Genome editing technique

Abstract

Provided is a method for deleting, substituting or inserting a target nucleotide sequence in a genome editing target region in an organism or a cell to be subjected to genome editing. The method comprises: (1) a step for introducing a type II restriction endonuclease recognition sequence into the genome editing target region; and (2) a step for expressing a type II restriction endonuclease capable of recognizing the introduced type II restriction endonuclease recognition sequence in the organism or the cell to cause double strand break (DSB) in the genome. In the method, after performing the steps (1) and (2), the target region is edited through homologous recombination repair with the genome editing gene sequence in the organism or the cell. In the organism or the cell, the GC content in the genome is 65% or more. As a result, it becomes possible to provide a genome editing technique for all organisms, particularly actinomycetes, wherein an organism or a cell obtained after the modification does not correspond to a recombinant and an off-target effect can be avoided.

Description

Genome editing technology

[0001] The present invention relates to genome editing technology for organisms in general, and actinomycetes in particular.

[0002] Actinomycetes have long played an important role in the pharmaceutical industry because they produce a wide variety of drug discovery seed compounds as secondary metabolites. However, because secondary metabolites are not essential for the growth of the producing bacteria themselves, their production is extremely small. Furthermore, because multiple metabolites are also produced, research has been actively conducted into breeding high-yielding mutant strains through genome modification, such as by closing co-producing secondary metabolic pathways or modifying metabolic fluxes, with the aim of improving production of the main product.

[0003] Homologous recombination, for example, is a widely known genome modification technique. However, while homologous recombination can modify target regions with high specificity, the foreign gene remains in the modified genome because the target gene is replaced with a drug resistance gene. Therefore, the modified organism or cell is considered a recombinant. Therefore, it is difficult to gain public acceptance for actual commercial production. Furthermore, the number of drug resistance genes available for genome modification is limited, making modification of multiple gene regions, or so-called multiple modification, difficult. It is widely known that multiple modifications can be performed using the Cre-LoxP system, which was subsequently developed, but the problem of LoxP sites remaining in the modified genome, which makes it a recombinant, remained unresolved.

[0004] On the other hand, genome editing systems using CRISPR-Cas9, which have recently attracted attention, can delete large-scale gene regions without leaving heterologous sequences in the modified genome. However, the Cas9 nuclease, which introduces double-strand breaks (DSBs) into the genome, requires a protospacer-adjacent motif (PAM) for sequence recognition. The PAM of the commonly used Cas9 from Streptococcus pyogenes is G-rich and has three bases (5'-NGG-3'). However, this sequence is distributed throughout the actinomycete genome, which has a GC content of approximately 70%, and is present at 260 locations per 1000 bp in the Streptomyces coelicolor genome (Non-Patent Document 1). Therefore, nonspecific genome cleavage leads to toxicity and frequently causes off-target effects, making its application difficult. To overcome this technical challenge, a genome editing technique specific to actinomycetes was developed using Cas9 and Cpf1 derived from Francisella novicida, which recognize the T-rich PAM sequence (5'-TTV-3') (Non-Patent Document 1). However, this PAM sequence is rarely found in actinomycete genomes, making it difficult to specifically target the nuclease to the target region, and therefore this technique has not been widely used (Non-Patent Document 2).

[0005] To selectively edit desired regions of actinomycete genomes with high GC content and many repeat sequences, a nuclease capable of specifically introducing DSBs into the target region is required, regardless of GC content or repeat sequence. Potential candidates for this nuclease include homing endonucleases, which are involved in RNA splicing in eukaryotes. Homing endonucleases recognize extremely long base sequences (14-40 bp) (Non-Patent Document 3). By pre-introducing a recognition sequence that is not present in genomic DNA into the target region, specific DSBs can be introduced. Therefore, homing endonucleases have been applied as genome editing technologies (Non-Patent Document 4). On the other hand, they have the property of requiring low recognition sequence stringency, allowing them to accurately cleave introns and inteins while avoiding toxicity even when target and host sequences are diverse (Non-Patent Document 3). Therefore, because differences of only a few bases are possible, it is considered difficult to completely avoid the off-target effects that occur with Cas9. Therefore, no genome editing technology using such homing endonucleases has been applied to genome editing of actinomycete genomes with high GC content.

[0006] Japanese Patent Application Laid-Open No. 2005-237335

[0007] Appl Environ Microbiol. 2018 Sep 15;84(18):e00827-18Biomolecules. 2020 May 8;10(5):734Quarterly reviews of biophysics. 2005;38(1):49-95Applied microbiology and biotechnology. 2020;104(8):3597-609

[0008] The present invention aims to provide a genome editing technology that targets organisms in general, and actinomycetes in particular, in which the modified organism or cell does not fall under the category of a recombinant organism and which can avoid off-target effects.

[0009] As a result of intensive research to solve the above problems, the present inventors came up with the idea of ​​introducing a type II restriction endonuclease recognition sequence that is not present in the genome into the region to be edited in the genome, and introducing a type II restriction endonuclease that recognizes the type II restriction endonuclease recognition sequence into an organism or cell from the outside. This led to the discovery that double-strand breaks (DSBs) can be induced in the DNA in the region to be edited, and that the organism's own DSB repair function by homologous recombination can be utilized to cause deletion, insertion, or substitution of DNA sequences in the region to be edited, thereby arriving at the present invention.

[0010] That is, the present invention is as follows: [1] A method for deleting, substituting, or inserting a targeted base sequence in a region to be edited in a genome editing target organism or cell, comprising: (1) introducing a type II restriction endonuclease recognition sequence into the region to be edited in the genome; and (2) causing a double-strand break (DSB) in the genome by expressing in the organism or cell a type II restriction endonuclease that recognizes the introduced type II restriction endonuclease recognition sequence, wherein after steps (1) and (2) are performed, the target region is edited in the organism or cell via homologous recombination repair with a gene sequence for genome editing, and wherein the GC content in the genome of the organism or cell is 65% or more. [2] The method according to [1], wherein the type II restriction endonuclease recognizes a base sequence consisting only of As and Ts. [3] The method according to [1] or [2], wherein the type II restriction endonuclease is PacI. [4] The method according to any one of [1] to [3], wherein the organism is a eukaryote or prokaryote that does not have the type II restriction endonuclease recognition sequence in its genome. [5] The method according to any one of [1] to [4], wherein the organism is an actinomycete. [6] The method according to [5], wherein the actinomycete does not have a PacI recognition sequence in its genome. [7] The method according to [5] or [6], wherein the actinomycete is an actinomycete selected from the group consisting of the genera Streptomyces, Saccharopolyspora, and Amycolaptosis. [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 coelicolor, Streptomyces rimosus, Streptomyces spectabilis, Streptomyces avermitilis, Amycolatopsis orientalis, and Saccharopolyspora spinosa.[9] The method of any of [1] to [8], wherein the introduction in step (1) is carried out using a vector for introducing a type II restriction endonuclease recognition sequence, and the introduction vector is a vector comprising (i) a genome editing gene sequence, (ii) a type II restriction endonuclease recognition sequence, (iii) a replication origin sequence that functions in a host organism used for cloning the introduction vector but does not function in the organism or cell that is the target of genome editing, and (iv) a drug resistance gene sequence.

