Method for editing target RNA

Abstract

To provide a method for converting an editing target C to U, or an editing target U to C, contained in a target RNA.SOLUTION: In this study, we show that a C-terminal DYW domain-containing portion of a PPR-DYW protein can be used as a modular editing domain for RNA editing. When each of the three kinds of DYW domains designed in this study was fused with PPR-P or PLS array, the DYW: PG and DYW: WW domains showed a C-to-U RNA editing activity and DYW:KP showed a U-to-C RNA editing activity.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an RNA editing technology using a protein capable of binding to a target RNA. The present invention is useful in a wide range of fields, such as medicine (drug discovery support, treatment), agriculture (agricultural, fishery and livestock production, breeding), and chemistry (biological substance production). [Background technology]

[0002] In plant mitochondria and chloroplasts, RNA editing, in which specific bases in the genome are replaced at the RNA level, occurs frequently, and it is known that this phenomenon is mediated by the RNA-binding protein pentatricopeptide repeat (PPR) protein.

[0003] PPR proteins are classified into two families, P and PLS, based on the structure of the PPR motif that constitutes the protein (Non-Patent Document 1). P class PPR proteins are composed of a simple repeat of the standard 35 amino acid PPR motif (P), while PLS proteins contain two similar motifs called L and S in addition to P. The PPR array (arrangement of PPR motifs) of PLS ​​proteins is composed of three PPR motifs, P1 (about 35 amino acids), L1 (about 35 amino acids), and S1 (about 31 amino acids), as PLS repeat units, and the PLS with slightly different sequences, namely, P2 (35 amino acids), L2 (36 amino acids), and S2 (32 amino acids) motifs follow on the C-terminal side of P1L1S1. In addition to those composed of PLS ​​repeat units, SS (31 amino acids) may also be repeated. Furthermore, this final P2L2S2 motif may be followed on the C-terminal side by two PPR-like motifs called E1 and E2, and a DYW domain having a 136 amino acid cytidine deaminase domain-like sequence (Non-Patent Document 2).

[0004] The interaction between PPR proteins and RNA is regulated by the PPR code, which specifies the binding RNA base by a combination of amino acids at several positions in each PPR motif, and these amino acids bind to the corresponding nucleotides via hydrogen bonds (Patent Document 1, Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6, Non-Patent Document 7, Non-Patent Document 8). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication WO2013 / 058404 [Patent Document 2] International Publication WO2014 / 175284 [Non-patent literature]

[0006] [Non-Patent Document 1] Lurin, C., Andres, C., Aubourg, S., Bellaoui, M., Bitton, F., Bruyere, C., Caboche, M., Debast, C., Gualberto, J., Hoffmann, B., et al. (2004). Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16:2089-2103. [Non-Patent Document 2] Cheng , S. , Gutmann , B. , Zhong , X. , Ye , Y. , Fisher , MF , Bai , F. , Castleden , I. , Song , Y. , Song , B. , Huang , J. , et al. (2016). Redefining the structural motifs that determine RNA binding and RNA editing by pentatricopeptide repeat proteins in land plants. Plant J. 85:532–547.

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[0007] In organelles of land plants, C-to-U RNA editing, in which the RNA base is replaced from cytidine to uridine, generally occurs. However, U-to-C RNA editing, in which uridine is replaced by cytidine, is also observed in some hornworts, microphylls, and ferns (Non-Patent Document 9). Two different bioinformatics studies have found a unique DYW domain with a sequence different from that of the standard DYW domain in plants with U-to-C RNA editing (Non-Patent Document 10, Non-Patent Document 11).

[0008] Two types of DYW:PG proteins derived from Physcomitrella patens have been reported to exhibit C-to-U RNA editing activity in Escherichia coli (Non-Patent Document 12). In the plant group that branched off early in the evolution of land plants, DYW domains are roughly classified into two groups. The first group is called the DYW:PG / WW group, which includes the standard DYW domain DYW:PG type and the DYW:WW type in which one tryptophan (W) is added to the PG box. The second group is called DYW:KP, and this DYW domain has a PG box and three amino acid sequences at the C-terminus that are different from those of the PG / WW group, and is present only in plants that have U-to-C RNA editing. The technology to change one base in an arbitrary RNA sequence to a specific base is useful in gene therapy and gene mutation introduction technology for industrial use. Although various RNA-binding molecules have been developed to date, a method has not been established for designing a molecule that can convert cytidine (C) in any target RNA to uridine (U), or vice versa, converting uridine (U) to cytidine (C) using a DYW domain and an artificial RNA-binding protein. [Means for solving the problem]

[0009] In this study, we show that the C-terminal DYW domain of the PPR-DYW protein can be used as a modular editing domain for RNA editing. When the three DYW domains designed in this study were fused to PPR-P or PLS arrays, the DYW:PG and DYW:WW domains showed C-to-U RNA editing activity, and the DYW:KP domain showed U-to-C RNA editing activity.

[0010] The present invention provides the following: [1] A method for editing a target RNA, comprising applying an artificial DYW protein to the target RNA, the artificial DYW protein comprising a DYW domain consisting of any one of the polypeptides a, b, c, and bc below. a.x a1 PGx a2 SWIEx a3 -xa16 HP … Hx aa E … Cx a17 x a18 A polypeptide having a sequence identity of at least 40% with the sequence of SEQ ID NO:1, and having C-to-U / U-to-C editing activity, comprising: CH...DYW. b.x b1 PGx b2 SWWTDx b3 -x b16 HP … Hx bb E … Cx b17 x b18 A polypeptide having a sequence identity of at least 40% with the sequence of SEQ ID NO:2, and having a C-to-U / U-to-C editing activity, comprising: CH...DYW. c. KPAx c1 Ax c2 IEx c3 … Hx cc E … Cx c4 x c5 CH...x c6 x c7 x c8 A polypeptide having at least 40% sequence identity with the sequence of SEQ ID NO: 3 and having C-to-U / U-to-C editing activity. b.c.x b1 PGx b2 SWWTDx b3 -x b16 HP … Hx cc E … Cx c4 x c5 CH...Dx bc1 x bc2 A polypeptide having at least 40% sequence identity with the sequence of SEQ ID NO: 90 of SEQ ID NO: 2 and having C-to-U / U-to-C editing activity. (In the sequences, x represents any amino acid and ... represents any polypeptide fragment.) [2] A DYW domain consisting of any one of the following polypeptides: a, b, c, and bc: a.x a1 PGx a2 SWIEx a3 -x a16 HP … Hx aa E … Cxa17 x a18 A polypeptide having a sequence identity of at least 40% with the sequence of SEQ ID NO:1, and having C-to-U / U-to-C editing activity, comprising: CH...DYW. b.x b1 PGx b2 SWWTDx b3 -x b16 HP … Hx bb E … Cx b17 x b18 A polypeptide having a sequence identity of at least 40% with the sequence of SEQ ID NO:2, and having a C-to-U / U-to-C editing activity, comprising: CH...DYW. c. KPAx c1 Ax c2 IEx c3 … Hx cc E … Cx c4 x c5 CH...x c6 x c7 x c8 A polypeptide having at least 40% sequence identity with the sequence of SEQ ID NO: 3 and having C-to-U / U-to-C editing activity. b.c.x b1 PGx b2 SWWTDx b3 -x b16 HP … Hx cc E … Cx c4 x c5 CH...Dx bc1 x bc2 A polypeptide having at least 40% sequence identity with the sequence of SEQ ID NO: 90 of SEQ ID NO: 2 and having C-to-U / U-to-C editing activity. [3] A DYW protein comprising an RNA-binding domain that contains at least one PPR motif and is capable of binding to a target RNA in a sequence-specific manner, and the DYW domain described in 2. [4] The DYW protein according to 2, wherein the RNA-binding domain is of the PLS type. [5] A method for editing a target RNA, comprising: A method comprising the step of applying a DYW domain consisting of the polypeptide c or bc below to a target RNA to convert an editing target U to C. c. KPAx c1 Ax c2 IEx c3 … Hx cc E … Cx c4 x c5 CH...x c6 x c7 x c8 A polypeptide having at least 40% sequence identity with the sequence of SEQ ID NO: 3 and having C-to-U / U-to-C editing activity. b.c.x b1 PGx b2 SWWTDx b3 -x b16 HP … Hx cc E … Cx c4 x c5 CH...Dx bc1 x bc2 A polypeptide having at least 40% sequence identity with the sequence of SEQ ID NO: 90 of SEQ ID NO: 2 and having C-to-U / U-to-C editing activity. [6] The method according to 5, wherein the DYW domain is fused to an RNA-binding domain that contains at least one PPR motif and is capable of sequence-specifically binding to a target RNA according to the rules of the PPR-code. [7] A composition comprising the DYW domain described in 2 for editing RNA in eukaryotic cells. [8] A nucleic acid encoding the DYW domain described in 2, or the DYW protein described in 3 or 4. [9] A vector comprising the nucleic acid described in 8.

[10] A cell (excluding a human individual) containing the vector described in 9.

[0011] [1] A method for editing a target RNA, comprising applying an artificial DYW protein containing a DYW domain consisting of any one of the following polypeptides a to c to the target RNA: a.x a1 PGx a2 SWIEx a3 -x a16 HP … HSE … Cx a17 x a18A polypeptide having a sequence identity of at least 40% with the sequence of SEQ ID NO:1, and having C-to-U / U-to-C editing activity, comprising: CH...DYW. b.x b1 PGx b2 SWWTDx b3 -x b16 HP … HSE … Cx b17 x b18 A polypeptide having a sequence identity of at least 40% with the sequence of SEQ ID NO:2, and having a C-to-U / U-to-C editing activity, comprising: CH...DYW. c. KPAx c1 Ax c2 IEx c3 … HAE … Cx c4 x c5 CH...Dx c6 x c7 A polypeptide having at least 40% sequence identity with the sequence of SEQ ID NO: 3 and having C-to-U / U-to-C editing activity. (In the sequences, x represents any amino acid and ... represents any polypeptide fragment.) [2] A DYW domain consisting of any one of the following polypeptides a to c: a.x a1 PGx a2 SWIEx a3 -x a16 HP … HSE … Cx a17 x a18 A polypeptide having a sequence identity of at least 40% with the sequence of SEQ ID NO:1, and having C-to-U / U-to-C editing activity, comprising: CH...DYW. b.x b1 PGx b2 SWWTDx b3 -x b16 HP … HSE … Cx b17 x b18 A polypeptide having a sequence identity of at least 40% with the sequence of SEQ ID NO:2, and having a C-to-U / U-to-C editing activity, comprising: CH...DYW. c. KPAx c1 Ax c2 IEx c3 … HAE … Cxc4 x c5 CH...Dx c6 x c7 A polypeptide having at least 40% sequence identity with the sequence of SEQ ID NO: 3 and having C-to-U / U-to-C editing activity. [3] A DYW protein comprising an RNA-binding domain that contains at least one PPR motif and is capable of binding to a target RNA in a sequence-specific manner, and the DYW domain described in 2. [4] The DYW protein according to 2, wherein the RNA-binding domain is of the PLS type. [5] A method for editing a target RNA, comprising: A method comprising the step of applying a DYW domain consisting of the polypeptide of c below to a target RNA to convert an editing target U to C. c. KPAx c1 Ax c2 IEx c3 … HAE … Cx c4 x c5 CH...Dx c6 x c7 A polypeptide having at least 40% sequence identity with the sequence of SEQ ID NO: 3 and having C-to-U / U-to-C editing activity. [6] The method according to 5, wherein the DYW domain is fused to an RNA-binding domain that contains at least one PPR motif and is capable of sequence-specifically binding to a target RNA according to the rules of the PPR-code. [7] A composition comprising the DYW domain described in 2 for editing RNA in eukaryotic cells. [8] A nucleic acid encoding the DYW domain described in 2, or the DYW protein described in 3 or 4. [9] A vector comprising the nucleic acid described in 8.

