Reverse transcriptase, DNA editing system, method for editing target DNA by using said reverse transcriptase and said DNA editing system, and method for producing cell in which target DNA has been edited

Abstract

This split-type reverse transcriptase includes an N-terminal fragment and a C-terminal fragment obtained by dividing a reverse transcriptase into two. The N-terminal fragment and the C-terminal fragment are at least one type selected from the group consisting of: (a) an N-terminal fragment and a C-terminal fragment obtained by splitting a polypeptide containing an amino acid sequence specified by SEQ ID NO: 1 at one location in the region of positions 114-133 in the amino acid sequence specified by SEQ ID NO: 1 or at one location in the region of positions 167-186 in the amino acid sequence specified by SEQ ID NO: 1; (b) an N-terminal fragment and a C-terminal fragment obtained by splitting a polypeptide, which contains the amino acid sequence specified by SEQ ID NO: 1 with one or a plurality of amino acid residues having been substituted, deleted, inserted, and / or added, at one location in a region corresponding to positions 114-133 in the amino acid sequence specified by SEQ ID NO: 1 or at one location in a region corresponding to positions 167-186 in the amino acid sequence specified by SEQ ID NO: 1; and (C) an N-terminal fragment and a C-terminal fragment obtained by splitting a polypeptide, which contains an amino acid sequence having at least 90% homology with the amino acid sequence specified by SEQ ID NO: 1, at one location in a region corresponding to positions 114-133 in the amino acid sequence specified by SEQ ID NO: 1 or at one location in a region corresponding to positions 167-186 in the amino acid sequence specified by SEQ ID NO: 1. The reverse transcriptase activity is regenerated owing to the presence of the N-terminal fragment and the C-terminal fragment.

Description

Reverse transcriptase, DNA editing system, and method for editing target DNA using them, and method for producing cells with edited target DNA

[0001] The present invention relates to a reverse transcriptase, a DNA editing system, and a method for editing target DNA using the same and a method for producing a cell in which the target DNA has been edited.

[0002] Genome editing technology is a technology that specifically cleaves target DNA using artificial DNA cleaving enzymes (e.g., zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN)) or RNA-guided DNA cleaving enzymes (e.g., clustered regularly interspaced short palindromic repeat and CRISPR-associated protein (CRISPR-Cas)), and modifies genes using the DNA repair system possessed by living organisms. Genome editing technology can theoretically be applied to all biological species and cell lines that have DNA, and allows editing (substitution, deletion, insertion) of anything from a single base to long chains. Therefore, it is used in a variety of fields, not only in agriculture and industry, but also in the medical field, such as gene therapy.

[0003] Among the many genome editing techniques, prime editing (PE) is a technique that specifically enables the substitution of all bases, the insertion / deletion (indel) of short chains, or a combination of these (Anzalone, A.V. et al., Nature 576, pp. 149-157, 2019 (Non-Patent Document 1)).

[0004] In prime editing, target DNA editing is thought to occur, for example, by using a Prime Editor, which is a fusion of a nickase-type Cas protein (nCas, for example, nicking Cas9 (nCas9)) that has partially deleted its nuclease activity with a reverse transcriptase (RTase), and a prime editing guide RNA (prime editing guide RNA (pegRNA)), according to the mechanism shown in Figures 1A to 1C. That is, first, the spacer sequence of the pegRNA recognizes the pegRNA recognition sequence on the target DNA and binds to the target DNA, guiding the Cas protein (nCas9 in Figures 1A to 1C) to the vicinity of the target site. The induced Cas protein (nCas9) recognizes a protospacer adjacent motif (PAM) and creates a nick in the strand where the PAM is located (sometimes referred to herein as the "target strand"). The 3'-terminal sequence of the target strand released by the binding of the PEGRNA and the nick is bound to the primer binding site (PBS) of the PEGRNA (Figure 1B). Then, using the template sequence (reverse transcription template (RTT)) contained on the 5' side following the PBS of the PEGRNA as a template, the reverse transcriptase (RTase) fused to the Cas protein (nCas9) performs reverse transcription. This creates an equilibrium state in the target DNA where the edited strand (the strand synthesized by reverse transcriptase) is released at the 3' flap ((a) in Figure 1C) and the non-edited strand is released at the 5' flap ((b) in Figure 1C). The 5' flap is removed by an intracellular enzyme, repairing the nick, and the edited strand is incorporated into the target DNA, replacing the target site; however, the incorporated edited strand is not complementary. Therefore, when repair is performed by the DNA repair mechanism using the edited strand as a template, edited DNA with the desired edit can be obtained ((c) in Figure 1C).

[0005] Non-Patent Document 1 also describes improved prime editing systems, such as PE2, in which five mutations are introduced into the reverse transcriptase to increase its processing ability and binding ability, and PE3, in which a nicking guide RNA (ngRNA) is additionally introduced to create a nick in the non-edited strand, thereby increasing the probability that the edited strand will be adopted during the DNA repair stage, and it has been reported that these systems have improved editing efficiency.

[0006] Furthermore, in an effort to increase the efficiency of prime editing, it has been reported that adding an 8-nt linker and a 51-nt RNA motif called tmpknot (trimmed mpknot) to the 3' end of pegRNA prevents the free 3' end from being cut off by exonuclease (Nelson, J.W. et al., Nat Biotechnol 40, pp. 402-410, 2022 (Non-Patent Document 2)).

[0007] In addition, a technology called PE3max has also been reported, in which two mutations are introduced into nCas9, a double nuclear localization signal (NLS) is placed between nCas9 and reverse transcriptase, the codons of the reverse transcriptase are optimized, and a c-Myc NLS is placed at the C-terminus of Prime Editor (Chen, P.J. et al., Nat Rev Genet 24, pp. 161-177, 2023 (Non-Patent Document 3)). Furthermore, it has also been found that the efficiency is further improved by using this in combination with the pegRNA described in Non-Patent Document 2.

[0008] It has also been reported that target DNA editing by prime editing was possible even when nCas9 and reverse transcriptase were not fused but were separated and expressed separately (Liu, B. et al., Nat Biotechnol 40, pp. 1388-1393, 2022 (Non-Patent Document 4)). Furthermore, in addition to the nickase-type Cas9 protein, for example, it has been reported that target DNA was edited by a double strand break (DSB) using a Cas9 protein that does not lose nuclease activity (having nuclease activity) through a mechanism basically similar to the above-mentioned prime editing (Li, X. et al., Nature Communications 14, Article number: 305, 2023, https: / / doi.org / 10.1038 / s41467-023-35870-0 (Non-Patent Document 5)).

[0009] Anzalone, A. V. et al. , Nature 576, p. 149-157, 2019 Nelson, J. W. et al. , Nat Biotechnol 40, p. 402-410, 2022Chen, P. J. et. al. , Nat Rev Genet 24, p. 161-177, 2023 Liu, B. et. al. , Nat Biotechnol 40, p. 1388-1393, 2022Li, X. etc. al. , Nature Communications 14, Article number: 305, 2023, https: / / doi. org / 10.1038 / s41467-023-35870-0

[0010] However, research into prime editing, which has only recently been developed, has not yet progressed sufficiently, and further improvements in editing efficiency and systems to suppress off-target events are needed. In particular, improvements to reverse transcriptase have not been sufficiently investigated.

[0011] The present invention has been made in consideration of the problems associated with the above-mentioned conventional technology, and aims to provide a novel reverse transcriptase that is active only under specific conditions as a reverse transcriptase useful for prime editing, more specifically, a novel split-type reverse transcriptase that is active only when all of its constituent fragments are present, a DNA editing system containing the same, and methods for editing target DNA using these and methods for producing cells in which target DNA has been edited.

[0012] As a result of extensive research to achieve the above-mentioned object, the present inventors have designed a new reverse transcriptase that is split into an N-terminal fragment and a C-terminal fragment, and its activity is restored only when all of the resulting fragments are present. Using this as the reverse transcriptase in a prime editing system is expected to improve the specificity of prime editing. The present inventors first split the reverse transcriptase into an N-terminal fragment and a C-terminal fragment at multiple positions, and compared the activity of each fragment when used alone with the activity of both fragments when used in the presence of a split GFP. As a result, they found that reverse transcription activity was not observed in the N-terminal fragment and the C-terminal fragment alone, but was specifically restored only when both fragments were present, leading to the completion of the present invention.

[0013] The present invention, based on this finding, provides the following aspects: [1] A split-type reverse transcriptase comprising an N-terminal fragment and a C-terminal fragment obtained by splitting a reverse transcriptase into two, the N-terminal fragment and the C-terminal fragment being selected from the following (a) to (c): (a) an N-terminal fragment and a C-terminal fragment obtained by splitting a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 at any one position within the region of positions 114 to 133 or any one position within the region of positions 167 to 186 of the amino acid sequence of SEQ ID NO: 1, (b) an N-terminal fragment and a C-terminal fragment obtained by splitting a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 in which one or more amino acid residues have been substituted, deleted, inserted and / or added at any one position within the region corresponding to positions 114 to 133 or any one position within the region corresponding to positions 167 to 186 of the amino acid sequence of SEQ ID NO: 1, (c) a polypeptide comprising an amino acid sequence having 90% or more homology to the amino acid sequence of SEQ ID NO: 1, wherein the polypeptide is divided at any one position within the region corresponding to positions 114 to 133 of the amino acid sequence of SEQ ID NO: 1 or at any one position within the region corresponding to positions 167 to 186 of the amino acid sequence of SEQ ID NO: 1; and an N-terminal fragment and a C-terminal fragment, wherein the reverse transcriptase activity is regenerated by the presence of the N-terminal fragment and the C-terminal fragment. [2] A DNA editing system comprising the following (A) to (C): (A) at least one selected from the group consisting of the reverse transcriptase described in [1], an expression vector for the reverse transcriptase, and a polynucleotide encoding the reverse transcriptase; (B) at least one Cas protein selected from the group consisting of Cas proteins (Cas) having nuclease activity and nickase-type Cas proteins (nCas) that are partially deficient in nuclease activity, an expression vector for the Cas protein, and at least one selected from the group consisting of polynucleotides encoding the Cas protein; and (C) at least one selected from the group consisting of a prime editing guide RNA (pegRNA) for the Cas protein, an expression vector for the pegRNA, and a polynucleotide encoding the pegRNA.[3] The DNA editing system of [2], further comprising (D) at least one selected from the group consisting of a nicking guide RNA (ngRNA) of the Cas protein that binds to an ngRNA recognition sequence that is different from the pegRNA recognition sequence to which the pegRNA binds, an expression vector for the ngRNA, and a polynucleotide encoding the ngRNA. [4] A method for editing target DNA, comprising the steps of contacting target DNA with the DNA editing system of [2] or [3] to edit a target site of the target DNA, wherein the pegRNA comprises, in order from the 5' side, a spacer sequence, a template sequence, and a primer binding sequence, the spacer sequence is a sequence that binds to a pegRNA recognition sequence that contains a sequence complementary to the target site, the template sequence is a sequence that serves as a template for the reverse transcriptase, and the primer binding sequence is a sequence that binds to a primer sequence 5' from the target site. [5] A method for producing a cell in which target DNA has been edited, comprising the steps of introducing the DNA editing system described in [2] or [3] into a cell, contacting it with the target DNA, and editing the target site of the target DNA, wherein the pegRNA comprises, in order from the 5' side, a spacer sequence, a template sequence, and a primer binding sequence, the spacer sequence is a sequence that binds to a pegRNA recognition sequence that contains a complementary sequence of the target site, the template sequence is a sequence that serves as a template for the reverse transcriptase, and the primer binding sequence is a sequence that binds to a primer sequence on the 5' side of the target site.

[0014] According to the present invention, it is possible to provide a novel split reverse transcriptase that is active only when all of its constituent fragments are present, a DNA editing system containing the same, a method for editing target DNA using the same, and a method for producing cells in which target DNA has been edited.