[10] The method of [9], wherein the replication origin sequence is a p15A ori sequence.

[11] The method of [9] or

[10] , wherein the introduction vector further comprises a constitutive expression promoter sequence in the organism or cell that is the target of genome editing and a negative selection gene sequence operably positioned downstream thereof, and optionally, the negative selection gene sequence is codon-optimized.

[12] The method according to

[11] , wherein the constitutive expression promoter sequence is an ermE promoter (PermE) sequence, and / or the negative selection gene sequence is a codA gene sequence.

[13] The method according to

[11] or

[12] , further comprising (3) a step of selecting cells into which a type II restriction endonuclease recognition sequence has been introduced using the negative selection gene.

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

[13] , wherein the step (2) is carried out using an expression vector for a type II restriction endonuclease that recognizes the introduced type II restriction endonuclease recognition sequence, the expression vector comprising a promoter sequence that functions in the organism or cell to be genome-edited and a type II restriction endonuclease sequence operably positioned downstream thereof, and a vector comprising an origin of replication sequence that functions in a host organism used for cloning of the expression vector but does not function in the organism or cell to be genome-edited, a drug resistance gene sequence, and a temperature-sensitive origin of replication sequence that functions in the organism or cell to be genome-edited.

[15] The method according to

[14] , wherein the promoter sequence is an inducible promoter sequence, the origin of replication sequence that functions in a host organism used for cloning the expression vector but does not function in the organism or cell to be genome-edited is a pUC ori sequence, and / or the temperature-sensitive origin of replication sequence that functions in the organism or cell to be genome-edited is a pSG5rep sequence.

[16] The method according to

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

[17] The method according to any one of

[14] to

[16] , wherein the expression vector comprises an operator sequence and a repressor sequence, the operator sequence is inserted between the promoter sequence and the transcription start site, and optionally, the type II restriction endonuclease sequence is codon-optimized.

[18] The method according to

[17] , wherein the operator sequence is a lacO sequence, and / or the repressor sequence is a lacI sequence.

[19] (4) The method of any of

[14] to

[18] , further comprising a step of removing the expression vector from the organism or cell based on the temperature sensitivity of the temperature-sensitive replication origin.

[20] A method of using a single vector to introduce a type II restriction endonuclease recognition sequence in the step (1) and to express a type II restriction endonuclease that recognizes the type II restriction endonuclease recognition sequence in the step (2), wherein the single vector is a vector comprising: (i) a genome editing gene sequence, (ii) a type II restriction endonuclease recognition sequence, (iii) a promoter sequence that functions in the organism or cell to be genome-edited and a type II restriction endonuclease sequence operably arranged downstream thereof, (iv) a replication origin sequence that functions in a host organism used for cloning the single vector but does not function in the organism or cell to be genome-edited, and (v) a drug resistance gene sequence.

[21] The method according to

[20] , wherein the promoter sequence is an inducible expression promoter sequence, and / or the replication origin sequence that operates in a host organism used for cloning the single vector and does not operate in an organism or cell that is the target of genome editing is a pUC ori sequence.

[22] The method according to

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

[23] The method according to any one of

[20] to

[22] , wherein the single vector comprises 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 codon-optimized.

[24] The method according to

[23] , wherein the operator sequence is a lacO sequence and / or the repressor sequence is a lacI sequence.

[25] A vector for introducing a type II restriction endonuclease recognition sequence, 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) an origin of replication sequence that functions in a host organism used for cloning the vector for introduction but does not function in an organism or cell that is a target for genome editing, and (iv) a drug resistance gene sequence.

[26] The introduction vector according to

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

[27] The introduction vector according to

[25] or

[26] , further comprising a constitutive expression promoter sequence in the organism or cell to be edited and a negative selection gene sequence operably positioned downstream thereof, and optionally, the negative selection gene sequence is codon-optimized.

[28] The introduction vector according to

[27] , wherein the constitutive expression promoter sequence is an ermE promoter (PermE) sequence and / or the negative selection gene sequence is a codA gene sequence.

[29] A type II restriction endonuclease expression vector comprising a promoter sequence that operates in the organism or cell to be edited and a type II restriction endonuclease sequence operably positioned downstream thereof, and comprising an origin of replication that operates in a host organism used for cloning the expression vector but does not operate in the organism or cell to be edited, a drug resistance gene sequence, and a temperature-sensitive origin of replication that operates in the organism or cell to be edited.

[30] The expression vector according to

[29] , wherein the promoter sequence is a tipA promoter (PtipA) sequence, the origin of replication sequence that functions in a host organism used for cloning the expression vector but does not function in the organism or cell to be subjected to genome editing is a pUC ori sequence, and / or the temperature-sensitive origin of replication sequence that functions in the organism or cell to be subjected to genome editing is a pSG5rep sequence.

[31] The expression vector according to

[29] or

[30] , wherein the expression vector comprises 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 codon-optimized.

[32] The expression vector according to

[31] , wherein the operator sequence is a lacO sequence and / or the repressor sequence is a lacI sequence.

[33] A single vector for introducing a type II restriction endonuclease recognition sequence and expressing a type II restriction endonuclease, the single vector 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 promoter sequence that functions in the organism or cell to be the target of genome editing and a type II restriction endonuclease sequence operably arranged downstream of the promoter sequence; (iv) an origin of replication that functions in a host organism used for cloning the vector but does not function in the organism or cell to be the target of genome editing; and (v) a drug resistance gene sequence.

[34] The single vector according to

[33] , wherein the promoter sequence is a tipA promoter (PtipA) sequence, and / or the origin of replication that functions in the host organism used for cloning the single vector but does not function in the organism or cell to be the target of genome editing is a pUC ori sequence.

[35] The single vector according to

[33] or

[34] , wherein the single vector comprises 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 codon-optimized.

[36] The single vector according to

[35] , wherein the operator sequence is a lacO sequence and / or the repressor sequence is a lacI sequence.

