Genome editing techniques

Abstract

By means of a method, a genome editing technique can be provided which is subject to a general organism, in particular actinomycetes, in which the engineered organism or cell does not conform to a recombinant and can avoid an off-target effect, in which the engineered organism or cell does not conform to the recombinant. The present invention relates to a method for deleting, substituting or inserting a target base sequence in a region to be subjected to genome editing in a living being or a cell to be subjected to genome editing, the method comprising: (1) a step for introducing a type II restriction endonuclease recognition sequence into the region to be subjected to genome editing; and (2) a step for causing a double-strand break (DSB) to occur in a genome by expressing in the organism or cell a type II restriction endonuclease that recognizes the introduced type II restriction endonuclease recognition sequence, after the steps (1) and (2) are performed, in the organism or cell, a sequence of the type II restriction endonuclease recognition sequence is introduced into the organism or cell, and the sequence of the type II restriction endonuclease recognition sequence is converted into a sequence of the type II restriction endonuclease recognition sequence into a sequence of the type II restriction endonuclease recognition sequence. The subject region is edited by homologous recombination repair with a gene sequence for genome editing, and the GC content in the genome in the organism or cell is 65% or more.

Description

Technical Field

[0001] This invention relates to a genome editing technology targeting general organisms, particularly actinomycetes. Background Technology

[0002] Actinomycetes produce a wide variety of lead compounds for drug development in the form of secondary metabolites, thus playing a vital role in the pharmaceutical industry since ancient times. However, since these secondary metabolites are not essential for the growth of the producing bacteria themselves, their yield is extremely small, and multiple metabolites are produced simultaneously. Therefore, research on breeding high-yielding mutant strains through genetic modification, such as shutting down concurrent secondary metabolic pathways or modifying metabolic flux, with the goal of increasing the production of the main product, has been actively ongoing.

[0003] Homologous recombination, as a genome editing technique, is well-known. However, while homologous recombination can modify target regions with high specificity, it also leaves foreign genes in the modified genome due to the substitution of the target gene with a drug resistance gene. Therefore, the modified organism or cell conforms to the recombinant. This makes it difficult to gain public acceptance in actual commercial production. Furthermore, the limited number of drug resistance genes available for genome editing makes multiplex editing—the so-called multiplex editing—difficult. While multiplex editing is known to be possible using the cyclorecombinase-P1 phage cross-site (Cre-LoxP) system, the LoxP site remains in the modified genome, and the problems arising from conforming to the recombinant remain unresolved.

[0004] On the other hand, in genome editing systems utilizing the recently popular clustered regularly interspaced short palindromic repeats-CRISPRassociated protein 9 (CRISPR-Cas9), no sequences of foreign biological origin remain on the modified genome, allowing for large-scale deletion of arbitrary gene regions. However, in the sequence recognition of Cas9 nucleases, which play a role in introducing double-strand breaks (DSBs) into the genome, a protospacer-adjacent motif (PAM) is required. The PAM of Cas9, typically derived from Streptococcus pyogenes, is a G-rich tribase (5'-NGG-3'). However, these sequences are distributed throughout the actinomycete genome, which contains approximately 70% guanine-cytosine (GC), with 260 occurrences per 1000 bp in the *Streptomyces coelicolor* genome (Non-Patent Literature 1). Therefore, non-specific gene breaks not only lead to toxic expression but also frequently cause off-target effects, making adaptation difficult. To address these technical challenges, a genome editing technology specific to actinomycetes was developed using CRISPR from *Prevotella* and *Francisella* 1 (Cpf1), a novel culprit that recognizes T-rich PAM sequences (5'-TTV-3'). However, the limited distribution of these PAM sequences in actinomycete genomes makes it difficult to specifically induce nucleases in the target region, thus limiting their widespread use (Non-Patent Literature 2).

[0005] To selectively edit desired regions of the actinomycete genome, which is characterized by high GC content and numerous repetitive sequences, nucleases capable of specifically introducing DSBs into the target region without being affected by GC content or repetitive sequences are needed. Homing endonucleases, which participate in ribonucleic acid (RNA) splicing in eukaryotes, are examples of potential candidates. Because homing endonucleases recognize extremely long base sequences (14 bp to 40 bp) (Non-Patent Reference 3), they can introduce specific DSBs by pre-introducing recognition sequences absent in the genomic deoxyribonucleic acid (DNA) into the target region, and are therefore practically used as a genome editing technology (Non-Patent Reference 4). On the other hand, even with diverse target or host sequences, they exhibit low precision in recognizing sequences in order to accurately break introns or inteins while avoiding toxicity (Non-Patent Reference 3). Therefore, even a difference of just a few bases can cause breakage, making it difficult to completely avoid off-target effects, similar to Cas9. Consequently, there are no examples of applying the genome editing technology using the aforementioned homing endonuclease to genome editing of actinomycetes with high GC content.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: Japanese Patent Application Publication No. 2005-237335

[0009] Non-patent literature

[0010] Non-Patent Literature 1: Applied and Environmental Microbiology (Appl Environ Microbiol.) 15 Sep 2018; 84(18):e00827-18

[0011] Non-Patent Literature 2: Biomolecules. May 8, 2020; 10(5): 734

[0012] Non-patent literature 3: Quarterly reviews of biophysics. 2005;38(1):49-95

[0013] Non-patent literature 4: Applied microbiology and biotechnology. 2020;104(8):3597-609 Summary of the Invention

[0014] The problem that the invention aims to solve

[0015] The subject of this invention is to provide a genome editing technology that targets general organisms, particularly actinomycetes, so that the modified organisms or cells do not conform to recombinants and can avoid off-target effects.

[0016] Technical means to solve the problem

[0017] To address the aforementioned problem, the inventors conducted extensive research and conceived of introducing a type II restriction endonuclease recognition sequence absent in the genome into the target region of the genome for editing, and introducing a type II restriction endonuclease that recognizes the recognition sequence from outside the organism or cell. This causes double-strand breaks (DSBs) in the DNA of the target region. Utilizing the DSB repair function based on homologous recombination inherent in the organism, deletions, insertions, and substitutions of DNA sequences are induced in the target region, thus leading to this invention.

[0018] That is, the present invention is as described below.

[0019] [1] A method for performing the deletion, substitution, or insertion of a target base sequence in a genome editing target region of an organism or cell that is the target of genome editing, comprising:

[0020] (1) The process of introducing a type II restriction endonuclease recognition sequence into the genome editing target region; and

[0021] (2) A process of causing double-strand breaks (DSBs) in the genome by expressing a type II restriction endonuclease that recognizes the introduced type II restriction endonuclease recognition sequence in the organism or cell.

[0022] After performing steps (1) and (2), in the organism or cell, the target region is edited through homologous recombination repair with the gene sequence used for genome editing.

[0023] The GC content in the genome of the organism or cell is above 65%.

[0024] [2] According to the method of [1], wherein the type II restriction endonuclease recognizes a base sequence containing only A and T.

[0025] [3] According to the method of [1] or [2], wherein the type II restriction endonuclease is PacI.

[0026] [4] The method according to any one of [1] to [3], wherein the organism is a eukaryote or prokaryote whose genome does not have the type II restriction endonuclease recognition sequence.

[0027] [5] The method according to any one of [1] to [4], wherein the organism is an actinomycete.

[0028] [6] According to the method of [5], wherein the actinomycete is an actinomycete whose genome does not have a recognition sequence of PacI.

[0029] [7] The method according to [5] or [6], wherein the actinomycete is selected from the group consisting of Streptomyces, Saccharopolyspora and Amycolaptosis.

[0030] [8] The method according to any one of [5] to [7], wherein the actinomycete is selected from the group consisting of Streptomyces fradiae, Streptomyces venezuelae, Streptomyces griseus, Streptomyces albulus, Streptomyces rimosus, Streptomyces spectabilis, Streptomyces avermitilis, Amycolatopsis orientalis, and Saccharopolyspora spinosa.

[0031] [9] The method according to any one of [1] to [8], wherein the introduction in step (1) is performed using a vector for introducing a type II restriction endonuclease recognition sequence.

[0032] The vector for introduction is a vector containing (i) a gene sequence for genome editing, (ii) a type II restriction endonuclease recognition sequence, (iii) a replication origin sequence that works in the host organism used to clone the vector for introduction and does not work in the organism or cell that is the target of the genome editing, and (iv) a drug resistance gene sequence.

[0033]

[10] According to the method described in [9], the replication origin sequence is the p15A replication origin (ori) sequence.

[0034]

[11] According to the method of [9] or

[10] , wherein the import vector further comprises a constant expression promoter sequence in the organism or cell to which the genome editing is intended and a negative selection gene sequence configured to function downstream thereof, and

[0035] Arbitrarily, the negative selection gene sequence is a codon-optimized gene sequence.

[0036]

[12] According to the method of

[11] , wherein the constant expression promoter sequence is an erythromycin resistance gene (ermE) promoter (erythromycin resistance gene promoter (PermE)) sequence, and / or

[0037] The negative selection gene sequence is the cytosine deaminase (codA) gene sequence.

[0038]

[13] The method according to

[11] or

[12] further includes (3) the step of selecting cells with type II restriction endonuclease recognition sequences introduced by the negative selection gene.

[0039]

[14] The method according to any one of [1] to

[13] , wherein step (2) is performed using an expression vector for a type II restriction endonuclease that recognizes the recognition sequence of the introduced type II restriction endonuclease.

[0040] The expression vector contains a promoter sequence that works in the organism or cell to which the genome editing is intended, and a type II restriction endonuclease sequence configured to work downstream theredown, and

[0041] It is a vector containing a replication origin sequence, an antibiotic resistance gene sequence, and a temperature-sensitive replication origin sequence that works in the host organism used to clone the expression vector but not in the organism or cell that is the target of the genome editing.

[0042]

[15] According to the method of

[14] , wherein the promoter sequence is an inducible expression promoter sequence,

[0043] The origin of replication sequence that works in the host organism used to clone the expression vector and not in the organism or cell that is the target of the genome editing is the plasmid origin of replication (pUC ori) sequence from the University of California, and / or

[0044] The temperature-sensitive origin of replication sequence used in the organism or cell that is the target of the genome editing is the pSG5rep sequence.

[0045]

[16] According to the method of

[15] , wherein the inducible expression promoter sequence is the Thiostrepton Induced Protein A (tipA) promoter (PtipA) sequence.