[10] A cell (excluding a human individual) containing the vector described in 9. Effect of the Invention

[0012] According to the present invention, an editing target C contained in a target RNA can be converted to U, or an editing target U can be converted to C. [Brief description of the drawings]

[0013] [Figure 1] Approximate maximum likelihood phylogenetic tree of the C-terminal domains of DYW proteins. Phylogenetic trees of (a) DYW:PG type, (b) DYW:WW type, and (c) DYW:KP type domains were constructed using FastTree. The DYW:PG domain was designed based on the DYW:PG protein from microphylla. The clades of proteins selected for designing the DYW:WW and DYW:KP domains are indicated by black lines. The phylogenetic tree was visualized using iTOL (Letunic, I. and Bork, P. (2016) Nucleic Acids Res. 44 W242-245), with red indicating proteins from hornworts, green indicating proteins from microphylla, and blue indicating proteins from ferns. [Diagram 2] The structure of the PPR protein designed in this study. (a) The PPR protein contains a thioredoxin, His-tag and TEV site on the N-terminus, followed by a PPR array consisting of a P or PLS motif, and finally a DYW domain (DYW:PG, DYW:WW or DYW:KP). The target sequence of the PPR protein (including the RNA editing site) was inserted downstream of the stop codon. (b) The 4th and iith amino acids involved in RNA recognition in the P, P1, L1, S1 and P2 motifs. The 4th and iith amino acids of L2, S2, E1 and E2 are identical to those of CLB19 (Chateigner-Boutin, AL, et al. (2008). Plant J. 56 590-602.). [Diagram 3] RNA editing assay using E. coli. (a) Target base (C or T) on DNA for each PPR-DYW. (b) C-to-U RNA editing activity of PPR-DYW:PG and WW, and (c) U-to-C RNA editing activity of PPR-DYW:KP. One of the results of three independent experiments is shown as an example. (d) C-to-U and (e) U-to-C RNA editing efficiencies were measured three times (except for P-DYW:KP, which was measured twice). Error bars indicate standard deviation. [Figure 4-1]The frequency of occurrence of amino acids in each selected DYW domain family. Visualized using sequence logos created with WebLogo. Same as Figure 4-2 and Figure 4-3. (a) DYW:PG [Figure 4-2] (b)DYW:WW [Figure 4-3] (c)DYW:KP [Diagram 5] RNA editing activity (C to U or U to C) in HEK293T cells. Sequencing results of the target site when the PLS type is fused with the PG1 or WW1 domain (a), or the KP1 domain (b). RNA editing activity in three independent experiments (c). Bar graphs show the average, and each point shows the RNA editing activity in three experiments. The left bar graph shows C to U editing activity, and the right bar graph shows U to C editing activity. [Figure 6] Improving KP domain performance by domain swapping. We investigated the RNA editing activity of Chimeric KP1a, in which the central part containing the active site (HxExnCxxCH) of KP1 was left intact and the front and rear domains were swapped with the WW1 domain. Schematic diagram of domain swapping (a), actual RNA editing activity (b, c). [Figure 7] Measurement of RNA editing activity of KP domain mutants. RNA editing activity of KP1, KP2, KP3, and KP4 in E. coli (a) and in HEK293T (b), and RNA editing activity of KP5 to KP23 in HEK293T (c). [Figure 8] Measurement of RNA editing activity of PG domain mutants. RNA editing activity of PG1 and PG2 in E. coli (a) and in HEK293T (b). RNA editing activity of PG3 to PG13 in HEK293T [Figure 9] Measurement of RNA editing activity of WW domain mutants. RNA editing activity of WW1 and WW2 in E. coli (a) and in HEK293T (b). RNA editing activity of WW3 to WW14 in HEK293T. [Figure 10] Human mitochondrial RNA editing. Target sequence information (a). RNA editing activity (b, c). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention relates to a method for editing a target RNA, which comprises applying a DYW protein containing a DYW domain consisting of any one of the following polypeptides a, b, c, and bc to the target RNA: a.x a1 PGx a2 SWIEx a3 -x a16 HP … Hx aa E … Cx a17 x a18 A polypeptide having a sequence identity of at least 40% with the sequence of SEQ ID NO:1, and having C-to-U / U-to-C editing activity, comprising: CH...DYW. b.x b1 PGx b2 SWWTDx b3 -x b16 HP … Hx bb E … Cx b17 x b18 A polypeptide having a sequence identity of at least 40% with the sequence of SEQ ID NO:2, and having a C-to-U / U-to-C editing activity, comprising: CH...DYW. c. KPAx c1 Ax c2 IEx c3 … Hx cc E … Cx c4 x c5 CH...x c6 x c7 x c8 A polypeptide having at least 40% sequence identity with the sequence of SEQ ID NO: 3 and having C-to-U / U-to-C editing activity. b.c.x b1 PGx b2 SWWTDx b3 -x b16 HP … Hx cc E … Cx c4 x c5 CH...Dx bc1 x bc2A polypeptide having at least 40% sequence identity with the sequence of SEQ ID NO: 90 of SEQ ID NO: 2 and having C-to-U / U-to-C editing activity.

[0015] In the present invention, C-to-U / U-to-C editing activity refers to the activity of converting an editing target C contained in a target RNA to U, or converting an editing target U to C, when an editing assay is performed by linking a subject polypeptide to the C-terminus of an RNA-binding domain capable of sequence-specifically binding to a target RNA. The conversion may be such that at least about 3%, preferably about 5%, of the editing target bases are replaced with the desired base under appropriate conditions.

[0016] [DYW domain] x a1 PGx a2 SWIEx a3 -x a16 HP … Hx aa E … Cx a17 x a18 CH…DYW,x b1 PGx b2 SWWTDx b3 -x b16 HP … Hx bb E … Cx b17 x b18 CH...DYW, and KPAx c1 Ax c2 IEx c3 … Hx cc E … Cx c4 x c5 CH...x c6 x c7 x c8 In the sequences, each x independently represents any amino acid, and each ... independently represents a polypeptide fragment of any length and any amino acid sequence. In the present invention, the DYW domain can be represented by any one of these three amino acid sequences. In particular, a1 PGx a2 SWIEx a3 -x a16 HP … Hx aa E … Cx a17 x a18The DYW domain consisting of CH … DYW is DYW:PG, x b1 PGx b2 SWWTDx b3 -x b16 HP … Hx bb E … Cx b17 x b18 The DYW domain consisting of CH … DYW is called DYW:WW, and KPAx c1 Ax c2 IEx c3 … Hx cc E … Cx c4 x c5 CH...x c6 x c7 x c8 The DYW domain consisting of the above is sometimes expressed as DYW:KP.

[0017] The DYW domain is made up of a 15 amino acid region at the N-terminus containing a PG box, a central zinc-binding domain (HxEx n CxxCH,x n is a sequence of any number of amino acids, n. The zinc-binding domain has three regions: HxE, CxxCH, and DYW at the C-terminus. The zinc-binding domain can be further divided into HxE and CxxCH regions. These regions of each DYW domain can be represented as shown in the table below.

[0018] [Table 1]

[0019] (DYW:PG) DYW:PG is x a1 PGx a2 SWIEx a3 -x a16 HP … Hx aa E … Cx a17 x a18 Preferably, x is a polypeptide consisting of a1 PGx a2 SWIEx a3 -x a16 HP … Hx aa E … Cx a17 xa18 CH ... DYW, a polypeptide having sequence identity to the sequence of SEQ ID NO: 1 (described in detail in the [Terminology] section), and having C-to-U / U-to-C editing activity. DYW:PG has the activity of converting the editing target C to U (C-to-U editing activity). SEQ ID NO: 1 shows the sequence of DYW:PG consisting of a full length of 136 amino acids, which was used in the experiment shown in the Examples section of this specification. This sequence is disclosed for the first time in this application and is novel.

[0020] The total length of DYW:PG is not particularly limited as long as it can exhibit C-to-U editing activity, but is, for example, 110 to 160 amino acids in length, preferably 124 to 148 amino acids in length, more preferably 128 to 144 amino acids in length, and even more preferably 132 to 140 amino acids in length.

[0021] DYW: Area including the PG box of PG (x a1 PGx a2 SWIEx a3 -x a16 HP: x a1 is not particularly limited as long as it can exert C-to-U editing activity as DYW:PG, but is preferably E (glutamic acid) or an amino acid having properties similar thereto, and more preferably G. x a2 is not particularly limited as long as it can exert C-to-U editing activity as DYW:PG, but is preferably C (cysteine) or an amino acid having properties similar thereto, and more preferably C. x a3 -x a16 Each of the amino acids is not particularly limited as long as it is capable of exerting C-to-U editing activity as DYW:PG, but is preferably the same as the corresponding amino acids at positions 9 to 22 of the sequence of SEQ ID NO:1 or an amino acid having similar properties to the corresponding amino acids, and more preferably the same as the corresponding amino acids at positions 9 to 22 of the sequence of SEQ ID NO:1.

[0022] In a preferred embodiment, the HxE region of DYW:PG is an HSE whatever the other regions may be.

[0023] DYW:PG CxxCH region, i.e. Cx a17 x a18 In CH: x a17 is not particularly limited as long as it can exert C-to-U editing activity as DYW:PG, but is preferably G (glycine) or an amino acid having properties similar thereto, and more preferably G. x a18 is not particularly limited as long as it can exert C-to-U editing activity as DYW:PG, but is preferably D (aspartic acid) or an amino acid having properties similar thereto, and more preferably D.