[0015]

[0023] Figure 1 is a schematic diagram showing one embodiment of a conventional prime editing mechanism, showing (a) target DNA, (b) Prime Editor, and (b) PEGRNA contained in the prime editing system. Figure 2 is a schematic diagram showing one embodiment of a conventional prime editing mechanism, showing the bound state of target DNA, Prime Editor, and PEGRNA. Figure 3 is a schematic diagram showing one embodiment of a conventional prime editing mechanism, showing (a) the 3' flap where the edited strand is released in the target DNA, (b) the 5' flap where the non-edited strand is released in the target DNA, and their equilibrium state; and (c) edited DNA obtained by repair using the edited strand as a template after the 5' flap has been removed and the edited strand has been replaced with the target site in the target DNA. Figure 4 is a schematic diagram showing the configuration of the vector "KS2" that expresses nCas9 under the EF1a promoter, prepared in <Preparation of vector>. Schematic diagram showing the structure of the vector "KS15" that expresses RTase under the CMV enhancer / promoter, prepared in <Vector Preparation>. Schematic diagram showing the structure of the vector "#1373" that expresses Prime Editor (a fusion protein of nCas9 and RTase) under the EF1a promoter, prepared in <Vector Preparation>. Schematic diagram showing the positional relationship of PAM, pegRNA recognition sequence (comp_pegRNA), and ngRNA recognition sequence (comp_ngRNA) present on the target gene RNF2, used in <Vector Preparation>. Schematic diagram showing the structure of pegRNA used in <Vector Preparation>. Schematic diagram showing the structures of (a) "KS2-1+ng+peg" and (b) "KS25-1+ng+peg," prepared in <Vector Preparation>. 1 is a graph showing the results of comparing the editing efficiency of "KS25-1 + ng + peg (all-in-one)" and the combination (separate) of "KS25 + ng" with a vector expressing pegRNA, measured in <Preparation of vector>. This is a schematic diagram showing the mechanism of DNA editing by cooperation of a split body (fusion protein with split GFP), nCas9, pegRNA, and ngRNA, designed in <Test Example 1>. This is a schematic diagram showing the method of (Preparation of N-terminal vector) in <Test Example 1> and <Test Example 2>. This is a schematic diagram showing the method of (Preparation of C-terminal vector) in <Test Example 1> and <Test Example 2>.1 is a graph showing the editing efficiency (%) measured in Test Example 1.

[0034] FIG. 2 is a graph showing the editing efficiency (%) measured in Test Example 2.

[0035] FIG. 3 is a diagram showing the results of alignment of the amino acid sequence shown in SEQ ID NO: 1 with the amino acid sequence of reverse transcriptase derived from various biological species (positions 3 to 62 of the amino acid sequence of SEQ ID NO: 1).

[0036] FIG. 4 is a diagram showing the results of alignment of the amino acid sequence shown in SEQ ID NO: 1 with the amino acid sequence of reverse transcriptase derived from various biological species (positions 63 to 122 / positions 123 to 182 of the amino acid sequence of SEQ ID NO: 1).

[0037] FIG. 5 is a diagram showing the results of alignment of the amino acid sequence shown in SEQ ID NO: 1 with the amino acid sequence of reverse transcriptase derived from various biological species (positions 183 to 242 / positions 243 to 302 of the amino acid sequence of SEQ ID NO: 1).

[0038] FIG. 6 is a diagram showing the results of alignment of the amino acid sequence shown in SEQ ID NO: 1 with the amino acid sequence of reverse transcriptase derived from various biological species (positions 303 to 362 / positions 363 to 422 of the amino acid sequence of SEQ ID NO: 1).

[0023] Figure 1 shows the results of an alignment of the amino acid sequence shown in SEQ ID NO: 1 with the amino acid sequences of reverse transcriptases derived from various organisms (positions 423-482 and 483-542 of the amino acid sequence of SEQ ID NO: 1). Figure 2 shows the results of an alignment of the amino acid sequence shown in SEQ ID NO: 1 with the amino acid sequences of reverse transcriptases derived from various organisms (positions 543-602 and 603-662 of the amino acid sequence of SEQ ID NO: 1).

[0016] Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to the drawings, but the present invention is not limited thereto. In the following description and drawings, the same or corresponding elements are designated by the same reference numerals, and redundant description will be omitted.

[0017] <Reverse Transcriptase> Reverse transcriptase (sometimes referred to as "RTase" in this specification) is an RNA-dependent DNA polymerase, and is an enzyme that catalyzes the reverse transcription reaction that synthesizes DNA using single-stranded RNA as a template.

[0018] The reverse transcriptase of the present invention is a split-type reverse transcriptase that is split into an N-terminal fragment and a C-terminal fragment and contains these N-terminal fragment and C-terminal fragment, and the presence of both the N-terminal fragment and the C-terminal fragment restores the reverse transcriptase activity that it had when not split. The N-terminal fragment refers to the fragment on the N-terminal side of the reverse transcriptase when not split, and the C-terminal fragment refers to the fragment on the C-terminal side of the reverse transcriptase when not split, and the "N-terminus (amino terminal, NH ２ The term "terminus," also referred to as the N-terminus, N-terminal end, or amine terminus, refers to the free amine (—NH ) of the first amino acid residue of a polypeptide. ２ ) group, and "C-terminus" (also called the carboxy terminus, COOH terminus, or C-terminal end) refers to the free carboxy (-COOH) group of the last amino acid residue of a polypeptide.

[0019] In the reverse transcriptase of the present invention, the N-terminal fragment and the C-terminal fragment are typically (a) N-terminal fragment and C-terminal fragment obtained by cleaving a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 at any one position within the region of positions 114 to 133 or any one position within the region of positions 167 to 186 of the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of a reverse transcriptase derived from murine leukemia virus (Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RTase)).

[0020] As used herein, "split at any one position" refers to cleavage between adjacent amino acids (to generate the C-terminus and N-terminus). For example, combinations of an N-terminal fragment and a C-terminal fragment (N-terminal fragment / C-terminal fragment) "split at any one position within the region of positions 114 to 133" include T1 (the first T in the amino acid sequence of SEQ ID NO: 1, the same applies below) to D114 / L115 to E691, T1 to L115 / R116 to E691, T1 to R116 / E117 to E691, T1 to E117 / V118 to E691, T1 to V118 / N119 to E691, T1 to N119 / K12 0~E691, T1~K120 / R121~E691, T1~R121 / V122~E691, T1~V122 / E123~E691, T1~E 123 / D124~E691, T1~D124 / I125~E691, T1~I125 / H126~E691, T1~H126 / P127~E6 91, T1 to P127 / T128 to E691, T1 to T128 / V129 to E691, T1 to V129 / P130 to E691, T1 to P130 / N131 to E691, T1 to N131 / P132 to E691, or T1 to P132 / Y133 to E691, and similarly in other regions.

[0021] In the reverse transcriptase of the present invention, the division position of the N-terminal fragment and the C-terminal fragment is preferably any one position within the region of positions 115 to 132 of the amino acid sequence of SEQ ID NO: 1, more preferably any one position within the region of positions 118 to 129, even more preferably any one position within the region of positions 120 to 127, particularly preferably any one position within the region of positions 122 to 125, or any one position within the region of positions 168 to 185 of the amino acid sequence of SEQ ID NO: 1, more preferably any one position within the region of positions 171 to 182, even more preferably any one position within the region of positions 173 to 180, and particularly preferably any one position within the region of positions 175 to 178.

[0022] The amino acid sequence of reverse transcriptase is highly conserved among biological species, as shown in Figures 14A to 14F, which show the results of alignment of the amino acid sequences of reverse transcriptase derived from each biological (viral) species shown in Table 1 below with the amino acid sequence set forth in SEQ ID NO: 1.

[0023]

[0024] Furthermore, as shown in the examples below, even split products of reverse transcriptase variants (e.g., S92R) can be cleaved at the above positions to restore reverse transcriptase activity in the presence of both the N-terminal and C-terminal fragments. Therefore, the reverse transcriptase of the present invention can be reverse transcriptases derived from other biological species or variants of reverse transcriptase (natural mutants and artificial mutants). In other words, the reverse transcriptase of the present invention also includes split products of reverse transcriptases consisting of an amino acid sequence highly homologous to the amino acid sequence set forth in SEQ ID NO: 1, as long as the reverse transcriptase activity is restored in the presence of both the N-terminal and C-terminal fragments when split at the above positions.

[0025] Therefore, aspects of the reverse transcriptase of the present invention also include an aspect in which the N-terminal fragment and the C-terminal fragment are (b) N-terminal fragments and C-terminal fragments obtained by splitting a polypeptide comprising an amino acid sequence in which one or more amino acid residues have been substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 1 at any one position within the region corresponding to positions 114 to 133 or any one position within the region corresponding to positions 167 to 186 of the amino acid sequence of SEQ ID NO: 1. In this aspect (b), preferred aspects of the split positions into the N-terminal fragment and the C-terminal fragment correspond to the preferred aspects in (a), respectively.

[0026] Here, in the amino acid sequence, "an amino acid sequence in which amino acid residues have been substituted, deleted, inserted, and / or added" refers to an amino acid sequence in which amino acid residues in the amino acid sequence have been substituted, deleted, inserted, or added, or an amino acid sequence in which two or more of these have been combined. Furthermore, "multiple" refers to an integer of 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2. In the amino acid sequence of the polypeptide (b), the one or more preferably refers to 1 to 30, 1 to 20 (e.g., 1 to 10, 1 to 5, 1 to 3, 2 or less) amino acid residues.

[0027] Further, embodiments of the reverse transcriptase of the present invention include an embodiment in which the N-terminal fragment and the C-terminal fragment are (c) N-terminal fragments and C-terminal fragments obtained by splitting a polypeptide comprising an amino acid sequence having 90% or more homology to the amino acid sequence of SEQ ID NO: 1 at any one position within the region corresponding to positions 114 to 133 or any one position within the region corresponding to positions 167 to 186 of the amino acid sequence of SEQ ID NO: 1. In this embodiment (c), preferred aspects of the split positions into the N-terminal fragment and the C-terminal fragment correspond to the preferred aspects in (a), respectively.

[0028] Here, when referring to amino acid sequence homology, when a control amino acid sequence and a target amino acid sequence are aligned using amino acid sequence analysis software, etc., the amino acid in the target amino acid sequence that is located at the same position as the amino acid in the control amino acid sequence (the control amino acid) may be the same amino acid as the control amino acid, or may have the same properties as the control amino acid. When referring to amino acid sequence identity, the target amino acid is the same amino acid as the control amino acid. Groups of amino acids with similar properties are well known in the technical field to which the present invention pertains, and can be classified into, for example, acidic amino acids (aspartic acid and glutamic acid); basic amino acids (lysine, arginine, histidine); neutral amino acids can be classified into amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), amino acids with hydroxyl groups (serine, threonine), amino acids containing sulfur (cysteine, methionine), amino acids with amide groups (asparagine, glutamine), amino acids with imino groups (proline), and amino acids with aromatic groups (phenylalanine, tyrosine, tryptophan).

[0029] The homology and identity of such amino acid sequences are determined by comparing two sequences aligned to maximize sequence identity. Methods for determining the numerical value (%) of sequence homology or identity are known to those skilled in the art. Any algorithm known to those skilled in the art (e.g., BLAST algorithm, FASTA algorithm, etc.) can be used to obtain optimal alignment and sequence identity, and the sequence homology or identity of amino acid sequences can be determined using sequence analysis software such as BLASTP or FASTA. Furthermore, the amino acid sequence of the polypeptide (c) may have a homology of 90% or more with the amino acid sequence of (a) (SEQ ID NO: 1), but is preferably 95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or more). More preferably, the identity is 90% or more, 95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or more).

