Intracellular assay to measure the base correction ability of base editors

Abstract

The present invention relates to a method for testing gene editing in an extracellular test tube environment (cell-free, in vitro system), and more specifically, to a method for confirming and quantifying target DNA mutations caused by base editors without the use of eukaryotic cells or other complicated testing processes. By using the present invention, target DNA mutations caused by base editors can be confirmed and quantified without the use of cells or other complicated testing processes. Furthermore, base editors that have the ability to correct target DNA mutations in actual cells can be screened through a simple extracellular testing method.

Description

[Technical Field]

[0001] The present invention relates to a method for testing gene base editing in an extracellular test tube environment (cell-free, in vitro system), specifically a method for confirming and quantifying target DNA mutations caused by base editors without using cells or additional complicated testing processes.

[0002] Target DNA can be amplified from cells, but more usefully, it can be used to mimic mutant DNA from organisms requiring base editing (e.g., patients with genetic diseases) through double-stranded DNA synthesis. Measuring DNA editing efficiency without cell lines or model animals carrying specific mutations can serve as a primary test for the base correction capabilities of various base editors, including adenine base editors, cytosine base editors, DdCBEs (DddA-derived cytosine base editors), TALEDs (TALE-linked deaminase), zinc finger deaminase (ZFD), and CRISPR systems. [Background technology]

[0003] Various proteins are used as genome editing tools. For example, base editing uses fusion proteins consisting of base-converting enzymes such as deaminase linked to Cas9 nickase (nCas9) or catalytically dead Cas9 (dCas9) to induce single-base mutations, such as A-to-G (adenine base editor) or C-to-T (cytosine base editor), without creating double-strand breaks (DSBs). Prime editing uses fusion proteins consisting of nCas9 linked to reverse transcriptase and prime editing guide RNA (pegRNA) containing an RNA template to induce various custom mutations, including base mutations and small insertions or deletions (indels), at specific locations in nuclear DNA. Furthermore, DdCBE and TALED technologies can edit cytosine or adenine bases in not only nuclear DNA but also organelle DNA, causing single-base mutations in thymine or guanine. Summary of the Invention [Problem to be solved by the invention]

[0004] While observing the efficiency of genome editing by applying it to endogenous genes in an organism is relatively easy because cell lines containing normal genes can be used, experiments targeting DNA containing mutations are not as straightforward. While it is possible to isolate cells from an organism model or patient carrying the desired mutation and directly conduct experiments on primary cells and / or create and test cell lines, this method requires difficult cell maintenance and takes a relatively long time (e.g., 3-4 months). Therefore, the present invention aims to provide a method for testing genome editing in an extracellular, in vitro environment (cell-free, in vitro system), which can confirm and quantify target DNA mutations caused by base editors without using cells or undergoing additional complex experimental procedures. The present invention also aims to provide a method for screening base editors capable of correcting target DNA mutations in actual cells through such a simple extracellular testing method. [Means for solving the problem]

[0005] The incellular test method according to the present invention involves contacting isolated double-stranded DNA serving as an editing target with a base editing protein in an extracellular environment to obtain base-corrected DNA, and then sequencing the resulting base-corrected DNA to measure base correction activity. Here, the base editing protein is mixed with the target DNA in the form of a polynucleotide (including DNA or mRNA) encoding the base editing protein or a vector containing the same in a single reaction tube (one pot) to form a base editing protein. This can be used in a reaction for DNA base correction without a separate protein purification process, or it can be expressed and mixed with the target DNA in a purified form and used. The target DNA can be nuclear DNA, mitochondrial DNA, or chloroplast DNA, and can be extracted from animal or plant cells, amplified, and / or synthesized. This is the first time that it has been confirmed that base correction in an extracellular environment without using cells exhibits substantially the same correction activity as base correction performed inside cells. Therefore, it is now possible to measure the base correction activity of a given base editor by confirming the target DNA mutation caused by the base editor in an extracellular environment and measuring the base correction ability without undergoing complicated experimental procedures using cells. This means that base editors that can correct target DNA mutations in actual cells can now be screened using only a simple extracellular test method. [Brief explanation of the drawings]

[0006] [Figure 1]These are the results of base editing of the human mitochondrial ND1 gene region using DdCBE (DddA-derived cytosine base editor) in an extracellular environment according to the present invention. 1397N indicates the N-terminal G1397DddAtox fragment, and 1397C indicates the C-terminal G1397DddAtox fragment. The box on the left indicates the DNA sequence recognized by the fusion protein containing 1397N (left DdCBE), and the box on the right indicates the DNA sequence recognized by the fusion protein containing 1397C (right DdCBE). The density indicates the efficiency of base correction. Base correction at guanine (G) base positions indicates that the cytosine (C) base pair at that position has also been corrected. [Figure 2] These are the results of base editing of the human mitochondrial ND1 gene region using TALED (TALE-linked deaminase) in an extracellular environment according to the present invention. 1397N indicates the N-terminal G1397DddAtox fragment, and 1397C indicates the C-terminal G1397DddAtox fragment. The box on the left indicates the DNA sequence recognized by the fusion protein (left split TALED: sTALED) containing 1397C and a TALE protein linked to TadA8e, while the box on the right indicates the DNA sequence recognized by the fusion protein (right split TALED: sTALED) containing a TALE protein linked to 1397N. The density indicates the efficiency of base correction. The indication of base correction at the thymine (T) base position indicates that the adenine (A) base pair at that position has also been corrected. The correction of the cytosine (C) base is the result of C-to-T correction by the DddAtox contained in TALED, and the fact that base correction also occurred at the guanine (G) base position indicates that the cytosine (C) base that forms a base pair at the corresponding position has been corrected. [Figure 3]The graph shows a comparison of the results of C-to-T correction using DdCBE and A-to-G correction using TALED at the human mitochondrial ND1 gene site in an extracellular environment according to the present invention with the results of C-to-T correction using the same DdCBE and A-to-G correction using the same TALED in HEK293T cells. IVTT indicates that base correction was performed using the extracellular test method according to the present invention, and HEK293T indicates that base correction was performed using HEK293T cells. [Figure 4] The C-to-T correction efficiency obtained using DdCBE and the A-to-G correction efficiency obtained using TALED targeting the ND1 gene site of human mitochondria in an extracellular environment according to the present invention were compared with the C-to-T correction efficiency obtained by applying the same DdCBE to HEK293T cells and the A-to-G correction efficiency obtained by applying the same TALED to HEK293T cells. [Figure 5] This figure shows the results of A-to-G base correction of the G3460A mutation (a mutation in which the 3460th base of the mitochondrial base is A instead of G) in the mitochondrial ND1 gene of a patient with Leber's hereditary optic neuropathy (LHON) using TALED in an extracellular environment according to the present invention. m.A3460G indicates A-to-G correction of the G3460A mutation. 1397N indicates the N-terminal G1397DddAtox fragment, and 1397C indicates the C-terminal DddAtox fragment. The boxed portion on the left indicates the DNA sequence recognized by the fusion protein (17C or 18C) containing TALE, 1397C, and TadA8e, and the boxed portion on the right indicates the DNA sequence recognized by the fusion protein (56N or 57N) containing TALE and 1397N. The boxed "A" indicates the G3460A mutation, and the intensity indicates the efficiency of base correction. The bar graph on the right shows the 3460A→G straightening efficiency. [Figure 6]This figure shows the results of A-to-G base correction of the G3460A mutation in the mitochondrial ND1 gene of a LHON patient using TALED in UDC cells. 1397N indicates the N-terminal G1397DddAtox fragment, and 1397C indicates the C-terminal G1397DddAtox fragment. The box on the left indicates the DNA sequence recognized by the fusion protein (17C or 18C) containing TALE, 1397C, and TadA8e. The box on the right indicates the DNA sequence recognized by the fusion protein (56N or 57N) containing TALE and 1397N. The boxed "A" indicates the G3460A mutation, and the density indicates the base correction efficiency. The bar graph on the right shows the 3460A→G correction efficiency. [Figure 7] A bar graph comparing the extracellular base correction efficiency obtained using the fusion protein 17C and the fusion protein 57N shown in Figure 5 as base editors with the intracellular base correction efficiency obtained using the same base editors shown in Figure 6. [Figure 8] A bar graph comparing the extracellular base correction efficiency obtained using the fusion protein 18C and the fusion protein 56N shown in Figure 5 as base editors with the intracellular base correction efficiency obtained using the same base editors shown in Figure 6. [Figure 9] The bar graph compares the base correction efficiency in cells obtained using the fusion protein 18C and the fusion protein 57N shown in Figure 5 as base editors with the intracellular base correction efficiency obtained using the same base editors shown in Figure 6. [Figure 10] A bar graph comparing the efficiency of 3460A→G base correction obtained using the base editor shown in FIG. 5 with the efficiency of 3460A→G base correction obtained using the same base editor shown in FIG. 6. DETAILED DESCRIPTION OF THE INVENTION

[0007] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the terms used herein are well known and commonly used in the art.

