Enzyme, enzyme complex, and use thereof

Abstract

To provide an enzyme having the activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in an animal cell; a complex of the enzyme; and use of the same.SOLUTION: The present invention employs an enzyme having the activity of converting guanosine in a polynucleotide into adenosine in an animal cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an enzyme and an enzyme complex having an activity of converting guanosine in RNA into a genetic code corresponding to adenosine in animal cells, and to the use of these. [Background technology]

[0002] DNA editing is one of the treatment methods for genetic diseases. Although DNA editing is an effective treatment method, there is a concern that unexpected mutations called off-target mutations may occur. Since DNA (genome) is a molecule that exists permanently within cells, if a mutation occurs in DNA, there is a concern that the mutation may be passed down through generations.

[0003] In recent years, RNA editing has been attracting attention as a method to solve such concerns. mRNA is not only a temporary molecule copied from DNA, but also has a mechanism in cells to degrade incorrect mRNA. Therefore, even if a mutation occurs in mRNA due to off-target, the mRNA is quickly degraded, so there is no concern that the mutation will be passed down through generations. Therefore, RNA editing is considered to be a highly safe and extremely effective method for treating genetic diseases and the like.

[0004] RNA editing is a physiological process. In mammals, RNA editing that converts adenosine to guanosine and cytidine to uridine are known, while in plants, RNA editing that converts cytidine to uridine and uridine to cytidine are known.

[0005] Among these RNA editing, the RNA editing that converts adenosine to guanosine and the RNA editing that converts cytidine to uridine are known to be hydrolytic deamination reactions of nucleic acid bases. In addition, among the enzymes that catalyze the conversion of cytidine to uridine, there are enzymes that not only have the activity of converting cytidine in RNA to uridine, but also have the activity of converting cytidine in DNA to uridine.

[0006] In RNA editing, adenosine in mRNA is converted to guanosine by adenosine deaminase (Adenosine Deaminase Acting on RNA; ADAR). Since inosine forms a Watson-Crick base pair with cytosine, the inosine is recognized as the same genetic code as guanosine in translation (Non-Patent Document 1).

[0007] In plants, there exists a mechanism for RNA editing that converts cytidine to uridine, and it is known that RNA editing is catalyzed by a type of PPR (pentatricopeptide repeat) protein that binds to RNA in a sequence-specific manner. PPR proteins are characterized by repeating PPR motifs with two α-helical structures consisting of approximately 35 amino acids. Among PPR proteins, PPR-DYW type PPR proteins, which have an E (extension) domain and a DYW (Asp-Tyr-Trp) domain at the carboxyl terminus, have deaminase activity and catalyze RNA editing that converts cytidine to uridine via hydrolytic deamination of cytidine.

[0008] The present inventors have discovered an enzyme from hornwort (Anthoceros agrestis) that catalyzes the reaction of converting uridine to cytidine. Furthermore, the present inventors have discovered that the GRP (Gly-Arg-Pro) domain of the enzyme catalyzes the reaction of converting uridine to cytidine (Patent Document 1).

[0009] Furthermore, the present inventors analyzed the base sequence of the total RNA of Arabidopsis thaliana, a model plant, and found that in addition to the above-mentioned base substitutions, there were many guanosine-to-adenosine substitutions, and that the occurrence rate of these substitutions was significantly higher than the occurrence frequency of mutations (Non-Patent Document 2). [Prior art documents] [Patent documents]

[0010] [Patent Document 1] WO2023 / 120658 A1 [Non-patent literature]

[0011] [Non-Patent Document 1] Md TA Azad et al., “Site-directed RNA editing by adenosine deaminase acting on RNA for correction of the genetic code in gene therapy” Gene Ther., 24(12) 779-786 (2017) [Non-Patent Document 2] Ruchika et al., “Genome-Wide Identification of U-to-C RNA Editing Events for Nuclear Genes in Arabidopsis thaliana” Cells, 10(3), 635 (2021) Summary of the Invention [Problem to be solved by the invention]

[0012] However, to date, the mechanism for converting guanosine in a polynucleotide into a genetic code equivalent to adenosine in animal cells and the enzymes that catalyze this conversion have not been identified, and it has not been possible to convert guanosine in a polynucleotide into a genetic code equivalent to adenosine in animal cells.

[0013] An object of one aspect of the present invention is to provide an enzyme and an enzyme complex having an activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in an animal cell, and uses of the enzyme and the enzyme complex. [Means for solving the problem]

[0014] In hornwort, PPR proteins involved in RNA editing are known to exist, including DYW-type PPR proteins that catalyze the deamination of cytidine, GRP-type PPR proteins that catalyze the amino group transfer to uridine, and DRH (Asp-Arg-His)-type PPR proteins. The function and role of DRH-type PPR proteins remained unknown.

[0015] As a result of intensive research, the inventors discovered that the DRH-type PPR protein has the activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in animal cells (e.g., human cells), thereby completing the present invention.

[0016] The activity of converting guanosine in polynucleotides into the genetic code equivalent to adenosine is believed to result from the deamination of guanosine to produce xanthosine, and more specifically, from the formation of a Watson-Crick base pair with uridine in the tRNA anticodon. Xanthosine, a product of the deamination of guanine, is known to be recognized as either guanosine or adenosine by Sanger sequencing analysis, and this is believed to be due to the fact that xanthosine forms base pairs with cytosine and thymine (Non-Patent Document 1).

[0017] In order to solve the above problems, one aspect of the present invention includes the following inventions.

[0018] [1] An enzyme that has the activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in animal cells.

[0019] [2] The enzyme according to [1], comprising any one of the following polypeptides (1) to (6): (1) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2; (2) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2 in which one or several amino acids have been substituted, deleted, inserted, and / or added; (3) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2; (4) A polypeptide encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 or 4; (5) A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 or 4; (6) A polypeptide having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a polypeptide encoded by a polynucleotide having the base sequence of SEQ ID NO: 3 or 4.