[0030] Furthermore, in an amino acid sequence, a region that "corresponds to" a region consisting of a specific amino acid sequence refers to a region consisting of an amino acid sequence that is located at the same position as the specific amino acid sequence (a control amino acid sequence, for example, positions 114 to 133 or positions 167 to 186 of the amino acid sequence set forth in SEQ ID NO: 1) when amino acid sequences are aligned using amino acid sequence analysis software (for example, GENETYX-MAC, Sequencher, ClustalW, etc.) (for example, parameters: default values ​​(i.e., initial settings)).

[0031] In the present invention, the "presence" of both the N-terminal fragment and the C-terminal fragment means that they are both present in the same reaction field, for example, within the same cell (more preferably, for example, within the same nucleus, the same mitochondrion, or the same chloroplast), and it is preferable that they are in close proximity to each other. In this case, "close proximity" means that they are close enough to interact with each other, and in this case, they may be associated by bonding (for example, bonding via intermolecular forces, hydrogen bonds, etc.).

[0032] In the present invention, the regeneration of reverse transcriptase activity due to the presence of both the N-terminal fragment and the C-terminal fragment can be appropriately confirmed by methods known to those skilled in the art. For example, as described in the Examples below, split GFP (GFP1-10 and GFP11, Berrios, K.N., et al., Nat Chem Biol 17, pp. 1262-1270, 2021 (Reference I)) was used to prepare a fusion protein of the N-terminal fragment and GFP1-10, and a fusion protein of the C-terminal fragment and GFP11, respectively. When these were introduced into cells together with the Cas protein (preferably nCas) and pegRNA described below, DNA editing did not occur or the editing efficiency was significantly low when each fusion protein was used alone, but when the two were combined, DNA editing occurred and the editing efficiency was high, which can be confirmed. The occurrence of DNA editing and its editing efficiency can be measured by conventionally known methods; for example, the EditR analysis described in the Examples below can be employed. In this method, for example, if the editing efficiency of the target DNA when using each single entity (N-terminal fragment or C-terminal fragment) is 10% or less compared to the editing efficiency (standard efficiency) of the target DNA when using a reverse transcriptase (unsplit reverse transcriptase) consisting of a polypeptide with the amino acid sequence set forth in SEQ ID NO: 1, it can be determined that DNA editing does not occur or that the editing efficiency is extremely low.On the other hand, if the editing efficiency when both are combined is 15% or more compared to the standard efficiency, it can be determined that DNA editing occurs and the editing efficiency is high.

[0033] The reverse transcriptase of the present invention may be tagged with an epitope tag (e.g., a flag tag, an HA tag, etc.) for purification or detection, or with various transport signals (e.g., a nuclear transport signal, a mitochondrial transport signal, or a plastid transport signal).

[0034] The N-terminal fragment and C-terminal fragment of the reverse transcriptase of the present invention can be obtained appropriately by conventionally known methods. For example, they can be prepared by artificial synthesis based on amino acid sequence information, or they can be prepared at the nucleic acid level. That is, the N-terminal fragment and C-terminal fragment of the reverse transcriptase of the present invention can be obtained in an appropriate host cell by introducing and expressing polynucleotides encoding the N-terminal fragment and C-terminal fragment, respectively, or an expression vector containing the polynucleotides into the cell. When using expression vectors, the polynucleotides encoding the N-terminal fragment and C-terminal fragment may be contained in separate vectors or in a single vector. Therefore, aspects of the reverse transcriptase of the present invention include expression vectors for the reverse transcriptase (i.e., N-terminal fragment expression vector, C-terminal fragment expression vector, and N- and C-terminal fragment expression vectors) and polynucleotides encoding the reverse transcriptase (i.e., polynucleotides encoding the N-terminal fragment and C-terminal fragment, respectively).

[0035] The expression vector can be appropriately selected from vectors used in the field, including, for example, plasmid vectors, viral vectors, phage vectors, phagemid vectors, BAC vectors, YAC vectors, MAC vectors, and HAC vectors. Furthermore, the expression vector preferably contains a regulatory element operably linked to the polynucleotide to be expressed. The regulatory element may include a promoter, an enhancer, an internal ribosome entry site (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and polyU sequences). Furthermore, the expression vector is preferably one that can stably express the encoded protein without being integrated into the host genome. Such an expression vector can be prepared according to a conventionally known method.

[0036] The host cells into which the expression vector is introduced can be appropriately selected in consideration of compatibility with the expression vector, and examples thereof include cells of prokaryotes such as Escherichia coli, actinomycetes, and archaea, and eukaryotes such as yeast, sea urchin, silkworm, zebrafish, mouse, rat, frog, tobacco, Arabidopsis thaliana, and rice. Note that "polynucleotide" encompasses both DNA and RNA (mRNA), and each polynucleotide may have its codons optimized for the purpose of increasing expression efficiency in cells, etc.

[0037] <DNA Editing System> The DNA editing system of the present invention comprises the following (A) to (C): (A) at least one selected from the group consisting of the reverse transcriptase of the present invention, an expression vector for the reverse transcriptase, and a polynucleotide encoding the reverse transcriptase, (B) at least one Cas protein selected from the group consisting of Cas proteins (Cas) with nuclease activity and nickase-type Cas proteins (nCas) partially lacking nuclease activity, an expression vector for the Cas protein, and at least one selected from the group consisting of polynucleotides encoding the Cas protein, (C) at least one selected from the group consisting of a prime editing guide RNA (pegRNA) for the Cas protein, an expression vector for the pegRNA, and a polynucleotide encoding the pegRNA. It is also preferable that the DNA editing system of the present invention further comprises: (D) at least one selected from the group consisting of a nicking guide RNA (ngRNA) for the Cas protein, an expression vector for the ngRNA, and a polynucleotide encoding the ngRNA. The reverse transcriptase, the expression vector for the reverse transcriptase, and the polynucleotide encoding the reverse transcriptase are as described above, including preferred embodiments thereof.

[0038] (Target DNA) In the present invention, DNA containing a target site to be subjected to DNA editing is referred to as "target DNA." Furthermore, when the target site is included in a region encoding a specific gene, the target DNA is also referred to as a "target gene." The target DNA according to the present invention is double-stranded DNA, and for convenience, the "target site" refers to the region of one of the strands that is replaced by the strand (edited strand) synthesized by reverse transcriptase. For convenience, in order to show the correspondence with the DNA editing system of the present invention, the target DNA according to the present invention has a structure in which one strand (first strand, target strand) contains, from the 5' side, a primer sequence, a target site, and a PAM (PAM_1), and the other strand (second strand) contains a pegRNA recognition sequence containing a sequence complementary to the target site. Furthermore, when the following ngRNA is used, an ngRNA recognition sequence is further present on the 3' side of PAM_1 of the first strand, and a PAM (PAM_2) is also present on the 5' side of the second strand. Here, when different Cas proteins are used for pegRNA and ngRNA, PAM_1 and PAM_2 may have the same sequence or different sequences.

[0039] A "PAM (protospacer adjacent motif)" is a sequence recognized by a Cas protein. Its length varies depending on the type of Cas protein, but typically consists of 2 to 5 bases adjacent to the 3' side of the complementary sequence of the pegRNA recognition sequence (and the ngRNA recognition sequence when an ngRNA is used). The nucleotide sequence of the PAM also varies depending on the type of Cas protein, but typically, for example, when the Cas protein is Cas9 or nCas9, the nucleotide sequence of the PAM recognized by this protein is 5'-NGG, etc. However, the nucleotide sequence of such a PAM can also be changed by modifying the Cas protein (e.g., by introducing a mutation) as described below, which can expand the range of target sites available.

[0040] As described above, the "target site" according to the present invention refers to the region of one strand of double-stranded DNA that is replaced by the edited strand. However, since the replaced edited strand is not complementary, when repair is performed using the edited strand as a template by the DNA repair mechanism, both strands are ultimately edited (FIG. 1C (c)). The target site according to the present invention is a site that is set according to the position of the PAM and is set to include at least one base of the site that is subject to cleavage by the Cas protein (preferably single-strand cleavage by nCas). The site that is subject to cleavage by the Cas protein depends on the type of Cas protein, but is usually between the third and fourth bases from the 5' side of the PAM. The length of such a target site is preferably 1 to 100 bases, more preferably 1 to 80 bases.

[0041] In the present invention, the "primer sequence" is a sequence located adjacent to the 5' side of the target site, to which the primer binding sequence of the PEG RNA described below binds, and its 3' end serves as the starting point for the reverse transcription reaction by reverse transcriptase. Therefore, the primer sequence is designed to include the 3' end of the target strand that is generated upon cleavage by the Cas protein (preferably single-strand cleavage by nCas). The length of such a primer sequence is preferably 4 to 30 bases, more preferably 6 to 25 bases, and even more preferably 8 to 20 bases.

[0042] In the present invention, a "pegRNA recognition sequence" is a sequence that is recognized and bound by the spacer sequence of the pegRNA described below. Furthermore, an "ngRNA recognition sequence" is a sequence that is recognized and bound by the spacer sequence of the ngRNA. These pegRNA recognition sequences and ngRNA recognition sequences are set depending on the positions of the PAM and target site described above. The lengths of the pegRNA recognition sequence and ngRNA recognition sequence according to the present invention are each independently dependent on the type of Cas protein, but in the case of Cas9 or nCas9, they are preferably 12 to 25 bases, more preferably 14 to 23 bases, and even more preferably 15 to 22 bases.

[0043] When the following ngRNA is used, the pegRNA recognition sequence and the ngRNA recognition sequence according to the present invention are sequences located at different positions. The length between the pegRNA recognition sequence and the ngRNA recognition sequence is preferably 0 (adjacent to each other) to 200 bases, more preferably 0 to 150 bases, and even more preferably 0 to 100 bases.

[0044] The nucleotide sequence of such target DNA is not particularly limited, and by designing the DNA editing system of the present invention (particularly pegRNA or ngRNA) to match that sequence, it can become the target of DNA editing of the present invention.

[0045] The target DNA according to the present invention is preferably DNA present in cells (endogenous DNA). The DNA present in cells may be endogenous DNA or exogenous DNA. Examples of endogenous DNA include genomic DNA in chromosomes, mitochondria, or chloroplasts, and examples of exogenous DNA include DNA introduced into cells (reporter genes, marker genes, genes of viruses that infect hosts, bacteria, or protozoa, etc.).

[0046] (Cas Protein) In the present invention, the term "Cas protein" refers to a CRISPR (clustered regularly interspaced short palindromic repeat)-associated enzyme, and is at least one selected from the group consisting of a Cas protein having nuclease activity and a nickase-type Cas protein partially deficient in nuclease activity. The Cas protein may be a class 1-CRISPR-associated enzyme or a class 2-CRISPR-associated enzyme. Among these, the Cas protein according to the present invention is preferably a class 2-CRISPR-associated enzyme, and more preferably a nickase-type Cas protein partially deficient in nuclease activity.

[0047] Among the Cas proteins, the "Cas protein having nuclease activity" is not particularly limited, and examples thereof include a Cas9 protein (Cas9, for example, a Cas9 protein derived from Streptococcus pyogenes (SpCas9), a Cas9 protein derived from Staphylococcus aureus (SaCas9), and a Cas9 protein derived from Campylobacter jejuni (CjCas9)), a Cas12a protein (Cas12, for example, a Cas12a (Cpf1) protein derived from Francisella novicida (FnCas12a), and a Cas12a (Cpf1) protein derived from Lachnospiraceae (Lachnospiraceae). Examples of Cas12a proteins include the Cas12a protein (LbCas12a) derived from Bacterium, the Cas12a protein (AsCas12a) derived from Acidaminococcus sp., the Cas12b (C2c1) protein (Cas12b), the Cas12e (CasX) protein (Cas12e), the Cas14 protein (Cas14), the Cas13 protein (Cas13), and the Cas3 protein (Cas3). Among these, the Cas according to the present invention is preferably a class 2-CRISPR-related enzyme, and more preferably Cas9, since it does not require a complex (cascade).

