ability of the attached protein among various MTSs, while ATP5G3, ADCK3, ATP9, and GHITM MTSs have been reported to have higher localization abilities. Considering this, the observation of such a remarkable increase in proofreading efficiency when ATP6, LOC, SDHD, NDUFAB1 MTSs, and especially SDHD MTSs, were used without tags can be said to be a result that surpasses previous predictions.

[0681] When these MTSs were combined with each of the Left and Right base editors, there was no significant change in the overall trend, and the highest correction efficiency was observed in the combination using SDHD MTS in both cases (see Fig. 6).

[0682] This increase in correction efficiency was observed not only in DddAtox-based sTALEDs but also in systems that introduced nickases, and the correction efficiency was also significantly improved when nickases were applied (see Figures 7 and 8).

[0683] Additionally, it was confirmed that when MTS was applied without using a tag in the correction of the G3460A mutation, the correction efficiency increased by approximately three times (see Figures 9 and 10).

[0684] In addition, we additionally applied SDHDcut and SDHA MTS, which are shortened variants of SDHD MTS, and compared their proofreading efficiency with the SDHD MTS-based base editor that showed the highest proofreading efficiency. As a result, both SDHA and SDHDcut MTS showed proofreading efficiency similar to that of SDHD MTS (see Figs. 11 and 12).

[0685] In addition, it was confirmed that the improved correction efficiency resulting from the application of SDHD MTS was maintained in a base editor that additionally incorporated a nuclear export signal (NES) (see Fig. 13).

[0686]

[0687] order

[0688] The amino acid sequences of the polypeptides used in the above examples are as follows.

[0689] SOD2_MTS:

[0690] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD (SEQ ID NO: 3)

[0691] COX8A_MTS:

[0692] MASVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 4)

[0693] crATP6 MTS:

[0694] MALQQAAPRVFGLLGRAPVALGQSGILTGSSGFKNQGFNGSLQSVENHVYAQAFSTSSQEEQAAPSIQGASGMKLPGMAGSMLLGKSRSGLRTGSMVPFAAQQAMNM (SEQ ID NO: 5)

[0695] hsADCK3 MTS:

[0696] MAAILGDTIMVAKGLVKLTQAAVETHLQHLGIGGELIMAARALQSTAVEQIGMFLGKVQGQDKHEEYFAENFGGPEGEFHFSVPHAAGASTDFSSASAPDQSAPPSLGHAHSEGPAPAYVASGPFREAGFPGQASSPLGRANGRLFANPRDFSAMGFQRRF (SEQ ID NO: 6)

[0697] hsATP5G3 MTS:

[0698] MFACAKLACTPSLIRAGSRVAYRPISASVLSRPEASRTGEGSTVFNGAQNGVSQLIQREFQTSAISR (SEQ ID NO: 7)

[0699] ncATP9 MTS:

[0700] MASTRVLASRLASQMAASAKVARPAVRVAQVSKRTIQTGSPLQTLKRTQMTSIVNATTRQAFQKRA (SEQ ID NO: 8)

[0701] zmLOC:

[0702] MALLRAAVSELRRRGRGALTPLPALSSLLSSLSPRSPASTRPEPNNPHADRRHVIALRRCPPLPASAVLAPELLHARGLLPRHWSHASPLSTSSSSSRPADKAQLTWVDKWIPEAARPY (SEQ ID NO: 9)

[0703] hsATP5G2 MTS:

[0704] MPELILYVAITLSVAERLVGPGHACAEPSFRSSRCSAPLCLLCSGSSSPATAPHPLKMFACSKFVSTPSLVKSTSQLLSRPLSAVVLKRPEILTDESLSSLAVSCPLTSLVSSRSFQTSAISRDIDTA (SEQ ID NO: 10)

[0705] hsGHITM MTS:

[0706] MLAARLVCLRTLPSRVFHPAFTKASPVVKNSITKNQWLLTPSRE (SEQ ID NO: 11)

[0707] hsSDHD MTS:

[0708] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH (SEQ ID NO: 12)

[0709] hsNDUFAB1 MTS:

[0710] MASRVLSAYVSRLPAAFAPLPRVRMLAVARPLSTALCSAGTQTRLGTLQPALVLAQVPGRVTQLCRQY (SEQ ID NO: 13)

[0711] hsOPA1 MTS:

[0712] MWRLRRAAVACEVCQSLVKHSSGIKGSLPLQKLHLVSRSIYHSHHPTLKLQRPQLRTSFQQFSSLTNLPLRKLKFSPIKYGYQPRRN (SEQ ID NO: 14)

[0713] hsSDHDcut MTS:

[0714] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQ (SEQ ID NO: 15)

[0715] hsSDHA MTS:

[0716] MSGVRGLSRLLSARRLALAKAWPTVLQTGTRGFHFTV (SEQ ID NO: 16)

[0717] 3X HA tag:

[0718] YPYDVPDYAGYPYDVPDYAGYPYDVPDYA (서열번호 17)

[0719] 3X Flag tags:

[0720] DYKDHDGDYKDHDIDYKDDDDK (서열번호 18)

[0721] ND1 Right TALE:

[0722] DLRTLGYSQQQQEKPKVRSTVAQHHEALVGHGFFTAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRG PPLQLDTGQLLKIAKRGGVTAAVHAWRNALTGAPLNLTQVVAIASNNGGKQALETVQRLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLPVLCQAHGLTPAQVVAIASNGGGKQALE TVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLPVLCQAHGLTPAQVVAIASNNGGQALETVQRLLPVLCQDHGLTPAQVVAIASNIGG KQALETVQRLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLPVLCQAHGLTPDQVVAIASNNGGQALETVQRLLPVLCQAHGLTPAQVVAIA SHDGGKQALETVQRLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLPVLCQAHGLTPDQ VVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHG LTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVQLSRPDPALALTNDHLVALACLGGRPALDAVKKGLGGS

[0723] ND1 Left TALE:

[0724] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS

[0725] ND4 Left TALE 90:

[0726] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS

[0727] ND4 Left TALE 322:

[0728] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS

[0729] ND4 Left TALE 326:

[0730] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS

[0731] ND4 Left TALE 329v:

[0732] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS

[0733] ND4 Left TALE 330v:

[0734] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS

[0735] ND4 Left TALE 329:

[0736] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS

[0737] ND4 Left TALE 330:

[0738] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS

[0739] ND4 Right TALE 384v:

[0740] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV

[0741] ND4 Right TALE 384:

[0742] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV

[0743] ND4 Right TALE 120:

[0744] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS

[0745] ND4 Right TALE 115

[0746] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS

[0747] ND4 Right TALE 110

[0748] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS

[0749] G3460A Left TALE 17:

[0750] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS

[0751] G3460A Left TALE 18:

[0752] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS

[0753] G3460A Right TALE 56:

[0754] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV

[0755] G3460A Right TALE 57:

[0756] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV

[0757] G3460A Right TALE 58:

[0758] DLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV

[0759] 2aa linker:

[0760] GS

[0761] 16 a.a linker:

[0762] SGSETPGTSESATPES

[0763] DddAtox 1397N:

[0764] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG (서열번호 19)

[0765] DddAtox 1397C:

[0766] AIPVKRGATGETKVFTGNSNSPKSPTKGGC (서열번호 20)

[0767] MutH*

[0768] SQPRPLLSPPETEEQLLAQAQQLSGYTLGELAALAGLVTPENLKRDKGWIGVLLEIWLGASAGSKPEQDFAALGVELKTIPVDSLGRPLATTAVCVAPLTGNSGVTWETSHVRHKLKRVLWIPVEGERSIPLAKRRVGSPLLWSPNEEEDRQLREDWEELMDMIVLGQIERITARHGEYLQIRPKAANAKALTEAIGARGERILTLPRGFYLKKNFTSALLARHFLIQ (서열번호 21)

[0769] BspD6I

[0770] RQLEEVIDLLEVYHEKKNVIEEKIKARFIANKNTVFEWLTWNGFIILGNALEYKNNFVIDEELQPVTHAAGNQPDMEIIYEDFIVLGEVTTSKGATQFKMESEPVTRHYLNKKKELEKQGVEKELYCLFIAPEINKNTFEEFMKYNIVQNTRIIPLSLKQFNMLLMVQKKLIEKGRRLSSYDIKNLMVSLYRTTIECERKYTQIKAGLEETLNNWVVDKEVRF (서열번호 22)

[0771] WC03_S257N:

[0772] NANCNQEKPVLPKYDGKTTEGVMVTPDGKQISFKSGNSSTPSGSYPQYKAQSASHVEGKAALYMRENGINEATVFHNNPNGTCGFCDRQVPALLPKGAKLTVVPPSNS (서열번호 23)

[0773] WC03_S257C:

[0774] VANNVRAIPVPKTYIGNSTVPKIK (SEQ ID NO: 24)

[0775] TadA8e:

[0776] SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN

[0777] TadA8e_V28R:

[0778] SEVEFSHEYWMRHALTLAKRARDERERPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN (SEQ ID NO: 25)

[0779] NES:

[0780] VDEMTKKFGTLTIHDTEK

[0781] The amino acid sequences of the individual fusion proteins used in the experiments are as follows. The parentheses in the sequence below indicate which protein the amino acid sequence before the parentheses represents and do not constitute part of the sequence.