[0046]

[17] The method according to any one of

[14] to

[16] , wherein the expression vector comprises an operator sequence and a repressor sequence,

[0047] The operation sequence is inserted between the promoter sequence and the transcription start site, and

[0048] Optionally, the type II restriction endonuclease sequence is a codon-optimized sequence.

[0049]

[18] According to the method of

[17] , wherein the operator sequence is a lactose operon (lacO) sequence, and / or

[0050] The repressor sequence is the lactose operon repressor (lacI) sequence.

[0051]

[19] The method according to any one of

[14] to

[18] further includes (4) the step of excluding the expression vector from the organism or cell based on the temperature sensitivity of the temperature-sensitive replication origin.

[0052]

[20] The method according to any one of [1] to [8] is a method for introducing the type II restriction endonuclease recognition sequence in step (1) using a single vector, and for expressing the type II restriction endonuclease that recognizes the type II restriction endonuclease recognition sequence in step (2).

[0053] The single vector is a vector comprising (i) a gene sequence for genome editing, (ii) a type II restriction endonuclease recognition sequence, (iii) a promoter sequence that works in the organism or cell to which the genome is to be edited and a type II restriction endonuclease sequence configured to work downstream thereto, (iv) a replication origin sequence that works in the host organism used to clone the single vector and does not work in the organism or cell to which the genome is to be edited, and (v) a drug resistance gene sequence.

[0054]

[21] According to the method of

[20] , wherein the promoter sequence is an inducible expression promoter sequence, and / or

[0055] The origin of replication sequence that works in the host organism used to clone the single vector and not in the organism or cell that is the target of the genome editing is the pUC ori sequence.

[0056]

[22] According to the method of

[21] , wherein the induced expression promoter sequence is a tipA promoter (PtipA) sequence.

[0057]

[23] The method according to any one of

[20] to

[22] , wherein the single vector comprises an operator sequence and a repressor sequence,

[0058] The operation sequence is inserted between the promoter sequence and the transcription start site, and

[0059] Optionally, the type II restriction endonuclease sequence is a codon-optimized sequence.

[0060]

[24] According to the method of

[23] , wherein the operation subsequence is a lacO sequence, and / or

[0061] The repressor sequence is a lacI sequence.

[0062]

[25] An introduction vector for a type II restriction endonuclease recognition sequence, comprising (i) a genome editing gene sequence or a region into which the genome editing gene sequence can be inserted, (ii) a type II restriction endonuclease recognition sequence, (iii) a replication origin sequence that works in the host organism for which the cloning introduction vector is used and does not work in the organism or cell that is the target of genome editing, and (iv) a drug resistance gene sequence.

[0063]

[26] According to the import vector described in

[25] , wherein the origin of replication sequence is a p15A ori sequence.

[0064]

[27] The delivery vector according to

[25] or

[26] further comprises a constant expression promoter sequence in the organism or cell to which the genome editing is intended, and a negative selection gene sequence configured to function downstream thereof, and

[0065] Arbitrarily, the negative selection gene sequence is a codon-optimized gene sequence.

[0066]

[28] According to the import vector described in

[27] , wherein the constant expression promoter sequence is an ermE promoter (PermE) sequence, and / or

[0067] The negative selection gene sequence is the codA gene sequence.

[0068]

[29] An expression vector for a type II restriction endonuclease, comprising a promoter sequence that works in an organism or cell intended for genome editing and a type II restriction endonuclease sequence configured to function downstream thereof, and

[0069] Includes replication origin sequences that function in the host organism used for cloning expression vectors but not in the organism or cell that is the target of the genome editing, drug resistance gene sequences, and temperature-sensitive replication origin sequences that function in the organism or cell that is the target of the genome editing.

[0070]

[30] According to the expression vector described in

[29] , wherein the promoter sequence is a tipA promoter (PtipA) sequence.

[0071] The origin of replication sequence that works in the host organism used to clone the expression vector and not in the organism or cell that is the target of the genome editing is a pUC ori sequence, and / or

[0072] The temperature-sensitive origin of replication sequence used in the organism or cell that is the target of the genome editing is the pSG5rep sequence.

[0073]

[31] The expression vector according to

[29] or

[30] , wherein the expression vector comprises an operator sequence and a repressor sequence.

[0074] The operation sequence is inserted between the promoter sequence and the transcription start site, and

[0075] Optionally, the type II restriction endonuclease sequence is a codon-optimized sequence.

[0076]

[32] The expression vector according to

[31] , wherein the operation subsequence is a lacO sequence, and / or

[0077] The repressor sequence is a lacI sequence.

[0078]

[33] A single vector for introducing a type II restriction endonuclease recognition sequence and for expressing a type II restriction endonuclease, comprising (i) a gene sequence for genome editing or a region into which the gene sequence for genome editing can be inserted, (ii) a type II restriction endonuclease recognition sequence, (iii) a promoter sequence that works in the organism or cell to which the genome editing is to be performed and a type II restriction endonuclease sequence that can be configured to work downstream thereto, (iv) a replication origin sequence that works in the host organism to which the cloning vector is used and does not work in the organism or cell to which the genome editing is to be performed, and (v) a drug resistance gene sequence.

[0079]

[34] According to the single vector described in

[33] , wherein the promoter sequence is a tipA promoter (PtipA) sequence, and / or

[0080] The origin of replication sequence that works in the host organism used to clone the single vector and not in the organism or cell that is the target of the genome editing is the pUC ori sequence.

[0081]

[35] The single vector according to

[33] or

[34] , wherein the single vector comprises an operator sequence and a repressor sequence.

[0082] The operation sequence is inserted between the promoter sequence and the transcription start site, and

[0083] Optionally, the type II restriction endonuclease sequence is a codon-optimized sequence.

[0084]

[36] According to the single vector described in

[35] , wherein the operation subsequence is a lacO sequence, and / or

[0085] The repressor sequence is a lacI sequence.

[0086]

[37] A kit comprising (i) an introduction vector according to any one of

[25] to

[28] , and an expression vector according to any one of

[29] to

[32] , or

[0087] Includes (ii) a single carrier according to any one of

[33] to

[36] ,

[0088] Used to perform deletion, substitution, or insertion of target base sequences in the genome editing target region of an organism or cell that is the target of genome editing.

[0089]

[38] One use is for the use of a vector according to any one of

[25] to

[28] for introducing a type II restriction endonuclease recognition sequence into a genome editing target region in an organism or cell that is a genome editing target.

[0090]

[39] One use is for expressing type II restriction endonucleases in organisms or cells that are the object of genome editing, using a vector according to any one of

[29] to

[32] .

[0091]

[40] One use is the use of the vector according to any one of

[33] to

[36] for introducing a type II restriction endonuclease recognition sequence into a genome editing target region of an organism or cell that is a genome editing target, and for expressing a type II restriction endonuclease in an organism or cell that is a genome editing target.

[0092]

[41] One use is for the use of a kit according to

[37] for the deletion, substitution or insertion of a target base sequence in a region of the genome editing target in an organism or cell that is the target of genome editing.

[0093] The effects of the invention

[0094] According to the present invention, a genome editing technology is provided that targets general organisms, particularly actinomycetes, such that the modified organisms or cells do not conform to recombinants and off-target effects are avoided. That is, since no foreign DNA remains in the edited genome, the genome-edited organisms or cells do not conform to recombinants. Furthermore, off-target effects are avoided by using type II restriction endonucleases that highly specifically recognize sequences absent in the genome of the microorganism targeted for editing. Attached Figure Description

[0095] [ Figure 1 [ ] This diagram illustrates genome editing using two different vectors.

[0096] [ Figure 2 [ ] Schematic diagram of the gene group for the biosynthesis of actinolite.

[0097] [ Figure 3 ] indicates pPacI int Vector graphics.

[0098] [ Figure 4 [Indicates pSET152-PtipA-gfp] uv -tsr vector graphics.

[0099] [ Figure 5 [Indicates pSET152-PtipA-gfp]uv -pSG5rep vector graphics.

[0100] [ Figure 6 The graph shows the concentration-dependent expression of GFP by thioseri in *Streptomyces lilacinus*. The left image is a photograph of a cell-free extract of *Streptomyces lilacinus* expressing GFP in a concentration-dependent manner under ultraviolet light irradiation, and the right image is a graph showing the fluorescence intensity.

[0101] [ Figure 7 The graph shows the concentration-dependent expression of GFP by thiostreptin in *Streptomyces albopictus*. The left image is a photograph of a cell-free extract of *Streptomyces albopictus* expressing GFP in a concentration-dependent manner under ultraviolet light irradiation, and the right image is a graph showing the fluorescence intensity.

[0102] [ Figure 8 The graph shows the concentration-dependent GFP expression of thiostreptin in *Streptomyces azureense*. The left image is a photograph of a cell-free extract of *Streptomyces azureense* expressing GFP in a concentration-dependent manner under ultraviolet light irradiation, and the right image is a graph showing the fluorescence intensity.

[0103] [ Figure 9 ] represents the vector graphic of pSET152-PtipA(lacO)-gfp-tsr-pSG5rep.

[0104] [ Figure 10 [] represents the vector image of pSET152-PtipA(lacO)-gfp-tsr-pSG5rep-lacI.

[0105] [ Figure 11 ] indicates pPacI int Vector graphic of Δact.

[0106] [ Figure 12 [] indicates the construction scheme of the editing intermediate strain in which the PacI recognition sequence is inserted into the genome.

[0107] [ Figure 13 [ ] is a diagram showing the transformants obtained by introducing pELIM into the editing intermediate strain.

[0108] [ Figure 14 [Image] is a photograph confirming the production of actinolite through liquid culture.

[0109] [ Figure 15 The top image shows a schematic diagram of the editing intermediate, the completed editing organism, and the wild-type revertant strain. The bottom image shows a photograph of the phenotype of the transformant (left) and a photograph of the genotype analysis (right).

[0110] [ Figure 16[Image] is a sequence analysis diagram of the region surrounding the genome editing region in the actinolite biosynthesis gene group.

[0111] [ Figure 17 ] indicates pPacI int Vector graphics for ΔactVB.

[0112] [ Figure 18 The diagram above shows the genotyping analysis of the pELIM-introduced strains. The top diagram illustrates the actVB deletion. The bottom diagram is a photograph showing the genotyping analysis of each sample.