[37] A kit for deleting, substituting, or inserting a targeted nucleotide sequence in a region to be edited in a genome-editing target organism or cell, the kit comprising (i) the introduction vector according to any of

[25] to

[28] and the expression vector according to any of

[29] to

[32] , or (ii) the single vector according to any of

[33] to

[36] .

[38] Use of the vector according to any of

[25] to

[28] for introducing a type II restriction endonuclease recognition sequence into a region to be edited in a genome-editing target organism or cell.

[39] Use of the vector according to any of

[29] to

[32] for expressing a type II restriction endonuclease in a genome-editing target organism or cell.

[40] 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 in an organism or cell to be genome-edited, and for expressing the type II restriction endonuclease in the organism or cell to be genome-edited.

[41] Use of the kit according to

[37] for deleting, substituting, or inserting a target nucleotide sequence in a genome-editing target region in an organism or cell to be genome-edited.

[0011] The present invention provides a genome editing technology for organisms in general, and actinomycetes in particular, in which the modified organism or cell does not qualify as a recombinant and off-target effects can be avoided. In other words, since no foreign DNA remains in the edited genome, the genome-edited organism or cell does not qualify as a recombinant, and off-target effects can be avoided by using a type II restriction endonuclease that highly specifically recognizes sequences not present in the genome of the microorganism to be edited.

[0012] A schematic diagram of genome editing using two types of vectors is shown. A schematic diagram of the actinorhodin biosynthetic gene cluster is shown. pPacI int The vector map of pSET152-PtipA-gfp is shown. uvThe vector map of pSET152-PtipA-gfp is shown. uv Figure 1 shows the vector map of pSET152-pSG5rep. Figure 1 shows thiostrepton concentration-dependent GFP expression in Streptomyces lividans. The left panel is a photograph of a cell-free extract of S. lividans expressing GFP in a thiostrepton concentration-dependent manner, observed under UV irradiation, and the right panel is a graph showing fluorescence intensity. Figure 1 shows thiostrepton concentration-dependent GFP expression in Streptomyces albulus. The left panel is a photograph of a cell-free extract of S. albulus expressing GFP in a thiostrepton concentration-dependent manner, observed under UV irradiation, and the right panel is a graph showing fluorescence intensity. Figure 1 shows thiostrepton concentration-dependent GFP expression in Streptomyces coelicolor. The left panel is a photograph of a cell-free extract of S. coelicolor expressing GFP in a thiostrepton concentration-dependent manner, observed under UV irradiation, and the right panel is a graph showing fluorescence intensity. Figure 1 shows the vector map of pSET152-PtipA(lacO)-gfp-tsr-pSG5rep. The vector map of pSET152-PtipA(lacO)-gfp-tsr-pSG5rep-lacI is shown. int The vector map of Δact is shown. The scheme for constructing an edited intermediate strain in which a PacI recognition sequence has been inserted into the genome is shown. This is a photograph showing a transformant obtained by introducing pELIM into an edited intermediate strain. This is a photograph confirming actinorhodin production in liquid culture. The upper panel shows a schematic diagram of the edited intermediate strain, edited strain, and revertant strain. The lower panel is a photograph showing the phenotype of the transformant (left) and a photograph showing genotype analysis (right). This is a sequence analysis diagram of the area around the genome edited region in the actinorhodin biosynthetic gene cluster. pPacI intΔactVB vector map. Figure 1 shows the genotype analysis of pELIM-introduced strains. The upper panel shows a schematic diagram of actVB deletion. The lower panel is a photograph showing the genotype analysis of each sample. This shows a sequence analysis of the genome-edited region surrounding the edited region in actVB deletion. This shows a schematic diagram of genome editing using a single vector (right), and a photograph (left) showing the results of restriction enzyme treatment of the prepared vector (pELIM-Δact1). This shows a schematic diagram of an editing intermediate, a fully edited organism, and a revertant strain (left), and a photograph (right) confirming actinorhodin production by liquid culture. In the photograph, the tube on the right shows the desired fully edited organism, and the tube on the left shows the revertant strain.

[0013] <Genome editing method> One embodiment of the present invention is a method for deleting, substituting, or inserting a targeted base sequence in a region to be edited in an organism or cell that is the target of genome editing, comprising the steps of: (1) introducing a type II restriction endonuclease recognition sequence into the region to be edited in the genome; and (2) causing a double-strand break (DSB) in the genome by expressing in the organism or cell a type II restriction endonuclease that recognizes the introduced type II restriction endonuclease recognition sequence. After steps (1) and (2) are performed, the target region is edited in the organism or cell via homologous recombination repair with a gene sequence for genome editing, and the GC content in the genome of the organism or cell is 65% or more.

[0014] In the present invention, genome editing refers to the deletion, substitution, or insertion of a target base sequence in a region to be edited. The region to be edited refers to a region containing a target gene for genome editing, and may be a region consisting of only the target gene, or may further include a region adjacent to the target gene.

[0015] In the present invention, the target organism or cell is not particularly limited as long as the genome of the organism or cell has a high GC content. In the present invention, a high GC content means that the ratio of G and C in the genome is 65% or more, or may be 68% or more, 70% or more, or 72% or more.

[0016] The organism or cell of interest is, for example, a eukaryote or prokaryote that does not have the type II restriction endonuclease recognition sequence in its genome.

[0017] Examples of organisms with a high GC content in their genomes include actinomycetes, which can be suitably used in the genome editing method of the present invention.

[0018] The actinomycete is not particularly limited as long as it can be used for genome editing according to the present invention, but it is preferable that the actinomycete does not have a recognition sequence for PacI in its genome.

[0019] As the actinomycete, for example, an actinomycete selected from the group consisting of the genera Streptomyces, Saccharopolyspora, and Amycolaptosis can be used.

[0020] Examples of bacteria belonging to the genus Streptomyces include Streptomyces fradiae, Streptomyces venezuelae, Streptomyces griseus, Streptomyces albulus, Streptomyces coelicolor, Streptomyces rimosus, Streptomyces spectabilis, Streptomyces avermitilis, etc. Examples of bacteria belonging to the genus Saccharopolyspora include Saccharopolyspora spinosa, etc. Examples of bacteria belonging to the genus Amycolaptosis include Amycolatopsis orientalis, etc.

[0021] For example, when using PacI (described below) as the type II restriction endonuclease, it is preferable that the target organism or cell does not have a PacI recognition sequence (i.e., a PacI site) in its genome. Examples of such bacteria include Streptomyces fradiae, Streptomyces venezuelae, Streptomyces griseus, Streptomyces albulus, Streptomyces coelicolor, Streptomyces rimosus, Streptomyces spectabilis, Streptomyces avermitilis, Saccharopolyspora spinosa, and Amycolatopsis orientalis.