[0024] In DYW:PG, the region including the PG box and Hx aa E bonded to the moiety, Hx aa The ... portion connecting the E domain and the CxxCH domain, and the ... portion connecting the CxxCH domain and the DYW domain are referred to as the first linking portion, the second linking portion, and the third linking portion, respectively (the same applies to other DYW domains).

[0025] The total length of the first linking part of DYW:PG is not particularly limited as long as it can exert C-to-U editing activity as DYW:PG, but is, for example, 39 to 47 amino acids long, preferably 40 to 46 amino acids long, more preferably 41 to 45 amino acids long, and even more preferably 42 to 44 amino acids long. The amino acid sequence of the first linking part is not particularly limited as long as it can exert C-to-U editing activity as DYW:PG, but is preferably the same as the part of positions 25 to 67 of the sequence of SEQ ID NO: 1, or a sequence in which 1 to 22 amino acids have been substituted, deleted, or added in that partial sequence, or a sequence having sequence identity to that partial sequence, and more preferably the same sequence as that partial sequence.

[0026] A preferred embodiment of the first junction of DYW:PG is a 43 amino acid long polypeptide represented by the following formula, regardless of the sequence of other parts of the DYW domain:

[0027] N a25 -N a26 -N a27 - … -N a65 -N a66 -N a67

[0028] The above polypeptide preferably has a sequence identical to the portion of positions 25 to 67 of the sequence of SEQ ID NO: 1 or a partial sequence thereof with multiple amino acid substitutions, and can exhibit C-to-U editing activity as DYW:PG. In this case, the amino acid substitution is an amino acid with a large bit value (e.g., N a29 , N a30 , N a32 , N a33 , N a35 , N a36 , N a40 , N a44 , N a45 , N a47 , N a48 , N a52 , N a53 , N a54 , N a55 , N a58 , N a61 , N a65 , N a67 ) is the same as in FIG. 4, and it is preferable that the other amino acids are substituted.

[0029] The total length of the second linking part of DYW:PG is not particularly limited as long as it can exert C-to-U editing activity as DYW:PG, but is, for example, 21 to 29 amino acids long, preferably 22 to 28 amino acids long, more preferably 23 to 27 amino acids long, and even more preferably 24 to 26 amino acids long. The amino acid sequence of the second linking part is not particularly limited as long as it can exert C-to-U editing activity as DYW:PG, but is preferably the same as the part of positions 71 to 95 of the sequence of SEQ ID NO: 1, or a partial sequence of the same with 1 to 13 amino acids substituted, deleted, or added, or a sequence having sequence identity to the partial sequence, and more preferably the same as the partial sequence.

[0030] A preferred embodiment of the second junction of DYW:PG is a 25 amino acid long polypeptide represented by the following formula, regardless of the sequence of other parts of the DYW domain:

[0031] N a71 -N a72 -N a73 - … -N a93 -N a94 -N a95

[0032] The above polypeptide preferably has a sequence identical to the portion of positions 71 to 95 of SEQ ID NO: 1 or a partial sequence thereof with multiple amino acid substitutions, and is capable of exerting C-to-U editing activity as DYW:PG. In this case, the amino acid substitution is performed by substituting an amino acid with a large bit value (e.g., N a71 , N a72 , N a73 , N a76 , N a77 , N a78 , N a79 , N a81 , N a82 , N a86 , N a88 , N a89 , N a91 , N a92 , N a93 , N a94) is the same as in FIG. 4, and it is preferable that the other amino acids are substituted.

[0033] The total length of the third linking part of DYW:PG is not particularly limited as long as it can exert C-to-U editing activity as DYW:PG, but is, for example, 29 to 37 amino acids long, preferably 30 to 36 amino acids long, more preferably 31 to 35 amino acids long, and even more preferably 32 to 34 amino acids long. The amino acid sequence of the third linking part is not particularly limited as long as it can exert C-to-U editing activity as DYW:PG, but is preferably the same as the part of positions 101 to 133 of the sequence of SEQ ID NO: 1, or a partial sequence of the same with substitution, deletion or addition of 1 to 17 amino acids, or a sequence having sequence identity to the partial sequence, and more preferably the same as the partial sequence.

[0034] A preferred embodiment of the third junction of DYW:PG is a 33 amino acid long polypeptide represented by the following formula, regardless of the sequence of other parts of the DYW domain:

[0035] N a101 -N a102 -N a103 - … -N a131 -N a132 -N a133

[0036] The above polypeptide preferably has a sequence identical to the portion of positions 101 to 133 of the sequence of SEQ ID NO: 1 or a partial sequence thereof with multiple amino acid substitutions, and can exhibit C-to-U editing activity as DYW:PG. In this case, the amino acid substitution is an amino acid with a large bit value (e.g., N a102 , N a104 , N a107 , N a112 , N a114 , N a117 , N a118 , N a121 , N a122 , N a123 , N a124 , N a125 , Na128 , N a130 , N a131 , N a132 ) is the same as in FIG. 4, and it is preferable that the other amino acids are substituted.

[0037] RNA editing activity can be improved by introducing a mutation into the PG domain consisting of the sequence of SEQ ID NO: 1. A preferred example of such a mutated domain is PG11 (a polypeptide consisting of the amino acid sequence of SEQ ID NO: 50) shown in the Examples section of this specification.

[0038] (DYW:WW) DYW:WW is x b1 PGx b2 SWWTDx b3 -x b16 HP … Hx bb E … Cx b17 x b18 Preferably, x is a polypeptide consisting of b1 PGx b2 SWWTDx b3 -x b16 HP … Hx bb E … Cx b17 x b18 CH ... DYW, a polypeptide having sequence identity to the sequence of SEQ ID NO: 2 and having C-to-U / U-to-C editing activity. DYW:WW has the activity of converting the editing target C to U (C-to-U editing activity). SEQ ID NO: 2 shows the sequence of DYW:WW consisting of a full length of 137 amino acids used in the experiment shown in the Examples section of this specification. This sequence is disclosed for the first time in this application and is novel.

[0039] The total length of DYW:WW is not particularly limited as long as it can exhibit C-to-U editing activity, but is, for example, 110 to 160 amino acids in length, preferably 125 to 149 amino acids in length, more preferably 129 to 145 amino acids in length, and even more preferably 133 to 141 amino acids in length.

[0040] The region containing the PG box of DYW:WW, i.e., x b1 PGx b2 SWWTDx b3 -x b16 In the HP, the portion consisting of WTD may be WSD.

[0041] In the area containing the PG box of DYW:WW: x b1 is not particularly limited as long as it can exert C-to-U editing activity as DYW:WW, but is preferably K (lysine) or an amino acid having properties similar thereto, and more preferably K. x b2 is not particularly limited as long as it can exert C-to-U editing activity as DYW:WW, but is preferably Q (glutamine) or an amino acid having properties similar thereto, and more preferably Q. x b3 -x b16 Each of the amino acids is not particularly limited as long as it is capable of exerting C-to-U editing activity as DYW:WW, but is preferably the same as the corresponding amino acids at positions 10 to 23 of the sequence of SEQ ID NO:2 or an amino acid with similar properties to the corresponding amino acids, and more preferably the same as the corresponding amino acids at positions 10 to 23 of the sequence of SEQ ID NO:2.

[0042] In a preferred embodiment, the HxE region of DYW:WW is an HSE, whatever the sequence of the other portions.

[0043] DYW:WW CxxCH region, i.e. Cx b17 x b18 In CH: x b17 is not particularly limited as long as it can exert C-to-U editing activity as DYW:WW, but is preferably D (aspartic acid) or an amino acid similar in properties thereto, and more preferably D. x b18 is not particularly limited as long as it can exert C-to-U editing activity as DYW:WW, but is preferably D or an amino acid having similar properties, and more preferably D.

[0044] The total length of the first linking part of DYW:WW is not particularly limited as long as it can exert C-to-U editing activity as DYW:WW, but is, for example, 39 to 47 amino acids long, preferably 40 to 46 amino acids long, more preferably 41 to 45 amino acids long, and even more preferably 42 to 44 amino acids long. The amino acid sequence of the first linking part is not particularly limited as long as it can exert C-to-U editing activity as DYW:WW, but is preferably the same as the part of positions 25 to 67 of the sequence of SEQ ID NO:2, or a partial sequence of the partial sequence in which 1 to 22 amino acids have been substituted, deleted, or added, or a sequence having sequence identity to the partial sequence, and more preferably the same sequence as the partial sequence.

[0045] A preferred embodiment of the first junction of DYW:WW is a 43 amino acid long polypeptide represented by the following formula, whatever the sequence of the other parts of the DYW domain:

[0046] N b26 -N b27 -N b28 - … -N b66 -N b67 -N b68

[0047] The above polypeptide preferably has a sequence identical to the portion of positions 26 to 68 of the sequence of SEQ ID NO: 2, or a partial sequence thereof with multiple amino acid substitutions, and is capable of exerting C-to-U editing activity as DYW:PG. In this case, the amino acid substitution is performed by substituting an amino acid with a large bit value (e.g., N b26 , N b30 , N b33 , N b34 , N b37 , N b41 , N b45 , N b46 , N b48 , N b49 , N b51 , N b52 , N b53 , N b55 , N b56 , N b57 , Nb59 , N b61 , N b62 , N b63 , N b64 , N b66 , N b67 , N b68 ) is the same as in FIG. 4, and it is preferable that the other amino acids are substituted.

[0048] The total length of the second linking part of DYW:WW is not particularly limited as long as it can exert C-to-U editing activity as DYW:WW, but is, for example, 21 to 29 amino acids long, preferably 22 to 28 amino acids long, more preferably 23 to 27 amino acids long, and even more preferably 24 to 26 amino acids long. The amino acid sequence of the second linking part is not particularly limited as long as it can exert C-to-U editing activity as DYW:WW, but is preferably the same as the part of positions 71 to 95 of the sequence of SEQ ID NO:2, or a partial sequence of the same with substitution, deletion or addition of 1 to 13 amino acids, or a sequence having sequence identity to the partial sequence, and more preferably the same as the partial sequence.

[0049] A preferred embodiment of the second junction of DYW:WW is a 25 amino acid long polypeptide represented by the following formula, whatever the sequence of the other parts of the DYW domain:

[0050] N b72 -N b73 -N b74 - … -N b94 -N b95 -N b96

[0051] The above polypeptide preferably has a sequence identical to the portion of positions 72 to 96 of the sequence of SEQ ID NO: 2, or a partial sequence thereof with multiple amino acid substitutions, and can exhibit C-to-U editing activity as DYW:WW. In this case, the amino acid substitution is performed by substituting an amino acid with a large bit value (e.g., N b72 , N b73 , N b74 , N b75 , Nb77 , N b78 , N b79 , N b81 , N b82 , N b84 , N b88 , N b89 , N b90 , N b91 , N b92 , N b93 , N b94 , N b95 , N b96 ) is the same as in FIG. 4, and it is preferable that the other amino acids are substituted.