[0048] Furthermore, in the present invention, a "nickase-type Cas protein partially deficient in nuclease activity" refers to a nickase-type Cas protein in which the nuclease activity of the Cas protein (Cas) is partially deficient and the Cas protein has nickase activity (single-stranded DNA cleavage activity), and in this specification, this may also be referred to as "nCas (nicking Cas)" in some cases.

[0049] Such nCas is not particularly limited, and examples thereof include nickase-type Cas9 proteins (nCas9, for example, nSpCas9 (nickase-type SpCas9) in which at least one selected from the group consisting of D10, E762, D839, H983, D986, H840, and N863 of SpCas9 is mutated; nSaCas9 (nickase-type SaCas9) in which at least one selected from the group consisting of D10, D556, H557, and N580 of SaCas9 is mutated). Examples of such a protein include: a nickase-type Cas12a protein (nCas12a, for example, nLbCas12a (a nickase type of LbCas12a) is R1138A; nAsCas12a (a nickase type of AsCas12a) is R1226A, etc.), a nickase-type Cas12b protein (nCas12b), a nickase-type Cas14 protein (nCas14), a nickase-type Cas3 protein (nCas3), and a nickase-type Cas10d protein (nCas10d). Among these, the nCas according to the present invention is preferably a nickase type of class 2-CRISPR-associated Cas, and more preferably nCas9.

[0050] The amino acid sequences encoding these Cas proteins are known and can be obtained from publicly available databases, such as GenBank (http: / / www.ncbi.nlm.nih.gov) or published literature.

[0051] Furthermore, the Cas protein according to the present invention is not limited to these, and further mutations, for example, mutations for modifying PAM recognition may be introduced (Benjamin, P. et al., Nature 523, pp. 481-485, 2015; Hirano, S. et al., Molecular Cell 61, pp. 886-894, 2016).

[0052] Furthermore, the Cas protein according to the present invention may be one that has been appropriately modified (e.g., by substitution, deletion, insertion, and / or addition of amino acid residues) based on the amino acid sequence of a known Cas protein, as long as its functions (e.g., the ability to form a complex with pegRNA and, if necessary, ngRNA, nuclease activity, nickase activity, etc.) are not inhibited. The ability of the Cas protein to form a complex and the nuclease activity or nickase activity can be confirmed, for example, by showing a target DNA editing efficiency equal to or greater than that (e.g., 50% or more, preferably 80% or more) before modification (e.g., a Cas protein consisting of a polypeptide with a known amino acid sequence) when introduced into a cell together with pegRNA and reverse transcriptase.

[0053] When the following ngRNA is used, the corresponding Cas protein is preferably nCas. In this case, the DNA editing system of the present invention may contain multiple Cas proteins, each different in type, for the pegRNA and the ngRNA, but from the standpoint of simplicity, it is preferable that the system contains one Cas protein common to both.

[0054] The Cas protein of the present invention may be added with an epitope tag (e.g., a flag tag, an HA tag, etc.) for purification or detection, or with various transport signals (e.g., a nuclear transport signal, a mitochondrial transport signal, a plastid transport signal).

[0055] The Cas protein of the present invention can be obtained appropriately by conventionally known methods. For example, it can be prepared by artificial synthesis based on amino acid sequence information, or it can be prepared at the nucleic acid level. That is, a polynucleotide encoding the Cas protein or an expression vector for the Cas protein containing the polynucleotide can be introduced into an appropriate host cell and expressed, thereby obtaining the Cas protein in the cell. The expression vector and host cell are the same as those described for the reverse transcriptase of the present invention. Therefore, aspects of the Cas protein of the present invention also include an expression vector for the Cas protein and a polynucleotide encoding the Cas protein.

[0056] (Prime editing guide RNA) In the present invention, a "prime editing guide RNA" is an RNA comprising, in order from the 5' side, a spacer sequence, a template sequence, and a primer binding sequence. It may be in a form consisting of a single nucleotide chain (RNA chain), or may be in a form consisting of two or more nucleotide chains joined together via bonds between complementary sequences. In this specification, "prime editing guide RNA" may also be referred to as "pegRNA (prime editing guide RNA)" in some cases.

[0057] A "spacer sequence," also referred to as a "protospacer," is originally a sequence derived from exogenous DNA incorporated during the adaptation process into the CRISPR structure of the bacterial genome from which the CRISPR-Cas system is derived. However, the spacer sequence of the present invention is a sequence designed to recognize and bind to the pegRNA recognition sequence. Such a spacer sequence is preferably a complementary sequence to the pegRNA recognition sequence, but it need not be completely complementary as long as it is a nucleotide sequence that can hybridize with the pegRNA recognition sequence to form a double strand. In the present invention, the sequence complementarity between such a spacer sequence and the pegRNA recognition sequence is preferably 80% or more, 85% or more, 90% or more, or 95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or more). Those skilled in the art can appropriately calculate the sequence complementarity using known methods (e.g., BLAST (NCBI)).

[0058] The spacer sequence of the pegRNA according to the present invention can be designed to correspond to the pegRNA recognition sequence according to the target site of interest, and its length, although depending on the type of Cas protein, is preferably 10 to 40 bases. For example, in the case of Cas9 or nCas9, it is more preferably 12 to 25 bases, even more preferably 14 to 23 bases, and even more preferably 15 to 22 bases.

[0059] In the present invention, the "template sequence" is also referred to as a "reverse transcription template (RTT)" and is a sequence that serves as a template for the reverse transcriptase. The template sequence according to the present invention can be designed appropriately depending on the nucleotide sequence desired to be replaced with the target site. The length of the template sequence according to the present invention is preferably 1 to 100 bases, more preferably 5 to 90 bases, and even more preferably 10 to 80 bases.

[0060] In the present invention, the "primer binding sequence," also referred to as a "primer binding site (PBS)," is a sequence designed to recognize and bind to a primer sequence at the 3' end generated on the target strand of a target DNA by a Cas protein (preferably nCas). Such a primer binding sequence is preferably a complementary sequence to the primer sequence, but it need not be completely complementary as long as it is a nucleotide sequence that can hybridize with the primer sequence to form a double strand. In the present invention, the sequence complementarity between the primer binding sequence and the primer sequence is preferably 80% or more, 85% or more, 90% or more, or 95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or more).

[0061] The length of the primer binding sequence according to the present invention is preferably 4 to 30 bases, more preferably 7 to 23 bases, and even more preferably 10 to 17 bases.

[0062] The pegRNA of the present invention preferably also includes a scaffold sequence as a tracrRNA structure that forms a complex with the Cas protein. The scaffold sequence may be a conventionally known sequence or a sequence similar thereto, as appropriate. The pegRNA of the present invention may further include a reverse transcriptase termination sequence (e.g., a sequence that forms a hairpin loop structure), a linker, an RNA motif (e.g., tmpknot, tevopreQ1, xrRNA), an RNA aptamer (e.g., MS2, PP7, boxB, com), or the like.

[0063] The pegRNA of the present invention can be obtained appropriately by conventionally known methods. For example, it can be prepared by artificial synthesis based on sequence information designed according to the pegRNA recognition sequence, primer sequence, and desired template sequence, or it can be prepared at the nucleic acid level. That is, pegRNA can be obtained in suitable host cells by introducing a polynucleotide encoding the pegRNA or an expression vector for pegRNA containing the polynucleotide into the cells and expressing it. The expression vector and host cell are the same as those described for the reverse transcriptase of the present invention. Therefore, aspects of the pegRNA of the present invention also include the expression vector for the pegRNA and the polynucleotide encoding the pegRNA.

[0064] (Nicking guide RNA) In the present invention, a "nicking guide RNA" is a guide RNA used to nick the unedited strand, i.e., the strand opposite the target site, by the Cas protein (preferably nCas), for the purpose of improving the probability of DNA repair occurring in the target DNA using the strand synthesized by reverse transcriptase (edited strand) as a template (Non-Patent Document 1), and is also referred to as "ngRNA (nicking guide RNA)" in this specification.

[0065] The ngRNA of the present invention comprises at least a spacer sequence designed as a sequence that recognizes and binds to the ngRNA recognition sequence. Such a spacer sequence is preferably a complementary sequence to the ngRNA recognition sequence, but it need not be completely complementary as long as it is a nucleotide sequence that can hybridize with the ngRNA recognition sequence to form a double strand, and does not have to be completely complementary. In the present invention, the sequence complementarity between such a spacer sequence and the pegRNA recognition sequence is preferably 80% or more, 85% or more, 90% or more, or 95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or more).

[0066] The spacer sequence of the ngRNA according to the present invention can be designed to correspond to the ngRNA recognition sequence according to the target site of interest, and its length, although depending on the type of Cas protein, is preferably 10 to 40 bases. For example, in the case of nCas9, it is more preferably 12 to 25 bases, even more preferably 14 to 23 bases, and even more preferably 15 to 22 bases.

[0067] The ngRNA according to the present invention preferably also includes a scaffold sequence as a tracrRNA structure that forms a complex with the Cas protein. As the scaffold sequence, a conventionally known scaffold sequence or a sequence similar thereto can be appropriately adopted.

[0068] The ngRNA of the present invention can be obtained appropriately by conventionally known methods. For example, it can be prepared by artificial synthesis based on sequence information designed according to the ngRNA recognition sequence, or it can be prepared at the nucleic acid level. That is, the ngRNA can be obtained in an appropriate host cell by introducing and expressing a polynucleotide encoding the ngRNA or an ngRNA expression vector containing the polynucleotide into the cell. The expression vector and host cell are the same as those described for the reverse transcriptase of the present invention. Therefore, aspects of the ngRNA of the present invention also include the ngRNA expression vector and the polynucleotide encoding the ngRNA.

[0069] <Method for Editing Target DNA> The present invention also provides a method for editing target DNA, comprising the steps of contacting the target DNA with the DNA editing system of the present invention and editing a target site in the target DNA. In the method for editing target DNA of the present invention, the DNA editing system and target DNA are each as described above for the DNA editing system of the present invention, including preferred embodiments thereof.

[0070] When the DNA editing system, i.e., the reverse transcriptase (N-terminal fragment, C-terminal fragment), Cas protein, pegRNA, and optionally ngRNA of the present invention, is contacted with the target DNA, the spacer sequence of the pegRNA recognizes and binds to the corresponding pegRNA recognition sequence on the target DNA, guiding the Cas protein to the vicinity of the target site on the target DNA. This causes the Cas protein to recognize the PAM and introduce a nick (a double-strand break (cut) or nick, preferably a nick) at or near the target site. Furthermore, the presence of both the N-terminal fragment and the C-terminal fragment restores reverse transcriptase activity. The primer binding sequence of the pegRNA recognizes the primer sequence containing the nick and binds to the 3' end of the target DNA (first strand, target strand) resulting from the introduction of the nick. Using the subsequent template sequence as a template, the reverse transcriptase synthesizes a DNA strand (edited strand) starting from the primer sequence. In the target DNA, the 5' flap (see (b) of Figure 1C) from the non-edited strand, which is in equilibrium with the 3' flap (see (a) of Figure 1C) from the edited strand, is cut by an intracellular enzyme, repairing the break and replacing the target site with the edited strand, resulting in DNA editing according to the present invention. Furthermore, as a result, the incorporated edited strand is not complementary and is in a mismatched state of double-stranded DNA. Therefore, when repair is carried out using the edited strand as a template by the intracellular DNA repair mechanism, edited DNA in which both strands have been edited can be obtained. In this case, if ngRNA is also used, a nick is introduced into the opposite strand (second strand) of the target site by the Cas protein (preferably nCas) induced by the ngRNA, thereby improving the probability of DNA repair using the edited strand as a template (Non-Patent Document 1). This mainly results in base substitutions at the target site and its complementary strand, but does not deny the possibility that various mutations may be introduced by further substitution with other bases or by deletion or insertion of one or several dozen bases during mismatch repair of double-stranded DNA.Furthermore, for example, by using a pair of DNA editing systems of the present invention, it is possible to perform editing over a wide area (for example, 16 megabases or less), such as deletion or substitution of a long chain between both cleavage sites (see Choi J. et al., Nature Biotechnology 40, p. 218-226, 2022; Tao R. et al., Signal Transduction and Targeted Therapy, 2022 Apr 20, 7(1): 108. doi: 10.1038 / s41392-022-00936-w.), suggesting that, for example, chromosomal translocations, inversions, and duplications are also possible. Therefore, DNA editing according to the present invention also includes the deletion of one or more bases, substitution with one or more other bases, or insertion of one or more bases, or a combination of these mutations, in the vicinity of the target site or in a long chain including the target site.