[0008] As used herein, the terms "correction," "editing," and "editing" are used interchangeably and refer to a method of altering a nucleic acid sequence by selective mutation of a specific game target, including, but not limited to, a gene, promoter, open reading frame, or any nucleic acid sequence.

[0009] As used herein, the terms "base editor," "base editing system," and "base correction system" are used interchangeably and refer to a substance that has the activity of altering a nucleic acid sequence by selectively mutating a game target, and include a combination of one or more different base editors. As used herein, the terms "base editor," "base editing system," or "base correction system" may be in the form of a polypeptide (which may be a fusion protein) or a polynucleotide, or a combination thereof, depending on the context, or may refer to a composition containing one or more polypeptides (which may be fusion proteins) or polynucleotides, or a combination thereof. Therefore, as used herein, the term "base correction system" or "base correction composition" may include one base editor or a combination of two or more different base editors, where the different base editors can be used simultaneously or separately.

[0010] The ability of the base correction system according to the present invention to correct a target DNA mutation is provided by a base correction protein, which is also called a "fusion protein" because it contains at least a DNA-binding protein and a base-converting protein. The "base correction system" or "base editor" used in the in vitro assay according to the present invention may be in the form of one or more fusion proteins, a polynucleotide (DNA or mRNA) encoding the one or more fusion proteins, or a combination of a protein and a polynucleotide (e.g., a case containing a base correction protein (fusion protein) and a guide RNA).

[0011] As used herein, the terms "target," "target," "target site," or "target region" refer to a pre-defined nucleic acid sequence of any composition and / or length. Such a target region includes, but is not limited to, a gene, a promoter, an open reading frame, or any nucleic acid sequence.

[0012] As used herein, the terms "cell-free, in vitro," "extracellular test tube environment," or "outside the cell" refer to a test tube environment that does not use cells (including tissues, organs, and organisms containing cells), and the term "extracellular test method" refers to a test method that does not use cells (including tissues, organs, and organisms containing cells). As used herein, the terms "in vitro coupled transcription / translation," "IVTT," or "in vitro transcription and translation reaction" refer to a system in which a polynucleotide (DNA or mRNA) encoding a base correction protein is transcribed or transcribed and translated in the "extracellular test tube environment" to exhibit base correction activity as a functional base correction protein.

[0013] The present invention provides (a) contacting isolated DNA containing a target DNA mutation in an extracellular environment with one or more base correction proteins used in a base correction system; (b) sequencing the DNA obtained from step (a) to measure the base correction activity of the base correction system; (c) providing information about the ability of the base correction system to correct the same DNA mutation in the cell; A method is provided that provides information about the ability of a base correction system to correct a DNA mutation.

[0014] The information provided by the method may be the ability of the base correction system to correct a DNA mutation. As used herein, the term "ability to correct a DNA mutation" refers to the ability to actually correct a DNA mutation in a cell. The information on the "ability to correct a DNA mutation" may be qualitative information on whether or not a target DNA mutation (or off-target mutation) occurs, or quantitative information on how much of a target DNA mutation (or off-target mutation) occurs. The quantitative information may be, for example, DNA base correction with an efficiency of 0.5% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, or 20% or more.

[0015] The base correction efficiency can be expressed as the ratio of sequencing reads that reflect the results of base correction among all sequencing reads for DNA that has undergone base correction, or it can be expressed as the frequency of base correction.

[0016] The method for providing information on the ability of the base correction system of the present invention to correct DNA mutations can be part of a method for screening base editors that can be used in actual cells.

[0017] Specifically, the present invention may provide a method for screening for a base editor capable of correcting a target mutation in a cell, comprising the following steps: (a) contacting isolated DNA comprising a target DNA mutation with candidate base editors in an extracellular environment, wherein the candidate base editors each independently comprise one or more base correcting proteins or polynucleotides encoding same; (b) sequencing the DNA molecules obtained from step (a) to measure the base correcting activity of the candidate base editors; (c) providing information regarding the ability of the candidate base editor to correct the same DNA mutation in the cell; and (d) selecting a base editor that can correct the same DNA mutation in the cell based on the information obtained from step (c).

[0018] The term "measurement" as used herein encompasses not only quantitative determination of the frequency of base mutations, but also qualitative confirmation of whether or not base mutations have occurred. Whether or not base mutations have occurred can typically be confirmed through DNA sequencing techniques widely known in the field of biotechnology.

[0019] As used herein, when isolated DNA containing a target DNA mutation is contacted in an extracellular environment with one or more base correction proteins used in the base correction system, this includes not only transferring the isolated DNA containing the target DNA mutation and the base correction protein into a reaction tube as reactants, but also mixing the isolated DNA containing the target DNA mutation with a polynucleotide encoding the base correction protein in the form of DNA or mRNA (or a vector containing the same), and reacting the base correction protein obtained as a product of the in vitro transcription and / or translation process with the target DNA.

[0020] In the method for providing information regarding the ability of the base correction system of the present invention to correct DNA mutations, the step of contacting isolated DNA containing a target DNA mutation with a base correction protein may involve mixing the isolated DNA containing the target DNA mutation with one or more polynucleotides encoding one or more base correction proteins, and allowing the polynucleotides (including DNA or mRNA) to be transcribed and translated extracellularly.

[0021] The base correction protein used in the present invention can be mixed in the form of a polynucleotide (including DNA or mRNA) or a vector containing the same with target DNA in a single reaction tube and used in reactions for base correction protein expression and DNA base correction without a separate protein purification process. For example, an expression plasmid can be used, and the plasmid can include, but is not limited to, a T7 promoter or an SP6 promoter. For example, the above method can be carried out by mixing the plasmid DNA and target DNA with reactants for in vitro transcription and translation in a single reaction tube, without a separate purification process for the synthesized protein. Expression of the base correction protein and DNA base correction can be carried out in a one-pot reaction in a single tube.

[0022] The reaction system for the extracellular assay according to the present invention may contain factors and enzymes for in vitro transcription and translation reactions, other enzymes required for the reaction system, various substrates, buffers, and salts. Enzymes and reactants related to DNA replication necessary for the DNA repair process may also be added. The components contained in the reaction system may include, for example, one or more selected from DNA polymerase, RNA polymerase, ribonucleotide triphosphate (NTP) mixtures for the four bases (adenine, cytosine, guanine, and uracil), cap analogs, ribonuclease inhibitors, protease inhibitors, amino acid mixtures, tRNA, aminoacyl-tRNA synthetases, methionyl-tRNA transformylase, ribosomes containing rRNA, initiation factors, elongation factors, termination factors, pyrophosphate hydrolase, MgCl2, antioxidants, buffers, polyamines, etc. All or some of the components (particularly those necessary for protein synthesis) may be provided in the form of cell lysates (e.g., reticulocyte lysates).

[0023] The transcription and translation reactions that can be used in the extracellular assay of the present invention can be carried out under commonly known reaction conditions, for example, at 20-40°C for 4-20 hours, preferably at 30°C for 6 hours followed by 37°C for 16 hours.

[0024] Examples of enzymes that can be added in addition to factors and enzymes for transcription or translation reactions include, but are not limited to, enzymes for regenerating energy in the reaction system, such as creatine kinase, myokinase, and nucleoside diphosphate kinase, and enzymes that hydrolyze inorganic pyrophosphate formed during transcription and translation reactions, such as inorganic pyrophosphatase.

[0025] Examples of various substrates include, but are not limited to, normal amino acids, nucleotide triphosphates as energy sources, creatine phosphate, and formyl folate, etc. Nucleotide triphosphates include ATP, GTP, CTP, and UTP.

[0026] Examples of the buffer solution that can be used include, but are not limited to, potassium phosphate buffer, and examples of the salt that can be used include, but are not limited to, potassium glutamate, ammonium chloride, magnesium acetate, calcium chloride, fthrescine, spermidine, and dithiothreitol.

[0027] In the in vitro assay method according to the present invention, the step of contacting isolated DNA containing a target DNA mutation with a base correction protein includes a process in which the target DNA mutation is corrected by the base correction protein. The base correction reaction is carried out by contacting the base correction protein with the target DNA followed by a DNA replication process.

[0028] When the base correction protein used in the in vitro assay according to the present invention is provided in the form of a polynucleotide (including DNA or mRNA) or a vector containing the same, expression of the base correction protein and DNA base correction can be performed in one tube through a one-pot reaction. For example, after completing the reactions for in vitro transcription and translation, the tube in which the reaction was completed can be directly provided to the PCR process.