[0113] [ Figure 19 [Image] is a sequence analysis diagram of the genome editing region surrounding the actVB deletion editing region.

[0114] [ Figure 20 [Right] is a schematic diagram of genome editing using a single vector, and a photograph showing the results of restriction enzyme treatment of the prepared vector (pELIM-Δact1) (left).

[0115] [ Figure 21 The image shows a schematic diagram (left) of the edited intermediate and the edited complete organism, the wild-type revertant strain, and a photograph (right) confirming the production of actinolite through liquid culture. In the photograph, the tube on the right represents the desired edited complete organism, and the tube on the left represents the wild-type revertant strain. Detailed Implementation

[0116] <Genome Editing Methods>

[0117] One embodiment of the present invention is a method.

[0118] The method involves deleting, replacing, or inserting target base sequences in the genome editing target region of an organism or cell that is the target of genome editing, including:

[0119] (1) The process of introducing a type II restriction endonuclease recognition sequence into the genome editing target region; and

[0120] (2) A process of causing double-strand breaks (DSBs) in the genome by expressing a type II restriction endonuclease that recognizes the introduced type II restriction endonuclease recognition sequence in the organism or cell.

[0121] After performing steps (1) and (2), in the organism or cell, the target region is edited through homologous recombination repair with the gene sequence used for genome editing.

[0122] The GC content in the genome of the organism or cell is above 65%.

[0123] In this invention, genome editing refers to the deletion, substitution, or insertion of target base sequences within a genome editing target region. The genome editing target region is the region containing the target gene for genome editing; it can be a region containing only the target gene or a region also containing a region adjacent to the target gene.

[0124] In this invention, the organism or cell being studied is not particularly limited as long as its genome has a high GC content. In this invention, "high GC content" means that the ratio of G to C in the genome is 65% or more, which can be 68% or more, 70% or more, or 72% or more.

[0125] The organism or cell being studied is, for example, a eukaryote or prokaryote whose genome does not contain the type II restriction endonuclease recognition sequence.

[0126] Organisms with high GC content in their genomes, such as actinomycetes, are suitable for use in the genome editing method of the present invention.

[0127] According to the present invention, there are no particular limitations on the actinomycetes as long as they are available for genome editing, but it is preferred that the actinomycetes do not have a PacI recognition sequence in their genome.

[0128] Actinomycetes can be selected from the group consisting of Streptomyces, Polysporus, and Amycetes.

[0129] Examples of fungi belonging to the genus Streptomyces include: Streptomyces freundii, Streptomyces venezulatus, Streptomyces grayi, Streptomyces whitei, Streptomyces azurei, Streptomyces fissulatus, Streptomyces spectabilis, and Streptomyces avermectinus.

[0130] Examples of fungi belonging to the genus *Saccharopolysporum* include *Saccharopolysporum spinosum*.

[0131] As bacteria belonging to the genus *Pseudomonas*, examples include *Pseudomonas orientalis*.

[0132] For example, when using PacI as a type II restriction endonuclease (described later), the organism or cell being studied preferably does not have a PacI recognition sequence (i.e., a PacI site) in its genome. Examples of such bacteria include: *Streptomyces freundii*, *Streptomyces venereum*, *Streptomyces griseus*, *Streptomyces alba*, *Streptomyces azureum*, *Streptomyces fissula*, *Streptomyces spectabilis*, *Streptomyces avermectin*, *Saccharomyces spp.*, and *Amycium orientalis*.

[0133] In this invention, there are no particular limitations on the type II restriction endonuclease, as long as it tightly recognizes the desired sequence. Here, the type II restriction endonuclease can recognize the sequence extremely tightly and cause double-stranded DNA breaks. Therefore, by using a type II restriction endonuclease, off-target effects can be avoided. Furthermore, since non-specific genomic breaks do not occur, toxic expression can also be avoided.

[0134] For example, when the target organism for genome editing is an actinomycete, the genome has a high GC content, so it is appropriate to use a type II restriction endonuclease that recognizes a sequence containing only A and T bases.

[0135] As a type II restriction endonuclease, PacI derived from *Pseudomonas salcaligenes* can be used, for example. PacI recognizes a palindromic double-stranded sequence containing only A and T (i.e., 5'-TTAATTAA-3'), which is not found in most actinomycete genomes. PacI is the smallest type II restriction endonuclease (142 amino acid residues), thus offering advantages such as miniaturization of expression vectors and ease of transformation.

[0136] In this embodiment, step (1) is the step of introducing the type II restriction endonuclease recognition sequence into the genome editing target region.

[0137] The method of introduction is not particularly limited; for example, a vector for introducing type II restriction endonuclease recognition sequences can be used. By introducing a vector for introducing type II restriction endonuclease recognition sequences into the host, a full-length editing intermediate containing the type II restriction endonuclease recognition sequence can be obtained by single crossover homologous recombination.

[0138] The introduction into the host can be carried out by methods known to those skilled in the art, such as conjugation transfer via Escherichia coli (E. coli) ET12567.

[0139] As an introduction vector for type II restriction endonuclease recognition sequences, a vector containing (i) a gene sequence for genome editing, (ii) a type II restriction endonuclease recognition sequence, (iii) a replication origin sequence that works in the host organism used for cloning the vector and does not work in the organism or cell that is the target of genome editing, and (iv) a drug resistance gene sequence can be used.

[0140] A genome editing sequence is a sequence to be edited within the target region of the genome, specifically a sequence resulting from the deletion, substitution, or insertion of target bases within that region. For example, in the case of deleting a target base sequence through genome editing, this is a sequence formed by linking the outer regions of the target base sequence on both the 5' and 3' sides. Furthermore, when performing genome editing aimed at eliminating (inactivating) the function of a gene, this linked sequence does not impede regions other than the protein-coding regions of that gene.

[0141] The length of the sequence in the outer region is arbitrary. For example, the lengths of the 5' and 3' regions can be approximately 1 kbp, 100 bp to 2 kbp, 500 bp to 1.5 kbp, or 800 bp to 1.2 kbp, respectively.

[0142] Alternatively, for example, in the case of replacing the target base sequence through genome editing, a sequence containing the replaced sequence and linked together between the outer regions on the 5' and 3' sides of the target base sequence in the genome editing target region can be used.

[0143] Alternatively, for example, in the case of inserting a target base sequence through genome editing, a sequence consisting of the outer regions on the 5' and 3' sides of the target base sequence inserted into the genome editing target region can be used and linked together.

[0144] As a type II restriction endonuclease recognition sequence, as described above, a base sequence containing only A and T can be used appropriately, and the PacI recognition sequence (i.e., 5'-TTAATTAA-3') can be used more appropriately.

[0145] The vector for introduction also contains the p15A ori sequence, which serves as the origin of replication in Escherichia coli, and the drug resistance gene sequence.

[0146] By having a replication origin sequence that functions in the host organism used for the cloning vector but not in the organism or cell targeted for genome editing, the insertion vector will not replicate independently within the organism or cell targeted for genome editing. When using *E. coli* to replicate the insertion vector, it is preferable to use a replication origin sequence suitable for replication in *E. coli*, such as the p15A ori sequence. The p15A ori sequence may, for example, have the base sequence described in Serial No. 68, provided that its function as p15A ori is not lost, or may have a base sequence that is 90% or more identical, 95% or more identical, or 98% or more identical to the base sequence described in Serial No. 68.

[0147] There are no particular limitations on the drug resistance gene sequence; for example, it can be a drug resistance gene against kanamycin, apramycin, neomycin, etc. Multiple drug resistance gene sequences may also be included as needed.

[0148] The vector for introduction may also contain a negative selection gene sequence. By introducing the negative selection gene into the editing intermediate strain, unedited intermediate strains can be excluded, and the edited strain can be selectively obtained. For example, the *E. coli*-induced cytosine deaminase (codA) gene is known as a negative selection gene that can be used for actinomycetes. There are no particular limitations on the negative selection gene; for example, the codA gene, the sucrose-6-fructosyltransferase (SacB) gene, etc., can be used. The codA gene may, for example, have a base sequence encoding the amino acid sequence described in Serial No. 69. In addition, as long as codA does not lose its function as a negative selection marker, codA may also have an amino acid sequence that is more than 90%, more than 95%, or more than 98% identical to the amino acid sequence described in Serial No. 69.

[0149] Furthermore, negative selection gene sequences can be used for codon optimization, which can improve translation efficiency within actinomycetes. For example, when using the codA gene as a negative selection gene, the base sequence for codon optimization can be the sequence described in sequence number 70 (codon assembly (codAsm)).

[0150] Negative selection genes are preferably constant expression promoter sequences in organisms or cells upstream of which genome editing targets can be configured functionally.

[0151] As a constant expression promoter sequence, the ermE promoter (PermE) sequence can be used. The PermE sequence may, for example, have the base sequence described in Serial No. 71, provided that it does not lose its function as a promoter, or it may have a base sequence that is more than 90%, more than 95%, or more than 98% identical to the base sequence described in Serial No. 71.

[0152] The principle of negative selection based on codA is as follows.

[0153] The codA gene is a gene in actinomycetes that functions as a negative selection marker in the presence of 5-fluorocytosine (5-FC).

[0154] While 5-fluorocytosine is harmless, it is converted into toxic 5-fluorouracil via codA. codA is persistently expressed in actinomycetes via the ermE promoter. If 5-fluorocytosine is added to the culture medium, strains containing codA cannot grow; only strains without codA can grow. Negative selection based on codA can be used to exclude intermediate editing strains that remain at a certain frequency during genome editing.

[0155] The negative selection can be implemented at any stage after step (2), or it can be omitted. By performing negative selection, the presence rate of the edited organism can be increased, that is, the number of intermediate strains can be reduced.

[0156] Therefore, the genome editing method of this embodiment may further include (3) the step of using the negative selection gene to select cells that have been introduced with a type II restriction endonuclease recognition sequence.

[0157] The vector used for introduction can also contain other genes as needed.

[0158] One type of vector can be used for importation, but multiple vectors can be used as needed, enabling multiple genome editing.

[0159] In this embodiment, step (2) is a step of causing a double-strand break (DSB) in the genome by expressing a type II restriction endonuclease that recognizes the introduced type II restriction endonuclease recognition sequence in the organism or cell.