[0020] [3] The enzyme according to [1] or [2], further comprising an E2 domain which is any one of the polypeptides (7) to (12): (7) A polypeptide consisting of the amino acid sequence of SEQ ID NO:5; (8) A polypeptide consisting of the amino acid sequence of SEQ ID NO:5 in which one or several amino acids have been substituted, deleted, inserted, and / or added; (9) A polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO:5; (10) A polypeptide encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO:6; (11) A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:6; (12) A polypeptide having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a polypeptide encoded by a polynucleotide consisting of the base sequence of SEQ ID NO:6.

[0021] [4] An enzyme complex comprising the enzyme according to any one of [1] to [3] and a base sequence recognition module that binds to a specific sequence in a polynucleotide, thereby allowing the enzyme to act on a specific guanosine in the polynucleotide sequence.

[0022] [5] The enzyme complex described in [4], wherein the base sequence recognition module (a) binds to a specific sequence in a polynucleotide involved in the cause or aggravation of a pathology, and / or (b) binds to a specific sequence in a polynucleotide having a mutation in which adenosine is replaced with guanosine.

[0023] [6] An expression vector for animals, comprising a polynucleotide encoding the enzyme complex according to [4] or [5].

[0024] [7] A genetic disease therapeutic agent for animals, comprising the enzyme complex according to [4] or [5], or the expression vector according to [6]. Effect of the Invention

[0025] According to one aspect of the present invention, there are provided an enzyme and an enzyme complex having an activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in an animal cell, and uses thereof. [Brief description of the drawings]

[0026] [Figure 1] FIG. 2 shows the structure of an expression vector in an embodiment of the present invention. [Diagram 2] FIG. 1 shows the function of the PPR domain of the PPR56 protein in an example of the present invention. [Diagram 3]1 is an image showing the activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in an example of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] An embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to each configuration described below, and various modifications are possible within the scope of the claims, and embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention. In addition, all academic literature and patent documents described in this specification are incorporated herein by reference. In addition, unless otherwise specified in this specification, "A to B" representing a numerical range means "A or more, B or less."

[0028] [1. Enzymes] The enzyme according to one embodiment of the present invention is an enzyme having an activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in an animal cell. The enzyme according to one embodiment of the present invention may be an enzyme having an activity of deaminating guanosine in a polynucleotide and converting it into xanthosine in an animal cell. Xanthosine in a polynucleotide can be translated as the same genetic code as adenosine during protein biosynthesis. In this specification, the term "polynucleotide" includes DNA and RNA.

[0029] Various intracellular mechanisms are different between microorganisms (e.g., E. coli) and animal cells (e.g., human cells), and therefore the metabolism and reactions occurring in microorganisms do not occur in the same way in animal cells. For example, codon usage frequency, protein stability, the presence or absence of organelles, the presence or absence of splicing, and gene expression mechanisms are different between microorganisms and animal cells, and therefore the metabolism and reactions occurring in microorganisms do not occur in the same way in animal cells. As demonstrated in the examples described below, the enzyme according to one embodiment of the present invention is expressed in animal cells. Therefore, the enzyme according to one embodiment of the present invention can convert guanosine in a polynucleotide into a genetic code corresponding to adenosine in an animal cell, and / or can deaminate guanosine in a polynucleotide to convert it to xanthosine in an animal cell.

[0030] There are no limitations on the method for confirming whether or not a desired enzyme (i) has the activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in an animal cell, or (ii) has the activity of deaminating guanosine in a polynucleotide and converting it to xanthosine in an animal cell.

[0031] For example, (i) converting the start codon "ATG" of a marker gene (e.g., a gene encoding a fluorescent protein) to "GTG", and then inserting the marker gene into an expression vector to prepare an expression vector A, (ii) introducing the expression vector A and an expression vector B for expressing a desired enzyme into an animal cell, and (iii) confirming the expression of a marker protein encoded by the marker gene in the animal cell. If the expression of the marker protein can be confirmed in the above (iii), it means that the start codon lost by mutation has been restored, that is, the mutated guanosine has been recognized as adenosine, and therefore it can be determined that the desired enzyme has the activity of converting guanosine in a polynucleotide into a genetic code equivalent to adenosine in an animal cell, or has the activity of deaminating guanosine in a polynucleotide into xanthosine in an animal cell.

[0032] For example, (iv) an expression vector B for expressing a desired enzyme is introduced into an animal cell, (v) a cDNA corresponding to the RNA of a specific gene is obtained in the animal cell, and (vi) the base sequence of the cDNA is decoded. If the frequency of a base that should be guanosine being decoded as adenosine in (vi) increases compared to when expression vector B is not introduced into the animal cell, it can be determined that the desired enzyme has an activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in the animal cell, or has an activity of deaminating guanosine in a polynucleotide into xanthosine in the animal cell.

[0033] The present invention has, for example, the following advantages (a) to (c): (a) It is possible to provide an enzyme and an enzyme complex having an activity for converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in an animal cell, and uses thereof.

[0034] (b) The mutated polynucleotide can be repaired to a normal polynucleotide by converting the guanosine that occurs in the polynucleotide due to the mutation into the genetic code equivalent to adenosine.