[0029] In the method for providing information about the ability of the base correction system according to the present invention to correct a DNA mutation, the step of contacting the isolated DNA containing the target DNA mutation with the base correction protein may involve directly mixing the isolated DNA containing the target DNA mutation with the base correction protein extracellularly. In the extracellular test method according to the present invention, the step of contacting the isolated DNA containing the target DNA mutation with the base correction protein includes a process in which the target DNA mutation is corrected by the base correction protein. The base correction reaction is carried out by a DNA replication process after contacting the base correction protein with the target DNA.

[0030] In this case, the base correction protein used in the present invention is expressed separately and / or mixed with the target DNA in a purified form. Methods for expressing and purifying base correction proteins are well known to those of ordinary skill in the field of biotechnology.

[0031] One aspect of the present invention relates to an IVTT kit (composition) for use in the extracellular base correction ability test method according to the present invention. The kit may contain factors and enzymes for in vitro transcription and translation reactions, other enzymes required for the reaction system, various substrates, buffers, and salts, and may also contain enzymes and reactants related to DNA replication required for the DNA correction process. The components included in the kit may include, for example, one or more selected from DNA polymerase, RNA polymerase, NTP (ribonucleotide triphosphate mixture) for the four bases (adenine, cytosine, guanine, and uracil), cap analogs, ribonuclease inhibitors, protease inhibitors, amino acid mixtures, tRNA, aminoacyl-tRNA synthetase, methionyl-tRNA transformylase, ribosomes including rRNA, initiation factors, elongation factors, termination factors, pyrophosphate hydrolase, MgCl, antioxidants, buffers, polyamines, etc. All or some of the components (especially those necessary for protein synthesis) may be provided in the form of a cell lysate (e.g., a reticulocyte lysate). The extracellular assay method according to the present invention may involve mixing DNA encoding a base correction protein used as a base correction system with the kit composition, followed by reaction under reaction conditions that allow DNA transcription, translation, and DNA base correction.

[0032] In the present invention, the reaction products (products obtained in step (a)) including base-corrected DNA obtained through the correction reaction using the base correction protein can be directly used in the step of measuring base correction activity (step (b)) through sequencing without further purification. DNA sequencing can be performed using conventional techniques widely known in the biotechnology field, such as targeted deep sequencing or Sanger sequencing.

[0033] Surprisingly, it was experimentally confirmed that the results of base correction performed in an extracellular environment without using cells and then measured were virtually identical to the results of base correction performed in cells. Here, "virtually identical" base correction results mean that the tendency of base correction, whether or not base correction occurs, and / or the frequency of base correction, depending on the base sequence, are identical. Specifically, it was confirmed that a base sequence confirmed to be corrected when base correction was performed in an extracellular environment was also corrected identically when base correction was performed in cells. Furthermore, it was confirmed that a base sequence that underwent base correction at a relatively high frequency in an extracellular environment also underwent base correction at a relatively high frequency when base correction was performed in cells. Therefore, if it is confirmed that a desired sequence correction was performed at a target sequence site when base correction was performed in an extracellular environment using a specific target DNA according to the present invention, this means that there is a very high possibility that the desired sequence correction will occur at the same sequence site in cells, and therefore the method can be very useful as a primary test method for various base editors. Conventionally, it has been believed that the base correction activity / ability of a base editor could not be determined without testing using cells, and therefore, no method for determining the base correction ability of a base editor through an extracellular test method such as that of the present invention has been tested and / or described.

[0034] To observe the efficiency of gene correction in cells, cells must first be cultured. This typically takes one to three days, or five to six days if subculture is required, to determine the appropriate number of cells for transfection depending on the cell type and the number of combinations of substances that cause gene correction, i.e., the number of screenings. To observe the efficiency of gene correction in extracellular conditions using an IVTT kit, the target DNA and DNA encoding the gene correction protein are mixed with the IVTT kit composition, incubated for 6 hours at 30°C and 16 hours at 37°C, and then PCR is performed. This incubation can also be performed using a PCR machine. While cell experiments require approximately three to twelve days, extracellular experiments can be performed in one day using the IVTT system of the present invention.

[0035] If cells are used in the library construction process to measure base correction ability, the cells must be lysed, and a nested primary PCR is performed using the cells as a template. Then, a secondary PCR is performed using the primary PCR product as a template, and a tertiary PCR is performed using index-containing primers and the secondary PCR product as a template to add the final index sequence. However, under extracellular conditions using an IVTT system, it is sufficient to incubate the PCR system at 30°C for 6 hours and 37°C for 16 hours, and then proceed with the secondary PCR using the resulting product as a template.

[0036] As such, the IVTT system of the present invention has the advantages of being simpler and time-saving than experiments using cells. Even for the purpose of developing therapeutic agents, mutated cells are generally required, and the methods and conditions for transfecting gene correction agents into cells often vary depending on the cell type. In contrast, the IVTT system of the present invention has the advantage that mutated DNA can be directly produced, omitting the transfection process and eliminating the need for condition optimization.

[0037] The target DNA used in the present invention may contain one or more single-base genetic mutations. Confirming the base correction ability of a base correction system to correct an inherited single-base mutation is particularly difficult because it requires isolating cells from a plant or animal (e.g., a patient with a genetic disease) with the corresponding genetic mutation. Therefore, the effect of being able to confirm substantially the same correction activity as when base correction is performed in cells by performing only the in-cell test method of the present invention is particularly useful in developing technologies for correcting genetic mutations such as those in genetic diseases.

[0038] The isolated target DNA used in the present invention can be nuclear DNA or organelle DNA. The organelle DNA can be mitochondrial DNA or chloroplast DNA, and can be extracted from animal or plant cells, amplified, and / or synthesized. For example, the isolated target DNA can be nuclear DNA and / or mitochondrial DNA from a human with a genetic disease. Methods for extracting and amplifying double-stranded DNA from cells and / or chemically synthesizing it based on its sequence are widely known to those of ordinary skill in the field of biotechnology.

[0039] The base correction system whose base correction ability can be measured through the in vitro assay according to the present invention may have the activity of correcting an adenine (A) base to guanine (G), cytosine (C), or thymine (T), and / or the activity of correcting a guanine (G) base to adenine (A), cytosine (C), or thymine (T), and / or the activity of correcting a cytosine (C) base to adenine (A), guanine (G), or thymine (T), and / or the activity of correcting a thymine (T) base to adenine (A), guanine (G), or cytosine (C), and / or may have one or more of these activities. For example, the base correction system whose base correction ability can be measured through the in vivo assay according to the present invention may have the activity of correcting an adenine (A) base to guanine (G), the activity of correcting a cytosine (C) base to thymine (T), and / or both of these activities.

[0040] The base correction protein contained in the base correction system that can be used in the extracellular assay according to the present invention can each independently contain one or more base-converting enzymes. The base correction protein contained in the base correction system that can be used in the extracellular assay according to the present invention is characterized in that it does not cause double-strand breaks in the target DNA.

[0041] As used herein, the term "base-converting enzyme" refers to an enzyme that catalyzes a reaction that converts a substituent on the purine or pyrimidine ring of a DNA base to another group or atom, and / or that catalyzes a reaction that removes a DNA base by hydrolyzing the N-glycosidic bond of DNA, ultimately converting (correcting) the target base to another type of base (through DNA replication and / or repair mechanisms). The class of base-converting enzymes that catalyzes a reaction that converts a substituent on the purine or pyrimidine ring of a DNA base to another group or atom includes deaminases, and the class of base-converting enzymes that catalyzes a reaction that removes a DNA base by hydrolyzing the N-glycosidic bond of DNA includes DNA glycosylases.

[0042] For example, one or more base correction proteins that can be included in the base correction system used in the extracellular assay according to the present invention can each independently include cytosine deaminase and / or adenine deaminase as a base-converting enzyme.

[0043] The cytosine deaminase that can be used in the base correction system used in the extracellular assay of the present invention refers to any amino-deaminase that has the activity of converting cytosine bases to uridine, and can be derived from any organism (e.g., eukaryote or prokaryote) including, but not limited to, algae, bacteria, fungi, plants, invertebrates, and mammals, and / or can be mutated (e.g., engineered and / or evolved). For example, the cytosine deaminase can be derived from and / or mutated by APOBEC (apolipoprotein B editing complex), AID (activation-induced deaminase), the bacterial adenine deaminase TadA (tRNA-specific adenosine deaminase), or its ortholog, or derived from and / or mutated by the bacterial cytosine deaminase DddA or its ortholog, or a fragment thereof. The cytosine deaminase mutated from TadA mentioned above may be, for example, a polypeptide in which one or more of the amino acid residues at positions 6, 26, 27, 28, 46, 48, 49, 61, 74, 76, 77, 82, 96, 107, 108, 112, 114, 115, 119, 122, 127, 142, 143, 151, 154, and 158 of the amino acid sequence of SEQ ID NO: 1 are mutated to other amino acids. For example, the polypeptide may be a polypeptide in which the amino acid at position 27 of the amino acid sequence of SEQ ID NO: 1 is mutated to lysine, the amino acid at position 28 to alanine, the amino acid at position 61 to isoleucine, and the amino acid at position 96 to asparagine. With regard to the configuration of cytosine deaminase that can be used in the present invention, reference may be made to content that was already known prior to the filing of this application, including International Patent Application Publications WO2022 / 060185, WO2023 / 086953, etc., which are incorporated by reference in their entirety in this application.