[0160] The expression method is not particularly limited; for example, an expression vector for a type II restriction endonuclease that recognizes the recognition sequence of the introduced type II restriction endonuclease can be used.

[0161] The introduction into the host can be carried out by methods known to those skilled in the art, such as conjugation transfer via Escherichia coli (E. coli ET12567).

[0162] As an expression vector for type II restriction endonuclease, a vector may be used that contains a promoter sequence that works in the organism or cell that is the target of genome editing and a type II restriction endonuclease sequence that can be configured to work downstream thereto, and contains a replication origin sequence that works in the host organism for cloning the expression vector but not in the organism or cell that is the target of genome editing, an antibiotic resistance gene sequence, and a temperature-sensitive replication origin sequence that works in the organism or cell that is the target of genome editing.

[0163] Regarding promoter sequences that work in organisms or cells that are the targets of genome editing, inducible expression promoter sequences can be used, particularly thioseriin-inducible promoter (PtipA) sequences. A thioseriin-inducible promoter is a promoter that induces the expression of a gene configured to function functionally downstream of thioseriin in the presence of thioseriin. As a thioseriin-inducible promoter, for example, it can have the base sequence described in sequence number 72, and can have a base sequence that is more than 90%, more than 95%, or more than 98% identical to the base sequence described in sequence number 72, provided that it does not lose its function as a thioseriin-inducible promoter.

[0164] A type II restriction endonuclease sequence is a sequence of a type II restriction endonuclease that recognizes a type II restriction endonuclease recognition sequence introduced into the introduction vector. For example, when a PacI recognition sequence is introduced into the introduction vector as a type II restriction endonuclease recognition sequence, the host expresses the PacI sequence as a type II restriction endonuclease sequence in the expression vector. The PacI sequence may, for example, have the base sequence described in Serial No. 22, and may also have a base sequence that is 90% or more identical, 95% or more identical, or 98% or more identical to the base sequence described in Serial No. 22, provided that its function as a PacI is not lost.

[0165] Furthermore, type II restriction endonuclease sequences can be codon-optimized sequences, which can improve translation efficiency in actinomycetes.

[0166] The replication origin sequence, which functions in the host organism used for cloning the expression vector but not in the organism or cell targeted for genome editing, does not function in the organism or cell targeted for genome editing. When using *E. coli* to replicate the expression vector, it is preferable to use a replication origin sequence suitable for replication in *E. coli*, such as a pUC ori sequence. The pUC ori sequence may, for example, have the base sequence described in Serial No. 73, and may also have a base sequence that is 90% or more identical, 95% or more identical, or 98% or more identical to the base sequence described in Serial No. 73, provided that its function as a pUC ori sequence is not lost.

[0167] As a drug resistance gene, there are no special restrictions, and it can contain any sequence.

[0168] For example, when using a thiostreptin-inducible promoter as the promoter in the expression vector, it is preferable to include a thiostreptin resistance gene (tsr) as the resistance gene sequence. Multiple resistance gene sequences may also be included as needed, for example, resistance genes against kanamycin, apramycin, neomycin, etc.

[0169] Regarding temperature-sensitive origin of replication sequences used in organisms or cells targeted for genome editing, for example, the pSG5rep gene sequence, which serves as the origin of replication for the temperature-sensitive plasmid pSG5, can be used. Vectors containing the pSG5rep gene sequence lose function above 39°C and therefore do not replicate. Consequently, during cell division, the plasmid does not transfer to daughter cells, and as culture progresses, almost all cells become plasmid-free. This property allows for the efficient removal of unwanted externally introduced vectors from the host after genome editing.

[0170] In *E. coli*, the repressor sequence overexpressed, combined with the operator sequence inserted between the promoter sequence and the transcription start site, can control gene expression directly below the promoter. Therefore, in order to suppress the basal-level expression of type II restriction endonuclease genes, expression vectors preferably contain both a repressor sequence and an operator sequence. lacI can be used as the repressor sequence, and lacO can be used as the operator sequence.

[0171] Therefore, the genome editing method of this embodiment may further include (4) the step of excluding the expression vector from the organism or cell based on the temperature sensitivity of the temperature-sensitive replication origin.

[0172] Expression vectors can contain other genes as needed.

[0173] One type of carrier can be used for expression, or multiple types can be used as needed.

[0174] In this embodiment, the methods for introducing the type II restriction endonuclease recognition sequence in step (1) and expressing the type II restriction endonuclease that recognizes the type II restriction endonuclease recognition sequence in step (2) are not particularly limited, and a single vector can be used. As a single vector, for example, a vector comprising (i) a gene sequence for genome editing, (ii) a type II restriction endonuclease recognition sequence, (iii) a promoter sequence that works in the organism or cell targeted for genome editing and a type II restriction endonuclease sequence that can be configured to work downstream thereon, (iv) a replication origin sequence that works in the host organism used to clone the single vector but not in the organism or cell targeted for genome editing, and (v) a drug resistance gene sequence. For details, refer to the descriptions of "vectors for introducing type II restriction endonuclease recognition sequences" and "vectors for expressing type II restriction endonucleases that recognize the introduced type II restriction endonuclease recognition sequence".

[0175] Regarding the promoter sequence for use in organisms or cells that are the targets of genome editing, inducible expression promoter sequences may be used, with the thiostreptin-inducible promoter (PtipA) sequence being particularly suitable. As a thiostreptin-inducible promoter, it may have, for example, the base sequence described in sequence number 72, and may have a base sequence that is more than 90%, more than 95%, or more than 98% identical to the base sequence described in sequence number 72, provided that it does not lose its function as a thiostreptin-inducible promoter.

[0176] As the origin of replication sequence that works in the host organism used to clone the single vector but not in the organism or cell that is the target of the genome editing, when using *E. coli* as the replication vector, it is preferable to use an origin of replication sequence suitable for replication in *E. coli*, such as a pUC ori sequence. The pUC ori sequence may, for example, have the base sequence described in sequence number 73, and may also have a base sequence that is 90% or more identical, 95% or more identical, or 98% or more identical to the base sequence described in sequence number 73, provided that its function as a pUC ori sequence is not lost.

[0177] To suppress the basal expression of type II restriction endonuclease genes, a preferred formulation includes both a repressor sequence and an operator sequence. lacI can be used as the repressor sequence, and lacO can be used as the operator sequence. The operator sequence can be inserted between the promoter sequence and the transcription start site.

[0178] There are no particular limitations on the type II restriction endonuclease sequence, as long as it can recognize the type II restriction endonuclease recognition sequence; it can be a codon-optimized sequence.

[0179] Single vectors can contain other genes as needed.

[0180] A single carrier can be used in a single manner, or multiple carriers can be used as needed.

[0181] The genome editing method of this embodiment may include additional steps as needed.

[0182] <Vector for introducing type II restriction endonuclease recognition sequences>

[0183] Another embodiment of the present invention is an introduction vector for a type II restriction endonuclease recognition sequence, comprising (i) a genome editing gene sequence or a region into which the genome editing gene sequence can be inserted, (ii) a type II restriction endonuclease recognition sequence, (iii) a replication origin sequence that works in the host organism used to clone the introduction vector and does not work in the organism or cell that is the target of genome editing, and (iv) a drug resistance gene sequence.

[0184] The vector for insertion in this embodiment can be used to insert a gene sequence for genome editing, or any genome editing gene sequence can be inserted as needed during use. Regions into which a genome editing gene sequence can be inserted include regions containing restriction enzyme sequences that can be processed using the desired restriction enzyme. Furthermore, the restriction enzyme sequence preferably does not include the sequence contained in the vector.

[0185] It is possible that, in the vector used for importation, the origin of replication sequence is the p15A ori sequence.

[0186] The vector for introduction may further comprise a constant expression promoter sequence in the organism or cell to which the genome editing is intended, and a negative selection gene sequence configured to function downstream thereof, and optionally, the negative selection gene sequence is a codon-optimized gene sequence.

[0187] In the vector used for introduction, the constant expression promoter sequence is the ermE promoter (PermE) sequence, and / or the negative selection gene sequence is the codA gene sequence.

[0188] The vector used for importation can contain any other arbitrary base sequence as needed.

[0189] The vector can be synthesized using methods known to those skilled in the art. There are no particular limitations on the synthesis method, as long as the desired vector can be synthesized. For example, a method can be listed as follows: amplifying the desired base sequence from a template sequence having the desired base sequence by polymerase chain reaction (PCR) in such a way that the ends are ligated with an arbitrary restriction enzyme sequence; on the other hand, treating the plasmid with the arbitrary restriction enzyme; and ligating the amplified product to the plasmid treated with the restriction enzyme using the Gibson assembly method.

[0190] In this embodiment, the description of the genome editing target and various sequences can be found in the section on "Genomic Editing Methods".

[0191] As another embodiment of this invention, the use of the vector for introducing a type II restriction endonuclease recognition sequence into a genome editing target region in an organism or cell that is a genome editing target can be listed.

[0192] <Expression vectors for type II restriction endonucleases>

[0193] Another embodiment of the present invention is a vector for expressing type II restriction endonuclease.

[0194] Includes the promoter sequence that works in the organism or cell that is the target of genome editing, and the type II restriction endonuclease sequence that can be configured to work downstream therein, and

[0195] Includes replication origin sequences that function in the host organism used for cloning expression vectors but not in the organism or cell that is the target of the genome editing, drug resistance gene sequences, and temperature-sensitive replication origin sequences that function in the organism or cell that is the target of the genome editing.

[0196] In the expression vector, the promoter sequence is the tipA promoter (PtipA) sequence, the origin of replication sequence that works in the host organism used to clone the expression vector but not in the organism or cell that is the target of the genome editing is the pUC ori sequence, and / or the temperature-sensitive origin of replication sequence that works in the organism or cell that is the target of the genome editing is the pSG5rep sequence.

[0197] The expression vector may contain an operator sequence and a repressor sequence, the operator sequence being inserted between the promoter sequence and the transcription start site, and optionally, the type II restriction endonuclease sequence being a codon-optimized sequence.

[0198] In the expression vector, the operator sequence may be a lacO sequence, and / or the repressor sequence may be a lacI sequence.

[0199] The expression vector can contain any other arbitrary base sequence as needed.