[0035] (c) By converting guanosine, which is important for the onset and / or progression of a disease, into a genetic code equivalent to adenosine, the onset and / or progression of a disease can be suppressed. For example, in animals, including humans, genetic information is divided in DNA by an intervening sequence called an intron, and after transcription, mature mRNA is generated from a pre-mRNA by RNA splicing. The sequences at the boundaries between exons and introns that code for protein information are well preserved, and are often Ag and gG. (Capital letters indicate exons and lowercase letters indicate introns.) If this conserved A mutates to guanosine, the exon will not be spliced. It is also known that g at the 5' end of an intron binds to a specific a in an intron in splicing, and if the a at this branch point mutates to g, splicing will also not occur. Many cases of diseases caused by such splicing abnormalities are known, and if these adenosines can be converted to guanosine, it is expected that the diseases will improve. In addition, high expression of TERRA (Telomeric repeat-containing RNA), a long non-coding RNA expressed from telomeres at the ends of chromosomes, is known to be involved in the progression of cancer. Furthermore, it is known that the function of TERRA is involved in a strong structure called a G-quadruplex. In a G-quadruplex, four molecules of guanine form hydrogen bonds with each other to form a rigid planar structure. By converting the guanosine in TERRA to a genetic code equivalent to adenosine (deamination of the guanosine in TERRA), the G-quadruplex structure formed by hydrogen bonds is broken, and as a result, it is expected that the progression of cancer can be suppressed.

[0036] An enzyme according to one embodiment of the present invention may be an enzyme containing any one of the following polypeptides (1) to (6). With this configuration, guanosine in a polynucleotide can be converted into a genetic code corresponding to adenosine.

[0037] (1) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2; (2) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2 in which one or several amino acids have been substituted, deleted, inserted, and / or added; (3) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2; (4) A polypeptide encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 or 4; (5) A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 or 4; (6) A polypeptide having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a polypeptide encoded by a polynucleotide having the base sequence of SEQ ID NO: 3 or 4.

[0038] Specific sequences of SEQ ID NOs: 1 to 4 are shown below. Note that a polynucleotide consisting of the base sequence of SEQ ID NO: 3 is presented as an example of a gene encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, and a polynucleotide consisting of the base sequence of SEQ ID NO: 4 is presented as an example of a gene encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

[0039] SEQ ID NO:1:KKPAKACIEVKNRVHEFTVGEERSDVASMLRDVSTKLKLGGHVPETWLVLKAASEQEKEDALCGHAEKLALAYGLLNTPDGTTLLVTKNLRMCHDCHSSTKIMSHVENREIIVRDVHRVHRFLNGACSCGDRH; SEQ ID NO:2:KKAAKACIEVQNIEVHEFTVGEDRDISSKLRSVNMRLKEEGGHVPQTQLVLKAMSEEKKEDALCGHAEKLALAYGLLNTPDGTTLVVTKNLRMCNDCHSSTKIMSRLEKREIIVRDAHRVHRFLDGACSCRDRH; Sequence number 3:AAGAAGCCAGCCAAGGCATGCATCGAGGTGAAGAACAGGTGCATGAGTTCACAGTGGGTGAAGAGAGGTCTGATGTTGCATCCATGCTAGAGGGATGTGAGTACGAAGTTGAAATTGGGCGGCCATGTTCCGGAAACATGGCTGGTCCTGAAGGCAGGAGAAGGAAGCAGCCTTTGTTGTGGACATGCCGAGAAGCTGGCTTGGCATATGGTCTGCTCCAACACTCCAGATGGTACAACTCTGCTAGTGACTAAAAACCTGCGCATGTGCCATGACTCCACAGCAGCACCAAGATTATGTCACATGTGGAGAATCGAGAAATCATAGTAAGAGATGTCACACCGAGTGCATCGTTTTCTGAATGGTGCTTGTCTCTGGGGATCCGCAC; Sequence number 4: AAGAAGGCAGCCAAGGCATGCATCGAGGTGCAGAACATAGTAGGAGGTGCATGAGTTCACAGTGGGTGAAGATAGGTCTGATAATTTCATCCAAGCTGAGGAGTGTGAATATGCGGTTGAAAGAGGAGGGCGGCCATGTTCCGCAAACACAGCTGGTCCTGAAGGCAATGTCTGAGGAGAAGAAAGGAAGAGGCCTTTGTTGTGGACATGCCGAGAAGCTGGCTTGGCATATGGTCTGCTACAACACTCCAGATGGTACAACTCTGGTAGTGACTAAAAACCTGCGCATGTGCAATGACTCCACAGCAGCACCAAGATTATGTCACGTCTGGAGAAAACGAGAAATCATAGTAAGAGATGCACACCGAGTGCATCGTTTTCTGGATGGTGCTTGCTTGTAGGGATCGCCAC.

[0040] The present inventors searched for PPR-DYW type homologs in the genome of Hornwort and identified a PPR protein having a DRH domain instead of the DYW domain. As shown in the following Examples, the DRH (E2-DRH) domain linked to the E2 domain is an enzyme that catalyzes a reaction in human-derived cells in which any guanosine is deaminated to convert it to xanthosine, resulting in the same genetic code as adenosine. The amino acid sequence of the E2-DRH domain is shown, for example, in the amino acid sequence of SEQ ID NO: 5 combined with SEQ ID NO: 1, or in the amino acid sequence of SEQ ID NO: 5 combined with SEQ ID NO: 2. As long as the enzyme has the activity of converting guanosine to xanthosine, it may be an E2-DRH-like protein of a plant other than Hornwort, an enzyme obtained by modifying cytidine deaminase or adenosine deaminase so as to act on guanosine, or an enzyme obtained by modifying an enzyme that deaminates free guanine or guanosine so as to act on guanosine in a polynucleotide.

[0041] The enzyme according to one embodiment of the present invention is more preferably an enzyme containing an E2 domain that is any one of the polypeptides (7) to (12). With this configuration, the activity of the enzyme can be increased, and guanosine in a polynucleotide can be more efficiently converted into a genetic code corresponding to adenosine.

[0042] (7) A polypeptide consisting of the amino acid sequence of SEQ ID NO:5; (8) A polypeptide consisting of the amino acid sequence of SEQ ID NO:5 in which one or several amino acids have been substituted, deleted, inserted, and / or added; (9) A polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO:5; (10) A polypeptide encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO:6; (11) A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:6; (12) A polypeptide having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a polypeptide encoded by a polynucleotide consisting of the base sequence of SEQ ID NO:6.