[0044] When the base correction system used in the extracellular assay according to the present invention includes cytosine deaminase, the cytosine deaminase may be in the form of a first segment and a second segment, or in the full-length form. When the cytosine deaminase has the form of a first segment and a second segment, the first segment and the second segment are linked to different DNA-binding proteins, respectively.

[0045] As used herein, when two proteins are "linked," they may be directly linked or indirectly linked via a linker or another protein.

[0046] The cytosine deaminase used in the present invention may be DddAtox, which is a part of a bacterial toxin derived from Burkholderia cenocepacia that exhibits enzymatic function and can deaminate cytosine in double-stranded DNA. DddAtox may comprise the amino acid sequence of SEQ ID NO: 2.

[0047] SEQ ID NO: 2: wild-type DddAtox GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC

[0048] Since DddAtox is toxic to cells, it can be used in the form of two inactivated split bodies, i.e., the first split body and the second split body, to avoid toxicity in host cells. When the cytosine deaminase used in the present invention is used in the form of the first split body and the second split body, each of the first split body and the second split body has no deamination activity.

[0049] The first segment of the DddAtox cytosine deaminase can comprise the sequence from the N-terminus to G33, G44, A54, N68, G82, N98, or G108 in the amino acid sequence of SEQ ID NO: 2. The second segment can comprise the sequence from G34, P45, G55, N69, T83, A99, or A109 in the amino acid sequence of SEQ ID NO: 2 to the C-terminus.

[0050] Preferably, the first segment of DddAtox cytosine deaminase comprises the sequence from the N-terminus to G44 of the amino acid sequence of SEQ ID NO: 2 (SEQ ID NO: 3 below), and the second segment comprises the sequence from P45 to the C-terminus (SEQ ID NO: 4 below).

[0051] SEQ ID NO: 3: wild-type DddAtox G1333-N GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGG

[0052] SEQ ID NO: 4: wild-type DddAtox G1333-C PTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC

[0053] Preferably, the first segment of DddAtox cytosine deaminase comprises the sequence from the N-terminus to G108 of the amino acid sequence of SEQ ID NO: 2 (SEQ ID NO: 5 below), and the second segment may comprise the sequence from A109 to the C-terminus (SEQ ID NO: 6 below).

[0054] SEQ ID NO: 5: wild-type DddAtox G1397-N GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG

[0055] SEQ ID NO: 6: wild-type DddAtox G1397-C AIPVKRGATGETKVFTGNSNSPKSPTKGGC

[0056] When the first and second segments of DddAtox are used as cytosine deaminase, one or more amino acids located on the surface where the first and second segments of cytosine deaminase bind to each other may be substituted with other amino acids. For example, the first and second segments of DddAtox may have the amino acid sequences of SEQ ID NO: 3 (G1333-N) and SEQ ID NO: 4 (G1333-C), respectively. In this case, one or more amino acids selected from the group consisting of positions 3, 5, 10, 11, 13, 14, 15, 16, 17, 18, 19, 28, 30, and 31 of SEQ ID NO: 3, or one or more amino acids selected from the group consisting of positions 13, 16, 17, 20, 21, 28, 29, 30, 31, 32, 33, 56, 57, 58, and 60 of SEQ ID NO: 4 may be substituted with other amino acids, but are not limited thereto. As another example, the first fragment of DddAtox may comprise the amino acid sequences of SEQ ID NO: 5 (G1397-N) and SEQ ID NO: 6 (G1397-C), in which case one or more amino acids selected from the group consisting of positions 87, 88, 91, 92, 95, 100, 101, 102, and 103 of SEQ ID NO: 5, or one or more amino acids selected from the group consisting of positions 13, 14, 15, and 16 of SEQ ID NO: 6 may be substituted with other amino acids, but is not limited thereto. The "other amino acids" refer to alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, aspartic acid, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartate, glutamic acid, arginine, histidine, lysine, and all known variants of these amino acids, excluding the amino acids present in the wild-type protein at the original mutated positions. By using such mutants, the pair of two DddAtox segments, each linked to a DNA-binding protein, does not function properly when not bound to DNA, preventing undesired non-targeted C-to-T correction, and thus enabling highly efficient and precise C-to-T correction.Examples of such mutants include the first fragment of DddAtox having the amino acid sequence of SEQ ID NO: 7 (which may be referred to as "G1397-N" or "G1397N"), and the second fragment of DddAtox having the amino acid sequence of SEQ ID NO: 8 (which may be referred to as "G1397-C" or "G1397C").

[0057] As used herein, the terms "G1333-N," "G1333N," or "1333N" can refer to the first fragment of wild-type DddAtox having the amino acid sequence of SEQ ID NO: 3 or an amino acid variant thereof, and the terms "G1333-C," "G1333C," or "1333C" can refer to the second fragment of wild-type DddAtox having the amino acid sequence of SEQ ID NO: 4 or an amino acid variant thereof.

[0058] As used herein, the terms "G1397-N," "G1397N," or "1397N" can refer to the first fragment of wild-type DddAtox having the amino acid sequence of SEQ ID NO: 5 or 7 or an amino acid variant thereof, and the terms "G1397-C," "G1397C," or "1397C" can refer to the second fragment of wild-type DddAtox having the amino acid sequence of SEQ ID NO: 6 or 8 or an amino acid variant thereof.

[0059] The cytosine deaminase used in the base correction system for the in vitro assay of the present invention can be used in its full-length form. In this case, the full-length cytosine deaminase (e.g., DddAtox) has its amino acid sequence modified to have no or only low toxicity. Positively charged amino acids are specifically concentrated at the C-terminus of DddAtox. Because DNA has a negative charge, it binds to positively charged amino acids in proteins. Substitution of these positively charged amino acids weakens the DNA-binding ability of DddAtox, thereby reducing and / or eliminating its intracellular toxicity. In other words, if the positively charged amino acids are substituted to eliminate toxicity, cloning in E. coli is possible, allowing the full-length DddAtox to be obtained. Such a non-toxic full-length cytosine deaminase can be obtained by substituting one or more, two or more, three or more, four or more, or five or more amino acids in the wild-type amino acid sequence of SEQ ID NO: 2 with other amino acids. The "other amino acid" refers to an amino acid selected from alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, aspartic acid, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartate, glutamic acid, arginine, histidine, lysine, and all known variants of the amino acids, excluding the amino acid that the wild-type protein has at the original mutation position. For example, the other amino acid may be alanine.

[0060] For example, a full-length cytosine deaminase mutant that can be included in the base correction system used in the in vitro assay of the present invention may have one or more amino acid substitutions selected from the group consisting of S to G substitution at position 37, G to S substitution at position 59, A to V substitution at position 109, and S to G substitution at position 129 in the amino acid sequence of SEQ ID NO: 2.

[0061] For example, a full-length cytosine deaminase mutant that can be included in the base correction system used in the extracellular assay method of the present invention may have all of the following substitutions in the amino acid sequence of SEQ ID NO: 2: S to G at position 37, G to S at position 59, A to V at position 109, and S to G at position 129.

[0062] The adenine deaminase that can be used in the base correction system used in the extracellular assay of the present invention refers to an amino-deaminase that converts adenine bases to hypoxanthine (or inosine as a nucleoside) and can be derived from any organism (e.g., eukaryote or prokaryote), including, but not limited to, algae, bacteria, fungi, plants, invertebrates, and mammals, such as E. coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, or C. crescentus, and / or can be mutated (e.g., engineered and / or evolved). Such adenine deaminase can be, for example, APOBEC, AID, or TadA, or a mutant thereof. The aforementioned TadA can be, for example, TadA8e (SEQ ID NO: 1) or a truncated or mutant thereof (e.g., a mutant that has been improved and evolved to be applicable to deoxynucleotides). The TadA8e mutants mentioned above may be those in which one or more of the amino acid residues at positions 23, 28, 30, 36, 46, 48, 49, 51, 76, 82, 84, 106, 108, 110, 111, 146, 147, 152, 154, 155, 156, and 157 of SEQ ID NO: 1 have been mutated to other amino acids. Regarding the construction of adenine deaminase that can be used in the present invention, reference may be made to International Patent Application Publications WO2022 / 060185, WO2023 / 086952, etc., which are incorporated herein by reference in their entirety. The adenine deaminase that can be used in the base correction system used in the extracellular assay method according to the present invention may have the amino acid sequence of SEQ ID NO: 1 or a conservative amino acid substitution thereof.