[0052] The total length of the third linking part of DYW:WW is not particularly limited as long as it can exert C-to-U editing activity as DYW:WW, but is, for example, 29 to 37 amino acids long, preferably 30 to 36 amino acids long, more preferably 31 to 35 amino acids long, and even more preferably 32 to 34 amino acids long. The amino acid sequence of the third linking part is not particularly limited as long as it can exert C-to-U editing activity as DYW:WW, but is preferably the same as the part of positions 101 to 133 of the sequence of SEQ ID NO:2, or a partial sequence of the same with 1 to 17 amino acids substituted, deleted or added, or a sequence having sequence identity to the partial sequence, and more preferably the same as the partial sequence.

[0053] A preferred embodiment of the third junction of DYW:WW is a 33 amino acid long polypeptide represented by the following formula, whatever the sequence of the other parts of the DYW domain:

[0054] N b102 -N b103 -N b104 - … -N b132 -N b133 -N b134

[0055] The above polypeptide preferably has a sequence identical to the portion of positions 102 to 134 of the sequence of SEQ ID NO: 2, or a partial sequence thereof with multiple amino acid substitutions, and can exhibit C-to-U editing activity as DYW:WW. In this case, the amino acid substitution is an amino acid with a large bit value (e.g., N b104 , N b105 , N b107 , N b108 , N b109 , N b110 , N b111 , N b113 , N b115 , N b116 , N b117 , N b118 , N b119 , N b121 , N b122 , N b123 , N b124 , N b126 , N b129 , N b131 , N b132 , N b133 N b134 ) is the same as in FIG. 4, and it is preferable that the other amino acids are substituted.

[0056] RNA editing activity can be improved by introducing a mutation into the WW domain consisting of the sequence of SEQ ID NO: 2. Preferred examples of domains into which such mutations have been introduced are WW2 to WW11 and WW13 shown in the Examples section of this specification, and WW11 (a polypeptide consisting of the amino acid sequence of SEQ ID NO: 63) has particularly high editing activity.

[0057] (DYW:KP) DYW:KP is KPAx c1 Ax c2 IEx c3 … Hx cc E … Cx c4 x c5 CH...x c6 x c7 x c8 Preferably, KPAx is a polypeptide consisting of c1 Ax c2 IEx c3… Hx cc E … Cx c4 x c5 CH...x c6 x c7 x c8 DYW:KP is a polypeptide having sequence identity to the sequence of SEQ ID NO: 3 and having C-to-U / U-to-C editing activity. DYW:KP has activity to convert the editing target U to C (U-to-C editing activity). SEQ ID NO: 3 shows the sequence of DYW:KP consisting of a full length of 133 amino acids, which was used in the experiment shown in the Examples section of this specification. This sequence is disclosed for the first time in this application and is therefore novel.

[0058] The total length of DYW:KP is not particularly limited as long as it can exhibit U-to-C editing activity, but is, for example, 110 to 160 amino acids in length, preferably 121 to 145 amino acids in length, more preferably 125 to 141 amino acids in length, and even more preferably 129 to 137 amino acids in length.

[0059] DYW:KP region including the PG box, i.e., KPAx c1 Ax c2 IEx c3 In: x c1 is not particularly limited as long as it can exert U-to-C editing activity as DYW:KP, but is preferably S (serine) or an amino acid having properties similar thereto, and more preferably S. x c2 is not particularly limited as long as it can exert U-to-C editing activity as DYW:KP, but is preferably L (leucine) or an amino acid having properties similar thereto, and more preferably L. x c3 is not particularly limited as long as it can exert U-to-C editing activity as DYW:KP, but is preferably V (valine) or an amino acid similar thereto, and more preferably V.

[0060] In a preferred embodiment, the HxE region of DYW:KP is HAE, whatever the sequence of the other portions.

[0061] DYW:KP CxxCH region, i.e., Cx c4 x c5 In CH: x c4 is not particularly limited as long as it can exert U-to-C editing activity as DYW:KP, but is preferably N (asparagine) or an amino acid having properties similar thereto, and more preferably N. x b5 is not particularly limited as long as it can exert U-to-C editing activity as DYW:KP, but is preferably D (aspartic acid) or an amino acid having properties similar thereto, and more preferably D.

[0062] DYW: The part of KP that corresponds to DYW, i.e., x c6 x c7 x c8 In: x c6 is not particularly limited as long as it can exert U-to-C editing activity as DYW:KP, but is preferably D (aspartic acid) or an amino acid having properties similar thereto, and more preferably D. x c7 is not particularly limited as long as it can exert U-to-C editing activity as DYW:KP, but is preferably M (methionine) or an amino acid having properties similar thereto, and more preferably M. x b8 is not particularly limited as long as it can exert U-to-C editing activity as DYW:KP, but is preferably F (phenylalanine) or an amino acid having properties similar thereto, and more preferably F. In one preferred embodiment, x c6 x c7 x c8 Regardless of the sequence of other parts, Dx c7 x c8 It is. In another preferred embodiment, x c6 x c7 x c8 is GRP, whatever the sequence of the other parts.

[0063] The total length of the first linking part of DYW:KP is not particularly limited as long as it can exert U-to-C editing activity as DYW:KP, but is, for example, 51 to 59 amino acids long, preferably 52 to 58 amino acids long, more preferably 53 to 57 amino acids long, and even more preferably 54 to 56 amino acids long. The amino acid sequence of the first linking part is not particularly limited as long as it can exert U-to-C editing activity as DYW:KP, but is preferably the same as the part of positions 10 to 64 of the sequence of SEQ ID NO: 3, or a partial sequence thereof in which 1 to 28 amino acids have been substituted, deleted, or added, or a sequence having sequence identity to the partial sequence, and more preferably the same sequence as the partial sequence.

[0064] A preferred embodiment of the first junction of DYW:KP is a 55 amino acid long polypeptide represented by the following formula, regardless of the sequence of other parts of the DYW domain:

[0065] N c10 -N c11 -N c12 - … -N c62 -N c63 -N c64

[0066] The above polypeptide preferably has a sequence identical to the portion of positions 10 to 64 of the sequence of SEQ ID NO: 3 or a partial sequence thereof with multiple amino acid substitutions, and can exhibit U-to-C editing activity as DYW::KP. In this case, the amino acid substitution is an amino acid with a large bit value (e.g., N c10 , N c13 , N c14 , N c15 , N c16 , N c17 , N c18 , N c19 , N c25 , N c26 , N c29 , N c30 , N c33 , N c34 , N c36 , N c38 , N c41 , Nc42 , N c44 , N c45 , N c47 , N 49 , N c55 , N c58 , N c59 , N c62 , N c63 , N c64 ) is the same as in FIG. 4, and it is preferable that the other amino acids are substituted.

[0067] The total length of the second linking part of DYW:KP is not particularly limited as long as it can exert U-to-C editing activity as DYW:KP, but is, for example, 21 to 29 amino acids long, preferably 22 to 28 amino acids long, more preferably 23 to 27 amino acids long, and even more preferably 24 to 26 amino acids long. The amino acid sequence of the second linking part is not particularly limited as long as it can exert U-to-C editing activity as DYW:KP, but is preferably the same as the part of positions 68 to 92 of the sequence of SEQ ID NO: 3, or a partial sequence of the same with substitution, deletion or addition of 1 to 13 amino acids, or a sequence having sequence identity to the partial sequence, and more preferably the same as the partial sequence.

[0068] A preferred embodiment of the second junction of DYW:KP is a 25 amino acid long polypeptide represented by the following formula, regardless of the sequence of other parts of the DYW domain:

[0069] N c68 -N c69 -N c70 - … -N c90 -N c91 -N c92

[0070] The above polypeptide preferably has a sequence identical to the portion of positions 68 to 92 of the sequence of SEQ ID NO: 3 or a partial sequence thereof with multiple amino acid substitutions, and is capable of exerting U-to-C editing activity as DYW:KP. In this case, the amino acid substitution is performed by substituting an amino acid with a large bit value (e.g., N c68 , Nc70 , N c71 , N c72 , N c73 , N c74 , N c75 , N c76 , N c77 , N c78 , N c79 , N c80 , N c81 , N c83 , N c84 , N c85 , N c86 , N c87 , N c88 , N c89 , N c90 , N c91 , N c92 ) is the same as in FIG. 4, and it is preferable that the other amino acids are substituted.

[0071] The total length of the third linking part of DYW:KP is not particularly limited as long as it can exert U-to-C editing activity as DYW:KP, but is, for example, 29 to 37 amino acids long, preferably 30 to 36 amino acids long, more preferably 31 to 35 amino acids long, and even more preferably 32 to 34 amino acids long. The amino acid sequence of the third linking part is not particularly limited as long as it can exert U-to-C editing activity as DYW:KP, but is preferably the same as the part of positions 98 to 130 of the sequence of SEQ ID NO: 3, or a partial sequence thereof in which 1 to 17 amino acids have been substituted, deleted, or added, or a sequence having sequence identity to the partial sequence, and more preferably the same sequence as the partial sequence.

[0072] A preferred embodiment of the third junction of DYW:KP is a 33 amino acid long polypeptide represented by the following formula, regardless of the sequence of other parts of the DYW domain:

[0073] N c98 -N c99 -N c100 - … -N c128 -N c129 -N c130

[0074] The above polypeptide preferably has a sequence identical to the portion of positions 98 to 130 of the sequence of SEQ ID NO: 3, or a partial sequence thereof with multiple amino acid substitutions, and can exhibit U-to-C editing activity as DYW:KP. In this case, the amino acid substitution is an amino acid with a large bit value (e.g., N c98 , N c100 , N c101 , N c103 , N c104 , N c105 , N c107 , N c109 , N c110 , N c111 , N c112 , N c114 , N c115 , N c117 , N c118 , N c119 , N c120 , N c121 , N c122 , N c123 , N c124 , N c125 , N c127 , N c129 , N c130 ) is the same as in FIG. 4, and it is preferable that the other amino acids are substituted.

[0075] Editing activity can be improved by introducing a mutation into the KP domain consisting of the sequence of SEQ ID NO: 3. Preferred examples of domains into which such mutations have been introduced are KP2 to 23 (SEQ ID NO: 68 to 89) shown in the Examples section of this specification. KP22 (SEQ ID NO: 88) has the highest U to C editing activity and the lowest C to U editing activity, and thus has improved RNA editing activity compared to the KP domain consisting of the sequence of SEQ ID NO: 3.