[0043] Specific sequences of SEQ ID NOs: 5 to 6 are shown below. The polynucleotide consisting of the base sequence of SEQ ID NO: 6 is an example of a gene encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:5.

[0044] SEQ ID NO:5:AATYVLLSNIYAEAGKWDMVSWVRTMMRERGIR; SEQ ID NO: 6: GCTGCTACCTATGTTCTCCTTTCCAACATCTATGCTGAAGCTGGAAAGTGGGACATGGTATCATGGGTGCGGACTATGATGCGCGAGAGAGGGATTCGC.

[0045] The E2 domains shown in SEQ ID NOs: 5 to 6 are derived from DRH-type PPR proteins of Hornwort. However, the E2 domain is not limited to the E2 domain derived from Hornwort. The E2 domain may also be derived from DYW-type or GRP-type PPR proteins.

[0046] From the viewpoint of increasing the activity of the enzyme, the E2 domain is preferably located on the amino terminal side of the DRH domain. The E2 domain and the DRH domain may be directly linked to each other or may be linked to each other via a desired linker (e.g., an amino acid or a polypeptide).

[0047] With regard to (2) and (8) above, "substitution, deletion, insertion, and / or addition of one or several amino acids" refers to the substitution, deletion, insertion, and / or addition of a number of amino acids (preferably 50 or less, more preferably 40 or less, more preferably 30 or less, more preferably 20 or less, more preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and most preferably 1 or less) that maintains the original function of the polypeptide. The "substitution, deletion, insertion, and / or addition of one or several amino acids" may include artificially induced mutations and naturally occurring mutations.

[0048] With respect to (3), (6), (9) and (12) above, the sequence identity is preferably 91% or more, more preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and most preferably 99% or more.

[0049] The determination of sequence identity between two sequences can, for example, be performed using a mathematical algorithm. Such mathematical algorithms include, but are not limited to, the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local homology algorithm of Smith et al (1981) Adv. Appl. Math. 2:482; the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the method of searching for similarity of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; and the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, with modifications as described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0050] In the above (5) and (11), the term "stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, the conditions include conditions under which DNAs with high homology (e.g., DNAs with a homology of 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more) hybridize with each other and DNAs with lower homology do not hybridize with each other, or conditions under which washing is performed once, preferably 2 to 3 times, at a salt concentration and temperature equivalent to the washing conditions of normal Southern hybridization, which are 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0051] 2. Enzyme Complex An enzyme complex according to an embodiment of the present invention includes an enzyme according to an embodiment of the present invention and a base sequence recognition module for binding to a specific sequence in a polynucleotide and thereby allowing the enzyme to act on a specific guanosine in the polynucleotide sequence, and is capable of converting a desired guanosine in a polynucleotide into a genetic code corresponding to adenosine in an animal cell, and of deaminating a desired guanosine in a polynucleotide into a xanthosine in an animal cell.

[0052] The guanosine on which the enzyme acts is not limited, and may be, for example, a guanosine closely related to the pathology of a disease (e.g., a guanosine in TERRA), or a guanosine present in a codon that has been mutated so as to code for an amino acid different from the wild-type amino acid.

[0053] The base sequence recognition module may be (a) one that binds to a specific sequence in a polynucleotide involved in the cause or aggravation of a pathological condition, and / or (b) one that binds to a specific sequence in a polynucleotide having a mutation in which adenosine is replaced with guanosine. With this configuration, it is possible to improve the pathological condition and / or to suppress the synthesis of mutant proteins and promote the synthesis of normal proteins.

[0054] The base sequence recognition module is not particularly limited as long as it allows the enzyme to act on guanosine present at a specific position in a polynucleotide. The base sequence recognition module may be a single molecule or a complex of multiple molecules. For example, the base sequence recognition module may include either a protein that binds to a polynucleotide in a sequence-specific manner or a polynucleotide that is complementary to at least a portion of the sequence.

[0055] More specifically, when the base sequence recognition module is a polynucleotide, examples of the base sequence recognition module include an MS2 system in which an RNA complementary to a portion of a target RNA is bound to the base sequence recognition module; a CRISPR-dCas system using dCas9 or 13, etc., whose nucleic acid cleavage ability has been inactivated; and an RNA or DNA complementary to a portion of a target RNA bound to a polynucleotide sequence that specifically binds to a specific nucleic acid binding protein.

[0056] When the base sequence recognition module is a protein, examples of the base sequence recognition module include a zinc finger motif, a TAL effector, a PPR protein, and a peptide nucleic acid (PNA) complementary to a target RNA.

[0057] In the CRISPR-dCas system, a dCas protein with no nuclease activity or nickase activity and a guide RNA are used. The guide RNA corresponds to CRISPR RNA (crRNA) and contains a complementary base sequence that forms a base pair with the target sequence and a base sequence that functions as a transactivating crRNA (tracrRNA) and serves as a scaffold for the binding of the dCas protein. The guide RNA binds to the dCas protein by containing the crRNA sequence, and a dCas-guide RNA complex is formed. The enzyme can be made to act specifically on the target guanosine by using a fusion protein of the enzyme and the dCas protein and a guide RNA designed for a guanosine closely related to the pathology of a disease or a guanosine generated in a polynucleotide by an A to G mutation.

[0058] The zinc finger motif is a combination of multiple different zinc finger units of Cys2His2 type, and has the property of binding specifically to a base sequence. The enzyme can be made to specifically act on the target guanosine by using a fusion protein of the enzyme and a zinc finger motif designed to bind to the vicinity of a guanosine closely related to the pathology of a disease or a guanosine generated in a polynucleotide by A to G mutation.