[0782] COX8A-3xFLAG-L-1397C-8e:

[0783] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)DYKDHDGDYKDHDIDYKDDDDK(3xFLAG)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND1 LeftTALE)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0784] COX8A-3xFLAG-L-1397C-V28R:

[0785] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)DYKDHDGDYKDHDIDYKDDDDK(3xFLAG)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND1 LeftTALE)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDERERPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(V28R)

[0786] SOD2-3xHA-R-1397N:

[0787] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD(SOD2)YPYDVPDYAGYPYDVPDYAGYPYDVPDYA(3xHA)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND1 Right TALE)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0788] COX8A-L-1397C-8e:

[0789] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND1 Left TALE)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0790] COX8A-L-1397C-V28R:

[0791] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND1 Left TALE)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDERERPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(V28R)

[0792] SOD2-R-1397N:

[0793] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD(SOD2)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND1 Right TALE)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0794] SDHD-L-1397C-8e:

[0795] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND1 Left TALE)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)。

[0796] SDHD-R-1397N:

[0797] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND1 Right TALE)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0798] SOD2-3xHA-L-1397N:

[0799] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD(SOD2)YPYDVPDYAGYPYDVPDYAGYPYDVPDYA(3xHA)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND1 Left TALE)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0800] COX8A-3xFLAG-R-1397C-8e:

[0801] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)DYKDHDGDYKDHDIDYKDDDDK(3xFLAG)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND1 RightTALE)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0802] SOD2-L-1397N:

[0803] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD(SOD2)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND1 Left TALE)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0804] COX8A-R-1397C-8e:

[0805] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND1 RightTALE)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0806] SDHD-L-1397N:

[0807] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND1 Left TALE)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0808] SDHD-R-1397C-8e:

[0809] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND1 RightTALE)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0810] SOD2-3xHA-90N:

[0811] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD(SOD2)YPYDVPDYAGYPYDVPDYAGYPYDVPDYA(3xHA)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE90)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0812] COX8A-3xFLAG-120C-8e:

[0813] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)DYKDHDGDYKDHDIDYKDDDDK(3xFLAG)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE120)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0814] SOD2-90N:

[0815] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD(SOD2)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 90)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0816] COX8A-120C-8e:

[0817] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE120)GSAIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0818] crATP6-90N:

[0819] MALQQAAPRVFGLLGRAPVALGQSGILTGSSGFKNQGFNGSLQSVENHVYAQAFSTSSQEEQAAPSIQGASGMKLPGMAGSMLLGKSRSGLRTGSMVPFAAQQAMNM(crATP6)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE90)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0820] crATP6-120C-8e:

[0821] MALQQAAPRVFGLLGRAPVALGQSGILTGSSGFKNQGFNGSLQSVENHVYAQAFSTSSQEEQAAPSIQGASGMKLPGMAGSMLLGKSRSGLRTGSMVPFAAQQAMNM(crATP6)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE120)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0822] hsADCK3-90N:

[0823] MAAILGDTIMVAKGLVKLTQAAVETHLQHLGIGGELIMAARALQSTAVEQIGMFLGKVQGQDKHEEYFAENFGGPEGEFHFSVPHAAGASTDFSSASAPDQSAPPSLGHAHSEGPAPAYVASGPFREAGFPGQASSPLGRANGRLFANPRDSFSAMGFQRRF(hsADCK3)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE90)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0824] hsADCK3-120C-8e:

[0825] MAAILGDTIMVAKGLVKLTQAAVETHLQHLGIGGELIMAARALQSTAVEQIGMFLGKVQGQDKHEEYFAENFGGPEGEFHFSVPHAAGASTDFSSASAPDQSAPPSLGHAHSEGPAPAYVASGPFREAGFPGQASSPLGRANGRLFANPRDSFSAMGFQRRF(hsADCK3)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE120)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0826] hsATP5G3-90N:

[0827] MFACAKLACTPSLIRAGSRVAYRPISASVLSRPEASRTGEGSTVFNGAQNGVSQLIQREFQTSAISR(hsATP5G3)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE90)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0828] hsATP5G3-120C-8e:

[0829] MFACAKLACTPSLIRAGSRVAYRPISASVLSRPEASRTGEGSTVFNGAQNGVSQLIQREFQTSAISR(hsATP5G3)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE120)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0830] ncATP9-90N:

[0831] MASTRVLASRLASQMAASAKVARPAVRVAQVSKRTIQTGSPLQTLKRTQMTSIVNATTRQAFQKRA(ncATP9)(ND4 Left TALE90)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0832] ncATP9-120C-8e:

[0833] MASTRVLASRLASQMAASAKVARPAVRVAQVSKRTIQTGSPLQTLKRTQMTSIVNATTRQAFQKRA(ncATP9)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE120)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0834] zmLOC-90N:

[0835] MALLRAAVSELRRRGRGALTPLPALSSLLSSLSPRSPASTRPEPNNPHADRRHVIALRRCPPLPASAVLAPELLHARGLLPRHWSHASPLSTSSSRPADKAQLTWVDKWIPEAARPY(zmLOC)(ND4 Left TALE90)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0836] zmLOC-120C-8e:

[0837] MALLRAAVSELRRRGRGALTPLPALSSLLSSLSPRSPASTRPEPNNPHADRRHVIALRRCPPLPASAVLAPELLHARGLLPRHWSHASPLSTSSSRPADKAQLTWVDKWIPEAARPY(zmLOC)(ND4 Right TALE120)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0838] hsATP5G2-90N:

[0839] MPELILYVAITLSVAERLVGPGHACAEPSFRSSRCSAPLCLLCSGSSSPATAPHPLKMFACSKFVSTPSLVKSTSQLLSRPLSAVVLKRPEILTDESLSSLAVSCPLTSLVSSRSFQTSAISRDIDTA(hsATP5G2)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE90)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0840] hsATP5G2-120C-8e:

[0841] MPELILYVAITLSVAERLVGPGHACAEPSFRSSRCSAPLCLLCSGSSSPATAPHPLKMFACSKFVSTPSLVKSTSQLLSRPLSAVVLKRPEILTDESLSSLAVSCPLTSLVSSRSFQTSAISRDIDTA(hsATP5G2)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE120)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C(SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0842] hsGHITM-90N:

[0843] MLAARLVCLRTLPSRVFHPAFTKASPVVKNSITKNQWLLTPSRE(hsGHITM)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE90)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0844] hsGHITM-120C-8e:

[0845] MLAARLVCLRTLPSRVFHPAFTKASPVVKNSITKNQWLLTPSRE(hsGHITM)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE120)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0846] hsSDHD-90N:

[0847] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(hsSDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE90)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0848] hsSDHD-120C-8e:

[0849] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(hsSDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE120)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0850] hsNDUFAB1-90N:

[0851] MASRVLSAYVSRLPAAFAPLPRVRMLAVARPLSTALCSAGTQTRLGTLQPALVLAQVPGRVTQLCRQY(hsNDUFAB1)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE90)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0852] hsNDUFAB1-120C-8e:

[0853] MASRVLSAYVSRLPAAFAPLPRVRMLAVARPLSTALCSAGTQTRLGTLQPALVLAQVPGRVTQLCRQY(hsNDUFAB1)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE120)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0854] hsOPA1-90N:

[0855] MWRLRRAAVACEVCQSLVKHSSGIKGSLPLQKLHLVSRSIYHSHHPTLKLQRPQLRTSFQQFSSLTNLPLRKLKFSPIKYGYQPRRN(hsOPA1)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE90)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0856] hsOPA1-120C-8e:

[0857] MWRLRRAAVACEVCQSLVKHSSGIKGSLPLQKLHLVSRSIYHSHHPTLKLQRPQLRTSFQQFSSLTNLPLRKLKFSPIKYGYQPRRN(hsOPA1)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE120)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0858] SOD2-3xHA-90-MutH*

[0859] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD(SOD2)YPYDVPDYAGYPYDVPDYAGYPYDVPDYA(3xHA)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 90)GS(Linker)SQPRPLLSPPETEEQLLAQAQQLSGYTLGELAALAGLVTPENLKRDKGWIGVLLEIWLGASAGSKPEQDFAALGVELKTIPVDSLGRPLATTAVCVAPLTGNSGVTWETSHVRHKLKRVLWIPVEGERSIPLAKRRVGSPLLWSPNEEEDRQLREDWEELMDMIVLGQIERITARHGEYLQIRPKAANAKALTEAIGARGERILTLPRGFYLKKNFTSALLARHFLIQ(MutH*)

[0860] COX8A-3xFLAG-120-8e:

[0861] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)DYKDHDGDYKDHDIDYKDDDDK(3xFLAG)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Right TALE120)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0862] COX8A-3xFLAG-115-8e:

[0863] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)DYKDHDGDYKDHDIDYKDDDDK(3xFLAG)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Right TALE115)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0864] COX8A-3xFLAG-110-8e:

[0865] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)DYKDHDGDYKDHDIDYKDDDDK(3xFLAG)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Right TALE110)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0866] COX8A-3xFLAG-90-8e:

[0867] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)DYKDHDGDYKDHDIDYKDDDDK(3xFLAG)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Right TALE90)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN

[0868] SOD2-3xHA-120-BspD6I

[0869] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD(SOD2)YPYDVPDYAGYPYDVPDYAGYPYDVPDYA(3xHA)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 120)GS(Linker)RQLEEVIDLLEVYHEKKNVIEEKIKARFIANKNTVFEWLTWNGFIILGNALEYKNNFVIDEELQPVTHAAGNQPDMEIIYEDFIVLGEVTTSKATQFKMESEPVTRHYLNKKKELEKQGVEKELYCLFIAPEINKNTFEEFMKYNIVQNTRIIPLSLKQFNMLLMVQKKLIEKGRRLSSDYDIKNLMVSLYRTTIECERKYTQIKAGLEETLNNWVVDKEVRF(BspD6I)