[0076] (Chimeric DYW) The DYW domain can be divided into several regions based on the conservation of the amino acid sequence. By exchanging each of these regions, the C-to-U editing activity or the U-to-C editing activity can be improved by Chimeric DYW. In one preferred embodiment, the region containing the PG box of DYW:WW, i.e., xb1 PGx b2 SWWTDx b3 -x b16 HP and DYW, DYW:KP other areas (... Hx cc E … Cx c4 x c5 CH …) in which x b1 , x b2 , x b3 -x b16 The first linking portion, the second linking portion, and the third linking portion are as described above for DYW:WW. The DYW portion is Dx bc1 x bc2 The total length is not particularly limited as long as it can exhibit U-to-C editing activity, and is, for example, 110 to 160 amino acids in length, preferably 125 to 149 amino acids in length, more preferably 129 to 145 amino acids in length, and even more preferably 133 to 141 amino acids in length.

[0077] A preferred chimeric domain is x b1 PGx b2 SWWTDx b3 -x b16 HP … Hx cc E … Cx c4 x c5 CH...Dx bc1 x bc2 and comprises a polypeptide having at least 40% sequence identity with the sequence of sequence 90 of SEQ ID NO:2 and having C-to-U / U-to-C editing activity.

[0078] One particularly preferred Chimeric domain is a polypeptide having sequence identity with the sequence of SEQ ID NO: 90 and having U-to-C editing activity. SEQ ID NO: 90 shows the sequence of the Chimeric domain used in the experiment shown in the Examples section of this specification. This domain has been observed to have a higher U to C editing activity than DYW:KP consisting of the sequence of SEQ ID NO: 3, and further has almost no C to U editing activity.

[0079] (Comparison with known sequences) As described above, the DYW domain consisting of the sequences of SEQ ID NOs: 1, 2, and 3 is novel. The results of an investigation using the PPR database (https: / / ppr.plantenergy.uwa.edu.au / onekp / ; see Non-Patent Document 11 above) are shown below.

[0080] [Table 2-1]

[0081] Thus, the present invention also provides any one of the following polypeptides d to i: d. a polypeptide having greater than 78% sequence identity to the sequence of SEQ ID NO:1, preferably greater than 80%, more preferably greater than 85%, even more preferably greater than 90%, even more preferably greater than 95%, and even more preferably greater than 97%, and having C-to-U editing activity; e. a polypeptide having greater than 84% sequence identity to the sequence of SEQ ID NO:2, preferably 85% or more, more preferably 90% or more, even more preferably 95% sequence identity, and even more preferably 97% sequence identity, and having C-to-U editing activity; f. A polypeptide having greater than 86% sequence identity to the sequence of SEQ ID NO:3, preferably 87% or more, more preferably 90% or more, even more preferably 95% sequence identity, and even more preferably 97% sequence identity, and having U-to-C editing activity. g. a polypeptide having a sequence in which 1 to 29, preferably 1 to 25, more preferably 1 to 21, even more preferably 1 to 17, even more preferably 1 to 13, even more preferably 1 to 9, and even more preferably 1 to 5 amino acids have been substituted, deleted, or added in the sequence of SEQ ID NO: 1, and having C-to-U editing activity; h. a polypeptide having a sequence in which 1 to 21, preferably 1 to 18, more preferably 1 to 15, even more preferably 1 to 12, even more preferably 1 to 9, and even more preferably 1 to 6 amino acids have been substituted, deleted, or added in the sequence of SEQ ID NO: 2, and having C-to-U editing activity; i. A polypeptide having a sequence in which 1 to 18, preferably 1 to 16, more preferably 1 to 14, even more preferably 1 to 12, even more preferably 1 to 10, even more preferably 1 to 8, and even more preferably 1 to 6 amino acids have been substituted, deleted, or added in the sequence of SEQ ID NO: 3, and having U-to-C editing activity.

[0082] The alignment of these sequences is shown below. The alignment was created using AliView (Larsson, A. (2014). AliView: a fast and lightweight alignment viewer and editor for large data sets. Bioinformatics30(22): 3276-3278. http: / / dx.doi.org / 10.1093 / bioinformatics / btu531). Identical amino acids are represented by dots.

[0083] [Table 2-2]

[0084] [RNA binding domain] In the present invention, when the editing target is converted using the DYW domain, an RNA-binding protein is used as an RNA-binding domain to target the RNA that contains the editing target.

[0085] A preferred example of an RNA-binding protein used as an RNA-binding domain is a PPR protein composed of a PPR motif.

[0086] (PPR motif) Unless otherwise specified, the PPR motif refers to a polypeptide consisting of 30 to 38 amino acids having an amino acid sequence in which the E value obtained for PF01535 in Pfam and PS51375 in Prosite is equal to or less than a predetermined value (preferably E-03) when the amino acid sequence is analyzed using a protein domain search program on the Web. The position number of the amino acid constituting the PPR motif defined in the present invention is almost synonymous with PF01535, but corresponds to the number obtained by subtracting 2 from the position of the amino acid in PS51375 (e.g., from No. 1 in the present invention to No. 3 in PS51375). However, when referring to the amino acid at No. "ii" (-2), it refers to the second amino acid from the end (C-terminal side) of the amino acid constituting the PPR motif, or the amino acid two amino acids N-terminal to the No. 1 amino acid of the next PPR motif, that is, the -2 amino acid. When the next PPR motif is not clearly identified, the amino acid two amino acids before the first amino acid of the next helix structure is designated as "ii". For Pfam, see http: / / pfam.sanger.ac.uk / ; for Prosite, see http: / / www.expasy.org / prosite / .

[0087] The conserved amino acid sequence of the PPR motif is not highly conserved at the amino acid level, but the two α-helices in the secondary structure are well conserved. A typical PPR motif consists of 35 amino acids, but its length varies from 30 to 38 amino acids.

[0088] More specifically, the PPR motif is composed of a polypeptide having a length of 30 to 38 amino acids and represented by formula 1.

[0089] [ka]

[0090] During the ceremony: Helix A is a 12 amino acid long portion capable of forming an α-helical structure and is represented by formula 2:

[0091] [ka]

[0092] In formula 2, A1~A 12 each independently represents an amino acid; X is absent or a moiety consisting of 1 to 9 amino acids in length; Helix B is a portion that can form an α-helical structure and is 11 to 13 amino acids long; L is a moiety represented by formula 3 that is 2 to 7 amino acids in length;

[0093] [ka]

[0094] In formula 3, each amino acid is numbered from the C-terminus as "i" (-1), "ii" (-2), etc. However, L iii ~L vii may not exist.

[0095] (PPR-code) In the PPR motif, the combination of the three amino acids 1, 4, and ii is important for specific binding to a base, and these combinations determine which base will bind. The relationship between the combination of the three amino acids 1, 4, and ii and the bases that can bind is known as the PPR-code (Patent Document 2, supra), and is as follows:

[0096] (1) A1, A4, and L ii When the combination of these three amino acids is, in order, valine, asparagine, and aspartic acid, the PPR motif has selective RNA base binding ability, binding strongly to U, then to C, and then to A or G. (2) A1, A4, and L iiWhen the three amino acid combinations are, in order, valine, threonine, and asparagine, the PPR motif has selective RNA base-binding ability, binding strongly to A, then to G, then to C, but not to U. (3) A1, A4, and L ii When the combination of these three amino acids is, in order, valine, asparagine, asparagine, the PPR motif has selective RNA base binding ability, binding strongly to C, then to A or U, but not to G. (4) A1, A4, and L ii When the combination of these three amino acids is, in order, glutamic acid, glycine, and aspartic acid, the PPR motif has selective RNA base-binding ability, binding strongly to G but not to A, U, or C. (5) A1, A4, and L ii When the combination of these three amino acids is, in order, isoleucine, asparagine, asparagine, the PPR motif has selective RNA base-binding ability, binding strongly to C, then to U, then to A, but not to G. (6) A1, A4, and L ii When the combination of these three amino acids is, in order, valine, threonine, and aspartic acid, the PPR motif has selective RNA base-binding ability, binding strongly to G, then to U, but not to A or C. (7) A1, A4, and L ii When the combination of these three amino acids is, in order, lysine, threonine, and aspartic acid, the PPR motif has selective RNA base-binding ability, binding strongly to G, then to A, but not to U or C. (8) A1, A4, and L ii When the combination of these three amino acids is, in order, phenylalanine, serine, and asparagine, the PPR motif has selective RNA base-binding ability, binding strongly to A, then to C, and then to G and U. (9) A1, A4, and L iiWhen the combination of these three amino acids is, in order, valine, asparagine, and serine, the PPR motif has selective RNA base-binding ability, binding strongly to C, then to U, but not to A or G. (10) A1, A4, and L ii When the combination of these three amino acids is, in order, phenylalanine, threonine, and asparagine, the PPR motif has selective RNA base binding ability, binding strongly to A but not to G, U, or C. (11) A1, A4, and L ii When the combination of these three amino acids is, in order, isoleucine, asparagine, and aspartic acid, the PPR motif has selective RNA base-binding ability, binding strongly to U, then to A, but not to G or C. (12) A1, A4, and L ii When the combination of these three amino acids is, in order, threonine, threonine, and asparagine, the PPR motif has selective RNA base binding ability, binding strongly to A but not to G, U, or C. (13) A1, A4, and L ii When the combination of these three amino acids is, in order, isoleucine, methionine, and aspartic acid, the PPR motif has selective RNA base-binding ability, binding strongly to U, then to C, but not to A or G. (14) A1, A4, and L ii When the three amino acid combinations are, in order, phenylalanine, proline, and aspartic acid, the motif is called PPR, and has selective RNA base-binding ability, binding strongly to U, followed by C, but not A or G. (15) A1, A4, and L ii When the combination of these three amino acids is, in that order, tyrosine, proline, and aspartic acid, the PPR motif has selective RNA base-binding ability, binding strongly to U but not to A, G, or C. (16) A1, A4, and L iiWhen the combination of these three amino acids is, in order, leucine, threonine, and aspartic acid, the PPR motif has selective RNA base-binding ability, binding strongly to G but not to A, U, or C.

[0097] (P Array) PPR proteins are classified into two families, P and PLS, based on the structure of the PPR motif they comprise. P-type PPR proteins are composed of simple repeats (P array) of a standard 35-amino acid PPR motif (P). The DYW domain of the present invention can be used by linking it to a P-type PPR protein.

[0098] (PLS array) The PPR motifs of PLS-type PPR proteins consist of a repeat unit of three PPR motifs, P1, L1, and S1, followed by P2, L2, and S2 motifs at the C-terminus of the repeat. In some cases, the final P2L2S2 motif is followed by two PPR-like motifs, E1 and E2, and a DYW domain at the C-terminus.