[0059] The TAL effector has a repeating structure of a module consisting of about 34 amino acids, and the binding stability and base specificity are determined by the 12th and 13th amino acid residues of each module. The enzyme can be made to act specifically on the target guanosine by using a fusion protein of the above enzyme and a TAL effector designed to bind to a guanosine closely related to the pathology of a disease or to the vicinity of a guanosine generated in a polynucleotide by A to G mutation.

[0060] Like the TAL effector, the PPR protein can be configured to recognize a specific base sequence by a series of PPR motifs that recognize one nucleic acid base. By using a fusion protein of the enzyme and a PPR protein designed to bind to a guanosine closely related to the pathology of a disease or to the vicinity of a guanosine generated in a polynucleotide by A to G mutation, the enzyme can be made to act specifically on the target guanosine.

[0061] In the backbone of PNA, N-(2-aminoethyl)glycine is bound by an amide bond, not a sugar. In PNA, the purine ring and pyrimidine ring corresponding to the nucleic acid base are bound to the backbone via a methylene group and a carbonyl group, and can bind to the target polynucleotide in a base sequence-specific manner, just like polynucleotides. The enzyme can be made to act specifically on the target guanosine by using a fusion protein of the enzyme and PNA designed to be complementary to the vicinity of a guanosine closely related to the pathology of a disease or a guanosine generated in a polynucleotide by A to G mutation.

[0062] In the enzyme complex, the enzyme and the base sequence recognition module may be linked via a covalent bond, or may be linked by a compound, a linker, or the like. The linker may be a functional group or a molecule that links the enzyme and the base sequence recognition module. For example, the linker may link the enzyme and a protein or a nucleic acid that binds to a polynucleotide in a sequence-specific manner. The linker may be disposed between, for example, two types of functional groups, two types of molecules, or two types of other moieties, and may be linked to each other by a covalent bond. The linker may be a peptide chain consisting of one amino acid or multiple amino acids, or may be an oligonucleotide. The linker may be an organic molecule, a functional group, a polymer, or the like.

[0063] In the following, a case where a PPR protein is used as a base sequence recognition module and a case where an MS2 system is used will be described. Note that these base sequence recognition modules are merely examples, and the present invention is not limited to these.

[0064] PPR proteins are RNA-binding proteins that are widely present in plants, and have repeats of PPR motifs with two α-helix structures consisting of about 35 amino acids. PPR proteins can be configured to recognize and bind to a specific base sequence by a series of PPR motifs that recognize one nucleic acid base, and therefore can be used as a base sequence recognition module. By fusing a fragment of a PPR protein in which a PPR motif is designed to recognize a base sequence near the target guanosine with the above-mentioned enzyme, it becomes possible to guide the enzyme to the target guanosine, and as a result, the target guanosine can be deaminated. A PPR motif that recognizes a base sequence up to just before the target guanosine is preferable. For example, a PPR motif that recognizes a sequence of 7 or more bases, such as up to 1 base upstream, 2 bases upstream, 3 bases upstream, 4 bases upstream, or 5 bases upstream of the target guanosine, may be used. A fusion protein of a PPR motif that recognizes a target base sequence and an enzyme binds to the vicinity of the target guanosine in a specific polynucleotide by the PPR motif in an animal cell (e.g., a human cell). For example, when the active site of an enzyme is induced to a guanosine that is closely related to the pathology of a disease or a guanosine that has been generated in a polynucleotide by an A to G mutation, conversion of the target nucleic acid base can be efficiently carried out.

[0065] The genome of the MS2 phage, an RNA virus, is a single-stranded RNA that functions as a positive strand mRNA. When the MS2 phage infects E. coli, negative strand RNA is synthesized following translation of the genes necessary for the proliferation of the MS2 phage, and positive strand RNA is further synthesized using this negative strand RNA as a template. The MS2 phage synthesizes various proteins of the MS2 phage based on the positive strand RNA, and among these proteins, the MS2 coat protein is a protein that constitutes the shell of the MS2 phage, and has the property of specifically and strongly binding to the loop RNA present in the replicated genome gene in order to insert the genome gene into the shell.

[0066] The MS2 system, which is used for visualization of RNA within cells, is composed of the above-mentioned MS2 coat protein and loop RNA, and the MS2 coat protein and loop RNA are specifically and strongly linked to each other within cells.

[0067] The guide RNA may be a fusion RNA of an RNA complementary to at least a part of a sequence of a polynucleotide containing guanosine closely related to the pathology of a disease, or guanosine generated in a polynucleotide by A to G mutation, and an MS2 loop RNA. If the nucleic acid base to be converted is a "target nucleic acid base", the base sequence of the guide RNA may be composed of a base sequence complementary to at least a part of a base sequence containing guanosine, which is a target nucleic acid base in a polynucleotide. As long as the guide RNA specifically hybridizes to a complementary base sequence, the guide RNA may be a base sequence containing a mismatch with respect to the complementary base sequence, that is, a base sequence complementary to a base sequence obtained by removing one or several bases from the complementary base sequence. When the guide RNA contains a mismatch, it is preferable that only the nucleic acid base in the guide RNA corresponding to the target nucleic acid base is a mismatch. In this case, the nucleic acid base in the guide RNA corresponding to the target nucleic acid base guanosine may be a base other than cytosine (C) which forms a Watson-Crick base pair with guanine.

[0068] The fusion RNA of the guide RNA and the MS2 loop RNA (guide RNA-MS2 RNA) can be prepared based on the base sequence of the guide RNA. For example, the fusion RNA may be one in which the 3' end (or 5' end) of the guide RNA is directly bound to one end of the MS2 loop RNA, or one indirectly bound to the fusion RNA via a linker sequence or the like. The fusion RNA may be one in which the MS2 loop RNA is bound to both ends of the guide RNA.