[0022] In the present invention, the type II restriction endonuclease is not particularly limited as long as it strictly recognizes the desired sequence. Here, type II restriction endonucleases recognize sequences with extremely high precision and are capable of cleaving double-stranded DNA. Therefore, by using type II restriction endonucleases, off-target effects can be avoided. Furthermore, toxicity can also be avoided because nonspecific genome cleavage does not occur. For example, when the target organism for genome editing is an actinomycete, the genome has a high GC content, so a type II restriction endonuclease that recognizes a base sequence consisting only of A and T can be suitably used.

[0023] An example of a type II restriction endonuclease that can be used is PacI derived from Pseudomonas alcaligenes. The sequence recognized by PacI is a palindromic double-stranded base sequence (i.e., 5'-TTAATTAA-3') consisting only of A and T, which is not present in many actinomycete genomes. PacI is the smallest type II restriction endonuclease (142 aa), which allows for the miniaturization of expression vectors and facilitates transformation.

[0024] Step (1) in this embodiment is a step of introducing a type II restriction endonuclease recognition sequence into the region to be edited in the genome. The method of introduction is not particularly limited, and can be carried out, for example, using a vector for introducing a type II restriction endonuclease recognition sequence. By introducing the vector for introducing a type II restriction endonuclease recognition sequence into a host, an editing intermediate is obtained in which the entire length of the vector containing the type II restriction endonuclease recognition sequence is inserted into the genome by single crossover homologous recombination. Introduction into the host can be carried out by methods known to those skilled in the art, such as conjugative transfer via Escherichia coli (E. coli ET12567).

[0025] As a vector for introducing a type II restriction endonuclease recognition sequence, a vector containing (i) a gene sequence for genome editing, (ii) a type II restriction endonuclease recognition sequence, (iii) a replication origin sequence that functions in the host organism used for cloning the introduction vector but does not function in the organism or cell that is the target of genome editing, and (iv) a drug resistance gene sequence can be used.

[0026] The gene sequence for genome editing refers to the desired post-edit sequence in the genome editing target region, i.e., the sequence after deletion, substitution, or insertion of the targeted base sequence in the genome editing target region. For example, when deleting a targeted base sequence by genome editing, the gene sequence refers to a sequence formed by linking the 5' and 3' outer regions of the targeted base sequence in the genome editing target region. Note that when genome editing is performed to eliminate (inactivate) the function of a gene, the linked sequence does not preclude the inclusion of regions other than the protein-coding region of the gene. The length of the outer region sequence is arbitrary; for example, the lengths of the 5' and 3' regions may each be approximately 1 kbp, 100 bp to 2 kbp, 500 bp to 1.5 kbp, or 800 bp to 1.2 kbp.

[0027] Furthermore, for example, when replacing a target base sequence by genome editing, a sequence can be used in which the replaced sequence is linked between the outer regions on the 5' and 3' sides of the target base sequence in the genome editing target region. Furthermore, for example, when inserting a target base sequence by genome editing, a sequence can be used in which the inserted sequence is linked between the outer regions on the 5' and 3' sides of the target base sequence in the genome editing target region.

[0028] As described above, a base sequence consisting only of A and T can be preferably used as the type II restriction endonuclease recognition sequence, and a PacI recognition sequence (i.e., 5'-TTAATTAA-3') can be more preferably used. Furthermore, the transfer vector contains the p15A ori sequence, which is the sequence of the replication origin in E. coli, and a drug resistance gene sequence.

[0029] By including an origin of replication sequence that functions in the host organism used for cloning the introduction vector but does not function in the organism or cell targeted for genome editing, the introduction vector does not autonomously replicate within the organism or cell targeted for genome editing. When E. coli is used to replicate the introduction vector, it is preferable to use an origin of replication sequence suitable for replication in E. coli, for example, a p15A ori sequence. The p15A ori sequence may have, for example, the nucleotide sequence set forth in SEQ ID NO: 68, and may have a nucleotide sequence that is 90% or more identical, 95% or more identical, or 98% or more identical to the nucleotide sequence set forth in SEQ ID NO: 68, as long as it does not lose its function as a p15A ori.

[0030] The drug resistance gene sequence is not particularly limited, and may be, for example, a drug resistance gene for kanamycin, apramycin, neomycin, etc. If necessary, multiple drug resistance gene sequences may be included.

[0031] The transfer vector may contain a negative selection gene sequence. By introducing the negative selection gene into an edited intermediate strain, unedited intermediate strains can be eliminated and fully edited strains can be selectively obtained. For example, the Escherichia coli-induced cytosine deaminase (codA) gene is known as a negative selection gene that can be used in actinomycetes. Negative selection genes are not particularly limited, but examples that can be used include the codA gene and the SacB gene. The codA gene may have a nucleotide sequence encoding the codA amino acid sequence set forth in SEQ ID NO: 69, for example. Furthermore, as long as codA does not lose its function as a negative selection marker, codA may have an amino acid sequence that is 90% or more identical, 95% or more identical, or 98% or more identical to the amino acid sequence set forth in SEQ ID NO: 69. Furthermore, the negative selection gene sequence may be codon-optimized, which can improve translation efficiency in actinomycetes. For example, when the codA gene is used as the negative selection gene, an example of a codon-optimized nucleotide sequence is that set forth in SEQ ID NO: 70 (codAsm).

[0032] Preferably, a constitutively expressing promoter sequence in the target organism or cell for genome editing is operably positioned upstream of the negative selection gene. The constitutively expressing promoter sequence can be the ermE promoter (PermE) sequence. The PermE sequence may have, for example, the nucleotide sequence set forth in SEQ ID NO: 71. As long as it does not lose its promoter function, it may have a nucleotide sequence that is 90% or more identical, 95% or more identical, or 98% or more identical to the nucleotide sequence set forth in SEQ ID NO: 71.

[0033] The principle of codA-mediated negative selection is as follows. The codA gene functions as a negative selection marker in actinomycetes in the presence of 5-fluorocytosine (5-FC). While 5-fluorocytosine is harmless, it is converted by codA to the toxic 5-fluorouracil. codA is constantly expressed in actinomycetes under the ermE promoter. When 5-fluorocytosine is added to the medium, strains containing codA cannot grow, and only strains lacking codA grow. Negative selection using codA can be used to eliminate editing intermediate strains that frequently remain during the genome editing process. Negative selection can be performed at any stage after step (2), but it is not necessary to perform negative selection. Negative selection can increase the rate of fully edited strains, thereby reducing the number of editing intermediate strains.

[0034] Therefore, the genome editing method of this embodiment may further include (3) a step of selecting cells into which a type II restriction endonuclease recognition sequence has been introduced using the negative selection gene.