[0063] The one or more base correction proteins included in the base correction system used in the extracellular assay of the present invention can each independently comprise cytosine deaminase (or a fragment thereof), adenine deaminase, or both.

[0064] The one or more base correction proteins included in the base correction system used in the in vitro assay according to the present invention may each independently comprise one or more DNA-binding proteins selected from the group consisting of zinc finger proteins, transcriptional activator-like effector (TALE) proteins, CRISPR-associated nucleases, and CRISPR-associated nickases. The base correction proteins included in the base correction system used in the in vivo assay according to the present invention are characterized in that they do not cause double-strand breaks in target DNA.

[0065] When the base correction system used in the present invention is in the form of a composition of one or more different base editors, one base editor may comprise a zinc finger protein, a TALE protein, a CRISPR-linked nuclease, or a CRISPR-linked nickase. Regarding the composition of zinc finger proteins, TALE proteins, CRISPR-linked nucleases, and CRISPR-linked nickases, reference may be made to International Patent Application Publications WO2022 / 060185, WO2023 / 086952, etc., which are incorporated herein by reference in their entirety.

[0066] Zinc fingers are a typical DNA-binding protein structure and form the main DNA-binding protein motif. The interaction between the α-helix of the zinc finger and the major groove of DNA enables strong and specific recognition of DNA sequences. One or more zinc finger motifs can be combined and used.

[0067] The DNA-binding protein used in the base correction system used in the extracellular assay of the present invention may be a TALE protein. The TALE protein of the present invention refers to a protein that binds to nucleotides in a sequence-specific manner through one or more TALE-repeat modules. The TALE protein includes at least one TALE-repeat module, preferably 1 to 30 TALE-repeat modules, but is not limited to this. A TALE-repeat module may also be called a "TALE array," and the term "TALE protein" refers to a configuration including an N-terminal domain and a C-terminal domain (which may include half domains) on both sides of the TALE array. As used herein, the term "TALE" refers to a "TALE protein" unless otherwise specified.

[0068] When a TALE protein is used as a DNA binding protein in the base correction system used in the extracellular test method of the present invention, a single-module TALE array or a multi-module TALE array (e.g., a dual-module TALE array consisting of a first TALE (or left TALE) array and a second TALE (or right TALE) array) can also be used.

[0069] When the base correction system used in the extracellular assay according to the present invention comprises two fusion proteins each containing a TALE protein, the two fusion proteins each contain a first TALE protein and a second TALE protein. The first TALE protein and the second TALE protein can each be independently linked, directly or indirectly (e.g., via a linker and / or other protein components) to one or more base-converting enzymes, such as at least one of cytosine deaminase and adenine deaminase. For example, the first fusion protein (a fusion protein that binds to DNA 5' upstream of the base correction target site) containing the first TALE protein (left TALE) can contain a first cleavage fragment of cytosine deaminase, and the second fusion protein (a fusion protein that binds 3' downstream of the base correction target site) containing the second TALE protein (right TALE) can contain a second cleavage fragment of cytosine deaminase. Either or both of the first and second fusion proteins can contain adenine deaminase. In another approach, a first fusion protein containing a first TALE protein (left TALE) may contain a second fragment of cytosine deaminase, and a second fusion protein containing a second TALE protein (right TALE) may contain a first fragment of cytosine deaminase, and either one or both of the first and second fusion proteins may contain adenine deaminase.In another approach, either one of the first fusion protein containing a first TALE protein and a second fusion protein containing a second TALE protein may contain the full-length form of cytosine deaminase, and either one or both of the first and second fusion proteins may contain adenine deaminase.

[0070] The DNA-binding protein used in the base correction system used in the in vitro assay of the present invention may be a CRISPR-associated protein called a "Cas protein." The "CRISPR system," a well-known DNA correction tool, refers to a ribonucleoprotein complex in which the Cas protein and associated RNA components work together to recognize specific bases in DNA and correct the corresponding site, and is also called the "CRISPR / Cas system."

[0071] "Cas protein" refers to an essential protein component of the CRISPR / Cas system. CRISPR-associated (cas) genes encoding Cas proteins are often associated with CRISPR repeat-spacer arrays. More than 40 distinct Cas protein families have been described, and information on known Cas genes and proteins is available, including but not limited to, in GenBank at the National Center for Biotechnology Information (NCBI). Cas proteins can be isolated from microorganisms, such as bacteria, and / or produced non-naturally, such as by recombinant synthesis or artificial synthesis.

[0072] The DNA-binding protein used in the base correction system employed in the in vitro assay of the present invention may be a CRISPR-linked nuclease. Here, the term "CRISPR-linked nuclease" refers to any Cas protein or variant thereof that has (endo)nuclease activity when complexed with a guide RNA. When a CRISPR-linked nuclease is used as part of a base correction protein in the in vivo assay of the present invention, a guide RNA can be added when isolated DNA containing a target DNA mutation is contacted with the corresponding base correction protein or a polynucleotide (DNA or mRNA) encoding it.

[0073] As used herein, the term "guide RNA" refers to a target DNA-specific RNA that can form a complex with a Cas protein and bring the Cas protein to the target DNA. The guide RNA can consist of two RNAs, i.e., a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA), or it can be a single-stranded RNA (sgRNA) generated by fusing the essential portions of a crRNA and a tracrRNA. The guide RNA can also be a double-stranded RNA (dual RNA) containing a crRNA and a tracrRNA. Any guide RNA can be used in the in vitro assay of the present invention, as long as it contains the essential portions of a crRNA and a tracrRNA and a portion complementary to the target.

[0074] The DNA-binding protein used in the base correction system employed in the in vitro assay of the present invention may be a CRISPR-linked nickase. In this context, the term "CRISPR-linked nickase" refers to any Cas protein or mutant thereof that has nickase activity when complexed with a guide RNA. The CRISPR-linked nickase used in the in vivo assay of the present invention may be a mutated Cas protein that has lost some or all of its endonuclease activity, which cleaves double-stranded DNA, to have nickase activity. "Nickase" activity refers to the activity of cleaving only one strand of a DNA molecule. Such mutants may be isolated from microorganisms such as bacteria and / or produced non-naturally, such as by recombinant synthesis and / or artificial synthesis. When a CRISPR-linked nickase is used as part of a base correction protein in the in vitro assay of the present invention, when isolated DNA containing a target DNA mutation is contacted with the base correction protein or a polynucleotide (DNA or mRNA) encoding it, a reverse transcriptase or a polynucleotide (DNA or mRNA) encoding the reverse transcriptase and a guide RNA (also called a prime editing guide RNA) can be added together.

[0075] When cytosine deaminase is included in a fusion protein comprising a DNA-binding protein, the cytosine deaminase can be linked directly or indirectly (e.g., via a linker and / or other protein components) to the N-terminus or C-terminus of the DNA-binding protein. When adenine deaminase is included in a fusion protein comprising a DNA-binding protein, the adenine deaminase can be linked directly or indirectly (e.g., via a linker and / or other protein components) to the N-terminus or C-terminus of the TALE protein. When cytosine deaminase and adenine deaminase are both included in a fusion protein comprising a DNA-binding protein, the adenine deaminase can be linked directly or indirectly (e.g., via a linker and / or other protein components) to the N-terminus or C-terminus (preferably the C-terminus) of the cytosine deaminase.

[0076] When the base correction system used in the extracellular assay of the present invention includes two fusion proteins, the fusion proteins may have different protein components (e.g., a DNA-binding protein and one or more base-converting enzymes) in composition and sequence. For example, the first fusion protein (the fusion protein that binds to DNA 5' upstream of the base correction target site) may contain adenine deaminase, while the second fusion protein (the fusion protein that binds to DNA 3' downstream of the base correction target site) may not contain adenine deaminase, or vice versa. For example, the first fusion protein (the fusion protein that binds to DNA 5' upstream of the base correction target site) may contain a TALE protein, while the second fusion protein (the fusion protein that binds to DNA 3' downstream of the base correction target site) may contain a zinc finger protein, or vice versa.

[0077] The protein components of the fusion protein can be arranged independently of each other in the first fusion protein (the fusion protein that binds to DNA 5' upstream of the base correction target site) and the second fusion protein (the fusion protein that binds to DNA 3' downstream of the base correction target site). For example, in the first fusion protein, adenine deaminase can be located after the C-terminus of cytosine deaminase, while in the second fusion protein, adenine deaminase can be located before the N-terminus of cytosine deaminase, or vice versa. For example, in the first fusion protein, the DNA-binding protein can be located after the C-terminus of cytosine deaminase, while in the second fusion protein, the DNA-binding protein can be located before the N-terminus of cytosine deaminase, or vice versa.