[0069] The enzyme may be a fusion protein with MS2 coat protein (MS2 coat protein-enzyme). The fusion protein may specifically and strongly bind to the guide RNA via the binding between the MS2 coat protein and the MS2 loop RNA. For example, the fusion protein may be a direct bond between the carboxyl terminal (or amino terminal) of the MS2 coat protein and the amino terminal (or carboxyl terminal) of the enzyme, or may be indirectly bonded via a linker peptide or the like. The fusion protein can be produced based on the gene sequences of the MS2 coat protein and the enzyme.

[0070] The MS2 loop RNA and the MS2 coat protein have strong binding properties. Therefore, by mixing the fusion protein and the fusion RNA or expressing them together in a cell, an enzyme complex consisting of "guide RNA-MS2 loop RNA-MS2 protein-enzyme" can be obtained. The enzyme complex binds complementarily to the polynucleotide targeted by the guide RNA in an animal cell. For example, the active site of the enzyme can be induced to guanosine closely related to the pathology of a disease or guanosine generated in a polynucleotide by A to G mutation, thereby efficiently converting the target nucleic acid base.

[0071] The enzyme complex according to one embodiment of the present invention can cause the enzyme (for example, an enzyme that converts guanosine to xanthosine) to act specifically on the target guanosine by the base sequence recognition module. Thus, by targeting guanosine closely related to the pathology of a disease or guanosine generated by adenosine-to-guanosine mutation, it is possible to convert the guanosine closely related to the pathology and / or repair the genetic code of a mutated polynucleotide. For example, the enzyme complex can convert guanosine in a polynucleotide generated by adenosine-to-guanosine mutation into a genetic code similar to adenosine. In addition, the enzyme complex can destroy the guanine-quadruplex structure by converting guanosine in the guanine-quadruplex of TERRA having a telomere sequence into xanthosine. These conversions make it possible to improve the pathology of a disease.

[0072] In another embodiment, a polynucleotide is provided that encodes a complex comprising the fusion protein and the fusion RNA.

[0073] When the base sequence recognition module includes a nucleic acid that binds to a polynucleotide in a sequence-specific manner, the nucleic acid may be RNA or DNA. In addition, the base sequence recognized by the nucleic acid may be in the vicinity of the target nucleic acid base, and the nucleic acid may or may not include the target nucleic acid base. In addition, when the base sequence recognition module includes a protein that binds to a polynucleotide in a sequence-specific manner, the base sequence in the polynucleotide that the protein recognizes may be in the vicinity of the target nucleic acid base, and the base sequence may or may not include the target nucleic acid base.

[0074] In addition, as long as the guide RNA binds to the target polynucleotide in a sequence-specific manner, a mutant of the MS2 coat protein or CRISPR-dCas protein may be used. In addition, as the base sequence recognition module, a known polynucleotide binding protein and polynucleotide, such as the λN system, may be used. The λN system utilizes the λN22 peptide and the base sequence (Box-B) of the RNA to which the λN22 peptide specifically binds. As long as it shows specific binding to the target, a mutant of the λN22 peptide may be used.

[0075] The polynucleotide-binding protein may also be a derivative of the above-mentioned PPR protein or TAL effector, etc. The RNA-binding protein may be, for example, fragile X mental retardation syndrome-related protein 1 (FMRR1), other known sequence-specific polynucleotide-binding proteins, fragments thereof, or derivatives thereof.

[0076] 3. Expression Vector An expression vector according to one embodiment of the present invention is an expression vector for animals, which comprises a polynucleotide encoding the enzyme complex according to one embodiment of the present invention.

[0077] The expression vector may be one that expresses the enzyme complex in an integrated state within an animal cell, or one that expresses the enzyme and the base sequence recognition module separately within an animal cell and then complexes them.

[0078] In detail, the expression vector may be one that expresses a fusion protein of the enzyme and the base sequence recognition module in an animal cell, or one that expresses a fusion RNA, which is the base sequence recognition module, and a fusion protein of the enzyme and the RNA-binding protein in a cell, and forms a complex by binding the two in the animal cell via specific binding between the fusion RNA and the RNA-binding protein.

[0079] The expression vector may contain a polynucleotide encoding a fusion protein between the enzyme and a protein that is a base sequence recognition module, or may contain a first polynucleotide encoding an RNA that recognizes a sequence and an RNA that specifically binds to an RNA-binding protein, and a second polynucleotide encoding a fusion protein between the enzyme and the RNA-binding protein in a single vector, or may contain the first polynucleotide and the second polynucleotide in separate vectors.

[0080] The base sequence of the first polynucleotide and the second polynucleotide can be determined based on the amino acid sequence that they code or the base sequence of the RNA that they code.The first polynucleotide and the second polynucleotide can be prepared according to a known method based on the base sequence.The first polynucleotide and the second polynucleotide can be introduced into various vectors using restriction enzymes, DNA ligase, or a plasmid construction kit by homologous recombination.

[0081] In addition, an intervening linker amino acid sequence may be included between the enzyme and the RNA-binding protein, and an intervening nucleic acid linker sequence may be included between the guide RNA and the RNA that specifically binds to the RNA-binding protein.

[0082] In one embodiment of the present invention, the expression vector preferably contains a promoter upstream of a polynucleotide encoding the enzyme complex for controlling the expression of the polynucleotide in order to express the enzyme complex in an animal cell. Examples of the promoter include CMV, CMV-IE, EF1α, and U6. Among these promoters, EF1α is preferred for polypeptide expression, and U6 is preferred for guide RNA expression, from the viewpoint of obtaining the advantage of stable and high expression.

[0083] The expression vector according to one embodiment of the present invention may be any expression vector capable of expressing the enzyme complex according to one embodiment of the present invention, and its form is not limited. The expression vector according to one embodiment of the present invention may be a DNA type, an RNA type, a circular type, a linear type, or a viral vector. If the expression vector is composed of DNA, which is more stable than RNA, the stability during storage etc. can be increased compared to the enzyme complex containing the above-mentioned guide RNA, and handling during use is also simplified.