[0200] The vector can be synthesized using methods known to those skilled in the art. There are no particular limitations on the synthesis method, as long as the desired vector can be synthesized. For example, a method can be listed as follows: amplifying the desired base sequence from a template sequence having the desired base sequence by PCR in such a way that an arbitrary restriction enzyme sequence is ligated to the ends; treating the plasmid with the arbitrary restriction enzyme; and ligating the amplified product to the restriction enzyme-treated plasmid using Gibson assembly.

[0201] In this embodiment, the description of the genome editing target and various sequences can be found in the section on "Genomic Editing Methods".

[0202] As another embodiment of this invention, the use of the vector for expressing type II restriction endonucleases in organisms or cells that are the object of genome editing can be listed.

[0203] <Single-carrier>

[0204] Another embodiment of the present invention is a single vector for introducing a type II restriction endonuclease recognition sequence and for expressing a type II restriction endonuclease, comprising (i) a gene sequence for genome editing or a region into which the gene sequence for genome editing can be inserted, (ii) a type II restriction endonuclease recognition sequence, (iii) a promoter sequence that works in the organism or cell to which the genome editing is to be performed and a type II restriction endonuclease sequence that can be configured to work downstream thereto, (iv) a replication origin sequence that works in the host organism to which the cloning vector is used and does not work in the organism or cell to which the genome editing is to be performed, and (v) a drug resistance gene sequence.

[0205] In a single vector, the promoter sequence may be a tipA promoter (PtipA) sequence, and / or the origin of replication sequence that works in the host organism used to clone the single vector but not in the organism or cell that is the target of the genome editing may be a pUC ori sequence.

[0206] The single vector may contain an operator sequence and a repressor sequence, wherein the operator sequence is inserted between the promoter sequence and the transcription start site, and optionally, the type II restriction endonuclease sequence is a codon-optimized sequence.

[0207] In a single vector, the operand sequence may be a lacO sequence, and / or the repressor sequence may be a lacI sequence.

[0208] The vector can be synthesized using methods known to those skilled in the art. There are no particular limitations on the synthesis method, as long as the desired vector can be synthesized. For example, a method can be listed as follows: amplifying the desired base sequence from a template sequence having the desired base sequence by PCR in such a way that an arbitrary restriction enzyme sequence is ligated to the ends; treating the plasmid with the arbitrary restriction enzyme; and ligating the amplified product to the restriction enzyme-treated plasmid using Gibson assembly.

[0209] As another embodiment of this invention, the use of the vector for introducing a type II restriction endonuclease recognition sequence into a genome editing target region of an organism or cell that is a genome editing target can be listed, as well as for expressing a type II restriction endonuclease in an organism or cell that is a genome editing target.

[0210] <Reagent Kit>

[0211] Another embodiment of the present invention is a reagent kit.

[0212] The vector included in (i) the vector described in the section for the introduction of type II restriction endonuclease recognition sequences, and the vector described in the section for the expression of type II restriction endonucleases, or

[0213] The carrier described in item (ii) of the <single carrier> includes,

[0214] Used to perform deletion, substitution, or insertion of target base sequences in the genome editing target region of an organism or cell that is the target of genome editing.

[0215] The kit of this embodiment may contain any other reagents and / or apparatus. Examples of reagents include, for example: primers, PCR reagents, controls, restriction enzymes, restriction enzyme treatment reagents, host cells for cloning, culture media, diluents, buffers, water, purification reagents, etc. Examples of apparatus include, for example, 96-well plates, reaction tubes, etc.

[0216] In this embodiment, the descriptions of genome editing targets and various vectors can be found in the sections on <Genomic Editing Methods>, <Vectors for Introducing Type II Restriction Endonuclease Recognition Sequences>, <Vectors for Expressing Type II Restriction Endonucleases>, and <Single Vectors>.

[0217] As other embodiments of this implementation, examples of the kit can be listed for use in performing deletions, substitutions, or insertions of target base sequences in genome editing target regions of organisms or cells that are genome editing targets.

[0218] Example

[0219] The present invention will now be described in more detail based on the embodiments, but the present invention is not limited to the following embodiments.

[0220] <Example 1. Confirmation of the number of PacI sites in the actinomycete genome and study on the principle of genome editing>

[0221] Actinomycete genomes are known to have high GC content. Therefore, theoretically, sequences of a certain length or longer containing only A and T bases have an extremely low frequency of occurrence.

[0222] For example, PacI from *Alcaligenes alkalitropha* is a type II restriction endonuclease that recognizes 5'-TTAATTAA-3'. Therefore, the number of PacI sites in major actinomycete genomes was investigated. The results showed that major actinomycete genomes either do not contain PacI sites, or if they do, they are very few, indicating that PacI does not cause genome breaks in most types of actinomycetes (Table 1).

[0223] [Table 1]

[0224] Table 1. Number of PacI sites in the genomes of model actinomycetes and lead compounds for major pharmaceutical products.

[0225] Based on the results, it is expected that by pre-introducing a PacI recognition sequence near the target gene and heterologously expressing PacI in the actinomycete host, a position-specific DSB can be induced. After subsequent homologous recombination repair, the desired region can be deleted without leaving any drug resistance gene or heterologous biologically derived sequence.

[0226] Here, Cas9, derived from Streptococcus pyogenes and frequently used in previous genome editing, allows for mismatches of adjacent motifs (PAMs) in protospacer regions to a certain extent. Therefore, in editing the genomes of actinomycetes, which have high GC content and numerous repetitive sequences, there is a risk of frequent off-target editing or toxic expression caused by non-specific breaks in the host genome. Furthermore, Cas9 has 1368 amino acid residues and a large molecular weight, inevitably leading to larger plasmid sizes for editing, which also poses an obstacle to genome editing in actinomycetes, a difficult-to-transform microorganism.

[0227] On the other hand, PacI is a homodimeric enzyme containing a polypeptide chain with only 142 amino acid residues, the smallest known nuclease, thus enabling significant miniaturization of editing plasmids. Therefore, genome editing using PacI methods allows for rational molecular breeding without causing off-target effects, and is expected to make a significant contribution to the future development of natural drug research.

[0228] Therefore, the inventors conducted research on genome editing using PacI and conceived of a method for genome editing based on the following examples ( Figure 1 ).

[0229] First, after the target base sequence is deleted through genome editing, the sequence formed by linking the outer regions of the 5' and 3' sides of the target base sequence in the genome editing target region is amplified by PCR to prepare homologous arms. These arms are then inserted into a mobilization vector containing a PacI site, thereby constructing a vector (hereinafter also referred to as the targeting vector) that introduces a type II restriction endonuclease recognition sequence into the editing target region. By introducing this vector into the host, a full-length edited intermediate containing a PacI site can be obtained by single crossover homologous recombination. Next, a PacI expression vector is added, and PacI expression is induced by adding an inducer, inducing specific DSB. Subsequently, edited complete organisms that have recovered through homologous recombination repair using the pre-introduced homologous arms are screened. Finally, the PacI expression vector carrying a temperature-sensitive origin of replication is detached from the edited complete organisms by high-temperature incubation, obtaining edited complete organisms without exogenous genes. In addition, multiple genome modification can also be achieved using a half-step method by adding a next target vector while maintaining the PacI expression vector.

[0230] In experiments verifying the principles of genome editing in actinomycetes, the method of selecting the biosynthetic genes of pigment antibiotics (actinol or undecylprodigiosin, RED) as target genes and visually distinguishing wild-type strains from edited organisms on a plate has been widely used. Therefore, the inventors used *Streptomyces violaceoruber* A3(2) (strain name: *Streptomyces violaceoruber* NBRC15146), a high-yield model of the blue antibiotic actinol, and selected the actinol biosynthetic gene group (21.6 kbp) in the actinomycete as the target gene. Figure 2 We will conduct scarless editing of the actinolite biosynthesis gene group to verify the genome editing principle.

[0231] <Example 2: Construction of Vectors for Genome Editing>

[0232] 1. Vector for introducing PacI recognition sequences (pPacI) int The construction of )

[0233] During genome editing, considering the possibility of intermediate strains remaining at a certain frequency, unedited intermediate strains can be excluded by pre-introducing negative selection marker genes along with the PacI recognition sequence into the intermediate strains, thus selectively obtaining the edited genome. Regarding negative selection systems in actinomycete strains, for example, the codon-optimized *E. coli*-induced cytosine deaminase (codA) gene is known to function as a negative selection marker in the presence of 5-fluorocytosine (5-FC) in several actinomycete strains.

[0234] DNA containing a codA gene configured as a negative selection marker downstream of a constant expression promoter (PermE) was chemically synthesized. The codA gene underwent codon optimization to improve translation efficiency within actinomycete cells. The synthesized gene (PermE-codAsm (Sequence No. 1)) was amplified by PCR using primers shown in Table 2 using methods known to those skilled in the art, and then inserted into the NheI-HindII site of pK18mob, thereby constructing pK18mob-codAsm.

[0235] [Table 2]

[0236] Table 2

[0237]

[0238] Next, using the primers in Table 3, the origin of replication p15A ori located in the 848 bp–1393 bp region of pACYC184 (Accession No.: X06403) was amplified by PCR. The primers were designed to confer a PacI recognition sequence upstream of the amplified product.

[0239] [Table 3]

[0240] Table 3

[0241]

[0242] pK18mob-codA-sm, pre-digested and opened using MseI, was mixed with p15A ori amplified by PCR and ligated using Gibson assembly. The vector was then set as pPacI. int (Serial number 6, Figure 3 (This will be used in future validations of the effectiveness of this genome editing system.)

[0243] 2. Construction of PacI expression vector (pELIM)

[0244] (1) Validation of the effectiveness of the thiosericin-inducible promoter based on green fluorescent protein (GFP) gene expression

[0245] To enable period-specific expression of PacI and guide DSB introduction within a wide range of actinomycete host cells, a broad-spectrum host-wide inducible promoter is required. To date, the effectiveness of the thioseriin-inducible PtipA has been known in several actinomycete strains. Therefore, based on the pIJ6021 plasmid (accession number AJ414669), the thioseriin-inducible promoter (PtipA (serial number 7)) was constructed using oligonucleotide assembly of the primers shown in Table 4.

[0246] [Table 4]

[0247] Table 4

[0248]

[0249] Next, the GFP that underwent codon optimization / chemical synthesis was... uv The gene (sequence number 12) was used as a template and amplified by PCR using the primers in Table 5.