[0035] The vector for transfer may contain other genes as needed.

[0036] Only one type of transfer vector may be used, but multiple types may be used as necessary, and the use of multiple types makes it possible to perform multiple genome modifications.

[0037] In this embodiment, step (2) is a step of generating 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. The expression method is not particularly limited, and can be carried out, for example, using an expression vector for a type II restriction endonuclease that recognizes the introduced type II restriction endonuclease recognition sequence. Introduction into the host can be carried out by methods known to those skilled in the art, such as conjugative transfer via Escherichia coli (E. coli ET12567).

[0038] As a type II restriction endonuclease expression vector, a vector can be used that includes a promoter sequence that operates in the organism or cells to be genome-edited and a type II restriction endonuclease sequence operably positioned downstream of the promoter sequence, as well as a replication origin sequence that operates in the host organism used for cloning the expression vector but does not operate in the organism or cells to be genome-edited, a drug resistance gene sequence, and a temperature-sensitive replication origin sequence that operates in the organism or cells to be genome-edited.

[0039] As a promoter sequence that operates in the organism or cell to be genome-edited, for example, an inducible promoter sequence can be used, and in particular, the thiostrepton-inducible promoter (PtipA) sequence can be preferably used. A thiostrepton-inducible promoter is a promoter that induces the expression of a gene operably located downstream thereof in the presence of thiostrepton. The thiostrepton-inducible promoter may have, for example, the nucleotide sequence set forth in SEQ ID NO: 72. It may also have a nucleotide sequence that is 90% or more identical, 95% or more identical, or 98% or more identical to the nucleotide sequence set forth in SEQ ID NO: 72, as long as it does not lose its function as a thiostrepton-inducible promoter.

[0040] The type II restriction endonuclease sequence is a type II restriction endonuclease sequence that recognizes the 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 the type II restriction endonuclease recognition sequence, the PacI sequence is expressed in the host as the type II restriction endonuclease sequence in the expression vector. The PacI sequence may have, for example, the nucleotide sequence set forth in SEQ ID NO: 22, or may have a nucleotide sequence that is 90% or more identical, 95% or more identical, or 98% or more identical to the nucleotide sequence set forth in SEQ ID NO: 22, as long as it does not lose its function as PacI. Furthermore, the type II restriction endonuclease sequence may be codon-optimized, which can increase the translation efficiency in actinomycetes.

[0041] The expression vector has an origin of replication sequence that functions in the host organism used for cloning but not in the organism or cell targeted for genome editing, thereby preventing it from functioning in the organism or cell targeted for genome editing. When using E. coli to replicate the expression 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 have, for example, the nucleotide sequence set forth in SEQ ID NO: 73, and may be 90% or more identical, 95% or more identical, or 98% or more identical to the nucleotide sequence set forth in SEQ ID NO: 73, as long as it does not lose its function as a pUC ori sequence.

[0042] The drug resistance gene is not particularly limited and may include any sequence. For example, when a thiostrepton-inducible promoter is used as the promoter in the expression vector, it is preferable to include a thiostrepton resistance gene (tsr) as the drug resistance gene sequence. If necessary, multiple drug resistance gene sequences may be included, and for example, drug resistance genes for kanamycin, apramycin, neomycin, etc. may be included.

[0043] The temperature-sensitive replication origin sequence that operates in the target organism or cell for genome editing can be, for example, the pSG5rep gene sequence, which is the replication origin of the temperature-sensitive plasmid pSG5. Vectors containing the pSG5rep gene sequence lose their function at temperatures above 39°C, preventing replication of the plasmid. Therefore, during cell division, the plasmid is not transmitted to daughter cells, and as the culture progresses, almost all cells become plasmid-free. This property can be used to efficiently eliminate exogenously introduced vectors from the host after genome editing.

[0044] The repressor sequence overexpressed in E. coli binds to an operator sequence inserted between the promoter sequence and the transcription start site, thereby controlling the expression of the gene directly under the promoter. Therefore, it is preferable that the expression vector contain a repressor sequence and an operator sequence to suppress the expression of the type II restriction endonuclease gene at a basal level. The repressor sequence can be lacI, and the operator sequence can be lacO.

[0045] Therefore, the genome editing method of this embodiment may further include (4) a step of eliminating the expression vector from the organism or cell based on the temperature sensitivity of the temperature-sensitive replication origin.

[0046] The expression vector may contain other genes as needed.

[0047] Only one type of expression vector may be used, but multiple types may also be used as necessary.

[0048] In this embodiment, the method for introducing a type II restriction endonuclease recognition sequence in step (1) and expressing a type II restriction endonuclease that recognizes the type II restriction endonuclease recognition sequence in step (2) is not particularly limited, and may be performed using a single vector. Examples of the single vector include: (i) a genome editing gene sequence; (ii) a type II restriction endonuclease recognition sequence; (iii) a promoter sequence that functions in the organism or cell to be genome-edited and a type II restriction endonuclease sequence operably located downstream thereof; (iv) a replication origin sequence that functions in a host organism used for cloning the single vector but does not function in the organism or cell to be genome-edited; and (v) a drug resistance gene sequence. For more details, the above descriptions regarding "vectors for introducing type II restriction endonuclease recognition sequences" and "vectors for expressing a type II restriction endonuclease that recognizes the introduced type II restriction endonuclease recognition sequence" may be used.

[0049] The promoter sequence operating in the target organism or cell for genome editing can be, for example, an inducible promoter sequence, and the thiostrepton-inducible promoter (PtipA) sequence is particularly suitable. The thiostrepton-inducible promoter may have, for example, the nucleotide sequence set forth in SEQ ID NO: 72, or may have a nucleotide sequence that is 90% or more identical, 95% or more identical, or 98% or more identical to the nucleotide sequence set forth in SEQ ID NO: 72, as long as it does not lose its function as a thiostrepton-inducible promoter.

[0050] When E. coli is used to replicate the vector, it is preferable to use an origin of replication sequence suitable for replication in E. coli, such as a pUC ori sequence, as the replication origin sequence that functions in the host organism used for cloning the single vector but does not function in the organism or cell that is the target of genome editing. The pUC ori sequence may, for example, have the nucleotide sequence set forth in SEQ ID NO: 73, and may have a nucleotide sequence that is 90% or more identical, 95% or more identical, or 98% or more identical to the nucleotide sequence set forth in SEQ ID NO: 73, as long as it does not lose its function as a pUC ori sequence.

[0051] To suppress the expression of the type II restriction endonuclease gene at a basal level, it is preferable to include a repressor sequence and an operator sequence. The repressor sequence can be lacI, and the operator sequence can be lacO. The operator sequence can be inserted between the promoter sequence and the transcription initiation site.