[0084] According to the expression vector of one embodiment of the present invention, the enzyme and the base sequence recognition module can be expressed as a fusion protein, or the enzyme and the base sequence recognition module can be expressed separately, and then the two can be self-assembled via an RNA-binding protein or the like to form a complex in an animal cell. This allows the enzyme according to one embodiment of the present invention to act specifically on guanosine, which is a target in a polynucleotide in an animal cell. By using the expression vector, guanosine closely related to the pathology of a disease, or guanosine generated in a polynucleotide by mutation from A to G, can be converted to adenosine. According to the expression vector of one embodiment of the present invention, the enzyme complex can be expressed in an animal cell, or the enzyme complex can be formed in an animal cell.

[0085] [4. Drugs for treating genetic disorders] A genetic disease therapeutic drug for animals according to one embodiment of the present invention comprises the enzyme complex according to one embodiment of the present invention or the expression vector according to one embodiment of the present invention.

[0086] The genetic disease may be a disease in which guanosine is involved in the cause or aggravation of the pathology, or may be a disease caused by a mutation from adenosine to guanosine.

[0087] The genetic disease therapeutic drug can be produced according to a known method. The genetic disease therapeutic drug may contain other pharmacologically acceptable components in addition to the enzyme complex or the expression vector as an active ingredient. The genetic disease therapeutic drug may contain, for example, the enzyme complex or the expression vector and a pharmacologically acceptable carrier. The pharmacologically acceptable carrier may be various organic carrier substances or inorganic carrier substances used as formulation materials. Examples of pharmacologically acceptable carriers include (i) excipients, lubricants, binders, and / or disintegrants in solid preparations, and (ii) solvents, solubilizers, suspending agents, isotonicity agents, buffers, and / or soothing agents in liquid preparations. In addition, additives such as preservatives, antioxidants, colorants, and / or sweeteners may be added to the genetic disease therapeutic drug as necessary.

[0088] The dosage of the genetic disease therapeutic drug can be appropriately determined depending on the sex, age, weight, and / or symptoms of the subject. In the genetic disease therapeutic drug, the enzyme complex or the expression vector is administered to the subject in an effective amount. An effective amount is the amount of the enzyme complex or expression vector required to obtain the desired result, and is the amount required to delay, inhibit, prevent, reverse, or cure the progression of the condition (symptoms) being treated or treated.

[0089] The administration route of the genetic disease therapeutic agent is not particularly limited. The genetic disease therapeutic agent is preferably used as an external preparation, an injection, an inhalant, or an oral preparation.

[0090] The above-mentioned genetic disease therapeutic drug can convert guanosine in a polynucleotide closely related to the pathology of a disease and / or convert mutated guanosine in a disease caused by A to G mutation into a genetic code similar to adenosine by specifically acting an enzyme according to one embodiment of the present invention on a target nucleic acid base in a polynucleotide. Therefore, the above-mentioned genetic disease therapeutic drug is effective in treating a disease caused by a gene mutation (e.g., a point mutation).

[0091] Another aspect of the present invention is that the above-mentioned enzyme complex or the above-mentioned expression vector can be administered to a subject to treat, improve, or prevent a genetic disease in the subject. For example, another aspect of the present invention is "a method for treating a genetic disease (or a method for improving a genetic disease, or a method for preventing a genetic disease) comprising a step of administering an enzyme complex according to one embodiment of the present invention or an expression vector according to one embodiment of the present invention to an animal (e.g., a human or a non-human animal)."

[0092] Another aspect of the present invention is the use of the enzyme complex or expression vector for the production of a genetic disease therapeutic drug. For example, another aspect of the present invention is "use of the enzyme complex according to one embodiment of the present invention or the expression vector according to one embodiment of the present invention for the production of a genetic disease therapeutic drug (or a genetic disease improving drug or a genetic disease preventive drug)."

[0093] The enzyme complex and / or the expression vector may be used as a reagent for in vitro, in vivo or ex vivo experiments.

[0094] [5. Other] The present invention according to one aspect includes the following inventions.

[0095] <1> An enzyme that has the activity of deaminating guanosine in polynucleotides and converting it to xanthosine in human cells.

[0096] <2> An enzyme that converts the genetic code of guanosine in polynucleotides to adenosine (the genetic code for adenosine) in human cells.

[0097] <3> (i) a region having an amino acid sequence as set forth in SEQ ID NO: 1 or 2, or (ii) a region having an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 2; <1> or <2> The enzyme described in

[0098] <4> <1> ~ <3> An enzyme according to any one of the above items, and a base sequence recognition module for allowing the enzyme to act specifically on guanosine in a polynucleotide that is involved in the cause or aggravation of a pathological condition.

[0099] <5> <1> ~ <3> An enzyme according to any one of the above items, and a base sequence recognition module for allowing the enzyme to act specifically on a guanosine generated in a polynucleotide by an A to G mutation.

[0100] <6> <4> or <5> 2. An expression vector for expressing and forming the enzyme complex described in claim 1 in human cells.

[0101] <7> <4> or <5> or <6> A therapeutic agent for genetic diseases comprising the expression vector according to claim 1.

[0102] An embodiment of the present invention has been described above. One embodiment of the present invention may be able to contribute to the achievement of Goal 3 of the Sustainable Development Goals (SDGs) advocated by the United Nations, "Ensure good health and promote well-being for all." EXAMPLES

[0103] The following examples will explain one embodiment of the present invention in more detail, but the present invention is not limited to these examples.

[0104] 1. Detection of polynucleotide editing activity in animal cells <1-1. Test method> A plasmid was prepared for expressing the "PPR56-E2-DRH" fusion protein in animal cells. The specific structure of the "PPR56-E2-DRH" fusion protein was such that the E2 domain was directly linked to the carboxyl terminus of the PPR domain of the PPR56 protein of Physcomitrium patens (NCBI identification number: LOC112295756), and the DRH domain was further linked to the carboxyl terminus of the E2 domain.