[0250] [Table 5]

[0251] Table 5

[0252]

[0253] Using the pLAE001 plasmid (serial number 15) constructed in Patent Document 1 as a template, the thiostreptin resistance gene (tsr (serial number 16)) was amplified by PCR using the primers in Table 6.

[0254] [Table 6]

[0255] Table 6

[0256]

[0257] The pSET152 plasmid (accession number AJ414670) was digested and opened using XbaI, and then combined with PtipA and gfp amplified by PCR. uv By mixing the tsr gene and linking them using the Gibson assembly method, a pSET152-PtipA-gfp gene capable of GFP-induced expression under PtipA control was constructed. uv -tsr (serial number 19) Figure 4 ).

[0258] After genome editing, the expression vector for type II restriction endonucleases needs to be detached from the cell. Here, it is known that modified plasmids carrying the origin of replication (pSG5rep) of the temperature-sensitive plasmid pSG5 can be efficiently detached from various actinomycete host strains by culturing at temperatures above 39°C. Therefore, pSET152-PtipA-gfp... uv The φC31 integrase element of -tsr was replaced with pSG5rep. Based on the sequence of pSG5 (accession number NC_008792.1), pSG5rep (sequence number 20) was chemically synthesized (commissioned to Eurofins Genomics, Inc.). The pSG5rep fragment was excised from the pEX-A2J2 plasmid containing the chemically synthesized pSG5rep using double restriction digestion with XbaI and SphI. This fragment was then compared with pSET152-PtipA-gfp, from which the φC31 integrase element was excised by the same restriction enzyme treatment. uv The -tsr link constructs pSET152-PtipA-gfp uv -pSG5rep (hereinafter, also recorded as pSET152-PtipA-gfp-tsr-pSG5rep (serial number 21)) Figure 5 ).

[0259] Next, the functionality of PtipA was evaluated based on GFPuv expression. The constructed pSET152-PtipA-gfp-tsr-pSG5rep was introduced into Streptomyces NBRC14147 (NITE Biological Resource Center, National Institute of Technology and Evaluation, Japan) via conjugation transfer from E. coli ET12567 (American Type Culture Collection, ATCC), Streptomyces lividans TK23 (ATCC), and Streptomyces A3(2) (strain name: Streptomyces lividans NBRC15146) (NITE Biological Resource Center, National Institute of Technology and Evaluation, Japan) via conjugation transfer from E. coli ET12567 (American Type Culture Collection, ATCC). Transformants were cultured in tryptic soy broth with yeast extract (TSBY) containing apramycin (Apr) (Sigma-Aldrich, 50 μg / ml) and incubated at 30°C with shaking until the stationary phase was reached. Subcultures were then performed in TSBY medium containing fresh Apr (50 μg / ml) and incubated at 30°C with shaking for 8 hours. Various concentrations of thiostreptin (Sigma-Aldrich) were then added to induce GFPuv expression. After the addition of thiostreptin, the culture was continued with shaking for 24 hours, and the cells were recovered by centrifugation.

[0260] The fluorescence of cell-free extracts prepared by ultrasonic disruption was measured at an excitation wavelength of 380 nm. The results confirmed that the fluorescence near 510 nm derived from GFPuv was enhanced in all strains in a concentration-dependent manner. Figures 6-8 Therefore, by using PtipA, the expression of downstream genes can be controlled in various actinomycete hosts in a concentration-dependent manner depending on the thiotetracyclin concentration.

[0261] (2) Confirmation of temperature sensitivity based on pSG5rep

[0262] Next, the temperature-sensitive replication of pSET152-PtipA-gfp-tsr-pSG5rep in various actinomycetes was verified. pSET152-PtipA-gfp uv -pSG5rep was introduced into *Streptomyces white* NBRC14147, *Streptomyces purpureus* TK23, and *Streptomyces aquamarine* A3(2) (strain name: *Streptomyces purpureus* NBRC15146) via conjugation transfer from *E. coli* ET12567. Transformants were selected based on apramycin resistance. Each transformant was cultured in apramycin-containing TSBY liquid medium with shaking for 48 hours (30°C). The culture medium that reached the stationary phase was spread onto antibiotic-free agar medium and incubated at 39°C for 72 hours to induce plasmid detachment.

[0263] In addition, as a control experiment, the same culture medium was spread on agar medium containing apramycin and incubated at 39°C for 3 days. The results showed that cell proliferation was significantly insufficient in the presence of apramycin, suggesting that the high-temperature incubation induced plasmid detachment. Furthermore, it was confirmed that the recombinant strains that induced plasmid detachment on antibiotic-free agar medium were apramycin-sensitive and did not retain the plasmid in PCR assays.

[0264] Based on the above results, it was confirmed that pSET152-PtipA-gfp-tsr-pSG5rep functions as a temperature-sensitive plasmid as designed.

[0265] (3) Research on vectors for pacI expression

[0266] Therefore, the pacI gene was cloned downstream of PtipA in the pSET152-PtipA-gfp-tsr-pSG5rep plasmid to construct a pacI expression vector. Based on the pacI sequence (accession number 3LDY_A) from *Pseudomonas alkaliflorus*, a synthetic pacI gene (accession number 22) optimized for actinomycetes was obtained. Using the synthetic gene as a template, PCR was performed using the primers in Table 7, thereby obtaining a pacI gene fragment with NdeI and BgllII recognition sequences linked at both ends.

[0267] [Table 7]

[0268] Table 7

[0269]

[0270] GF was extracted from pSET152-PtipA-gfp-tsr-pSG5rep through dual restriction digestion based on NdeI and BglII. uvThe pacI gene, digested with NdeI and BglII, was ligated using T4 DNA ligase. The ligation product was then used to transform the E. coli host NEB10β (New England Biolabs) for cloning, but few transformants were obtained. Furthermore, sequence confirmation of plasmids obtained from multiple transformants revealed unexpected base substitutions or deletions in the pacI gene region. This suggests that the pacI gene, positioned downstream of PtipA, is expressed at a basal level within the E. coli host for cloning, potentially resulting in cleavage of the host genome.

[0271] Here, it is known that gene expression directly below the promoter can be controlled by binding an overexpressed lac repressor (lacI (SEQ ID NO: 25)) to an operator sequence (lacO (SEQ ID NO: 26)) inserted between the -10 region of the promoter and the transcription start site. Therefore, this study investigated the control of basal expression from PtipA by inserting lacO between the -10 region of PtipA and the transcription start site.

[0272] Using the constructed pSET152-PtipA-gfp-tsr-pSG5rep as a template, PCR was used to detect PtipA (PtipA(lacO) (serial number 27)) and gfp containing the inserted lacO sequence. uv The gene and tsr gene were amplified. The primers used in the amplification based on the PCR method are shown in Table 8.

[0273] [Table 8]

[0274] Table 8 PtipA (PtipA(lacO)), gfp uv Primers used in the amplification of genes, including the tsr gene.

[0275]

[0276] By mixing these DNA fragments with pSET152 that had been digested and opened using EcoRV and XbaI, and then ligating them using the Gibson assembly method, pSET152-PtipA(lacO)-gfp was constructed. uv -tsr. Furthermore, by replacing the φC31 integrase element of the vector backbone with pSG5rep according to the method described, pSET152-PtipA(lacO)-gfp-tsr-pSG5rep (sequence number 34) was constructed. Figure 9 ).

[0277] Next, pSET152-PtipA(lacO)-gfp uvThe lacI gene was appended to pSG5 rep. Using the primers in Table 9, the lacI gene (containing the promoter region (sequence number 35)) was amplified from pET21b(+) (Merck Millipore) by PCR and inserted into pSET152-PtipA(lacO)-gfp. uv The NheI site of -pSG5rep is used to construct pSET152-PtipA(lacO)-gfp. uv -pSG5rep-lacI ( Figure 10 )

[0278] [Table 9]

[0279] Table 9

[0280]

[0281] Next, the pacI gene was cloned into pSET152-PtipA-gfp, which carries an expression control system based on the lac operator and lac repressor. uv The chemically synthesized pacI gene was used as a template and amplified by PCR using the primers in Table 7, thereby obtaining the pacI gene with NdeI and BglII sites ligated at both ends. T4-DNA ligase was used to ligate the pacI gene, which had undergone restriction digestion with NdeI and BglII, and pSET152-PtipA(lacO)-gfp. uv -pSG5rep-lacI. After converting a portion of the reaction solution to E. coli NEB10β, transformants were selected based on apramycin resistance. At this point, 5% (w / v) glucose was added to Luria-Bertani (LB) liquid medium and LB agar medium used for cloning to control basal expression of PacI. For the plasmid extracted and purified from the transformants, the construction of pSET152-PtipA(lacO)-pacI-pSG5rep-lacI (serial number 38) was confirmed by restriction mapping based on EcoRV and NheI double breaks and DNA sequence analysis, and it was used as the pacI expression vector (pELIM) in subsequent validation experiments.

[0282] <Example 3: Seamless Deletion of Actinoporin Biosynthetic Gene Cluster Using the PacI Genome Editing System (Principle Verification Experiment)>

[0283] (1) Introducing PacI recognition sequence into the actinolite biosynthesis gene group

[0284] Using the genome extracted from Streptomyces NBRC15146 (strain name: Streptomyces azureus A3(2)) as a template, and with the primers shown in Table 10, approximately 1 kbp was amplified from the outer regions of the 5' and 3' sides of the actinol biosynthesis gene group (sequence number 39) by PCR (the peripheral regions of the sco5071 and sco5092 genes (sequence numbers 40 and 41, respectively)).

[0285] [Table 10]

[0286] Table 10

[0287]

[0288] pPacI, which had been pre-digested and ring-opened using EcoRI and PstI, was used. int The mixture was combined with two 1kbp fragments amplified by PCR and ligated using the Gibson assembly method. A portion of the reaction solution was used to transform *E. coli* NEB10β competent cells, and clones exhibiting kanamycin (Fujifilm and Koden) resistance were screened. The plasmid, purified using methods known to those skilled in the art, was subjected to NdeI and NheI-based restriction mapping to identify a target vector (pPacI) specific to the actinol biosynthesis gene group. int The construction of Δact (serial number 46) Figure 11 ).