[0099] The total length of P1 is not particularly limited as long as it can bind to the target base, but is, for example, 33 to 37 amino acids, preferably 34 to 36 amino acids, and more preferably 35 amino acids. The total length of L1 is not particularly limited as long as it can bind to the target base, but is, for example, 33 to 37 amino acids, preferably 34 to 36 amino acids, and more preferably 35 amino acids. The total length of S1 is not particularly limited as long as it can bind to the target base, but is, for example, 30 to 33 amino acids, preferably 30 to 32 amino acids, and more preferably 31 amino acids.

[0100] The total length of P2 is not particularly limited as long as it can bind to the target base, but is, for example, 33 to 37 amino acids, preferably 34 to 36 amino acids, and more preferably 35 amino acids. The total length of L2 is not particularly limited as long as it can bind to the target base, but is, for example, 34 to 38 amino acids, preferably 35 to 37 amino acids, and more preferably 36 amino acids. The total length of S2 is not particularly limited as long as it can bind to the target base, but is, for example, 30 to 34 amino acids, preferably 31 to 33 amino acids, and more preferably 32 amino acids. SEQ ID NOs: 17, 22, and 27 show the sequences of P2 used in the Examples section of this specification. SEQ ID NOs: 18, 23, and 28 show the sequences of L2 used in the Examples section of this specification. SEQ ID NOs: 19, 24, and 29 show the sequences of S2 used in the Examples section of this specification.

[0101] The total length of E1 is not particularly limited as long as it can bind to the target base, but is, for example, 32 to 36 amino acids long, preferably 33 to 35 amino acids long, and more preferably 34 amino acids long. SEQ ID NOs: 20, 25, and 30 show the sequences of E1 used in the Examples section of this specification.

[0102] The total length of E2 is not particularly limited as long as it can bind to the target base, but is, for example, 30 to 34 amino acids long, preferably 31 to 33 amino acids long, and more preferably 33 amino acids long. SEQ ID NOs: 21, 26, and 31 show the sequences of E2 used in the Examples section of this specification.

[0103] In a PLS-type PPR protein, the P1L1S1 repeat portion and the portion up to P2 can be designed according to the sequence of the target RNA, following the rules of the PPR-code described above.

[0104] The S2 motif correlates with the nucleotide corresponding to the amino acid ii (the 31st N in SEQ ID NO: 19) (Non-Patent Document 8 cited above). In addition, the C or U four to the right of the target base in the S2 motif can be incorporated into a PLS-type PPR protein, keeping in mind that it is the editing target base by the DYW domain.

[0105] In the E1 motif, correlation with the nucleotide is only seen in the fourth amino acid (the fourth G in SEQ ID NO:20) (Ruwe et al. (2019) New Phytol. 222 218-229.).

[0106] The fourth (the fourth V in SEQ ID NO: 21) and last (the 33rd K in SEQ ID NO: 21) amino acids in the E2 motif are highly conserved and are not involved in specific PPR-RNA recognition (Non-Patent Document 2, supra).

[0107] The number of repeats of P1L1S1 is not particularly limited as long as it can bind to the target base sequence, but is, for example, 1 to 5, preferably 2 to 4, and more preferably 3. In principle, even one unit (3 repeats) can be used. MEF8 (L1-S1-P2-L2-S2-E-DYW), which is composed of five PPR motifs, is known to be involved in editing at approximately 60 sites.

[0108] In natural PPR proteins, the first and last P1L1S1 have clear differences in amino acid residues at specific positions and are different from the internal P1L1S1. From the viewpoint of designing an artificial PLS array that is as close as possible to the naturally occurring one, it is advisable to design three types of P1L1S1 according to the position of the PPR motif, dividing it into the first (N-terminal) P1L1S1, the internal P1L1S1, and the last (C-terminal) P1L1S1 located just before P2L2S2. In addition to those composed of PLS ​​repeating units, there are also cases in which SS (31 amino acids) is repeated in nature, and such ones can also be used in the present invention.

[0109] [DYW protein] The present invention provides a DYW protein for editing a target RNA, comprising an RNA-binding domain that contains at least one PPR motif and is capable of sequence-specifically binding to a target RNA according to the rules of the PPR code, and a DYW domain that is any one of the aforementioned DYW:PG, DYW:WW, or DYW:KP.

[0110] Such a DYW protein may be an artificial product. Artificial means that it is not a natural product but is artificially synthesized. For example, artificial means that it has a DYW domain with a sequence that does not occur in nature, that has a PPR binding domain with a sequence that does not occur in nature, that has a combination of an RNA binding domain and a DYW domain that does not occur in nature, or that a protein for RNA editing in an animal cell further includes a portion that does not exist in a natural DYW protein derived from a plant, such as a nuclear localization signal or a human mitochondrial localization signal. Examples of nuclear localization signal sequences include PKKKRKV (SEQ ID NO: 32) derived from SV40 large T antigen, and KRPAATKKAGQAKKKK (SEQ ID NO: 33), which is an NLS of nucleoplasmin.

[0111] The number of PPR motifs can be appropriately determined depending on the sequence of the target RNA. The number of PPR motifs may be at least one, and may be two or more. It is known that two PPR motifs can bind to RNA (Nucleic Acids Research, 2012, Vol. 40, No. 6, 2712-2723).

[0112] A preferred embodiment of the DYW protein is as follows: An artificial DYW protein for editing a target RNA, comprising at least one, preferably 2 to 25, more preferably 5 to 20, and even more preferably 10 to 18 PPR motifs, an RNA-binding domain capable of sequence-specifically binding to a target RNA according to the rules of the PPR-code, and a DYW domain which is any one of the aforementioned DYW:PG, DYW:WW, or DYW:KP.

[0113] One preferred embodiment of the other DYW proteins is as follows: A DYW protein for editing a target RNA, comprising at least one, preferably 2 to 25, more preferably 5 to 20, and even more preferably 10 to 18 PPR motifs, an RNA-binding domain (preferably an RNA-binding domain that is a PLS-type PPR protein) capable of sequence-specifically binding to an animal's target RNA in accordance with the rules of the PPR-code, and a DYW domain that is any one of the aforementioned DYW:PG, DYW:WW, or DYW:KP.

[0114] The DYW domain, the PPR protein which is the RNA-binding domain, and the DYW protein of the present invention can be prepared in relatively large quantities by methods well known to those skilled in the art. Such methods may include determining the nucleic acid sequence encoding the target domain or protein from its amino acid sequence, cloning it, and preparing a transformant that produces the target domain or protein.

[0115] [Use of DYW protein] (Nucleic acids, vectors, and cells encoding DYW proteins, etc.) The present invention also provides the above-mentioned PPR motif, DYW protein, or nucleic acid encoding the DYW protein, and vectors containing the nucleic acid (e.g., vectors for amplification, expression vectors). Vectors also include viral vectors. Vectors for amplification may use Escherichia coli or yeast as a host. In this specification, an expression vector means a vector containing, for example, from upstream, DNA having a promoter sequence, DNA encoding a desired protein, and DNA having a terminator sequence, but they do not necessarily have to be arranged in this order as long as they exert the desired function. In the present invention, various vectors that can be normally used by those skilled in the art can be recombined and used.

[0116] Specifically, the present invention provides a nucleotide sequence encoding a DYW protein, which includes an RNA-binding domain (preferably an RNA-binding domain that is a PLS-type PPR protein) that contains at least one PPR motif and is capable of sequence-specifically binding to a target RNA (preferably an animal target RNA) according to the rules of the PPR code, and a DYW domain that is any one of the aforementioned DYW:PG, DYW:WW, or DYW:KP.

[0117] More specifically, the present invention provides a vector for editing a target RNA, comprising an RNA-binding domain (preferably an RNA-binding domain that is a PLS-type PPR protein) that includes at least one PPR motif and is capable of sequence-specifically binding to a target RNA (preferably an animal target RNA) according to the rules of the PPR code, and a nucleotide sequence encoding a DYW protein that includes a DYW domain that is any one of the aforementioned DYW:PG, DYW:WW, or DYW:KP.

[0118] The DYW protein of the present invention can function in eukaryotic cells (e.g., animals, plants, microorganisms (yeast, etc.), protists). The DYW protein of the present invention can function in particular in animal cells (in vitro or in vivo). Examples of animal cells into which the DYW protein of the present invention or a vector expressing the DYW protein can be introduced include cells derived from humans, monkeys, pigs, cows, horses, dogs, cats, mice, and rats. Examples of cultured cells into which the DYW protein of the present invention or a vector expressing the DYW protein can be introduced include, but are not limited to, Chinese hamster ovary (CHO) cells, COS-1 cells, COS-7 cells, VERO (ATCC CCL-81) cells, BHK cells, canine kidney-derived MDCK cells, hamster AV-12-664 cells, HeLa cells, WI38 cells, HEK293 cells, HEK293T cells, and PER.C6 cells.

[0119] (Application) The DYW protein of the present invention can convert the editing target C contained in the target RNA to U, or convert the editing target U to C. RNA-binding PPR proteins are involved in all RNA processing steps found in organelles, including cleavage, RNA editing, translation, splicing, and RNA stabilization.

[0120] In addition, the DYW protein of the present invention can edit mitochondrial RNA1 bases. Mitochondria have their own genome, which encodes the constituent proteins of important complexes involved in respiration and ATP production. It is known that mutations in these proteins cause various diseases. Mutation repair using the present invention is expected to be useful in treating various diseases.

[0121] On the other hand, the CRISPR-Cas system has been developed as a C-to-U RNA editing tool in the cytoplasm by fusing Cas proteins to modified ADAR domains (Abudayyeh et al., 2019). However, the CRISPR-Cas system is composed of a protein and a guide RNA, and efficient mitochondrial transport of the guide RNA is difficult. On the other hand, a single PPR protein molecule can edit RNA, and proteins can generally be delivered to mitochondria by fusing a mitochondrial localization signal sequence to the N-terminus. Therefore, to confirm whether this technology can be used for mitochondrial RNA editing, we designed PPRs targeting MT-ND2 and MT-ND5 and created genes fused with the PG or WW domain (Figure 10a).

[0122] These proteins, consisting of a mitochondrial targeting sequence (MTS) and a PPR-P sequence, target the third position of the 178th and 301st codons of MT-ND2 and MT-ND5 to avoid adverse effects on HEK293T cells (Fig. 10a). After plasmid introduction into HEK293T cells, editing was confirmed in the mRNAs of MT-ND2 and MT-ND5 (Fig. 10b, c). These four proteins showed up to 70% editing activity against the target, and no off-target mutations were detected against the same mRNA molecules (Fig. 10bc).

[0123] Therefore, the method for editing RNA bases provided by the present invention is expected to be used in various fields as follows.

[0124] (1) Medical care Recognize and edit specific RNA associated with a specific disease. By using the present invention, genetic diseases caused by single base mutations can be treated. The direction of many mutations in genetic diseases is from C to U. Therefore, the method of the present invention, which can convert U to C in particular, can be useful.