[0078] One or more fusion proteins included in the base correction system used in the in vitro assay according to the present invention may each independently further contain a UGI (uracil glycosylase inhibitor). UGI can increase the efficiency of base correction by inhibiting the activity of UDG (uracil DNA glycosylase), an enzyme that catalyzes the removal of uracil (U) bases from DNA and repairs mutated DNA. When UGI is used in the base correction protein included in the in vivo assay according to the present invention, its position may vary, for example, it may be linked directly or indirectly (e.g., via a linker and / or other protein components) to the C-terminus of cytosine deaminase, but is not limited thereto.

[0079] One or more fusion proteins included in the base correction system used in the extracellular assay of the present invention may each independently further include an NES (nuclear export signal). The NES sequence may be any signal sequence that confers the ability to translocate out of the nucleus, and a natural NES or an artificially synthesized NES may be used. For example, it may be derived from, but is not limited to, MVM (mirute virus of mice). When an NES is used in the base correction system used in the extracellular assay of the present invention, its position may vary. For example, it may be linked directly or indirectly (e.g., via a linker and / or other protein components) to the N-terminus of cytosine deaminase, but is not limited thereto.

[0080] One or more fusion proteins included in the base correction system used in the extracellular assay of the present invention may each independently further include an MTS (mitochondrial targeting sequence). The MTS may be any signal sequence capable of translocating into mitochondria, and may be a natural MTS present at the N-terminus of various mitochondrial proteins, or an artificially synthesized MTS may be used. When an MTS is used in the base correction system used in the extracellular assay of the present invention, its position may vary, for example, it may be linked directly or indirectly (e.g., via a linker and / or other protein components) to the N-terminus of a DNA-binding protein or the N-terminus of an NES, but is not limited thereto.

[0081] The term "fusion protein" as used herein refers to a polypeptide formed by linking two or more different polypeptides via peptide bonds. The fusion protein contained in the base correction system used in the extracellular assay of the present invention includes a DNA-binding protein and a base-converting enzyme, and may additionally include a UGI and / or an NES and / or an MTS. These protein components may be linked via a linker or directly. A tag sequence may also be added, if necessary. Methods for designing and constructing a fusion protein (or a polynucleotide encoding the fusion protein) may be any method known in the field of biotechnology, and the polynucleotide may be inserted into a vector.

[0082] Based on the above, the present invention can relate to the following (1) to (44), but is not limited thereto.

[0083] (1) A method for providing information about the ability of a base correction system to correct a DNA mutation, comprising the steps of: (a) contacting isolated DNA containing a target DNA mutation in an extracellular environment with one or more base correction proteins used in a base correction system; (b) sequencing the DNA obtained from step (a) to measure the base correction activity of the base correction system; (c) providing information about the ability of the base correction system to correct the same DNA mutation in a cell;

[0084] (2) In (1), step (a) comprises mixing isolated DNA containing the target DNA mutation with one or more polynucleotides encoding the one or more base-correcting proteins, and accompanying extracellular transcription and translation of the polynucleotides.

[0085] (3) The method according to (2), wherein the polynucleotide encoding the base-correcting protein is DNA or mRNA.

[0086] (4) In any one of (1) to (3), the method of expressing the base correction protein in step (a) and correcting the DNA base is carried out through a one-pot reaction.

[0087] (5) The method of (1), wherein step (a) involves mixing isolated DNA containing the target DNA mutation with a base-correcting protein outside the cell.

[0088] (6) In any one of (1) to (5), the reaction product of step (a) is used in step (b) without further purification.

[0089] (7) A method according to any one of (1) to (6), wherein the target DNA mutation comprises one or more single base mutations.

[0090] (8) A method according to any one of (1) to (7), wherein the isolated DNA containing the target DNA mutation is extracted and amplified from animal or plant cells and / or artificially synthesized.

[0091] (9) In any one of (1) to (8), the isolated DNA containing the target DNA mutation is nuclear DNA or organelle DNA.

[0092] (10) The method according to (9), wherein the organelle DNA is mitochondrial DNA or chloroplast DNA.

[0093] (11) The method according to any one of (1) to (10), wherein the isolated DNA containing the target DNA mutation is nuclear DNA and / or mitochondrial DNA of a human having a genetic disease.

[0094] (12) A method according to any one of (1) to (11), wherein the base correction is correcting an adenine (A) base to a guanine (G), and / or correcting a cytosine (C) base to a thymine (T), and / or both.

[0095] (13) In any one of (1) to (12), the one or more base correction proteins each independently comprise one or more DNA binding proteins selected from the group consisting of zinc finger proteins, TALE (transcriptional activator-like effector) proteins, CRISPR-associated nucleases, and CRISPR-associated nickases.

[0096] (14) The method of (13), wherein the one or more DNA-binding proteins include a CRISPR-linked nuclease, and in step (a), the guide RNA is additionally contacted with isolated DNA containing the target DNA mutation.

[0097] (15) The method of (13), wherein the one or more DNA-binding proteins include a CRISPR-associated nickase, and in step (a), a reverse transcriptase or a polynucleotide encoding a reverse transcriptase and a guide RNA are additionally contacted with isolated DNA containing a target DNA mutation.

[0098] (16) The method according to any one of (1) to (15), wherein the one or more base proteins each independently comprise a base-converting enzyme.

[0099] (17) The method according to (16), wherein the one or more base-converting enzymes each independently comprise one or more of APOBEC (apolipoprotein B editing complex), AID (activation-induced deaminase), TadA (tRNA-specific adenosine deaminase), or DddAtox, or a mutant thereof.

[0100] (18) The method according to (17), wherein DddAtox or a mutant thereof is used in the form of two split bodies.

[0101] (19) The method according to (17), wherein DddAtox or a mutant thereof is used in the form of a full-length protein.

[0102] (20) In any one of (1) to (19), the method wherein one or more base correcting proteins each independently contain a UGI (uracil glycosylase inhibitor).

[0103] (21) A method for screening for a base editor capable of correcting a target DNA mutation in a cell, comprising the steps of: (a) contacting isolated DNA comprising a target DNA mutation with candidate base editors in an extracellular environment, wherein the candidate base editors each independently comprise one or more base correcting proteins or one or more polynucleotides encoding the same; (b) sequencing the DNA molecules obtained from step (a) to measure the base correcting activity of the candidate base editors; (c) providing information regarding the ability of the candidate base editor to correct the same DNA mutation in the cell; and (d) selecting a base editor that can correct the same DNA mutation in the cell based on the information obtained from step (c).

[0104] (22) The method of (21), comprising mixing the isolated DNA containing the target DNA mutation of step (a) with one or more polynucleotides encoding the one or more base-correcting proteins, and allowing the polynucleotides to be transcribed and translated extracellularly.

[0105] (23) The method according to (22), wherein the polynucleotide encoding the base correction protein is DNA or mRNA.

[0106] (24) In any one of (21) to (23), the method of expressing the base correction protein in step (a) and correcting the DNA base is carried out through a one-pot reaction.

[0107] (25) The method of (21), wherein step (a) involves mixing isolated DNA containing the target DNA mutation with a base-correcting protein outside the cell.

[0108] (26) In any one of (21) to (25), the reaction product of step (a) is used in step (b) without further purification.

[0109] (27) The method according to any one of (21) to (26), wherein the target DNA mutation includes one or more single base mutations.

[0110] (28) The method according to any one of (21) to (27), wherein the isolated DNA containing the target DNA mutation is extracted from an animal or plant cell, amplified, and / or artificially synthesized.

[0111] (29) The method according to any one of (21) to (28), wherein the isolated DNA containing the target DNA mutation is nuclear DNA or organelle DNA.

[0112] (30) The method according to (29), wherein the organelle DNA is mitochondrial DNA or chloroplast DNA.

[0113] (31) The method according to any one of (21) to (30), wherein the isolated DNA containing the target DNA mutation is nuclear DNA and / or mitochondrial DNA of a human having a genetic disease.

[0114] (32) In any one of (21) to (31), the base correction is correcting an adenine (A) base to a guanine (G), and / or correcting a cytosine (C) base to a thymine (T) base, and / or performing both.

[0115] (33) In any one of (21) to (32), The method, wherein the one or more base correction proteins each independently comprise one or more DNA binding proteins selected from the group consisting of zinc finger proteins, TALE (transcriptional activator-like effector) proteins, CRISPR-associated nucleases, and CRISPR-associated nickases.

[0116] (34) The method of (33), wherein the one or more DNA-binding proteins include a CRISPR-linked nuclease, and in step (a), the guide RNA is additionally contacted with isolated DNA containing the target DNA mutation.

[0117] (35) The method of (33), wherein the one or more DNA-binding proteins include a CRISPR-associated nickase, and in step (a), a reverse transcriptase or a polynucleotide encoding a reverse transcriptase and a guide RNA are additionally contacted with isolated DNA containing a target DNA mutation.