[0071] The method for editing target DNA of the present invention is preferably carried out intracellularly. The "intracellular" where the method for editing target DNA of the present invention is carried out may be a eukaryotic cell or a prokaryotic cell, but is preferably a eukaryotic cell. Examples of the eukaryotic cells include animal cells (such as cells of mammals, fish, birds, reptiles, amphibians, and insects), plant cells, algae cells, and yeast. Examples of the prokaryotic cells include Escherichia coli, Salmonella, Bacillus subtilis, lactic acid bacteria, and extreme thermophiles.

[0072] "Animal cells" include, for example, cells constituting an individual animal, cells constituting organs or tissues extracted from an animal, and cultured cells derived from animal tissues. Specific examples include germ cells such as oocytes and sperm; germ cells of various stages of embryos (e.g., 1-cell embryos, 2-cell embryos, 4-cell embryos, 8-cell embryos, 16-cell embryos, morula embryos, etc.); stem cells such as induced pluripotent stem (iPS) cells and embryonic stem (ES) cells; and somatic cells such as fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, hepatocytes, pancreatic cells, brain cells, and kidney cells. Pre- and post-fertilization oocytes can be used as the oocytes, but post-fertilization oocytes, i.e., fertilized eggs, are preferred. Pronuclear stage embryos are particularly preferred. Oocytes can be used by thawing cryopreserved oocytes.

[0073] "Plant cells" include, for example, cells that constitute an individual plant, cells that constitute organs or tissues separated from a plant, cultured cells derived from plant tissue, etc. Examples of plant organs and tissues include leaves, stems, shoot tips (growing points), roots, tubers, calluses, etc.

[0074] The method for contacting the DNA editing system with the target DNA is not particularly limited, and examples thereof include a method for introducing the DNA editing system into cells containing the target DNA, such as the method for producing cells with edited target DNA described below.

[0075] <Method for producing cells with edited target DNA> The present invention also provides a method for producing cells with edited target DNA, comprising the steps of introducing the DNA editing system of the present invention into a cell, contacting it with the target DNA, and editing the target site of the target DNA.

[0076] In the method of the present invention for producing cells in which target DNA has been edited (hereinafter sometimes simply referred to as the "production method"), the DNA editing system and target DNA are as described above for the method of editing target DNA of the present invention, including preferred embodiments thereof. The target DNA in the production method of the present invention is genomic DNA, and pegRNA and, if necessary, ngRNA can be designed depending on the purpose of editing the genomic DNA.

[0077] Furthermore, in the production method of the present invention, the DNA editing system, i.e., the reverse transcriptase of the present invention, the Cas protein, the pegRNA, and, if necessary, the ngRNA, can be contacted with the target DNA by introducing the DNA editing system into a cell in the form of a protein or RNA, or by introducing the DNA editing system into a cell in the form of a polynucleotide encoding the protein or RNA, and / or by introducing the DNA editing system into a cell in the form of an expression vector and expressing it within the cell. Thus, the DNA editing systems may be independently introduced into a cell in the form of a protein or RNA, or may be introduced into a cell in the form of DNA or RNA (polynucleotide) encoding these and expressed within the cell, or may be introduced into a cell in the form of an expression vector and expressed within the cell.

[0078] Furthermore, when the DNA editing system is introduced into a cell in the form of an expression vector, for example, a vector that expresses each protein or RNA separately may be introduced into the cell, or a vector that expresses a combination of these may be introduced into the cell.

[0079] As a method for introducing each of the proteins or RNAs, polynucleotides encoding the proteins or RNAs, and expression vectors into cells, known methods for introducing proteins or polynucleotides into cells can be appropriately adopted depending on the type of cell, and examples of such methods include electroporation, microinjection, particle gun, calcium phosphate, polyethyleneimine (PEI), liposome (lipofection), DEAE-dextran, cationic lipid-mediated transfection, viruses (adenovirus, lentivirus, adeno-associated virus, baculovirus, etc.), Agrobacterium, lithium acetate, spheroplast, and heat shock methods (calcium chloride, rubidium chloride). Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology, New York: Elsevier, 1986.

[0080] When the DNA editing system is introduced into a cell, each introduced component or each component expressed in the cell comes into contact with the target DNA in the cell, and the DNA editing described in the method for editing the target DNA of the present invention above causes the desired base to be replaced at the target site, resulting in the production of a cell in which the target DNA has been edited.

[0081] The present invention also provides a method for producing a non-human individual containing cells in which the target DNA has been edited. This method includes a step of producing a non-human individual from cells obtained by the above-mentioned production method. Examples of the non-human individual include non-human animals and plants. Examples of the non-human animal include mammals (e.g., mice, rats, guinea pigs, hamsters, rabbits, monkeys, pigs, cows, goats, sheep), fish, birds, reptiles, amphibians, and insects. When producing a model animal, the mammal is preferably a rodent such as a mouse, rat, guinea pig, or hamster, with mice being particularly preferred. Examples of the plant include grains, oilseed crops, forage crops, fruits, and vegetables. Specific examples of crops include rice, corn, banana, peanut, sunflower, tomato, rapeseed, tobacco, wheat, barley, potato, soybean, cotton, and carnation.

[0082] Known methods can be used to create non-human individuals from cells in which the target DNA has been edited. When creating non-human individuals from cells in animals, germ cells or pluripotent stem cells are typically used. For example, the DNA editing system is microinjected into the oocytes, and the resulting oocytes are implanted into the uterus of a pseudopregnant female non-human mammal, after which offspring can be obtained. It has long been known that somatic cells of plants possess totipotency. For example, a plant in which the desired DNA has been edited can be obtained by microinjecting the DNA editing system into plant cells and regenerating a plant from the resulting plant cells. Furthermore, from the resulting non-human individuals, offspring or clones in which the desired DNA has been edited can also be obtained.

[0083] The presence or absence of target DNA editing and the determination of the genotype can be performed based on conventionally known techniques, such as PCR, sequencing, Southern blotting, etc.

[0084] <Kit> The present invention also provides a kit for use in the method of editing target DNA of the present invention, the production method of the present invention, or the method of producing a non-human individual of the present invention, comprising: (A) at least one selected from the group consisting of the reverse transcriptase of the present invention, an expression vector for the reverse transcriptase, and a polynucleotide encoding the reverse transcriptase. The kit of the present invention preferably further comprises at least one selected from the group consisting of the following (B) to (C): (B) at least one selected from the group consisting of a Cas protein, an expression vector for the Cas protein, and a polynucleotide encoding the Cas protein, (C) at least one selected from the group consisting of a pegRNA, an expression vector for the pegRNA, and a polynucleotide encoding the pegRNA, and more preferably (D) at least one selected from the group consisting of an ngRNA, an expression vector for the ngRNA, and a polynucleotide encoding the ngRNA.

[0085] When the kit of the present invention comprises (B) to (C) or (B) to (D), and when (A) to (D) are in the form of expression vectors, these may be in an embodiment where two or more components are combined and contained in a single vector. Furthermore, when (C) and (D) are in the form of expression vectors, these may be in a form where the user can design the pegRNA and ngRNA according to the target site of the target DNA, and the pegRNA expression vector may be (C') a pegRNA expression vector including an insertion site for a spacer sequence, an insertion site for a template sequence, and an insertion site for a primer binding sequence, and the ngRNA expression vector may be (D') a ngRNA expression vector including an insertion site for a spacer sequence.

[0086] The kit of the present invention may further include one or more additional reagents. Examples of such additional reagents include, but are not limited to, a dilution buffer, a nucleic acid introduction reagent, a protein introduction reagent, and a control reagent (e.g., a complete reverse transcriptase consisting of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1). The kit may also further include instructions for carrying out the method of the present invention.

[0087] Each component included in the kit of the present invention may be contained in a separate container or may be contained in the same container. Each component may be contained in a single-use amount in a container, or multiple doses may be contained in a single container. Each component may be contained in a container in a dry form, or in a form dissolved in an appropriate solvent (a solvent containing a buffer, stabilizer, preservative, antiseptic, etc.).

[0088] The present invention will be explained in more detail below based on test examples including working examples, but the present invention is not limited to the following test examples.

[0089] <Basic Operations> The basic operations performed in this test example are shown below.

[0090] 1. Agarose electrophoresis: 1-3% by mass of Agarose S was added to 1x TAE and dissolved by heating. This solution was poured into a gel maker, a comb was inserted, and the gel was allowed to stand at room temperature to solidify. The solidified gel was placed in an electrophoresis tank, and an appropriate amount of 1x TAE was poured in. Loading buffer was added to the sample to a concentration of 1x or higher, and the sample was applied to the gel wells along with a DNA size marker. If the gel was to be excised after electrophoresis and DNA extracted, electrophoresis was performed at 50 V for approximately 1 hour; if not, electrophoresis was performed at 100 V for approximately 30 minutes. The gel after electrophoresis was placed in ion-exchanged water, 2 μL of ethidium bromide was added, and the gel was shaken for approximately 20 minutes. The gel was photographed using a UV transilluminator to confirm the bands. If DNA was to be extracted, the gel was excised at the position where the target band was located.

[0091] 2. DNA extraction using Wizard SV Gel and PCR Clean-Up System (Promega) The weight of the gel excised in step 1 above was measured, and 100 μL of Membrane Binding Solution was added to 0.1 g of 1% agarose gel. This was vortexed for approximately 15 seconds and heated in a 55°C heat block for approximately 10 to 15 minutes. After confirming that each gel had dissolved, the solution was transferred to a collection tube containing a column and allowed to stand at room temperature for 1 minute. The mixture was centrifuged at 20°C and 16,000 g for 1 minute, and the filtrate was discarded. 700 μL of Membrane Wash solution was added to the column, and the column was centrifuged again at 20°C and 16,000 g for 1 minute, and the filtrate was discarded. Furthermore, 500 μL of Membrane Wash solution was added to the column, and the column was centrifuged at 20°C and 16,000 g for 5 minutes, and the filtrate was discarded. The column was transferred to a new 1.5 mL tube, and 15 μL of nuclease-free water was added. The column was then left to stand at room temperature for 1 minute, and then centrifuged at 20°C and 16,000 g for 1 minute. 15 μL of nuclease-free water was added again, and the column was left to stand at room temperature for 1 minute, and then centrifuged at 20°C and 16,000 g for 1 minute. The concentration of the filtrate was measured using Nanodrop (Thermo Fisher Scientific), adjusted to 200 ng / μL, and then stored at −20°C.

[0092] 3. PCR using PrimeSTAR Max DNA Polymerase (Takara Bio Inc.): 5 μL of PrimeSTAR Max Premix (2x), 0.3 μL of 10 μM forward primer, 0.3 μL of 10 μM reverse primer, 0.2 μL of 1 ng / μL template, and 4.2 μL of autoclaved ultrapure water were mixed in a PCR tube. Using a thermal cycler, the tube was preheated to 94°C for 2 minutes, followed by 35 cycles of thermal denaturation at 98°C for 10 seconds, annealing at 60-68°C (depending on the Tm value of the primer) for 5 seconds, and extension at 72°C for 5-70 seconds (depending on the number of bases to be amplified), and then stored at 4°C.