[0052] The type II restriction endonuclease sequence is not particularly limited as long as it can recognize the above-mentioned type II restriction endonuclease recognition sequence, and may be codon-optimized.

[0053] The single vector may contain other genes as needed.

[0054] Only one type of single vector may be used, but multiple types may also be used as needed.

[0055] The genome editing method of this embodiment may include additional steps as necessary.

[0056] <Vector for Introducing a Type II Restriction Endonuclease Recognition Sequence> Another embodiment of the present invention is 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) an origin of replication sequence that functions in a host organism used for cloning the introduction vector but does not function in an organism or cell that is the target of genome editing, and (iv) a drug resistance gene sequence. The introduction vector of this embodiment may have a genome editing gene sequence inserted therein, or any genome editing gene sequence may be inserted therein as desired during use. Examples of regions into which a genome editing gene sequence can be inserted include regions containing a restriction enzyme sequence that can be treated with a desired restriction enzyme. It is preferable that the restriction enzyme sequence does not include a sequence contained in the vector.

[0057] In the transfer vector, the replication origin sequence may be the p15A ori sequence.

[0058] The introduction vector further comprises a constitutive expression promoter sequence in the organism or cell to be genome edited and a negative selection gene sequence operably positioned downstream thereof, and optionally, the negative selection gene sequence may be codon-optimized.

[0059] In the transfer vector, the constitutive expression promoter sequence may be an ermE promoter (PermE) sequence, and / or the negative selection gene sequence may be a codA gene sequence.

[0060] The vector for introduction may contain any other base sequence as necessary.

[0061] Methods known to those skilled in the art can be used to synthesize vectors. There are no particular limitations on the synthesis method, as long as the desired vector can be synthesized. For example, a method can be used in which a template sequence having a desired nucleotide sequence is amplified by PCR so that a desired nucleotide sequence is ligated to the end of the desired nucleotide sequence, a plasmid is treated with the desired restriction enzyme, and the amplified product and the restriction enzyme-treated plasmid are ligated by Gibson assembly.

[0062] In this embodiment, the descriptions regarding the genome editing target and various sequences can be found in the above <Genome editing method> section.

[0063] Another aspect of this embodiment is the use of the above vector to introduce a type II restriction endonuclease recognition sequence into a region to be edited in an organism or cell to be edited.

[0064] <Vector for expressing type II restriction endonuclease> Another embodiment of the present invention is a vector for expressing a type II restriction endonuclease, comprising: a promoter sequence that operates in an organism or cell that is a target for genome editing; and a type II restriction endonuclease sequence operably positioned downstream of the promoter sequence; and an origin of replication that operates in a host organism used for cloning of the expression vector but does not operate in the organism or cell that is a target for genome editing; a drug resistance gene sequence; and a temperature-sensitive origin of replication that operates in the organism or cell that is a target for genome editing.

[0065] In the expression vector, the promoter sequence may be a tipA promoter (PtipA) sequence, the replication origin sequence that functions in a host organism used for cloning the expression vector but does not function in the organism or cell to be genome-edited may be a pUC ori sequence, and / or the temperature-sensitive replication origin sequence that functions in the organism or cell to be genome-edited may be a pSG5rep sequence.

[0066] The expression vector comprises 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 may be codon-optimized.

[0067] In the expression vector, the operator sequence may be a lacO sequence and / or the repressor sequence may be a lacI sequence.

[0068] The expression vector may contain any other base sequence as needed.

[0069] Methods known to those skilled in the art can be used to synthesize vectors. There are no particular limitations on the synthesis method, as long as the desired vector can be synthesized. For example, a method can be used in which a template sequence having a desired nucleotide sequence is amplified by PCR so that a desired nucleotide sequence is ligated to the end of the desired nucleotide sequence, a plasmid is treated with the desired restriction enzyme, and the amplified product and the restriction enzyme-treated plasmid are ligated by Gibson assembly.

[0070] In this embodiment, the descriptions regarding the genome editing target and various sequences can be found in the above <Genome editing method> section.

[0071] Another aspect of this embodiment is the use of the vector described above to express a type II restriction endonuclease in an organism or cell to be genome-edited.

[0072] <Single Vector> 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 functions in the organism or cell that is the target of genome editing and a type II restriction endonuclease sequence operably arranged downstream of the promoter sequence; (iv) an origin of replication sequence that functions in a host organism used for cloning the vector but does not function in the organism or cell that is the target of genome editing; and (v) a drug resistance gene sequence.

[0073] In the single vector, the promoter sequence may be a tipA promoter (PtipA) sequence, and / or the replication origin sequence that functions in a host organism used for cloning the single vector but does not function in the organism or cell to be genome edited may be a pUC ori sequence.

[0074] The single vector comprises 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 codon-optimized.

[0075] In a single vector, the operator sequence may be a lacO sequence and / or the repressor sequence may be a lacI sequence.

[0076] Methods known to those skilled in the art can be used to synthesize vectors. There are no particular limitations on the synthesis method, as long as the desired vector can be synthesized. For example, a method can be used in which a template sequence having a desired nucleotide sequence is amplified by PCR so that a desired nucleotide sequence is ligated to the end of the desired nucleotide sequence, a plasmid is treated with the desired restriction enzyme, and the amplified product and the restriction enzyme-treated plasmid are ligated by Gibson assembly.

[0077] Other aspects of this embodiment include the use of the vector to introduce a type II restriction endonuclease recognition sequence into a region to be genome-edited in an organism or cell to be genome-edited, and to express a type II restriction endonuclease in an organism or cell to be genome-edited.

[0078] <Kit> Another embodiment of the present invention is a kit for deleting, substituting or inserting a targeted base sequence in a region to be edited in an organism or cell to be genome-edited, the kit comprising: (i) a vector described above in the section <Vector for introducing a type II restriction endonuclease recognition sequence> and a vector described above in the section <Vector for expressing a type II restriction endonuclease>, or (ii) a vector described above in the section <Single vector>.

[0079] The kit of this embodiment may also include any other reagents and / or tools. Examples of the reagents include primers, PCR reagents, controls, restriction enzymes, restriction enzyme treatment reagents, cloning host cells, culture media, diluents, buffers, water, purification reagents, etc. Examples of the tools include 96-well plates, reaction tubes, etc.

[0080] In this embodiment, the descriptions regarding the genome editing target and various vectors can be referenced from the descriptions in the above sections <Genome editing method>, <Vector for introducing type II restriction endonuclease recognition sequence>, <Vector for expressing type II restriction endonuclease>, and <Single vector>.