[0118] (36) The method according to any one of (21) to (35), wherein the one or more base proteins each independently comprise a base-converting enzyme.

[0119] (37) The method according to (36), wherein the one or more base-converting enzymes each independently comprise one or more of APOBEC (apolipoprotein B editing complex), AID (activation-induced deaminase), TadA (tRNA-specific adenosine deaminase), or DddAtox, or a mutant thereof.

[0120] (38) The method according to (37), wherein DddAtox or a mutant thereof is used in the form of two split bodies.

[0121] (39) The method according to (37), wherein DddAtox or a mutant thereof is used in the form of a full-length protein.

[0122] (40) In any one of (21) to (39), the method wherein the one or more base correcting proteins each independently contain a UGI (uracil glycosylase inhibitor).

[0123] (41) In any one of (1) to (40), A method in which a composition comprising a DNA polymerase, an RNA polymerase, a ribonucleotide triphosphate mixture, an amino acid mixture, a ribosome containing tRNA and rRNA, an initiation factor, an elongation factor, a termination factor, and a buffer is used in step (a).

[0124] (42) A base editor selected by any one of (21) to (41).

[0125] (43) A base editor selected by any one of (21) to (41), comprising one or more base correction proteins used in step (a) or polynucleotides encoding the same.

[0126] (44) A kit for the method according to any one of (1) to (41), comprising a DNA polymerase, an RNA polymerase, a ribonucleotide triphosphate mixture, an amino acid mixture, a ribosome containing tRNA and rRNA, an initiation factor, an elongation factor, a termination factor, and a buffer solution.

[0127] The present invention will be described in more detail with reference to the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0128] Example 1: Template DNA amplification

[0129] A double-stranded DNA sequence mimicking the human mitochondrial ND1 gene was synthesized as a gBlock DNA fragment by IDT (Integrated DNA Technologies) as a target DNA for DNA correction. The resulting template DNA sequence is as follows:

[0130] TACTATTGCCAGCATTGCTGC CCTCAACCTAGGCCTCCTATTTATTCTAGCCACCTCTAGCCTAGCCGTTTACTCAATCCTCTGATCAGGGTGAGCATCAAACTCAAACTACGCCCTGATCGGCGCACTGCGAGCAGTAGCCCAAACAATCTCA AGATGGTGCACGATGCACAG (The underlined sequences are optional PCR-amplified sequences to prevent contamination.)

[0131] Template DNA was amplified using the forward primer (TACTATTGCCAGCATTGCTGC) and reverse primer (CTGTGCATCGTGCACCATCT) and purified using a PCR purification kit (GeneAll). The DNA was freshly diluted to 10 ng / ul in distilled water just before the experiment.

[0132] Example 2: In vitro transcription and translation (IVTT) and base correction

[0133] The template DNA obtained in Example 1 was quantified at 10 ng / μL, and the template DNA (1 ng, 5 ng, or 10 ng), 0.5 μg of a plasmid containing DNA encoding Left DdCBE (or sTALED), 0.5 μg of a plasmid containing DNA encoding Right DdCBE (or sTALED), and 20 μL of an in vitro coupled transcription / translation (IVTT) kit mixture (distilled water was added up to 25 μL) were mixed in a tube and incubated at 30°C for 6 hours and then at 37°C for 16 hours. The IVTT kit used was TNT® Quick Coupled Transcription / Translation Systems (Promega), and TNT® Quick Master Mix was used, which contains TNT® Rabbit Reticulocyte Lysate, TNT® Reaction Buffer, TNT® RNA polymerase, Amino Acid Mixture, and RNasin® Ribonuclease Inhibitor.

[0134] The amino acid sequences of the left DdCBE and sTALED, and the right DdCBE and sTALED are as follows: The proteins used are located between the T7 promoter (TAATACGACTCACTATAG) and the termination sequence.

[0135] Left DdCBE MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDYPYDVPDYAGYPYDVPDYAGYPYDVPDYAGIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALLVGSGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML(SEQ ID NO: 9)

[0136] Right DdCBE MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDDDDKGIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALLVGSAIPVKRGATGETKVFTGNSNSPKSPTKGGCSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML(SEQ ID NO: 10)

[0137] Left sTALED (query number 11)

[0138] Right sTALED MASVLTPLLLRGLTGSARRLPVPRAKIHSLDYKDHDGDYKDHDIDYKDDDDKGIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSGSGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG(SEQ ID NO: 12)

[0139] Example 3: Sequencing to confirm base correction and measurement of base correction efficiency

[0140] The reaction product obtained in Example 2 was used as a template without purification, and the sequence was analyzed using targeted deep sequencing (Sanger sequencing can be used for sequencing). The template was amplified using a DNA polymerase with uracil or hypoxanthine (inosine as the nucleoside) error tolerance. The base correction results are shown in Figures 1 and 2. The base correction results in Figure 1 were obtained using the left DdCBE and right DdCBE in the base editor, while the base correction results in Figure 2 were obtained using the left sTALED and right sTALED in the base editor.

[0141] Example 4: Intracellular base correction

[0142] HEK293T cells were cultured in TC-Treated Multiple Well Plates (Corning) supplemented with 10% fetal bovine serum. HEK293T cells were cultured in DMEM (Welgene) culture medium containing 1% antibiotic-antimycotic solution at 37°C in 5% CO2. 0.75 x 10 cells were grown in 48-well plates. 5The plates were dispensed at a cell / well ratio and mixed with 0.5 μg of a plasmid containing DNA encoding left DdCBE (or sTALED), followed by 25 μl of Opti-MEM (Mixture 1). Then, 1.5 μl of lipofectamine 2000 and 25 μl of Opti-MEM were mixed (Mixture 2). Mixture 1 and Mixture 2 were mixed at a 1:1 volume ratio and incubated at room temperature for 15 minutes. 50 μl of the culture was added per well and incubated for 4 days, after which the cell culture medium was removed. Cell lysis was carried out by adding 100 μl of cell lysis buffer (50 mM Tris, 1 mM EDTA, 0.05% SDS, and Proteinase K at a 1:20 volume ratio) per well. The plates were then incubated at 50°C for 1 hour and 80°C for 20 minutes, followed by storage at 4°C. The DNA was amplified using deep-sequencing primers that specifically differentiate mitochondrial DNA and then sequenced. The obtained base correction results were compared with the base correction results obtained through the extracellular test method in Example 3 (FIGS. 3 and 4).

[0143] As can be seen in Figure 3, all base sequences corrected to C-to-T by DdCBE in the in-cell assay were also corrected in the cell assay. In addition, it was confirmed that all base sequences corrected to A-to-G by TALED were also corrected identically in the cell assay. C The underlined base sequence in the TGATC sequence shows a relatively high frequency of C-to-T base correction, and ACTCAA T CC T C T A-to-G base correction (when the underlined base is T, the A base paired with it is corrected) occurs relatively frequently in the underlined base sequence of the GATC sequence, and this tendency for base correction was also confirmed through cell experiments.

[0144] Example 5: Template DNA amplification for m.A3460G correction

[0145] To screen for a base editor that corrects the G3460A mutation in the ND1 gene, which is the cause of LHON, a typical mitochondrial genetic disease, a double-stranded DNA sequence containing the G3460A mutation was synthesized as a gBlock DNA fragment by IDT (Integrated DNA Technologies). The sequence of the resulting template DNA is as follows:

[0146] CTTCAAGGACGACGGCAAC CTAGGCTATATACACTACGCAAAGGCCCCAACGTTGTAGGCCCCTACGGGCTACTACAACCCTTCGCTGACACCATAAAACTCTTCACCAAAGAGCCCCTAAAACCCGCCACATCTACCATCACCCTCTACATCACCGCCCCGACCTTAGC GGCATCAAGGTGAACTTCA (The underlined sequences are PCR-amplified sequences to prevent contamination. The double-byte A indicates the G3460A mutation.)

[0147] The template DNA was amplified by PCR using the forward primer (CTTCAAGGACGACGGCAACCTAGGCTATATACAACTACGC) and the reverse primer (TGAAGTTCACCTTGATGCCGCTAAGGTCGGGGCGGTGATG), and then amplified using the PCR SV mini kit (GeneAll). The antibody was freshly diluted in distilled water to 10 ng / μl immediately before the experiment and used.

[0148] Example 6: Construction of a base editor for m.A3460G correction

[0149] A TALED containing the T7 promoter (TAATACGACTCACTATAG) was constructed. Specifically, the TAL effector array plasmid and expression plasmid were constructed using the Golden Gate cloning system. Competent DH5α (enzynomics) E. coli cells were transformed by heat shock (42°C), and single colonies were cultured in LB medium at 37°C overnight with shaking. Plasmid DNA was purified using the Plasmid SV mini kit (GeneAll) according to the manufacturer's protocol. The nucleotide sequence of the purified plasmid DNA, i.e., the TALED, was confirmed by Sanger sequencing (Macrogen).