[0125] - Creating cells with controlled RNA suppression and expression. Such cells include stem cells (e.g., iPS cells) with differentiated or undifferentiated states monitored, model cells for evaluating cosmetics, and cells in which the expression of functional RNA can be turned on and off for the purpose of elucidating the mechanisms of drug discovery and pharmacological testing.

[0126] (2) Agriculture, forestry and fisheries Improve yields and quality of agricultural, forestry and fishery products. -Breeding organisms with improved disease resistance, improved environmental tolerance, and improved or new functionalities.

[0127] For example, with regard to first generation hybrid (F1) crops, it may be possible to artificially produce F1 crops by editing mitochondrial RNA with DYW proteins, thereby improving yield and quality. RNA editing with DYW proteins makes it possible to selectively breed and genetically improve organisms more accurately and quickly than conventional techniques. Furthermore, because RNA editing with DYW proteins does not transform traits using foreign genes as in genetic modification, it is closer to traditional breeding methods such as mutant selection and backcrossing. Therefore, it may be possible to respond reliably and quickly to global food and environmental problems.

[0128] (3)Chemistry In the production of useful substances using microorganisms, cultured cells, plants, and animals (e.g. insects), the amount of protein expression is controlled by manipulating RNA. This makes it possible to improve the productivity of useful substances. Examples of useful substances include proteinaceous substances such as antibodies, vaccines, and enzymes, as well as relatively low molecular weight compounds such as pharmaceutical intermediates, fragrances, and dyes.

[0129] -Improve the efficiency of biofuel production by modifying metabolic pathways in algae and microorganisms.

[0130] [term] Numerical ranges from x to y include the endpoints x and y, unless otherwise specified.

[0131] With respect to the amino acid sequence of a protein or polypeptide, an amino acid residue may be simply referred to as an amino acid.

[0132] Unless otherwise specified, "identity" in relation to a base sequence (sometimes called a nucleotide sequence) or an amino acid sequence means the percentage of the number of matching bases or amino acids shared between two sequences when the two sequences are aligned in an optimal manner. In other words, identity can be calculated as follows: identity = (number of matching positions / total number of positions) x 100, and can be calculated using a commercially available algorithm. In addition, such an algorithm is incorporated in the NBLAST and XBLAST programs described in Altschul et al., J.Mol.Biol. 215(1990) 403-410. More specifically, searches and analyses regarding the identity of base sequences or amino acid sequences can be performed using algorithms or programs (e.g., BLASTN, BLASTP, BLASTX, ClustalW) well known to those skilled in the art. When using a program, parameters can be appropriately set by those skilled in the art, and the default parameters of each program may be used. Specific techniques for these analysis methods are also well known to those skilled in the art.

[0133] With respect to the base sequence or amino acid sequence, the higher the sequence identity, unless otherwise specified, the more preferable. Specifically, it is preferably 40% or more, more preferably 45% or more, even more preferably 50% or more, even more preferably 55% or more, even more preferably 60% or more, and even more preferably 65% ​​or more. It is also preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, and even more preferably 97.5% or more.

[0134] With respect to a polypeptide or protein, the number of amino acids to be substituted, deleted, or added when referring to a "sequence having substitutions, deletions, or additions" is not particularly limited in any motif or protein, unless otherwise specified, so long as the motif or protein consisting of that amino acid sequence has the desired function, but may be about 1 to 9 or 1 to 4, or may have a greater number of substitutions, etc., so long as the substitutions are with amino acids with similar properties. Means for preparing polynucleotides or proteins relating to such amino acid sequences are well known to those skilled in the art.

[0135] Amino acids with similar properties refer to amino acids that have similar physical properties such as hydropathicity, charge, pKa, and solubility, and include, for example, the following: Hydrophobic (non-polar) amino acids: alanine, valine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan, tyrosine Non-hydrophobic amino acids; arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine, cysteine, histidine, methionine; Hydrophilic amino acids; arginine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine, threonine; Acidic amino acids: aspartic acid, glutamic acid; Basic amino acids: lysine, arginine, histidine; Neutral amino acids: alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine; Sulfur-containing amino acids: methionine, cysteine; Aromatic ring-containing amino acids: tyrosine, tryptophan, phenylalanine. EXAMPLES

[0136] [1. Design of DYW domain and measurement of RNA editing activity in E. coli] [result] (DYW Domain Design) Currently, most of the available plant genome information is from angiosperms, so the development of artificial DYW proteins is limited to the DYW:PG type. On the other hand, the transcriptome data includes information on early land plant species that have both C-to-U and U-to-C RNA editing, although they are partial sequences. Therefore, we designed a complete artificial DYW domain using a database of PPR proteins (see above, Non-Patent Document 11) built based on the transcriptome dataset created by the 1000 Plants (1KP) International Consortium.

[0137] In designing the artificial DYW domain, in addition to the cytidine deaminase-like DYW domain, we added PPR (P2 and L2) motifs and PPR-like (S2, E1 and E2) motifs to the design. This is because the importance of these motifs to RNA editing activity is currently unknown. We created a phylogenetic tree of DYW domains from hornworts, microphyllous plants and ferns, and classified them into three groups: DYW:PG, DYW:WW and DYW:KP. We then created a new phylogenetic tree for the DYW domains of each group and selected phylogenetic groups of proteins that can be used to design the artificial DYW domain (Fig. 1). The DYW:PG group had a large difference between the sequences, making it difficult to select a phylogenetic group of proteins to use for the design (Fig. 1a). We decided to focus on microphyllous proteins (PG1) because of their high sequence diversity and large number. For the design of the DYW:WW domain, we selected a phylogenetic group of short-branched proteins found only in hornworts (Fig. 1b, WW1). Because this phylogenetic group has short branches, it is assumed that there is little genetic variation between proteins. For the design of the DYW:KP domain, we focused on a phylogenetic group of DYW domains specific to ferns. The protein sequences in this phylogenetic group show large amino acid variations but are conserved in length (Fig. 1c, KP1).

[0138] (Design of RNA-binding domain) We fused each DYW domain to an artificial P or PLS array designed based on the PPR motif sequence identified from plant genome information (see above, Non-Patent Document 2) (Figure 2a). The PPR motif of the P array was constructed based on the consensus sequence obtained from the alignment of 35 amino acid P motifs, with some amino acids replaced to enhance RNA recognition. Standard length 35 amino acid motifs (P1 and L1) and 31 amino acid motif (S1) were selected for the design of the PLS array. In natural PPR proteins, P1L1S1 located at the beginning and end has clear differences in amino acid residues at specific positions and is different from internal P1L1S1. In order to design an artificial PLS array as close as possible to the one that exists in nature, we divided it into P1L1S1 located at the first (N-terminus), P1L1S1 located inside, and P1L1S1 located at the end (C-terminus) just before P2L2S2, and designed three types of P1L1S1 according to the position of the PPR motif. Hereafter, these proteins will be named based on their structure, for example, the DYW:WW domain fused to the P array will be called P-DYW:WW.

[0139] (Artificial DYW proteins can specifically edit target sequences.) In Arabidopsis, the PPR protein CLB19 recognizes the RNA editing sites on chloroplast rpoA and clpP RNAs. We decided to design a PPR protein that targets the rpoA editing site. The 4th and iith amino acids in the P1, L1, S1, and P2 motifs follow the PPR code. On the other hand, since the PPR code for the C-terminal PPR-like motifs (L2, S2, E1, and E2) is unknown, we used the 4th and iith amino acids of CLB19 for the L2, S2, E1, and E2 motifs (Fig. 2b).

[0140] The gene region encoding the recombinant artificial DYW protein was cloned into an expression vector, and a target sequence was added downstream of the stop codon. Based on previous research on two types of PPR proteins (PPR56, PPR65) from Physcomitrella patens (Non-Patent Document 12), a method was developed to test the RNA editing activity of the designed PPR protein in E. coli. The designed PLS-DYW:PG1 and PLS-DYW:WW1 showed no editing activity for DNA, while they replaced cytidine with uridine with an editing efficiency of over 90% for RNA (Figures 3a, b, d). When the P array was used instead of the PLS domain, the editing efficiency decreased by 10 to 40%. On the other hand, both P-DYW:KP and PLS-DYW:KP showed U-to-C RNA editing activity (Figures 3c, e). However, the editing activity was reduced compared to DYW:PG and DYW:WW.

[0141] [method] (phylogenetic tree) The P2-L2-S2-E1-E2-DYW region, including the DYW domain (minimum 132 amino acids), was extracted from the PPR database (https: / / ppr.plantenergy.uwa.edu.au / onekp / ; see above, Non-Patent Document 11). The resulting sequence alignment was generated using MAFFT L-INS-i (v7.407 automatic mode) (K. Katoh, DM Standley (2013). Mol Biol Evol. 30(4): 772-780.), and then trimmed using trimAl (v.1.4.rev15) (Salvador Capella-Gutierrez, et al. (2009). Bioinformatics. 25(15): 1972-1973.). The trimming parameters were gt 0.2 cons 20. The active site (HxEx n Sequences with mutations in CxxCH) were removed from the alignment (Fig. 4 ).

[0142] A phylogenetic tree was constructed from the remaining sequences using FastTree (v2.1.10) (Price, MN, et al. (2010). PLoS One 5:e9490.) to identify DYW:PG, DYW:WW, and DYW:KP using parameters wag and cat 8.

[0143] (Cloning of Trx-PPR-DYW protein and target sequence) The consensus sequences of each DYW domain (including P2, L2, S2, E1, and E2) were designed using EMBOSS:cons (v.6.6.0.0). The protein expression vector was modified from pET21b+PA to remove the original Esp3I and BpiI restriction enzyme sites and add two Esp3I sites as cloning sites. The gene was divided into four sections (Trx, PPR array, DYW domain, and RNA editing site) and constructed using the two-step Golden Gate method. First, three parts were cloned into the Esp3I site of the modified pET21b vector: 1) the thioredoxin-6×His-TEV gene region (containing a BpiI restriction enzyme site at the 3'), 2) the P2-L2-S2-E1-E2-DYW gene region (containing a BpiI restriction enzyme site at the 5'), and 3) the coding sequence region of the RNA editing site. Next, the full-length PLS domain or P domain was cloned into the BpiI site to prepare the PLS:DYW (SEQ ID NOs: 35 to 37) or P:DYW (SEQ ID NOs: 38 to 40) protein.