[0081] Another aspect of this embodiment is the use of the above kit to perform deletion, substitution, or insertion of a targeted base sequence in a genome editing target region in an organism or cell to be genome edited.

[0082] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0083] Example 1: Confirmation of the number of PacI sites in actinomycete genomes and investigation of the principles of genome editing. Actinomycete genomes are known to have a high GC content. Therefore, theoretically, the frequency of base sequences consisting only of A and T beyond a certain length is extremely low. For example, PacI derived from Pseudomonas alcaligenes is a type II restriction endonuclease that recognizes 5'-TTAATTAA-3'. Therefore, the number of PacI sites in the genomes of major actinomycetes was investigated. The results showed that the genomes of major actinomycetes do not have PacI sites, or if they do, they have very few, indicating that PacI does not cleave the genomes of many types of actinomycetes (Table 1).

[0084]

[0085] These results suggest that by introducing a PacI recognition sequence near the target gene and heterologously expressing PacI in an actinomycete host, a site-specific DSB can be induced, followed by homologous recombination repair, allowing the desired region to be deleted without leaving behind any drug resistance genes or sequences derived from the heterologous organism.

[0086] The Cas9 enzyme derived from Streptococcus pyogenes, which has been widely used for genome editing, tolerates a certain level of mismatches in the protospacer-adjacent motif (PAM). Therefore, editing the genomes of actinomycetes, which have a high GC content and many repetitive sequences, carries the risk of frequent off-target editing and toxicity due to nonspecific cleavage of the host genome. Furthermore, Cas9 has a length of 1368 amino acid residues and a large molecular weight, which inevitably increases the size of the editing plasmid, posing another obstacle to genome editing in actinomycetes, which are difficult to transform.

[0087] On the other hand, PacI is a homodimeric enzyme consisting of a polypeptide chain of only 142 amino acid residues, the smallest among known nucleases, which allows for significant miniaturization of editing plasmids. Therefore, genome editing using PacI will enable rational molecular breeding without causing off-target effects, which is expected to greatly contribute to the future development of natural product drug discovery.

[0088] Therefore, the present inventors have investigated genome editing using PacI and have conceived of genome editing based on the method exemplified below (Figure 1). First, when deleting a target nucleotide sequence by genome editing, a sequence linking the 5' and 3' outer regions of the target nucleotide sequence in the genome editing target region is amplified by PCR, homologous arms are prepared, and the amplified vector is inserted into a mobilization vector containing a PacI site to construct a vector (hereinafter also referred to as a targeting vector) that introduces a type II restriction endonuclease recognition sequence into the editing target region. By introducing this vector into a host, an editing intermediate is obtained in which the full length of the vector containing the PacI site is inserted into the genome by single-crossover homologous recombination. Next, a PacI expression vector is introduced and PacI expression is induced by adding an inducer, inducing specific DSBs. Subsequently, a fully edited mutant is selected that has been restored by homologous recombination repair using the previously introduced homologous arms. Finally, the PacI expression vector, which contains a temperature-sensitive replication origin, is removed from the edited mutants by incubation at high temperature, yielding edited mutants that do not contain foreign genes. Furthermore, by retaining the PacI expression vector and introducing the next targeting vector, this design allows for multiple genome modifications in a single step. In experiments to verify the principles of genome editing in actinomycetes, a widely used method is to select biosynthetic genes for pigmented antibiotics (actinorhodin and undecylprodigiosin (RED)) as target genes and visually distinguish the wild-type strain from the edited mutants on a plate. Therefore, the inventors used the model actinomycete Streptomyces coelicolor A3(2) (strain name: Streptomyces violaceoruber NBRC15146), which is a high-yield producer of the blue antibiotic actinorhodin, to select the actinorhodin biosynthetic gene cluster (21.6 kbp) in this actinomycete as the target gene (Figure 2), and conducted a verification experiment of the above-mentioned genome editing principle by performing scarless editing of the actinorhodin biosynthetic gene cluster.

[0089] Example 2: Construction of a genome editing vector 1. Vector for introducing a PacI recognition sequence (pPacIint During the genome editing process, edited intermediate strains may remain at a certain frequency. However, by introducing a negative selection marker gene into the edited intermediate strain along with the PacI recognition sequence, unedited intermediate strains can be eliminated and fully edited strains can be selectively obtained. Regarding negative selection systems in actinomycete strains, for example, the codon-optimized Escherichia coli-inducible cytosine deaminase (codA) gene is known to function as a negative selection marker in the presence of 5-fluorocytosine (5-FC) in some actinomycete strains. DNA was chemically synthesized containing the codA gene, used as a negative selection marker, downstream of a constitutive promoter (PermE). This codA gene was codon-optimized to increase translation efficiency within actinomycete cells. Using a method known to those skilled in the art, this synthetic gene (PermE-codAsm (SEQ ID NO: 1)) was amplified by PCR using the primers shown in Table 2, and then inserted into the NheI-HindII site of pK18mob to construct pK18mob-codAsm.

[0090]

[0091] Next, the replication origin p15A ori located in the 848-1393 bp region of pACYC184 (Accession No.: X06403) was amplified by PCR using the primers listed in Table 3. The primers were designed to add a PacI recognition sequence upstream of the amplified product.

[0092]

[0093] pK18mob-codA-sm, which had been digested and opened with MseI in advance, was mixed with p15A ori amplified by PCR and ligated by the Gibson assembly method. int (SEQ ID NO: 6, Figure 3) and was used in subsequent validation of the effectiveness of this genome editing system.

[0094] 2. Construction of PacI Expression Vector (pELIM) (1) Confirmation of the Efficacy of a Thiostrepton-Inducible Promoter Based on gfp Gene Expression A broad-host-range inducible promoter is required to induce DSB introduction by time-specifically expressing PacI in a wide range of actinomycete host cells. The effectiveness of the thiostrepton-inducible PtipA promoter has been demonstrated in several actinomycete strains. Therefore, a thiostrepton-inducible promoter (PtipA (SEQ ID NO: 7)) was constructed by oligonucleotide assembly using the primers shown in Table 4, based on the sequence of the pIJ6021 plasmid (Accession No. AJ414669).

[0095]

[0096] Subsequently, codon-optimized / chemically synthesized GFP uv The gene (SEQ ID NO: 12) was used as a template and amplified by PCR using the primers in Table 5.

[0097]

[0098] Using the pLAE001 plasmid (SEQ ID NO: 15) constructed in Patent Document 1 as a template and the primers in Table 6, the thiostrepton resistance gene (tsr (SEQ ID NO: 16)) was amplified by PCR.