[0870] SOD2-3xHA-115-BspD6I

[0871] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD(SOD2)YPYDVPDYAGYPYDVPDYAGYPYDVPDYA(3xHA)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 115)GS(Linker)RQLEEVIDLLEVYHEKKNVIEEKIKARFIANKNTVFEWLTWNGFIILGNALEYKNNFVIDEELQPVTHAAGNQPDMEIIYEDFIVLGEVTTSKATQFKMESEPVTRHYLNKKKELEKQGVEKELYCLFIAPEINKNTFEEFMKYNIVQNTRIIPLSLKQFNMLLMVQKKLIEKGRRLSSDYDIKNLMVSLYRTTIECERKYTQIKAGLEETLNNWVVDKEVRF(BspD6I)

[0872]

[0873] SOD2-3xHA-110-BspD6I

[0874] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD(SOD2)YPYDVPDYAGYPYDVPDYAGYPYDVPDYA(3xHA)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 110)GS(Linker)RQLEEVIDLLEVYHEKKNVIEEKIKARFIANKNTVFEWLTWNGFIILGNALEYKNNFVIDEELQPVTHAAGNQPDMEIIYEDFIVLGEVTTSKGATQFKMESEPVTRHYLNKKKELEKQGVEKELYCLFIAPEINKNTFEEFMKYNIVQNTRIIPLSLKQFNMLLMVQKKLIEKGRRLSSYDIKNLMVSLYRTTIECERKYTQIKAGLEETLNNWVVDKEVRF(BspD6I)

[0875] SOD2-3xHA-322-MutH*

[0876] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD(SOD2)YPYDVPDYAGYPYDVPDYAGYPYDVPDYA(3xHA)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 322)GS(Linker)SQPRPLLSPPETEEQLLAQAQQLSGYTLGELAALAGLVTPENLKRDKGWIGVLLEIWLGASAGSKPEQDFAALGVELKTIPVDSLGRPLATTAVCVAPLTGNSGVTWETSHVRHKLKRVLWIPVEGERSIPLAKRRVGSPLLWSPNEEEDRQLREDWEELMDMIVLGQIERITARHGEYLQIRPKAANAKALTEAIGARGERILTLPRGFYLKKNFTSALLARHFLIQ(MutH*)

[0877] SOD2-3xHA-326-BspD6I

[0878] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD(SOD2)YPYDVPDYAGYPYDVPDYAGYPYDVPDYA(3xHA)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 326)GS(Linker)RQLEEVIDLLEVYHEKKNVIEEKIKARFIANKNTVFEWLTWNGFIILGNALEYKNNFVIDEELQPVTHAAGNQPDMEIIYEDFIVLGEVTTSKGATQFKMESEPVTRHYLNKKKELEKQGVEKELYCLFIAPEINKNTFEEFMKYNIVQNTRIIPLSLKQFNMLLMVQKKLIEKGRRLSSYDIKNLMVSLYRTTIECERKYTQIKAGLEETLNNWVVDKEVRF(BspD6I)

[0879]

[0880] SDHD-90-MutH*

[0881] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 90)GS(Linker)SQPRPLLSPPETEEQLLAQAQQLSGYTLGELAALAGLVTPENLKRDKGWIGVLLEIWLGASAGSKPEQDFAALGVELKTIPVDSLGRPLATTAVCVAPLTGNSGVTWETSHVRHKLKRVLWIPVEGERSIPLAKRRVGSPLLWSPNEEEDRQLREDWEELMDMIVLGQIERITARHGEYLQIRPKAANAKALTEAIGARGERILTLPRGFYLKKNFTSALLARHFLIQ(MutH*)

[0882] SDHD -120-8e:

[0883] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Right TALE120)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0884] SDHD -115-8e:

[0885] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Right TALE115)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0886] SDHD -110-8e:

[0887] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Right TALE110)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0888] SDHD -90-8e:

[0889] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Right TALE90)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN

[0890] SDHD -120-BspD6I

[0891] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 120)GS(Linker)RQLEEVIDLLEVYHEKKNVIEEKIKARFIANKNTVFEWLTWNGFIILGNALEYKNNFVIDEELQPVTHAAGNQPDMEIIYEDFIVLGEVTTSKATQFKMESEPVTRHYLNKKKELEKQGVEKELYCLFIAPEINKNTFEEFMKYNIVQNTRIIPLSLKQFNMLLMVQKKLIEKGRRLSSDYDIKNLMVSLYRTTIECERKYTQIKAGLEETLNNWVVDKEVRF(BspD6I)

[0892] SDHD-115-BspD6I

[0893] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 115)GS(Linker)RQLEEVIDLLEVYHEKKNVIEEKIKARFIANKNTVFEWLTWNGFIILGNALEYKNNFVIDEELQPVTHAAGNQPDMEIIYEDFIVLGEVTTSKATQFKMESEPVTRHYLNKKKELEKQGVEKELYCLFIAPEINKNTFEEFMKYNIVQNTRIIPLSLKQFNMLLMVQKKLIEKGRRLSSDYDIKNLMVSLYRTTIECERKYTQIKAGLEETLNNWVVDKEVRF(BspD6I)