[0093] 4. PCR using PrimeSTAR GXL DNA Polymerase (Takara Bio Inc.) PrimeSTAR GXL DNA Polymerase 0.5 μL, 5× PrimeSTAR GXL Buffer 5 μL, 2 μL of 2.5 mM each dNTP mixture, 0.75 μL of 10 μM forward primer, 0.75 μL of 10 μM reverse primer, 1.25 μL of 1 to 10 ng / μL template, and 14.75 μL of autoclaved ultrapure water were mixed in a PCR tube. Using a thermal cycler, the mixture was preheated at 94°C for 2 minutes, followed by 45 cycles of thermal denaturation at 98°C for 10 seconds, annealing at 60 to 68°C (depending on the Tm value of the primer) for 15 seconds, and extension at 68°C for 300 to 350 seconds (depending on the number of bases to be amplified), and then stored at 4°C.

[0094] 5. In-Fusion reaction 0.8 μL of insert DNA and 0.8 μL of vector DNA, or 1.6 μL of vector DNA alone, were mixed with 0.4 μL of 5x In-Fusion Premix (Takara Bio Inc.) in a PCR tube. The reaction was carried out at 50°C for 15 minutes using a thermal cycler and then stored at 4°C.

[0095] 6. Restriction enzyme treatment: 200 ng / μL of plasmid, 0.3 μL of restriction enzyme (0.3 μL each if two restriction enzymes were used), 1 μL of buffer corresponding to the restriction enzyme, and 7.7 μL of autoclaved ultrapure water (7.4 μL if two restriction enzymes were used) were mixed in a 1.5 mL tube. This was left to stand in a 37°C incubator for at least 1 hour to allow the restriction enzyme reaction to occur. After this, if ligation (see 7 below) was to be performed, 0.5 μL of rAPid Alkaline Phosphatase 2 (ROCHE) was added, and the mixture was again left to stand in a 37°C incubator for 1 hour to allow phosphorylation.

[0096] 7. Ligation 1 μL of insert DNA, 1 μL of vector DNA, and 1.0 μL of 2×Ligation Mix (manufactured by Takara Bio Inc.) were mixed in a PCR tube, and the mixture was reacted at 16° C. for 30 minutes using a thermal cycler.

[0097] 8. Sequence analysis using SeqStudio Genetic Analyzer (Thermo Fisher Scientific) 8.1 Cycle sequencing reaction 1 μL of 200 ng / μL or 100 ng / μL plasmid, 1 μL of BigDye Terminator v3.1 Ready Reaction Mix (Applied Biosystems), 1.5 μL of 5× Sequencing Buffer, 0.32 μL of 10 μM primer, and 6.18 μL of autoclaved ultrapure water were mixed. Using a thermal cycler, the mixture was preheated at 96°C for 2 minutes, followed by 25 cycles of heat denaturation at 96°C for 10 seconds, annealing at 50°C for 5 seconds, and extension at 60°C for 4 minutes, and then stored at 4°C.

[0098] 8.2 Purification of cycle sequencing products using Optima DTR 8-Well Strip Kit (EdgeBio) The 8-well strip was removed from the 96-well holder, placed on a flat bottom waste plate, and centrifuged at 2600 rpm for 3 minutes. The 8-well strip was placed on a 96-well v-bottom collection plate, and 10 μL of cycle sequencing product was added to the 8-well strip and centrifuged at 2600 rpm for 5 minutes.

[0099] 8.3 Sequencing Analysis 10 μL of the product purified in 8.2 above was added to a sequencing plate, spun down, and subjected to sequencing analysis using a SeqStudio Genetic Analyzer (Applied Biosystems).

[0100] 9. Transformation into E. coli The sample (all transformed samples in the following test examples have the Ampicillin resistance gene) was mixed with at least 10 times the amount of competent cells (XL10-Gold) in a 1.5 mL tube. After leaving it to stand on ice for at least 10 minutes, it was incubated at 42°C for 30 seconds using a heat block, and then cooled on ice for approximately 2 minutes. In a safety cabinet, the bacterial solution was spread on an LB + Ampicillin plate, which was then placed in a 37°C incubator and cultured for 16 to 18 hours.

[0101] 10. Colony PCR A PCR reaction solution was prepared in a PCR tube by mixing 4 μL of 2× SapphireAmp Fast PCR Master Mix (manufactured by Takara Bio Inc.), 0.16 μL of 10 μM forward primer, 0.16 μL of 10 μM reverse primer, and 3.68 μL of autoclaved ultrapure water. A colony on the plate was picked with a 200 μL pipette tip, touched to the replica LB + Ampicillin plate, and then the tip was immersed in the PCR reaction solution. The chip was discarded, and the mixture was preheated at 94°C for 2 minutes using a thermal cycler, followed by 27 cycles of heat denaturation at 98°C for 5 seconds, annealing at 52-68°C (depending on the Tm value of the primer) for 5 seconds, and extension at 72°C for 10-40 seconds (depending on the number of bases to be amplified), and then stored at 4°C. The presence of the target band was confirmed by agarose electrophoresis as described in 1 above.

[0102] 11. Plasmid Extraction Using GenElute Plasmid Miniprep Kit (Sigma-Aldrich) 11.1 Small Culture 15 μL of 25 mg / mL Ampicillin was added to 3 mL of LB liquid medium in a test tube. An E. coli colony was picked with a 200 μL pipette tip, and the tip was dropped into the test tube. The mixture was then cultured with shaking at 37°C for at least 16 hours.

[0103] 11.2 Plasmid extraction from E. coli Approximately half of the culture medium after small culture in 11.1 above was decanted into a collection tube and centrifuged at 20°C and 10,000 g for 1 minute, after which the supernatant was discarded. The remaining culture medium was also placed in a tube and centrifuged at 20°C and 10,000 g for 1 minute, after which the supernatant was discarded. 200 μL of Resuspension solution stored at 4°C was added to each collection tube, and the precipitate was suspended by vortexing. 200 μL of Lysis solution was added to each collection tube, mixed by inversion, and then the lid was opened and left at room temperature for 3 minutes. Next, 350 μL of Neutralization / Binding Buffer was added, mixed by inversion, and centrifuged at 20°C and 12,000 g for 10 minutes. Meanwhile, the column was placed in a collection tube, and 500 μL of Column Preparation Solution was added. The column was centrifuged at 20°C and 12,000 g for 1 minute. The filtrate was discarded, and the lysate supernatant was transferred to the column and centrifuged at 20°C and 12,000 g for 1 minute. The filtrate was discarded, and 500 μL of Wash solution 1 was added, followed by centrifugation at 20°C and 12,000 g for 1 minute. The filtrate was then discarded, and 750 μL of Wash solution 2 was added. The mixture was then centrifuged at 20°C and 12,000 g for 1 minute. The filtrate was discarded, and the mixture was again centrifuged at 20°C and 12,000 g for 1 minute. The column was placed in a DNA LoBind tube, and 15 μL of Elution solution was added. The mixture was left for 1 minute, and then centrifuged at 20°C and 12,000 g for 1 minute. 15 μL of Elution solution was added again, and the mixture was left for 1 minute, and then centrifuged at 20°C and 12,000 g for 1 minute. The DNA concentration of the filtrate was measured using NanoDrop2000 (manufactured by Thermo Fisher Scientific), and the filtrate was diluted with Elution solution to 200 ng / μL (100 ng / μL if the measured concentration was below 200 ng / μL).

[0104] 12. Annealing of Oligo DNA: 0.5 μL each of the sense strand and antisense strand oligo DNA (100 μM) synthesized for the template of the ngRNA target sequence was added to 10× Annealing Buffer (400 μM Tris-HCl (pH 8.0), 200 μM MgCl ２ 1 μL of the solution (prepared with 500 μM NaCl) and 8 μL of autoclaved ultrapure water were mixed. After incubating at 95°C for 5 minutes using a thermal cycler, the temperature was lowered to 25°C over 90 minutes to anneal the oligo DNA.

[0105] 13. Insertion into Plasmid by Golden Gate Reaction 0.3 μL of 25 ng / μL plasmid, 0.5 μL of the oligo DNA annealed in step 12 above, 0.1 μL of BpiI, 0.1 μL of Quick Ligase (New England Biolabs), 0.2 μL of 10× T4 DNA Ligase Buffer (Takara Bio Inc.), and 0.8 μL of autoclaved ultrapure water were mixed. Using a thermal cycler, three cycles of restriction enzyme reaction at 37°C for 5 minutes followed by ligation reaction at 16°C for 10 minutes were performed, followed by storage at 4°C.

[0106] <Cell Experiment> The cell experiment in this test example was carried out according to the following procedures: All cell experiment procedures were carried out in a clean bench, and the instruments used had been previously treated with UV light for 15 minutes or more in the clean bench.

[0107] 1. Preparation of complete medium To 500 mL of L-glutamine and phenol red-containing D-MEM medium (High Glucose), 5.5 mL of 10x Non-Essential Amino Acid (NEAA), 5.5 mL of Penicillin-Streptomycin (ST-PN), and 55 mL of fetal bovine serum (FBS) inactivated at 56°C for 30 minutes were added, mixed well, and then stored at 4°C.

[0108] 2. Culturing HEK293T cells The culture medium in a 100 mm dish was removed using an aspirator, and 10 mL of new complete medium was added. At this time, the cells were detached from the dish by pipetting with an electric pipettor, and a cell suspension was prepared. Approximately 9 mL of complete medium and approximately 1 mL of cell suspension were added to a new 100 mm dish, and the dish was shaken to disperse the cells uniformly. The dish was then incubated at 37°C and 5% CO. ２ The amount of complete medium and cell suspension was adjusted depending on the state of cell growth, and this procedure was carried out once every 2 to 3 days to prevent the cells from overgrowing in the dish.

[0109] 3. Lipofection using Lipofectamin LTX (Thermo Fisher Scientific) The day before or on the day of lipofection, the plasmid to be introduced was diluted with autoclaved ultrapure water to a concentration of 90 ng / 6 μL or 200 ng / 6 μL and a liquid volume of 7.2 μL. 25 μL of D-MEM was added to each well of a 96-well plate, and 6 μL of pre-prepared plasmid was added. Both reagents were mixed in a reservoir so that 0.7 μL of Lipofectamin LTX and 25 μL of D-MEM were added to each well, and then incubated at room temperature for 30 minutes.

[0110] During this time, cells were prepared as follows: The culture medium of HEK293T cells cultured in a 100 mm dish was removed using an aspirator, and 3 mL of TrypLE was added. ＴＭ Express was added to the dish and incubated for 30 seconds. 7 mL of complete medium was added to the dish, and the cells were detached using an electric pipettor. 3 mL of the cell suspension was transferred to a 50 mL tube. This tube was centrifuged at 20°C and 1000 rpm for 3 minutes, the supernatant was removed using an aspirator, and 1.5 mL of complete medium was added for resuspension. 10 μL of the cell suspension was placed in a LUNA automated cell counter (Logos Biosystems) to measure the cell concentration, which was 1.5 x 10 ５ The cells were diluted with complete medium to give a concentration of 1000 cells / mL.

[0111] After the 30-minute incubation, 100 μL of the cell suspension was added to each well and incubated at 37°C, 5% CO ２ The cells were cultured for 72 hours under the conditions.

[0112] <Measurement of Editing Efficiency Using EditR> In this test example, the editing efficiency was measured using EditR as follows.

[0113] 1. Cell Recovery When the cells were transfected with a GFP expression vector, the 96-well plate was observed under a fluorescence microscope before cell recovery to confirm whether the plasmid had been introduced into the cells. Then, the cell medium was removed, and TrypLE was added to each well. ＴＭ 50 μL of Express was added and the mixture was incubated at 37°C for 5 minutes. 150 μL of 1x PBS(-) was added to all wells, and the cells were detached by pipetting. The entire cell suspension was transferred to a PCR tube and centrifuged at 20°C and 13,000 rpm for 3 minutes. The supernatant was discarded, and 150 μL of 1x PBS(-) was added again. The tubes were centrifuged at 20°C and 13,000 rpm for 3 minutes. The supernatant was discarded, and 20 μL of Cell Lysis Buffer (Invitrogen) and 0.8 μL of Protein Degrader (Invitrogen) were added to each tube. A thermal cycler was run with a cycle program of 60°C for 15 minutes followed by 95°C for 10 minutes, and the tubes were then stored at -20°C.