[0144] (Assay of RNA editing activity in E. coli) To analyze the RNA editing activity of the recombinant protein in E. coli, we modified the protocol developed by Oldenkott et al. (Non-Patent Document 12, supra). The plasmid DNA prepared above was introduced into E. coli Rosetta 2 strain and cultured overnight at 37°C in 1 mL of LB medium (containing 50 μg / mL carbenicillin and 17 μg / mL chloramphenicol). 5 mL of LB medium containing the appropriate antibiotics was prepared in a deep-bottom 24-well plate, and 100 μL of the preculture was inoculated into it. The culture was analyzed by optical density (OD 600The cells were grown at 37°C and 200 rpm until the β-amino acid residue (NA) reached 0.4-0.6, after which the plates were cooled at 4°C for 10-15 min. Then, 0.4 mM ZnSO4 and 0.4 mM IPTG were added, and the cells were further grown at 16°C and 180 rpm for 18 h. 750 μL of the culture was taken and centrifuged, after which the bacterial pellet was frozen in liquid nitrogen and stored at -80°C.

[0145] The frozen cell mass was resuspended in 200 μL of 1-thioglycerol / homogenization solution and sonicated at 40 W for 10 seconds to separate the cells, after which 200 μL of lysis buffer was added. RNA was extracted using the Maxwell® RSC simplyRNA Tissue Kit (Promega). RNA was treated with DNaseI (Takara Bio), and cDNA was synthesized using 1 μg of treated RNA and 1.25 μM random primers (6 mer) using SuperScript® III Reverse Transcriptase (Invitrogen). The region containing the editing site was amplified using NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs), 1 μL of cDNA, and primers for thioredoxin and T7 terminator sequences. The PCR product was purified using NucleoSpin® Gel and PCR Cleanup (Takara Bio), and sequence analysis was performed using a forward primer specific to the DYW domain sequence to determine the base of the RNA editing site. The RNA editing efficiency was measured using the ratio of the heights of the waveform peaks of C and U at the editing site, and the C-to-U RNA editing efficiency was calculated as U / (C+U) × 100, and the U-to-C editing efficiency was calculated as C / (C+U) × 100. Independent experiments were repeated three times.

[0146] [List of cited sequences]

[0147] [Table 3-1]

[0148] [Table 3-2]

[0149] [Table 3-3]

[0150] [2. Example of cultured animal cells] [result] Genes fusing PLS-type PPR with each DYW domain (PG1 (sequence number: 1), WW1 (sequence number: 2), KP1 (sequence number: 3)) and a plasmid containing the target sequence were transfected into HEK293T cells, and RNA was collected after cultivation. The conversion efficiency of cytidine (C) to uridine (U) or uridine (U) to cytidine (C) at the target site was analyzed by Sanger sequencing (Figure 5). When the PG1 or WW1 domain was fused, there was more than 90% C to U activity, while no U to C activity was detected (Figure 5a, c). When the KP1 domain was fused, 25% U to C activity was detected, but about 10% C to U activity was also detected (Figure 5b, c). These results indicate that the editing enzyme functions in animal cultured cells.

[0151] [method] (Preparation of PPR expression plasmid for animal cell culture tests) From the plasmid used in Figure 3, the 6xHis-PPR-DYW protein (the same protein (SEQ ID NO: 35-37) used in the experiment with E. coli) gene sequence and the region containing the editing site (SEQ ID NO: 34) were amplified by PCR and cloned into an animal cell expression vector by the Golden Gate Assembly method. The vector expresses PPR under the control of a promoter containing a CMV promoter and a human β-globin chimeric intron, and a poly A signal is provided by the SV40 polyadenylation signal.

[0152] (HEK293T cell culture) HEK293T cells were cultured at 37°C and 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) containing high glucose, glutamine, phenol-RED, and sodium pyruvate (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 10% fetal bovine serum (Capricom) and 1% penicillin-streptomycin (Fujifilm Wako Pure Chemical Industries, Ltd.). Cells were passaged every 2–3 days when they reached 80–90% confluence.

[0153] (Transfection) For the RNA editing assay, approximately 8.0 x 10 HEK293T cells were plated in each well of a 24-well flat-bottom cell culture plate. 4 The wells were incubated at 37°C, 5% CO2 for 24 hours. 500 ng of plasmid was added to each well in 18.5 μl of Opti-MEM® I Reduced Serum Medium (ThermoFisher) and 1.5 μl of FuGENE® HD Transfection Reagent (Promega) for a final volume of 25 μl. The mixture was incubated at room temperature for 10 minutes before being added to the cells. Cells were harvested 24 hours after transfection.

[0154] (RNA extraction, reverse transcription, sequencing) The assay was carried out in the same manner as in the above-mentioned assay using E. coli. In the following examples, unless otherwise specified, experiments were carried out in the same manner as in the assay using E. coli and in this example.

[0155] [3. Effect of domain swapping on RNA editing activity] The DYW domain can be divided into several regions based on the conservation of amino acid sequences, but the relationship between these regions and RNA editing activity is unknown. Here, we swapped parts of the KP1 and WW1 domains and examined the effect on RNA editing activity in HEK239T cells (Figure 6). In a product in which the PG box and DYW of the WW1 domain were fused to the central site containing the active site of the KP1 domain (chimKP1a sequence number: 90), we found that the U to C activity was nearly 50% higher than that of the KP1 domain, and that the C to U activity was almost completely eliminated. By domain swapping, we succeeded in improving the U to C editing performance of the KP domain.

[0156] [4. Improvement of RNA editing activity of KP domain by mutation introduction] We aimed to improve the RNA editing activity of the KP domain by introducing various mutations into the KP domain. We designed KP2 to KP23 (sequence numbers: 68 to 89) and examined the RNA editing activity of C to U or U to C in E. coli (Fig. 7a) and HEK293T cells (Fig. 7b, c). KP22 (sequence number: 88) had the highest U to C editing activity and the lowest C to U editing activity, and we succeeded in improving the RNA editing activity compared to KP1.

[0157] [5. Improvement of RNA editing activity of PG domain by mutation introduction] We aimed to improve the RNA editing activity of the PG domain by introducing various mutations into the PG domain. We designed PG2 to PG13 (sequence numbers: 41 to 53), and examined the RNA editing activity of C to U in E. coli (Fig. 8a) and HEK293T cells (Fig. 8b, c). PG11 (sequence number: 50) had the highest editing activity for C to U, and we succeeded in improving the RNA editing activity.

[0158] [6. Improvement of RNA editing activity of WW domains by mutation introduction] We aimed to improve the RNA editing activity of the WW domain by introducing various mutations into the WW domain. WW2 to WW14 were designed, and the RNA editing activity of C to U was examined in E. coli (Figure 9a) and HEK293T cells (Figures 9b, c). WW11 (sequence number: 63) had the highest editing activity for C to U, and we succeeded in improving the RNA editing activity compared to WW1.

[0159] [7. Human mitochondrial RNA editing using PPR proteins] [result] Mitochondria have their own genome, which encodes the proteins that make up the important complexes involved in respiration and ATP production. Mutations in these proteins are known to cause various diseases, and methods to repair these mutations are needed.

[0160] The CRISPR-Cas system has been developed as a C-to-U RNA editing tool in the cytoplasm by fusing a modified ADAR domain to a Cas protein (Abudayyeh et al. 2019 Science Vol.365, Issue 6451, pp.382-386). However, the CRISPR-Cas system is composed of a protein and a guide RNA, and efficient mitochondrial transport of the guide RNA is difficult. On the other hand, a single PPR protein can edit RNA, and proteins can generally be delivered to mitochondria by fusing a mitochondrial localization signal sequence to the N-terminus. Therefore, to confirm whether this technology can be used for mitochondrial RNA editing, we designed PPRs targeting MT-ND2 and MT-ND5 and created genes fused with the PG1 or WW1 domain (Figure 10a).

[0161] These proteins, consisting of a mitochondrial targeting sequence (MTS) and a P-DYW sequence, target the third position of the 178th and 301st codons of MT-ND2 and MT-ND5 to avoid adverse effects on HEK293T cells (Fig. 10a). After plasmid introduction into HEK293T cells, editing was confirmed in MT-ND2 and MT-ND5 mRNA (Fig. 10b, c). These four proteins showed up to 70% editing activity on the target, and no off-target mutations were detected on the same mRNA molecule (Fig. 10bc).

[0162] [method] (Cloning for mitochondrial editing) The mitochondrial targeting sequence from the Zea mays LOC100282174 protein (Chin et al. 2018), 10 PPR-P and PPR-like motifs, and the DYW domain portion (DYW domain uses PG1 and WW1) were cloned into an expression plasmid under the control of the CMV promoter by Golden Gate Assembly (sequence numbers 91–94).

[0163] [List of sequences cited]

[0164] [Table 4-1]

[0165] [Table 4-2]

[0166] [Table 4-3]

[0167] [Table 4-4]

Claims

1. A method for editing an editing target U to C by applying an artificial DYW protein containing a DYW domain consisting of any one of the polypeptides c1 and bc1 below to a target RNA. c1. A polypeptide having at least 90% sequence identity to the sequence of SEQ ID NO: 3 and having U-to-C editing activity; or a polypeptide consisting of the sequence of SEQ ID NO: 68, 69, 70, 72, 74, 76, 77, 78, 80, 81, 82, 84, 86, 87, 88, or 89. bc1. A polypeptide having at least 90% sequence identity to the sequence of SEQ ID NO: 90 and having U-to-C editing activity.

2. A DYW domain consisting of any one of the following polypeptides: A polypeptide consisting of the sequence of SEQ ID NO: 3; a polypeptide having at least 90% sequence identity with the sequence of SEQ ID NO: 3 and having C-to-U editing activity; a polypeptide having a sequence in which 1 to 12 amino acids are substituted, deleted or added in the sequence of SEQ ID NO: 3 and having U-to-C editing activity; or a polypeptide consisting of the sequence of SEQ ID NO: 68, 69, 70, 72, 74, 76, 77, 78, 80, 81, 82, 84, 86, 87, 88, or 89 A polypeptide consisting of the sequence of SEQ ID NO: 90; a polypeptide having at least 90% sequence identity with the sequence of SEQ ID NO: 90 and having U-to-C editing activity.

3. A DYW domain consisting of any one of the following polypeptides according to claim 2: A polypeptide consisting of the sequence of SEQ ID NO:3 A polypeptide consisting of the sequence of SEQ ID NO: 90

4. A DYW protein comprising an RNA-binding domain that contains at least one PPR motif and is capable of binding to a target RNA in a sequence-specific manner, and a DYW domain according to claim 2 or 3.

5. The DYW protein of claim 4, wherein the RNA binding domain is of the PLS type.

6. A composition comprising the DYW protein of claim 4 or 5 for editing RNA in a eukaryotic cell.

7. A nucleic acid encoding the DYW protein of claim 4 or 5.

8. A vector comprising the nucleic acid of claim 7.

9. A cell (excluding a human individual) comprising the vector according to claim 8.

10. A composition comprising the nucleic acid of claim 7 or the vector of claim 8 for editing RNA in a eukaryotic cell.

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