[0894]

[0895] SDHD-110-BspD6I

[0896] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 110)GS(Linker)RQLEEVIDLLEVYHEKKNVIEEKIKARFIANKNTVFEWLTWNGFIILGNALEYKNNFVIDEELQPVTHAAGNQPDMEIIYEDFIVLGEVTTSKGATQFKMESEPVTRHYLNKKKELEKQGVEKELYCLFIAPEINKNTFEEFMKYNIVQNTRIIPLSLKQFNMLLMVQKKLIEKGRRLSSYDIKNLMVSLYRTTIECERKYTQIKAGLEETLNNWVVDKEVRF(BspD6I)

[0897] SDHD -322-MutH*

[0898] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 322)GS(Linker)SQPRPLLSPPETEEQLLAQAQQLSGYTLGELAALAGLVTPENLKRDKGWIGVLLEIWLGASAGSKPEQDFAALGVELKTIPVDSLGRPLATTAVCVAPLTGNSGVTWETSHVRHKLKRVLWIPVEGERSIPLAKRRVGSPLLWSPNEEEDRQLREDWEELMDMIVLGQIERITARHGEYLQIRPKAANAKALTEAIGARGERILTLPRGFYLKKNFTSALLARHFLIQ(MutH*)

[0899] SDHD -326-BspD6I

[0900] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 326)GS(Linker)RQLEEVIDLLEVYHEKKNVIEEKIKARFIANKNTVFEWLTWNGFIILGNALEYKNNFVIDEELQPVTHAAGNQPDMEIIYEDFIVLGEVTTSKATQFKMESEPVTRHYLNKKKELEKQGVEKELYCLFIAPEINKNTFEEFMKYNIVQNTRIIPLSLKQFNMLLMVQKKLIEKGRRLSSDYDIKNLMVSLYRTTIECERKYTQIKAGLEETLNNWVVDKEVRF(BspD6I)

[0901] SOD2-3xHA-17N:

[0902] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD(SOD2)YPYDVPDYAGYPYDVPDYAGYPYDVPDYA(3xHA)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(G3460A Left TALE 17)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0903] SOD2-3xHA-18N:

[0904] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD(SOD2)YPYDVPDYAGYPYDVPDYAGYPYDVPDYA(3xHA)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(G3460A Left TALE 18)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0905]

[0906] COX8A-3xFLAG-56C-8e:

[0907] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)DYKDHDGDYKDHDIDYKDDDDK(3xFLAG)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(G3460A Right TALE56)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0908] COX8A-3xFLAG-57C-8e:

[0909] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)DYKDHDGDYKDHDIDYKDDDDK(3xFLAG)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(G3460A Right TALE57)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0910] COX8A-3xFLAG-58C-8e:

[0911] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)DYKDHDGDYKDHDIDYKDDDDK(3xFLAG)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(G3460A Right TALE58)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0912] SOD2-17N:

[0913] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD(SOD2)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(G3460A Left TALE 17)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0914] SOD2-18N:

[0915] MALSRAVCGTSRQLAPVLGYLGSRQKHSLPD(SOD2)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(G3460A Left TALE 18)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0916] COX8A-56C-8e:

[0917] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(G3460A Right TALE 56)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0918] COX8A-57C-8e:

[0919] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(G3460A Right TALE57)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0920] COX8A-58C-8e:

[0921] MASVLTPLLLRGLTGSARRLPVPRAKIHSL(COX8A)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(G3460A Right TALE58)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0922]

[0923] SDHD-17N:

[0924] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(G3460A Left TALE 17)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0925] SDHD-18N:

[0926] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(G3460A Left TALE 18)GS(Linker)GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(1397N)

[0927] SDHD-56C-8e:

[0928] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(G3460A Right TALE56)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0929] SDHD-57C-8e:

[0930] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(G3460A Right TALE57)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0931] SDHD-58C-8e:

[0932] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(G3460A Right TALE58)GS(Linker)AIPVKRGATGETKVFTGNSNSPKSPTKGGC(1397C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(TadA8e)

[0933] SDHD-329v-WC03_S257N

[0934] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 329v)GS(Linker) NANCNQEKPVLPKYDGKTTEGVMVTPDGKQISFKSGNSSTPSGSYPQYKAQSASHVEGKAALYMRENGINEATVFHNNPNGTCGFCDRQVPALLPKGAKLTVVPPSNS(WC03_S257N)

[0935] SDHD-330v-WC03_S257N

[0936] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 330v)GS(Linker)NANCNQEKPVLPKYDGKTTEGVMVTPDGKQISFKSGNSSTPSGSYPQYKAQSASHVEGKAALYMRENGINEATVFHNNPNGTCGFCDRQVPALLPKGAKLTVVPPSNS(WC03_S257N)