[0150] The amino acid sequence of the base correction protein is as follows:

[0151] 17C: (query number 13)

[0152] [Chemical formula] (Accession number 14)

[0153] 56N: MALSRAVCGTSRQLAPVLGYLGSRQKHSLPDYPYDVPDYAGYPYDVPDYAGYPYDVPDYAGIRIQDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPAQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNNGGKQALETVQRLLPVLCQDHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGGSGSGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG (Accession number 15)

[0154] 57N: (query number 16)

[0155] Example 7: In vitro transcription and translation (IVTT) and base correction for m.A3460G correction

[0156] The template DNA obtained in Example 5 was quantified at 10 ng / μL, and a reaction mixture containing 10 ng of template DNA, 0.5 μg of a plasmid containing DNA encoding a base-corrected protein having the amino acid sequence of SEQ ID NO: 13 or 14, and 0.5 μg of a plasmid containing DNA encoding a base-corrected protein having the amino acid sequence of SEQ ID NO: 15 or 16, and 20 μL of an in vitro coupled transcription / translation (IVTT) kit mixture (distilled water was added up to 25 μL) was mixed in a tube and incubated at 30°C for 6 hours and then at 37°C for 16 hours. The IVTT kit used was the TNT® Quick Coupled Transcription / Translation Systems (Promega), and TNT® Quick Master Mix, which contains TNT® Rabbit Reticulocyte Lysate, TNT® Reaction Buffer, TNT® RNA polymerase, Amino Acid Mixture, and RNasin® Ribonuclease Inhibitor, was used.

[0157] The results of the base correction are shown in FIG.

[0158] Example 8: Intracellular base correction for m.A3460G correction

[0159] Primary cells were isolated from the urine of a LHON patient with a G3460A point mutation in the mitochondrial ND1 gene and cryopreserved. These were aliquoted at passage 2 and cryopreserved. Urine-derived cells (UDCs) were cultured in 12-well Clear TC-Treated Multiple Well Plates (Corning) coated with 0.1% gelatin (Welgene) in Renal Epithelial Cell Growth Medium Bullet Kit (REGM, Lonza) culture medium at 37°C with 5% CO2. UDCs were plated at 0.6 x 10 per well in 96-well Clear TC-Treated Multiple Well Plates (Corning). 4 The cells were seeded to the desired cell number. After 20-24 hours, 250 ng of a plasmid encoding the base-correcting protein 17C or 18C and 250 ng of a plasmid encoding the base-correcting protein 56N or 57N were transfected using Lipofectamine LTX reagent (Invitrogen) and then placed in the pre-seeded 96-well plate. The transfected cells were maintained and cultured at 37°C in 5% CO2 with medium changes.

[0160] The transfected UDC cells were placed in 100 μl of cell lysis buffer (50 mM Tris-HCl pH 7.4 (Welgene), 1 mM EDTA pH 8.0 (Welgene), 0.005% sodium dodecyl sulfate (Welgene), and Proteinase K (Qiagen) in a 1:20 volume ratio) per well, then incubated in a PCR machine at 50°C for 1 hour and 80°C for 20 minutes and stored at 4°C.

[0161] Cell lysates were used as templates without purification, and sequences were analyzed using targeted deep sequencing to analyze the base correction ratio at the target site. To prepare a deep sequencing library, nested primary PCR was performed using deep-sequencing primers capable of resolving mitochondrial DNA alone, and secondary PCR was performed using the primary PCR as a template using PrimeSTAR® GXL DNA Polymerase (TAKARA). A final index sequence was then added using index-containing primers in a tertiary PCR. The tertiary PCR reaction containing the index sequence was purified using a PCR SV mini kit (GeneAll) and paired-end sequencing was performed using a MiniSeq Mid Output Kit (Illumina) on a MiniSeq system (Illumina).

[0162] The base correction results are shown in Figure 6 and are compared with the base correction results obtained through the in-cell assay in Example 7 (Figures 7 to 10). As shown in Figure 10, all base editors confirmed to have the ability to correct the G3460A mutation (on-target correction) through the in-cell assay also demonstrated the same on-target correction ability in in-cell experiments. Furthermore, as shown in Figures 7 to 9, it was confirmed that not only the on-target correction ability but also the bystander (non-target) base correction efficiency showed similar trends between the in-cell assay and the in-cell assay.

Claims

1. (a) contacting isolated DNA containing a target DNA mutation in an extracellular environment with one or more base correction proteins used in a base correction system; (b) sequencing the DNA obtained from step (a) to measure the base correction activity of the base correction system; (c) providing information about the ability of the base correction system to correct the same DNA mutation in the cell. A method for providing information about the ability of a base correction system to correct DNA mutations.

2. The method of claim 1, wherein step (a) comprises mixing isolated DNA containing the target DNA mutation with one or more polynucleotides encoding the one or more base-correcting proteins, and accompanying extracellular transcription and translation of the polynucleotides.

3. 3. The method of claim 2, wherein the polynucleotide encoding the base-correcting protein is DNA or mRNA.

4. 4. The method according to claim 1, wherein in step (a), expressing the base correction protein and correcting the DNA base are carried out through a one-pot reaction.

5. 10. The method of claim 1, wherein step (a) involves mixing isolated DNA containing the target DNA mutation with a base-correcting protein outside the cell.

6. 6. The process of any one of claims 1 to 5, wherein the reaction product of step (a) is used in step (b) without further purification.

7. 7. The method of claim 1, wherein the target DNA mutation comprises one or more single base mutations.

8. 8. The method of any one of claims 1 to 7, wherein the isolated DNA containing the target DNA mutation is extracted and amplified from an animal or plant cell and / or artificially synthesized.

9. 9. The method of any one of claims 1 to 8, wherein the isolated DNA containing the target DNA mutation is nuclear DNA or organelle DNA.

10. 10. The method of claim 9, wherein the organelle DNA is mitochondrial DNA or chloroplast DNA.

11. 11. The method of any one of claims 1 to 10, wherein the isolated DNA containing the target DNA mutation is nuclear DNA and / or mitochondrial DNA of a human with a genetic disease.

12. 12. The method of claim 1, wherein the base correction comprises correcting an adenine (A) base to a guanine (G) and / or correcting a cytosine (C) base to a thymine (T) base, and / or both.

13. The one or more base correction proteins each independently comprise one or more DNA binding proteins selected from the group consisting of zinc finger protein, TALE (transcriptional activator-like effector) protein, CRISPR-associated nuclease, and CRISPR-associated nickase. The method of any one of claims 1 to 12.

14. 14. The method of claim 13, wherein the one or more DNA binding proteins comprise a CRISPR-linked nuclease, and wherein in step (a) the guide RNA is additionally contacted with isolated DNA comprising the target DNA mutation.

15. 14. The method of claim 13, wherein the one or more DNA binding proteins comprise a CRISPR-linked nickase, and wherein in step (a) the reverse transcriptase or a polynucleotide encoding the reverse transcriptase and the guide RNA are additionally contacted with isolated DNA comprising the target DNA mutation.

16. 16. The method of any one of claims 1 to 15, wherein the one or more base proteins each independently comprise a base-converting enzyme.

17. The method of claim 16, wherein the one or more base-converting enzymes each independently comprise one or more of APOBEC (apolipoprotein B editing complex), AID (activation-induced deaminase), TadA (tRNA-specific adenosine deaminase), or DddAtox, or a mutant thereof.

18. 18. The method of claim 17, wherein DddAtox or a variant thereof is used in the form of two split bodies.

19. 18. The method of claim 17, wherein DddAtox or a variant thereof is used in the form of a full-length protein.

20. 20. The method of claim 1, wherein one or more base correcting proteins each independently contain a UGI (uracil glycosylase inhibitor).

21. (a) contacting isolated DNA comprising a target DNA mutation with candidate base editors in an extracellular environment, wherein the candidate base editors each independently comprise one or more base correcting proteins or one or more polynucleotides encoding the same; (b) sequencing the DNA molecules obtained from step (a) to measure the base correcting activity of the candidate base editors; (c) providing information regarding the ability of the candidate base editor to correct the same DNA mutation in the cell; and (d) selecting a base editor that can correct the same DNA mutation in the cell based on the information obtained from step (c); A method for screening for base editors that can correct target DNA mutations in cells.

22. A base editor selected by the method of claim 21.

23. The kit includes a DNA polymerase, an RNA polymerase, a ribonucleotide triphosphate mixture, an amino acid mixture, a ribosome containing tRNA and rRNA, an initiation factor, an elongation factor, a termination factor, and a buffer solution. A kit for use in the method of any one of claims 1 to 21.

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