[0289] pPacI int Δact was introduced into *Streptomyces purpureus* NBRC15146 via conjugative transfer via *E. coli* S17-1 strain (ATCC). Genomic DNA extracted from edited intermediate candidate strains exhibiting resistance to neomycin (Fujifilm and Kodenki) was used as a template, and the results were confirmed by PCR using primers shown in Table 11. Figure 12 The following paragraph shows the construction of an editing intermediate for inserting the PacI recognition sequence into the genome.

[0290] [Table 11]

[0291] Table 11

[0292]

[0293] (2) Seamless deletion of the actinolite biosynthesis gene cluster based on PacI expression

[0294] The constructed PacI expression plasmid pELIM was introduced into an intermediate strain via conjugation transfer from E. coli S17-1. PacI expression from pELIM was engineered to be induced by the addition of thiostreptin. However, using apramycin resistance as an indicator to screen for intermediate strains introduced with pELIM, it was confirmed that strains lacking blue pigment production were produced without expression induction via the addition of thiostreptin. Figure 13 (The circled colonies are used to produce the blue pigment for the missing strain).

[0295] The results suggest that, shortly after the vector is introduced, PacI is expressed at the basal level even without induction by the addition of thiotetracycline. The introduction of DSB into the genome could potentially induce homologous recombination repair, resulting in edit-complete variants. Therefore, two clones each of the blue and brown colonies were cultured in TSB (containing 10% sucrose) liquid medium for 3 days to confirm the presence or absence of pigment production. The results strongly suggest that the blue clones exhibit vigorous production of actinol, while the brown clones do not produce actinol. Therefore, it is possible to generate edit-complete variants with a deletion of the actinol biosynthetic gene group simply by introducing pELIM. Figure 14 The two samples on the left are actinol-producing strains, while the two samples on the right are non-actinol-producing strains.

[0296] Therefore, genomic DNA was extracted from these bacteria and validated by PCR using primers designed to amplify the central region (serial number 49) of the actinol biosynthesis gene group (Table 12) (genotyping analysis).

[0297] [Table 12]

[0298] Table 12

[0299]

[0300] Furthermore, the production of the pigment in the secondary metabolite production medium, R5 medium, was confirmed again (phenotypic analysis). Genotypic analysis showed that amplification was confirmed by PCR only in colonies displaying blue color, completely consistent with the phenotypic analysis results in R5 medium. Figure 15 ).

[0301] Based on the above results, it was confirmed that approximately 92% of the blue clones, representing all transformants, were wild-type revertant strains, while the remaining 8% of the brown clones were completed edited strains. Furthermore, since all blue and brown clones were neomycin-sensitive, it was also confirmed that no editing intermediates remained after pELIM introduction.

[0302] Next, to confirm whether the target region could be accurately deleted, sequence analysis of the surrounding region was performed. The sequence analysis used the primers shown in Table 13.

[0303] [Table 13]

[0304] Table 13

[0305]

[0306] For the wild-type revertant strains, the 5' and 3' lateral regions of the actinoplasmin biosynthesis gene group were analyzed. For the edited strains, the junctional regions of the 5' and 3' lateral regions were analyzed. The results confirmed that the blue clones completely reverted to the wild-type sequence, while the target regions of the brown clones were accurately deleted as designed. Figure 16 Furthermore, by culturing the edited somatic strain at 40°C for 5 days, apramycin-sensitive clones with pELIM detachment can also be obtained.

[0307] The verification experiments based on the above principles demonstrate that this method, using the type II restriction enzyme PacI, can specifically introduce DSB into the actinomycete genome, achieving accurate genome editing without leaving any foreign gene residue.

[0308] Next, to confirm the impact of deletion length on genome editing efficiency, a deletion of actVB (560 bp) located at the end of the biosynthetic gene group was performed. Using the genome extracted from *Streptomyces purpureus* NBRC15146 as a template, and with the primers shown in Table 14, approximately 1 kbp (sequence numbers 57 and 58, respectively) was amplified by PCR on the outer regions of the 5' and 3' sides of actVB (sequence number 56).

[0309] [Table 14]

[0310] Table 14

[0311]

[0312] The two obtained PCR fragments were combined with a mobile vector (pPacI) that had been pre-digested and opened using EcoRI and PstI. int The mixture was ligated using the Gibson assembly method. A portion of the reaction solution was used to transform E. coli NEB10β competent cells, and transformants were screened based on kanamycin resistance. The actVB gene cluster-specific target vector (pPacI) was identified by performing restriction mapping based on NdeI and NheI using plasmids purified from the transformants. int The construction of ΔactVB (serial number 63) Figure 17 ).

[0313] pPacI int ΔactVB was introduced into *Streptomyces purpureus* NBRC15146 via conjugative transfer via *E. coli* S17-1. Genomic DNA extracted from neomycin-resistant editing intermediate candidate strains was used as a template, and PCR was performed using the primers shown in Table 15 to confirm the genomic editing intermediate *Streptomyces purpureus* NBRC15146 / pPacI with the PacI recognition sequence inserted. int The construction of ΔactVB.

[0314] [Table 15]

[0315] Table 15

[0316]

[0317] Next, in order to induce PacI-based genome editing, pELIM was introduced into Streptomyces purpureus NBRC15146 / pPacI via conjugation transfer via E. coli S17-1 strain. int ΔactVB. Since the deletion of only the actVB gene, located at the end of the biosynthetic gene group, does not result in the loss of pigment production capacity, the success or failure of phenotype-based genome editing could not be confirmed. Therefore, 20 pELIM-introduced strains exhibiting apramycin resistance were randomly screened and validated by PCR using the primers shown in Table 16.

[0318] [Table 16]

[0319] Table 16

[0320]

[0321] The results confirmed that 12 clones were wild-type reverting strains and 8 clones were actVB gene deletion strains. Figure 18 Furthermore, it was confirmed that, similar to the case of full-length deletion of the actinolite biosynthesis gene group, no intermediate editing residue remained after pELIM was introduced.

[0322] In addition, such as Figure 19 As shown, sequence analysis around the edited region also confirmed that the blue clones were not all editing intermediates, but completely reverted to the original wild-type sequence, and the brown clones had accurate deletions of the actVB gene region without any single-base errors.

[0323] In the deletion of the 3' end gene *actVB* (560 bp) of the actinoporosis biosynthesis gene group, 8 out of 20 clones were edited successfully. In contrast, in the full-length deletion of the actinoporosis biosynthesis gene group, which can be visually identified by pigment, only 4 out of 48 clones showed a brown edit. Based on these results, a tendency to produce wild-type revertant strains is confirmed in long-distance gene deletions.

[0324] According to the present invention, a new foundation for genome editing technology has been successfully established to avoid off-target effects, the biggest challenge in genome editing of actinomycetes. In this invention, because promoters or temperature-sensitive replication origins that function in a wide range of actinomycete species are combined, the method can be applied not only to the breeding of the actinomycete strains exemplified in the examples, but also to the breeding of a wide range of industrial actinomycete strains. Therefore, further improvements to this method are expected to pave the way for the molecular breeding of a wide range of industrial actinomycete strains.

[0325] <Example 4: Construction of a single-vector PacI genome editing system and seamless deletion of the actinolite biosynthesis gene group using the system>

[0326] It can be proven that pPacI was used. int The effectiveness of two-stage genome editing using plasmids and pELIM plasmids was investigated, but the process of introducing two plasmids into recalcitrant actinomycetes, followed by screening of the edited organisms and the shedding of pELIM, takes approximately 40 days. Therefore, a one-stage genome editing process was attempted, concentrating only the necessary elements into a single vector, to accelerate the overall process.

[0327] By assigning pPacI to pELIM int The target sequence of the actinol biosynthesis gene group of Δact (serial number 46) (the surrounding regions of the sco5071 and sco5092 genes (serial numbers 40 and 41, respectively)) and the PacI recognition sequence were used to construct an actinol biosynthesis gene group-specific editing vector. The actinol biosynthesis gene group-specific editing vector can perform the following operations with a single vector: inserting the PacI recognition sequence into the genome, inducing double-strand breaks through PacI expression, and repairing the required region through homologous recombination.

[0328] Using the primers shown in Table 17, approximately 1 kbp was amplified from the outer regions of the 5' and 3' sides of the actinol biosynthesis gene group (sequence number 39) by PCR (the peripheral regions of the sco5071 and sco5092 genes (sequence numbers 74 and 75, respectively)).

[0329] [Table 17]

[0330] Table 17

[0331]

[0332] pELIM, pre-digested and open-ringed with XbaI and SphI, was mixed with two 1 kbp fragments amplified by PCR and ligated using Gibson assembly. A portion of the reaction solution was used to transform E. coli NEB10β competent cells, and clones exhibiting apramycin resistance were screened. Restriction mapping based on XbaI and SphI was performed on the plasmid, which had been extracted and purified using methods known to those skilled in the art, thus confirming the construction of a single actinol biosynthesis gene-specific editing vector (pELIM-Δact1 (serial number 78)). Figure 20 ).

[0333] pELIM-Δact1 was introduced into *Streptomyces purpureus* NBRC15146 via conjugative transfer via *E. coli* S17-1 strain (ATCC). Single cross-recombinant strains exhibiting apramycin resistance were selected as editing intermediates for the full-length pELIM-Δact1 insertion into the genome.

[0334] The selected colonies were scraped multiple times using the tip of a sterile toothpick and then transferred by streaking to agar medium containing 2 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG). After incubation at 30°C for 3 days, the strain was confirmed to produce the expected blue pigment-deficient material.

[0335] Two clones each of the blue and brown colonies were cultured in TSB (containing 10% sucrose) liquid medium for 3 days to confirm the presence or absence of pigment production. The results strongly suggested that the blue clones exhibited vigorous production of actinol, while the brown clones did not produce actinol. Therefore, it is possible to eliminate the actinol biosynthetic gene group through a single editing vector. Figure 21 ).

[0336] Therefore, genomic DNA was extracted from these bacterial cells and validated by PCR (genotyping analysis) using primers designed to amplify the central region (sequence number 49) of the actinol biosynthesis gene cluster. The results showed that the region was not detected. Furthermore, sequence analysis of the surrounding region was performed using the primers shown in Table 13, confirming that the target region was accurately deleted, similar to the two-stage editing.