[0937] SDHD-384v-WC03_S257C-V28R

[0938] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE384v)GS(Linker)VANNVRAIPVPKTYIGNSTVPKIK(WC03_S257C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDERERPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(V28R)

[0939] SDHDcut-329v-WC03_S257N

[0940] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQ(SDHDcut)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 329v)GS(Linker) NANCNQEKPVLPKYDGKTTEGVMVTPDGKQISFKSGNSSTPSGSYPQYKAQSASHVEGKAALYMRENGINEATVFHNNPNGTCGFCDRQVPALLPKGAKLTVVPPSNS(WC03_S257N)

[0941] SDHDcut-330v-WC03_S257N

[0942] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQ(SDHDcut)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 330v)GS(Linker)NANCNQEKPVLPKYDGKTTEGVMVTPDGKQISFKSGNSSTPSGSYPQYKAQSASHVEGKAALYMRENGINEATVFHNNPNGTCGFCDRQVPALLPKGAKLTVVPPSNS(WC03_S257N)

[0943] SDHDcut-384v-WC03_S257C-V28R

[0944] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQ(SDHDcut)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE384v)GS(Linker)VANNVRAIPVPKTYIGNSTVPKIK(WC03_S257C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDERERPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(V28R)

[0945] SDHA-329v-WC03_S257N

[0946] MSGVRGLSRLLSARRLALAKAWPTVLQTGTRGFHFTV(SDHA)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 329v)GS(Linker) NANCNQEKPVLPKYDGKTTEGVMVTPDGKQISFKSGNSSTPSGSYPQYKAQSASHVEGKAALYMRENGINEATVFHNNPNGTCGFCDRQVPALLPKGAKLTVVPPSNS(WC03_S257N)

[0947] SDHA-330v-WC03_S257N

[0948] MSGVRGLSRLLSARRLALAKAWPTVLQTGTRGFHFTV(SDHA)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 330v)GS(Linker)NANCNQEKPVLPKYDGKTTEGVMVTPDGKQISFKSGNSSTPSGSYPQYKAQSASHVEGKAALYMRENGINEATVFHNNPNGTCGFCDRQVPALLPKGAKLTVVPPSNS(WC03_S257N)

[0949] SDHDcut-384v-WC03_S257C-V28R

[0950] MSGVRGLSRLLSARRLALAKAWPTVLQTGTRGFHFTV(SDHA)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKYHGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE384v)GS(Linker)VANNVRAIPVPKTYIGNSTVPKIK(WC03_S257C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDERERPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(V28R)

[0951]

[0952] SDHD-329-WC03_S257N

[0953] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 329v)GS(Linker) NANCNQEKPVLPKYDGKTTEGVMVTPDGKQISFKSGNSSTPSGSYPQYKAQSASHVEGKAALYMRENGINEATVFHNNPNGTCGFCDRQVPALLPKGAKLTVVPPSNS(WC03_S257N)

[0954] SDHD-384-WC03_S257C-V28R

[0955] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE384v)GS(Linker)VANNVRAIPVPKTYIGNSTVPKIK(WC03_S257C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDERERPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(V28R)

[0956] SDHDcut-329-WC03_S257N

[0957] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQ(SDHDcut)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 329v)GS(Linker) NANCNQEKPVLPKYDGKTTEGVMVTPDGKQISFKSGNSSTPSGSYPQYKAQSASHVEGKAALYMRENGINEATVFHNNPNGTCGFCDRQVPALLPKGAKLTVVPPSNS(WC03_S257N)

[0958] SDHDcut-384-WC03_S257C-V28R

[0959] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQ(SDHDcut)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE384v)GS(Linker)VANNVRAIPVPKTYIGNSTVPKIK(WC03_S257C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDERERPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(V28R)

[0960] SDHD-329-WC03_S257N-NES

[0961] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 329v)GS(Linker)NANCNQEKPVLPKYDGKTTEGVMVTPDGKQISFKSGNSSTPSGSYPQYKAQSASHVEGKAALYMRENGINEATVFHNNPNGTCGFCDRQVPALLPKGAKLTVVPPSNS(WC03_S257N)VDEMTKKFGTLTIHDTEK(NES)

[0962]

[0963] SDHD-384-WC03_S257C-V28R-NES

[0964] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQDRPIPEWCGVQHIHLSPSHH(SDHD)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE384v)GS(Linker)VANNVRAIPVPKTYIGNSTVPKIK(WC03_S257C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDERERPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(V28R)VDEMTKKFGTLTIHDTEK(NES)

[0965] SDHDcut-329-WC03_S257N-NES

[0966] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQ(SDHDcut)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGS(ND4 Left TALE 329v)GS(Linker) NANCNQEKPVLPKYDGKTTEGVMVTPDGKQISFKSGNSSTPSGSYPQYKAQSASHVEGKAALYMRENGINEATVFHNNPNGTCGFCDRQVPALLPKGAKLTVVPPSNS(WC03_S257N)VDEMTKKFGTLTIHDTEK(NES)