[0114] 2. Genomic PCR and ExoSAP-IT Treatment Following the basic procedure 3 above, the region surrounding the target site was amplified by PCR using PrimeSTAR Max DNA Polymerase (Takara Bio Inc.). In experiments targeting the RNF2 locus, primers RNF2-KN-F1 (SEQ ID NO: 2, Tm: 62.1°C) and RNF2-KN-R1 (SEQ ID NO: 3, Tm: 63.9°C) were used. Of the 10 μL of PCR product, 3 μL was used for agarose electrophoresis, and the remaining 7 μL was used for analysis. 1 μL of ExoSAP-IT (Applied Biosystems) was added to the 7 μL PCR product, and a thermal cycler was run with a program consisting of 37°C for 15 minutes or more followed by 80°C for 15 minutes, followed by storage at -20°C.

[0115] 3. Sequence Analysis According to the basic operation 8 above, sequence analysis was performed using SeqStudio Genetic Analyzer (manufactured by Thermo Fisher Scientific) to obtain waveform data of the nucleotide sequence including the edited site.

[0116] 4. Measurement of genome editing efficiency using EditR The waveform data obtained in 3 above was entered into EditR (https: / / moriaritylab.shinyapps.io / editr_v10 / , Kluesner MG et al., 2018), and the genome editing efficiency (editing efficiency (%)) was calculated.

[0117] <Preparation of vectors> 1. Preparation of nCas9 vector, RTase vector, and PE vector First, a vector "KS2" (nCas9 vector, SEQ ID NO: 4) that expresses nCas9 under the EF1a promoter and a vector "KS15" (RTase vector, SEQ ID NO: 5) that expresses RTase under the CMV enhancer / promoter were prepared according to Non-Patent Document 4. Schematic diagrams of the structures of "KS2" and "KS15" are shown in Figures 2 and 3, respectively. Here, "KS2" has an ngRNA promoter and a scaffold sequence, and in Figure 2, "Cas9" indicates nCas9 (hereinafter the same in Figures 4 and 7). The amino acid sequence of the RTase encoded by "KS15" is the amino acid sequence described in SEQ ID NO: 1, in which S at position 92 has been replaced with R (S92R).

[0118] In addition, for use as a positive control, a vector "#1373" (PE vector, SEQ ID NO: 6) expressing Prime Editor (a fusion protein of nCas9 and RTase) under the EF1a promoter was also prepared according to Non-Patent Document 4. A schematic diagram of the structure of "#1373" is shown in Figure 4. Here, "#1373" has a ngRNA promoter and a scaffold sequence.

[0119] The target gene was RNF2, the effectiveness of which was verified in Non-Patent Document 1, the original paper on prime editing. Figure 5 shows a schematic diagram of the positional relationship between PAM, a pegRNA recognition sequence (comp_pegRNA), and the ngRNA recognition sequence (comp_ngRNA) to which the ngRNA binds, which are present on RNF2.

[0120] 2. Construction of ngRNA and insertion into PE vector ngRNA was designed separately from pegRNA, based on PE3max described in Non-Patent Document 3. First, an ngRNA expression cassette encoding ngRNA (SEQ ID NO: 9) obtained by annealing the oligo DNA "RNF2-nick-s (sense strand, SEQ ID NO: 7)" and "RNF2-nick-as (antisense strand, SEQ ID NO: 8)" according to the above basic procedure 12. was inserted into the above nCas9 vector "KS2" and PE vector "#1373" by the Golden Gate reaction of the above basic procedure 13. Next, 0.1 μL of BpiI and 0.1 μL of 10× Buffer G were added to the total amount of sample, and the restriction enzyme reaction was carried out using a thermal cycler at 37°C for 1 hour, followed by heat inactivation of the restriction enzyme at 80°C for 5 minutes, thereby digesting the plasmid into which the ngRNA expression cassette had not been inserted.

[0121] Next, transformation was performed as in Basic Procedure 9 above, followed by plasmid extraction as in Basic Procedure 11 above, and the extracted plasmid was subjected to restriction enzyme treatment with BpiI as in Basic Procedure 6 above. The sample after restriction enzyme treatment was subjected to agarose electrophoresis as in Basic Procedure 1 above, and the BpiI recognition sequence was lost, no cleavage occurred, and multiple bands appeared, confirming that the ngRNA expression cassette had been inserted.

[0122] The vector in which the ngRNA expression cassette was inserted into the nCas9 vector "KS2" was designated "KS2+ng", and the vector in which the ngRNA expression cassette was inserted into the PE vector "#1373" was designated "KS25+ng".

[0123] 3. Design of pegRNA and insertion into PE vector (preparation of all-in-one type vector) pegRNA is expressed from the U6 promoter and, from the 5' side, comprises a 20nt spacer sequence (protospacer), a scaffold sequence (gRNA scaffold), a 14nt template sequence (RTT), a 15nt primer binding sequence (PBS), a linker (pegLIT linker), tmpknot, and a U6 terminator. A schematic diagram of the structure of pegRNA is shown in Figure 6, and the nucleotide sequence of pegRNA is shown in SEQ ID NO: 10. Of the nucleotide sequence (191 nt) shown in SEQ ID NO: 10, bases 1 to 20 represent a spacer sequence, bases 21 to 96 represent a scaffold sequence, bases 97 to 110 represent a template sequence, bases 111 to 125 represent a primer binding sequence, and bases 134 to 184 represent tmpknot.

[0124] First, a vector expressing a pegRNA targeting RNF2 was subjected to restriction enzyme digestion with Esp3I according to the above-mentioned basic procedure 6. The restriction enzyme-digested sample was subjected to agarose electrophoresis according to the above-mentioned procedure 1. The gel at the position where the band of the pegRNA expression cassette (SEQ ID NO: 11) encoding the pegRNA was located was excised, and DNA was extracted according to the above-mentioned basic procedure 2. The "KS2+ng" or "KS25+ng" prepared in the above-mentioned procedure 2. was used as the vector DNA, and ligation according to the above-mentioned procedure 7. was carried out. After transformation according to the above-mentioned basic procedure 9., colony PCR was carried out according to the above-mentioned procedure 10. using primers pegRNA-ColP-R (SEQ ID NO: 12, Tm value: 60.7°C) and EF1α-proseq-R (SEQ ID NO: 13, Tm value: 59.2°C). Next, after plasmid extraction according to the above-mentioned basic procedure 11., the plasmid was extracted according to the above-mentioned basic procedure 6. The plasmid was subjected to restriction enzyme treatment with HindIII and PmeI according to the above procedure, and the restriction enzyme-treated sample was subjected to agarose electrophoresis as described in the same procedure as in 1. It was confirmed that the pegRNA expression cassette had been inserted into the plasmid, resulting in the appearance of a HindIII recognition site and cleavage at two sites within the plasmid.

[0125] The vector in which a pegRNA expression cassette was inserted into "KS2+ng" was named "KS2-1+ng+peg (SEQ ID NO: 14)," and the vector in which a pegRNA expression cassette was inserted into "KS25+ng" was named "KS25-1+ng+peg (SEQ ID NO: 15)." Schematic diagrams of the structures of "KS2-1+ng+peg" and "KS25-1+ng+peg," respectively, are shown in Figure 7(a) and (b).

[0126] 4. Verification of PE Vector Efficiency The "KS25-1+ng+peg (all-in-one)" prepared in Section 3 above, or the combination of "KS25+ng" prepared in Section 2 above and a vector expressing pegRNA (separate) was introduced into HEK293T cells by Lipofection as described in Cell Experiment 3 above, and the editing efficiency was measured using EditR as described above. The editing efficiency was also measured in the same manner using the plasmid pcDNA, which does not act on cells, and the plasmid pGFP, which expresses only GFP. The results are shown in Figure 8. As shown in Figure 8, it was confirmed that there was no significant difference in editing efficiency between the all-in-one type and the separate type.

[0127] <Test Example 1> (1) Design of Split Enzyme RTase A split-type RTase (hereinafter sometimes referred to as "split enzyme") was designed in which RTase is split and prime editing occurs only when all of the constituent fragments are present. First, based on the description in Berrios, K. N., et al., Nat Chem Biol 17, pp. 1262-1270, 2021 (Reference I), GFP was split into two fragments, GFP1-10 on the N-terminus and GFP11 on the C-terminus, and the regeneration of the activity of the split RTase was verified using the association between these two fragments. That is, a fusion protein was prepared by linking the N-terminal fragment of RTase split into two and GFP1-10 with a GSAGSAAGSG linker (SEQ ID NO: 16), and the remaining C-terminal fragment of RTase split into two and GFP11 with a (GGGGS)3 linker (SEQ ID NO: 17). This split protein (in this test example, a fusion protein with split GFP) was made to cooperate with nCas9 and pegRNA, and as shown in Figure 9, when both fusion proteins were present, the target DNA was edited by prime editing. In addition, in this test, ngRNA was also made to cooperate with the aim of improving the probability of adopting the edited strand during the DNA repair stage (Non-Patent Documents 1 and 3).

[0128] The split forms include RTases obtained by splitting a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which S at position 92 is replaced with R (S92R) at the following seven positions: Split between E123 and D124: "SPL RT1" Split between E176 and M177 (S92R): "SPL RT2" Split between E233 and L234 (S92R): "SPL RT3" Split between T293 and P294 (S92R): "SPL RT4" Split between G405 and W406 (S92R): "SPL RT5" Split between L21 and G22 (S92R): "SPL RT+N" The product divided between E488 and E489 (S92R): "SPL RT+C" was examined.

[0129] (Preparation of N-terminal vector) First, in accordance with Basic Procedure 3 above, inverse-PCR was performed using PrimeSTAR Max DNA Polymerase (manufactured by Takara Bio Inc.) with the RTase-expressing plasmid "KS15 (S92R)" or "KS16 (KS15 with R92 reverted to S92)" as a template to amplify the region of the split N-terminal fragment of RTase that did not contain the unnecessary portion (here, the C-terminal fragment). The primer combinations used in each inverse-PCR, as well as their Tm values ​​and SEQ ID NOs indicating their nucleotide sequences, are shown in Table 2 below. Furthermore, a region on the plasmid encoding GFP1-10 (SEQ ID NO: 18) was used as a template to amplify the region encoding GFP1-10 according to Basic Procedure 3 above. The primer combinations used here, as well as their Tm values ​​and SEQ ID NOs indicating their nucleotide sequences, are also shown in Table 2 below. The primers were designed so that after amplification, a 15-base homologous sequence would be generated at the end of each fragment, and the N-terminal fragment of RTase and GFP1-10 would be linked by the GSAGSAAGSG linker (FIG. 10).

[0130]

[0131] The procedures after PCR were the same as those in the above vector preparation 2., except that EcoRI and AgeI were used in the restriction enzyme treatment to confirm the insertion of GFP1-10. In the same manner as in the insertion of the ngRNA expression cassette of 1. above into the PE vector, expression vectors for fusion proteins (N) in which the N-terminal fragments of each split RTase and GFP1-10 are linked with a GSAGSAAGSG linker were prepared: "SPL RT1(N)-GFP1-10 (SEQ ID NO: 35)," "SPL RT2(N)-GFP1-10 (SEQ ID NO: 36)," "SPL RT3(N)-GFP1-10 (SEQ ID NO: 37)," "SPL RT4(N)-GFP1-10 (SEQ ID NO: 38)," "SPL RT5(N)-GFP1-10 (SEQ ID NO: 39)," "SPL RT+N(N)-GFP1-10 (SEQ ID NO: 40)," and "SPL RT+C(N)-GFP1-10 (SEQ ID NO: 41)."