[0105] Specifically, the amino acid sequence of the "PPR56-E2-DRH" fusion protein is such that, from the amino terminus, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence of SEQ ID NO: 1 are linked without a linker. Meanwhile, the base sequence of the "PPR56-E2-DRH" fusion protein is such that, from the 5' terminus, the base sequence of SEQ ID NO: 8, the base sequence of SEQ ID NO: 6, and the base sequence of SEQ ID NO: 3 are linked without a linker. In this plasmid, PPR56 is a sequence recognition module, and E2-DRH exhibits enzyme activity.

[0106] A DNA encoding the "PPR56-E2-DRH" fusion protein was inserted into a plasmid (pSC101), and a CMV promoter for expressing the "PPR56-E2-DRH" fusion protein in animal cells was inserted upstream of the DNA. The plasmid thus prepared was named pC101-PPR56-E2-DRH (see FIG. 1).

[0107] As shown in Figure 2, the PPR domain of the PPR56 protein recognizes a polynucleotide having the base sequence "UAUAGACGGUAUCUCU" (SEQ ID NO: 9) in the mRNA transcribed from the Nad4 gene and specifically binds to the polynucleotide. Therefore, the "E2-DRH" domain in the "PPR56-E2-DRH" fusion protein can approach the "G" present on the 3'-terminal side of "UAUAGACGGUAUCUCU".

[0108] The target genes used in this study were the EGFP gene in which the initiation codon "ATG" had been converted to "GTG" and DNA corresponding to the PPR56 recognition sequence at the 5' end of the EGFP gene had been inserted.

[0109] In the above target gene, since the initiation codon has been lost due to a mutation, the EGFP protein cannot be normally biosynthesized, and the fluorescence derived from the EGFP cannot be emitted.

[0110] On the other hand, if the first "G" of the "GTG" resulting from the mutation is converted to xanthosine by deamination, the xanthosine will be recognized as "A." In other words, if the first "G" of the "GTG" resulting from the mutation is converted to xanthosine by deamination, the "GTG" will be synonymous with the start codon "ATG" in the genetic code. As a result, the EGFP protein is synthesized and emits fluorescence derived from the EGFP.

[0111] HEK293 cells, a human cell line, were cultured at 6.4 × 10 5 Before transfection, the culture medium in the glass-bottom dish was removed, and 2 mL of fresh FBS-containing DMEM was added to the glass-bottom dish.

[0112] Next, (i) a tube containing 100 μL of Opti-MEM and 4 μL of Lipofectamine 3000 reagent, and (ii) a tube containing 100 μL of Opti-MEM and 4 μL of a plasmid solution ((a) a mixture of 1600 ng pC101-PPR56-E2-DRH and 400 ng of mutated EGFP plasmid, or (b) 400 ng of mutated EGFP plasmid only) were prepared. The solutions in the two tubes were mixed and incubated at room temperature for 10 minutes, and then the mixed solution was added to a glass-bottom dish. 24 hours after transfection, green fluorescence was observed with a confocal laser microscope to confirm whether or not green fluorescence had returned.

[0113] <2-2. Test Results> The test results are shown in Figure 3. 301 and 302 in Figure 3 are images of human cells into which only the mutated EGFP plasmid was introduced, and 303 and 304 in Figure 3 are images of human cells into which the mutated EGFP plasmid and pC101-PPR56-E2-DRH were introduced, respectively.

[0114] As is clear from 302 in FIG. 3, in human cells into which only the mutated EGFP plasmid was introduced, no fluorescence derived from EGFP was observed. On the other hand, as is clear from 304 in FIG. 3, in human cells into which the mutated EGFP plasmid and pC101-PPR56-E2-DRH were introduced, fluorescence derived from EGFP was observed. This indicates that the first "G" of "GTG" generated by the mutation was deaminated and converted to xanthosine by the "DRH domain", and the xanthosine was recognized as "A", in other words, "GTG" was converted and repaired to the start codon "ATG". [Industrial Applicability]

[0115] The present invention can be used in medicines or research reagents.

Claims

1. An enzyme that converts guanosine in a polynucleotide into the genetic code equivalent to adenosine in animal cells.

2. The enzyme according to claim 1, comprising any one of the following polypeptides (1) to (6): (1) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2; (2) A polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 1 or 2; (3) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2; (4) A polypeptide encoded by a polynucleotide consisting of the base sequence of SEQ ID NO: 3 or 4; (5) A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 or 4; (6) A polypeptide having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a polypeptide encoded by a polynucleotide having the base sequence of SEQ ID NO: 3 or 4.

3. The enzyme according to claim 2, further comprising an E2 domain which is any one of the polypeptides (7) to (12): (7) A polypeptide consisting of the amino acid sequence of SEQ ID NO:5; (8) A polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO:5; (9) A polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO:5; (10) A polypeptide encoded by a polynucleotide consisting of the base sequence of SEQ ID NO:6; (11) A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the polynucleotide consisting of the nucleotide sequence of SEQ ID NO:6; (12) A polypeptide having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a polypeptide encoded by a polynucleotide consisting of the base sequence of SEQ ID NO:

6.

4. 10. An enzyme complex comprising the enzyme according to claim 1 and a base sequence recognition module that binds to a specific sequence within a polynucleotide, thereby allowing the enzyme to act on a specific guanosine within the polynucleotide sequence.

5. The enzyme complex described in claim 4, wherein the base sequence recognition module (a) binds to a specific sequence in a polynucleotide involved in the cause or aggravation of a pathology, and / or (b) binds to a specific sequence in a polynucleotide having a mutation in which adenosine is replaced by guanosine.

6. An expression vector for animals, comprising a polynucleotide encoding the enzyme complex according to claim 4.

7. A therapeutic agent for genetic diseases in animals, comprising the enzyme complex according to claim 4 or the expression vector according to claim 6.

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