[0967] SDHDcut-384-WC03_S257C-V28R-NES

[0968] MAVLWRLSAVCGALGGRALLLRTPVVRPAHISAFLQ(SDHDcut)GIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLLV(ND4 Right TALE384v)GS(Linker)VANNVRAIPVPKTYIGNSTVPKIK(WC03_S257C)SGSETPGTSESATPES(Linker)SEVEFSHEYWMRHALTLAKRARDERERPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN(V28R)VDEMTKKFGTLTIHDTEK(NES)

Claims

1. (1) DNA binding protein, (2) one or more deaminase selected from adenine deaminase and cytosine deaminase, and (3) Contains a mitochondrial targeting sequence (MTS), Does not include affinity tags, A mitochondrial DNA base editing system comprising a base editor or a polynucleotide encoding the same.

2. In paragraph 1, The above base editor A cytosine deaminase is included, and the cytosine deaminase is a double-stranded DNA-specific cytosine deaminase, which exists in one full-length form or in two split forms, and when it exists in two split forms, it exhibits cytosine deaminizing activity through their dimerization. Adenine deaminase is optionally included, Mitochondrial DNA base editing system.

3. In paragraph 2, The above base editor (1) DNA binding protein, (2) cytosine deaminase, and (3) MTS in the form of two fusion proteins, The two fusion proteins each independently comprise one DNA binding protein, The cytosine deaminase exists in the form of two splits, one of which is included in each of the fusion proteins, In the case where adenine deaminase is included, it is included in either or both of the two fusion proteins. Mitochondrial DNA base editing system.

4. A mitochondrial DNA base editing system according to claim 1, wherein the base editor further comprises (4) a nickase.

5. In the fourth paragraph, the base editor comprises (1) a DNA binding protein, (2) a deaminase, (3) MTS, and (4) a nickase in the form of two fusion proteins, The two fusion proteins each independently comprise one DNA binding protein, One of the two fusion proteins comprises a nickase, and the other fusion protein comprises an adenine deaminase or a cytosine deaminase. Mitochondrial DNA base editing system.

6. A mitochondrial DNA base correction system according to claim 1, wherein the DNA binding proteins included in the base editor are each independently selected from the group consisting of a zinc finger protein, a TALE (transcription activator-like effector) protein, and a CRISPR-associated nuclease.

7. A mitochondrial DNA base editing system in which the MTS included in the base editor in paragraph 1 is directly linked to a DNA binding protein.

8. A mitochondrial DNA base correction system in which less than 5 cationic amino acids exist between the MTS and the DNA binding protein in the first paragraph.

9. A mitochondrial DNA base editing system according to claim 1, wherein the base editor comprises two fusion proteins, each of the two fusion proteins comprising MTS, and the MTS included in the two fusion proteins have the same amino acid sequence.

10. A mitochondrial DNA base editing system according to claim 1, wherein the base editor comprises two fusion proteins, each of the two fusion proteins comprising MTS, and the MTS included in the two fusion proteins have different amino acid sequences.

11. A mitochondrial DNA base editing system in the first paragraph, wherein the MTS included in the base editor is one or more identical or different MTS sequences connected in series.

12. A mitochondrial DNA base editing system, wherein the MTS included in the base editor independently comprises an amino acid sequence of an MTS of SDHA, SDHB, SDHC, SDHD, ATP6, LOC, GHITM, ADCK3, ATP5G2, ATP5G3, ATP9, OPA1, COX8A or SOD2, or a truncated form thereof, or a conservative amino acid substitution thereof.

13. A mitochondrial DNA base correction system according to claim 12, wherein MTS comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 15, and 16.

14. A mitochondrial DNA base correction system having improved base correction efficiency compared to a corresponding base system comprising an affinity tag or a polynucleotide encoding the same in the first paragraph.

15. A polynucleotide encoding the base editor described in paragraph 1.

16. A mitochondrial DNA base correction composition comprising the base correction system described in paragraph 1.

17. A delivery system for delivering the base correction system, comprising the base correction system described in paragraph 1, to a cell, tissue or organism containing target DNA for mitochondrial DNA base correction.

18. In the 28th paragraph, the base correction system comprises a polynucleotide, and is a carrier that is a plasmid vector, a viral vector, a lipid nanoparticle, or a polymeric nanoparticle.

19. A method for base correction of mitochondrial DNA, comprising introducing the base correction system described in paragraph 1 into a cell containing target DNA for base correction. 20.(1) Measuring the base correction efficiency of a base editor comprising a DNA binding protein, (2) one or more deaminase selected from adenine deaminase and cytosine deaminase, (3) MTS, and (4) an affinity tag for target mitochondrial DNA, Providing a base correction system comprising a tag-free base editor having the same amino acid sequence as the base editor but not including an affinity tag, or a polynucleotide encoding the same. A method for improving the efficiency of mitochondrial DNA base editing.

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