[0132] (Preparation of C-terminal vector) First, inverse-PCR was performed using PrimeSTAR Max DNA Polymerase (manufactured by Takara Bio Inc.) or PrimeSTAR GXL DNA Polymerase (manufactured by Takara Bio Inc.) with the RTase-expressing plasmid "KS15" or "KS16" as a template, according to Basic Procedure 3 or 4 above, to amplify the C-terminal fragment of the split RTase that did not contain the unnecessary portion (here, the N-terminal fragment). The primer combinations used in each inverse-PCR, as well as their Tm values ​​and SEQ ID NOs indicating their nucleotide sequences, are shown in Table 3 below. Furthermore, the region encoding GFP11 (SEQ ID NO: 42) on the plasmid was used as a template, and the region encoding GFP11 was amplified according to Basic Procedure 3 or 4 above. The primer combinations used here, along with their Tm values ​​and sequence numbers indicating their nucleotide sequences, are shown in Table 3. The primers were designed so that after amplification, a 15-base homologous sequence would be generated at the end of each fragment, and the C-terminal fragment of RTase and GFP11 would be linked by the (GGGGS)3 linker (FIG. 11).

[0133]

[0134] The procedures after PCR were the same as those for the preparation of the N-terminal vector described above, and expression vectors for fusion proteins (C) in which the C-terminal fragments of each split form of RTase and GFP11 were linked with a (GGGGS)3 linker: "GFP11-SPL RT1(C) (SEQ ID NO: 59)," "GFP11-SPL RT2(C) (SEQ ID NO: 60)," "GFP11-SPL RT3(C) (SEQ ID NO: 61)," "GFP11-SPL RT4(C) (SEQ ID NO: 62)," "GFP11-SPL RT5(C) (SEQ ID NO: 63)," "GFP11-SPL RT+N(C) (SEQ ID NO: 64)," and "GFP11-SPL RT+C(C) (SEQ ID NO: 65)" were prepared.

[0135] (2) Introduction into cells, verification of editing efficiency Each split expression vector prepared in (1) above (expression vector for fusion protein (N), expression vector for fusion protein (C), or a set thereof) was introduced into HEK293T cells by Lipofection as described in Cell Experiment 3. above, together with the "KS2-1+ng+peg (vector expressing nCas9, ngRNA, and pegRNA)" prepared in Vector Preparation 3. above, and the editing efficiency was measured using EditR as described above. In addition, as a positive control, the combination of "KS25+ng" prepared in 2. above and a vector expressing pegRNA (separate), the plasmid pcDNA that does not act on cells, and the plasmid pGFP that expresses only GFP were also used to measure the editing efficiency. The results are shown in Figure 12.

[0136] As shown in Figure 12, in the split forms other than "SPL RT+N" split near the N-terminus and "SPL RT+C" split near the C-terminus, the editing efficiency of the N-terminal fragment (fusion protein (N)) alone or the C-terminal fragment (fusion protein (C)) alone was significantly low, and it was confirmed that each fragment alone did not exhibit reverse transcription activity ("(N)" and "(C)" in Figure 12). Furthermore, in three of the seven split forms, "SPL RT3," "SPL RT4," and "SPL RT5," the editing efficiency remained low even when the N-terminal fragment and the C-terminal fragment were combined ("(N)+(C)" in Figure 12). On the other hand, in the two split forms of "SPL RT1" and "SPL RT2," the editing efficiency was improved by combining the N-terminal fragment (fusion protein (N)) and the C-terminal fragment (fusion protein (C)) ("(N) + (C)" in Figure 12). These results confirmed that "SPL RT1" and "SPL RT2" specifically did not exhibit reverse transcription activity on their own, and that reverse transcription activity could be restored only when both the N-terminal fragment and the C-terminal fragment were present.

[0137] <Test Example 2> (1) Design of Additional Split Enantiomers As split enantiomers, a polypeptide (S92R) consisting of an amino acid sequence in which S at position 92 of the amino acid sequence set forth in SEQ ID NO: 1 is replaced with R was used as an RTase, and the following were further split at the following three positions: Split enantiomer (S92R) between P141 and S142: "SPL RT1.1" Split enantiomer (S92R) between Q165 and P166: "SPL RT1.2" Split enantiomer (S92R) between R205 and D206: "SPL RT2.1" were also examined.

[0138] (Preparation of N-terminal vector) In the same manner as in the preparation of the N-terminal vector in Test Example 1 above, a region of the split N-terminal fragment of RTase that does not contain the unnecessary portion (here, the C-terminal fragment) was amplified. The primer combinations used in each inverse-PCR, as well as their Tm values ​​and SEQ ID NOs indicating their nucleotide sequences, are shown in Table 4 below. Similarly, a region encoding GFP1-10 was amplified. The primer combinations used in this amplification, as well as their Tm values ​​and SEQ ID NOs indicating their nucleotide sequences, are also shown in Table 4 below. The primers were designed so that, after amplification, a 15-base homologous sequence would be generated at the end of each fragment, and the N-terminal fragment of RTase and GFP1-10 would be linked by the GSAGSAAGSG linker ( FIG. 10 ).

[0139]

[0140] Furthermore, the PCR and subsequent steps were carried out in the same manner as in the preparation of the N-terminal vector in Test Example 1 above to prepare expression vectors for fusion proteins (N) in which the N-terminal fragments of each split RTase and GFP1-10 were linked via a GSAGSAAGSG linker: "SPL RT1.1(N)-GFP1-10 (SEQ ID NO: 74)," "SPL RT1.2(N)-GFP1-10 (SEQ ID NO: 75)," and "SPL RT2.1(N)-GFP1-10 (SEQ ID NO: 76)."

[0141] (Preparation of C-terminal vector) In the same manner as in the preparation of the C-terminal vector in Test Example 1 above, a region of the split C-terminal fragment of RTase that does not contain the unnecessary portion (here, the N-terminal fragment) was amplified. The primer combinations used in each inverse-PCR, as well as their Tm values ​​and SEQ ID NOs indicating their nucleotide sequences, are shown in Table 5 below. Similarly, a region encoding GFP11 was amplified. The primer combinations used in this case, as well as their Tm values ​​and SEQ ID NOs indicating their nucleotide sequences, are also shown in Table 5 below. The primers were designed so that, after amplification, a 15-base homologous sequence would be generated at the end of each fragment, and the C-terminal fragment of RTase and GFP11 would be linked by the (GGGGS)3 linker ( FIG. 11 ).

[0142]

[0143] Furthermore, the PCR and subsequent steps were carried out in the same manner as in the preparation of the C-terminal vector in Test Example 1 above to prepare expression vectors for fusion proteins (C) in which the C-terminal fragments of each split form of RTase and GFP11 were linked via a (GGGGS)3 linker: "GFP11-SPL RT1.1(C) (SEQ ID NO: 85)," "GFP11-SPL RT1.2(C) (SEQ ID NO: 86)," and "GFP11-SPL RT2.1(C) (SEQ ID NO: 87)."

[0144] (2) Introduction into cells The expression vectors of each split body prepared in (1) above (expression vector for fusion protein (N), expression vector for fusion protein (C), or a set thereof) were introduced into HEK293T cells by Lipofection as described in Cell Experiment 3. above, together with the "KS2-1+ng+peg (vector expressing nCas9, ngRNA, and pegRNA)" prepared in Vector Preparation 3. above, and the editing efficiency was measured using EditR as described above. In addition, as a positive control, the combination of "KS25+ng" prepared in 2. above and a vector expressing pegRNA (separate), the plasmid pcDNA which does not act on cells, and the plasmid pGFP which expresses only GFP were also used to measure the editing efficiency. The results are shown in Figure 13.

[0145] As shown in Figure 13, for all of the additionally prepared split bodies, the editing efficiency was significantly low when the N-terminal fragment (fusion protein (N)) alone or the C-terminal fragment (fusion protein (C)) alone was used ("(N)" and "(C)" in Figure 13), and the editing efficiency did not improve when the N-terminal fragment and the C-terminal fragment were combined ("(N) + (C)" in Figure 13). Thus, it was confirmed that reverse transcription activity was specifically restored in "SPL RT1" and "SPL RT2" only when both the N-terminal fragment and the C-terminal fragment were present.

[0146] As described above, the present invention provides a novel split reverse transcriptase that is active only when all of its constituent fragments are present, a DNA editing system containing the same, a method for editing target DNA using the same, and a method for producing cells with edited target DNA. Such a reverse transcriptase is useful as a reverse transcriptase for prime editing and can improve the specificity of the prime editing.

Claims

1. A split-type reverse transcriptase comprising an N-terminal fragment and a C-terminal fragment obtained by splitting the reverse transcriptase into two, the N-terminal fragment and the C-terminal fragment being selected from the following (a) to (c): (a) an N-terminal fragment and a C-terminal fragment obtained by splitting a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 at any one position within the region of positions 114 to 133 or any one position within the region of positions 167 to 186 of the amino acid sequence of SEQ ID NO: 1; (b) an N-terminal fragment and a C-terminal fragment obtained by splitting a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 in which one or more amino acid residues have been substituted, deleted, inserted and / or added at any one position within the region corresponding to positions 114 to 133 or any one position within the region corresponding to positions 167 to 186 of the amino acid sequence of SEQ ID NO: 1; (c) a polypeptide comprising an amino acid sequence having 90% or more homology to the amino acid sequence of SEQ ID NO: 1, wherein the polypeptide is divided at any one position within the region corresponding to positions 114 to 133 of the amino acid sequence of SEQ ID NO: 1 or at any one position within the region corresponding to positions 167 to 186 of the amino acid sequence of SEQ ID NO: 1; and an N-terminal fragment and a C-terminal fragment, wherein the reverse transcriptase activity is regenerated by the presence of the N-terminal fragment and the C-terminal fragment.

2. A DNA editing system comprising the following (A) to (C): (A) at least one selected from the group consisting of the reverse transcriptase of claim 1, an expression vector for the reverse transcriptase, and a polynucleotide encoding the reverse transcriptase; (B) at least one Cas protein selected from the group consisting of Cas proteins (Cas) having nuclease activity and nickase-type Cas proteins (nCas) that are partially deficient in nuclease activity, an expression vector for the Cas protein, and at least one selected from the group consisting of polynucleotides encoding the Cas protein; (C) at least one selected from the group consisting of a prime editing guide RNA (pegRNA) for the Cas protein, an expression vector for the pegRNA, and a polynucleotide encoding the pegRNA.

3. The DNA editing system of claim 2, further comprising: (D) at least one selected from the group consisting of a nicking guide RNA (ngRNA) for the Cas protein that binds to an ngRNA recognition sequence that is a different sequence from the pegRNA recognition sequence to which the pegRNA binds; an expression vector for the ngRNA; and a polynucleotide encoding the ngRNA.

4. A method for editing target DNA, comprising the steps of contacting target DNA with the DNA editing system described in claim 2 or 3 and editing a target site in the target DNA, wherein the pegRNA comprises, in order from the 5' side, a spacer sequence, a template sequence, and a primer binding sequence, the spacer sequence is a sequence that binds to a pegRNA recognition sequence that contains a complementary sequence of the target site, the template sequence is a sequence that serves as a template for the reverse transcriptase, and the primer binding sequence is a sequence that binds to a primer sequence on the 5' side of the target site.

5. A method for producing cells in which target DNA has been edited, comprising the steps of introducing the DNA editing system of claim 2 or 3 into cells, bringing it into contact with the target DNA, and editing the target site of the target DNA, wherein the pegRNA comprises, in order from the 5' side, a spacer sequence, a template sequence, and a primer binding sequence, the spacer sequence is a sequence that binds to a pegRNA recognition sequence that contains a complementary sequence of the target site, the template sequence is a sequence that serves as a template for the reverse transcriptase, and the primer binding sequence is a sequence that binds to a primer sequence 5' from the target site.

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