[0337] In one-stage genome editing based on the single vector, all operations up to the editing stage can be performed with a single plasmid introduction. Furthermore, the introduced editing plasmid does not remain in the cell after editing, thus eliminating the need for plasmid curing. In two-stage genome editing using two plasmids, the construction of the editing vector specific to the edited region takes approximately 40 days from start to finish. However, with one-stage genome editing, this can be achieved in about 20 days.

[0338] In one-stage genome editing, the editing efficiency is the same as in two-stage genome editing. This example discloses a specific, seamless deletion (knockout) of the actinomycete genome using the type II restriction enzyme PacI, but arbitrary sequences can also be inserted into the desired region (knockin) by pre-inserting arbitrary sequences between the homologous arms of the editing vector.

Claims

1. A method for performing the deletion, substitution, or insertion of a target base sequence in a genome editing target region of an organism or cell that is the object of genome editing, comprising: (1) The process of introducing a type II restriction endonuclease recognition sequence into the genome editing target region; as well as (2) A process of causing double-strand breaks (DSBs) in the genome by expressing a type II restriction endonuclease that recognizes the introduced type II restriction endonuclease recognition sequence in the organism or cell. After performing steps (1) and (2), in the organism or cell, the target region is edited through homologous recombination repair with the gene sequence used for genome editing. The genome of the organism or cell contains more than 65% guanine-cytosine.

2. The method according to claim 1, wherein, The type II restriction endonuclease recognizes sequences containing only A and T bases.

3. The method according to claim 1, wherein, The type II restriction endonuclease is PacI.

4. The method according to claim 1, wherein, The organism is a eukaryote or prokaryote whose genome does not contain the type II restriction endonuclease recognition sequence.

5. The method according to claim 4, wherein, The organism in question is an actinomycete.

6. The method according to claim 5, wherein, The actinomycetes mentioned are actinomycetes whose genomes do not contain the PacI recognition sequence.

7. The method according to claim 5, wherein, The actinomycetes are selected from the group consisting of Streptomyces, Polysporus, and Amylopectinus.

8. The method according to claim 5, wherein, The actinomycetes are selected from the group consisting of *Streptomyces freundii*, *Streptomyces venereum*, *Streptomyces grayi*, *Streptomyces whitei*, *Streptomyces azurei*, *Streptomyces fissula*, *Streptomyces spectabilis*, *Streptomyces avermectin*, *Streptomyces orientalis*, and *Polysporium spp.* 9. The method according to any one of claims 1 to 8, wherein, The introduction in step (1) is performed using a vector for introducing a type II restriction endonuclease recognition sequence. The vector for introduction is a vector containing (i) a gene sequence for genome editing, (ii) a type II restriction endonuclease recognition sequence, (iii) a replication origin sequence that works in the host organism used to clone the vector for introduction and does not work in the organism or cell that is the target of the genome editing, and (iv) a drug resistance gene sequence.

10. The method according to claim 9, wherein, The replication origin sequence is the p15A replication origin sequence.

11. The method according to claim 9, wherein, The import vector further comprises a constant expression promoter sequence in the organism or cell from which the genome editing is intended, and a negative selection gene sequence configured to function downstream theredown, and Arbitrarily, the negative selection gene sequence is a codon-optimized gene sequence.

12. The method according to claim 11, wherein, The constant expression promoter sequence is the erythromycin resistance gene promoter (PermE) sequence, and / or The negative selection gene sequence is the cytosine deaminase gene sequence.

13. The method according to claim 11, further comprising (3) the step of using the negative selection gene to select cells that have been introduced with a type II restriction endonuclease recognition sequence.

14. The method according to any one of claims 1 to 8, wherein, Step (2) is performed using an expression vector for a type II restriction endonuclease that recognizes the recognition sequence of the introduced type II restriction endonuclease. The expression vector contains a promoter sequence that works in the organism or cell to which the genome editing is intended, and a type II restriction endonuclease sequence configured to work downstream theredown, and It is a vector containing a replication origin sequence, an antibiotic resistance gene sequence, and a temperature-sensitive replication origin sequence that works in the host organism used to clone the expression vector but not in the organism or cell that is the target of the genome editing.

15. The method according to claim 14, wherein, The promoter sequence is an inducible expression promoter sequence. The origin of replication sequence that works in the host organism used to clone the expression vector and not in the organism or cell that is the target of the genome editing is the University of California plasmid origin of replication sequence, and / or The temperature-sensitive origin of replication sequence used in the organism or cell that is the target of the genome editing is the pSG5rep sequence.

16. The method according to claim 15, wherein, The inducible expression promoter sequence is the thiostreptin-induced protein A promoter (PtipA) sequence.

17. The method of claim 14, wherein, The expression vector contains an operator sequence and a repressor sequence. The operation sequence is inserted between the promoter sequence and the transcription start site, and Optionally, the type II restriction endonuclease sequence is a codon-optimized sequence.

18. The method according to claim 17, wherein, The operation subsequence is a lactose operon sequence, and / or The repressor sequence is the lactose operon repressor protein sequence.

19. The method of claim 14, further comprising (4) the step of excluding the expression vector from the organism or cell based on the temperature sensitivity of the temperature-sensitive replication origin.

20. The method according to any one of claims 1 to 8 is a method for introducing the type II restriction endonuclease recognition sequence in step (1) using a single vector, and for expressing the type II restriction endonuclease that recognizes the type II restriction endonuclease recognition sequence in step (2). The single vector is a vector comprising (i) a gene sequence for genome editing, (ii) a type II restriction endonuclease recognition sequence, (iii) a promoter sequence that works in the organism or cell to which the genome is to be edited and a type II restriction endonuclease sequence configured to work downstream thereto, (iv) a replication origin sequence that works in the host organism used to clone the single vector and does not work in the organism or cell to which the genome is to be edited, and (v) a drug resistance gene sequence.

21. The method according to claim 20, wherein, The promoter sequence is an inducible expression promoter sequence, and / or The origin of replication sequence that works in the host organism used to clone the single vector and not in the organism or cell that is the target of the genome editing is the University of California plasmid origin of replication sequence.

22. The method according to claim 21, wherein, The inducible expression promoter sequence is the thiostreptin-induced protein A promoter (PtipA) sequence.

23. The method of claim 20, wherein, The single vector contains an operator sequence and a repressor sequence. The operation sequence is inserted between the promoter sequence and the transcription start site, and Optionally, the type II restriction endonuclease sequence is a codon-optimized sequence.

24. The method according to claim 23, wherein, The operation subsequence is a lactose operon sequence, and / or The repressor sequence is the lactose operon repressor protein sequence.

25. A vector for introducing a type II restriction endonuclease recognition sequence, comprising (i) a genome editing gene sequence or a region into which the genome editing gene sequence can be inserted, (ii) a type II restriction endonuclease recognition sequence, (iii) a replication origin sequence that works in a host organism used for cloning the vector and does not work in an organism or cell that is the target of genome editing, and (iv) a drug resistance gene sequence.

26. The carrier for introduction according to claim 25, wherein, The replication origin sequence is the p15A replication origin sequence.

27. The vector for introduction according to claim 25, further comprising a constant expression promoter sequence in the organism or cell from which the genome editing is intended, and a negative selection gene sequence configured functionally downstream thereof, and Arbitrarily, the negative selection gene sequence is a codon-optimized gene sequence.

28. The carrier for introduction according to claim 27, wherein, The constant expression promoter sequence is the erythromycin resistance gene promoter (PermE) sequence, and / or The negative selection gene sequence is the cytosine deaminase gene sequence.

29. An expression vector for a type II restriction endonuclease, comprising a promoter sequence that functions in an organism or cell intended for genome editing and a type II restriction endonuclease sequence configured functionally downstream thereof, and Includes replication origin sequences that function in the host organism used for cloning expression vectors but not in the organism or cell that is the target of the genome editing, drug resistance gene sequences, and temperature-sensitive replication origin sequences that function in the organism or cell that is the target of the genome editing.

30. The expression carrier according to claim 29, wherein, The promoter sequence is the PtipA promoter sequence, which is a thiotetracycline-induced protein A promoter sequence. The origin of replication sequence that works in the host organism used to clone the expression vector and not in the organism or cell that is the target of the genome editing is the University of California plasmid origin of replication sequence, and / or The temperature-sensitive origin of replication sequence used in the organism or cell that is the target of the genome editing is the pSG5rep sequence.

31. The expression carrier according to claim 29, wherein, The expression vector contains an operator sequence and a repressor sequence. The operation sequence is inserted between the promoter sequence and the transcription start site, and Optionally, the type II restriction endonuclease sequence is a codon-optimized sequence.

32. The expression carrier according to claim 31, wherein, The operation subsequence is a lactose operon sequence, and / or The repressor sequence is the lactose operon repressor protein sequence.

33. A single vector for introducing a type II restriction endonuclease recognition sequence and for expressing a type II restriction endonuclease, comprising (i) a gene sequence for genome editing or a region capable of being inserted into the gene sequence for genome editing, (ii) a type II restriction endonuclease recognition sequence, (iii) a promoter sequence that works in the organism or cell to which the genome editing is intended and a type II restriction endonuclease sequence configured to work downstream thereto, (iv) a replication origin sequence that works in the host organism for which the cloning vector is used and does not work in the organism or cell to which the genome editing is intended, and (v) a drug resistance gene sequence.

34. The single carrier according to claim 33, wherein, The promoter sequence is the PtipA (thiosericin-induced protein A) promoter sequence, and / or The origin of replication sequence that works in the host organism used to clone the single vector and not in the organism or cell that is the target of the genome editing is the University of California plasmid origin of replication sequence.

35. The single carrier according to claim 33, wherein, The single vector contains an operator sequence and a repressor sequence. The operation sequence is inserted between the promoter sequence and the transcription start site, and Optionally, the type II restriction endonuclease sequence is a codon-optimized sequence.

36. The single carrier according to claim 35, wherein, The operation subsequence is a lactose operon sequence, and / or The repressor sequence is the lactose operon repressor protein sequence.

37. A kit comprising (i) an introduction vector as described in any one of claims 25 to 28, and an expression vector as described in any one of claims 29 to 32, or Includes (ii) a single carrier as described in any one of claims 33 to 36, Used to perform deletion, substitution, or insertion of target base sequences in the genome editing target region of an organism or cell that is the target of genome editing.

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