[0099]

[0100] The pSET152 plasmid (Accession No. AJ414670) was digested and opened with XbaI, and PCR-amplified PtipA and gfp uv pSET152-PtipA-gfp, which allows inducible expression of GFP under the control of PtipA, was created by mixing the tsr gene and ligating it using the Gibson assembly method. uv -tsr (SEQ ID NO: 19) was constructed (Figure 4).

[0101] After genome editing is complete, the type II restriction endonuclease expression vector must be removed from the cells. It is known that modified plasmids carrying the replication origin of the temperature-sensitive plasmid pSG5 (pSG5rep) are efficiently removed from various actinomycete host strains by culturing them at temperatures above 39°C. Therefore, pSET152-PtipA-gfp uv The φC31 integrase element of -tsr was replaced with pSG5rep. pSG5rep (SEQ ID NO: 20) was chemically synthesized (outsourced to Eurofins Genomics) based on the sequence of pSG5 (Accession No. NC_008792.1). The pSG5rep fragment was excised from the pEX-A2J2 plasmid into which the chemically synthesized pSG5rep had been cloned by double restriction digestion with XbaI and SphI. This was then treated with the same restriction enzymes to create pSET152-PtipA-gfp, from which the φC31 integrase element had been excised. uv -tsr and pSET152-PtipA-gfp uv -pSG5rep (hereinafter also referred to as pSET152-PtipA-gfp-tsr-pSG5rep (SEQ ID NO: 21)) was constructed (Figure 5).

[0102] Next, the functionality of PtipA was evaluated based on the expression of GFPuv. The constructed pSET152-PtipA-gfp-tsr-pSG5rep was transfected into Streptomyces albulus NBRC14147 (National Institute of Technology and Evaluation (NITE) Biotechnology Center (NBRC)), Streptomyces lividans TK23 (ATCC), and Streptomyces coelicolor A3(2) (strain: Streptomyces violaceoruber NBRC15146) (National Institute of Technology and Evaluation (NITE) Biotechnology Center (NBRC)) via conjugal transfer via E. coli ET12567 (ATCC). The transformants were inoculated into TSBY liquid medium (TSB medium (Becton, Dickinson and Company) supplemented with 0.5% yeast extract) containing apramycin (Apr) (Sigma-Aldrich, 50 μg / ml) and cultured at 30°C with shaking until stationary phase was reached. The transformants were then subcultured into fresh TSBY liquid medium containing Apr (50 μg / ml) and cultured at 30°C with shaking for 8 hours. After the addition of thiostrepton, various concentrations of thiostrepton (Sigma-Aldrich) were added to induce GFPuv expression. After the addition of thiostrepton, the transformants were cultured for an additional 24 hours with shaking, and the cells were harvested by centrifugation. Fluorescence at an excitation wavelength of 380 nm was measured for cell-free extracts prepared by sonication. The GFPuv-derived fluorescence at around 510 nm increased in a thiostrepton concentration-dependent manner in all strains (Figures 6–8). This confirmed that PtipA can be used to control downstream gene expression in a thiostrepton concentration-dependent manner in various actinomycete hosts.

[0103] (2) Confirmation of temperature sensitivity based on pSG5rep Next, we also verified the temperature-sensitive replication of pSET152-PtipA-gfp-tsr-pSG5rep in various actinomycetes. uvpSG5rep was introduced into Streptomyces albulus NBRC14147, Streptomyces lividans TK23, and Streptomyces coelicolor A3(2) (strain: Streptomyces violaceoruber NBRC15146) via conjugation with E. coli ET12567, and transformants were selected for apramycin resistance. Each transformant was cultured in TSBY liquid medium containing apramycin for 48 hours with shaking at 30°C. The stationary-phase cultures were plated onto antibiotic-free agar plates and incubated at 39°C for 72 hours to induce plasmid loss. As a control, the same cultures were plated onto apramycin-containing agar plates and incubated at 39°C for 3 days. Cell growth in the presence of apramycin was significantly reduced, suggesting that high-temperature incubation induced plasmid loss. Furthermore, the recombinant strains in which the plasmid was induced to be lost on antibiotic-free agar medium were apramycin-sensitive, and PCR analysis confirmed that the plasmid was not retained. These results confirmed that pSET152-PtipA-gfp-tsr-pSG5rep functions as a temperature-sensitive plasmid as designed.

[0104] (3) Study of pacI Expression Vectors We then cloned the pacI gene downstream of PtipA in the pSET152-PtipA-gfp-tsr-pSG5rep plasmid to construct a pacI expression vector. A synthetic pacI gene (SEQ ID NO: 22) was obtained, codon-optimized for actinomycetes, based on the sequence of pacI (Accession No. 3LDY_A) derived from Pseudomonas alcaligenes. Using this synthetic gene as a template, PCR was performed with the primers listed in Table 7 to obtain a pacI gene fragment with NdeI and BgllII recognition sequences ligated to both ends.

[0105]

[0106] pSET152-PtipA-gfp-tsr-pSG5rep was double-restricted with NdeI and BglII to obtain gfp uvThe gene was excised, and the pacI gene digested with NdeI and BglII was ligated using T4 DNA ligase. The ligation product was transformed into the cloning host E. coli NEB10β (New England Biolabs), but only a few transformants were obtained. Furthermore, sequencing of the plasmids obtained from several transformants revealed unexpected base substitutions and deletions in the pacI gene region. This suggests that the pacI gene placed downstream of PtipA may be cleaved in the host genome as a result of basal expression in the cloning host E. coli.

[0107] It is known that the lac repressor (lacI (SEQ ID NO: 25)) overexpressed in E. coli can control gene expression directly downstream of a promoter by binding to an operator sequence (lacO (SEQ ID NO: 26)) inserted between the -10 region of the promoter and the transcription start site. Therefore, we investigated the possibility of controlling basal expression from PtipA by inserting lacO between the -10 region and the transcription start site of PtipA. Using the constructed pSET152-PtipA-gfp-tsr-pSG5rep as a template, we inserted the lacO sequence into PtipA (PtipA(lacO) (SEQ ID NO: 27)), gfp uv The tsr gene and the tsr gene were amplified by PCR. The primers used for PCR amplification are shown in Table 8.

[0108]

[0109] These DNA fragments were mixed with pSET152 that had been digested and opened with EcoRV and XbaI, and ligated by the Gibson assembly method to obtain pSET152-PtipA(lacO)-gfp uv Furthermore, by following the method described above, the φC31 integrase element in the vector backbone was replaced with pSG5rep to construct pSET152-PtipA(lacO)-gfp-tsr-pSG5rep (SEQ ID NO: 34) ( FIG. 9 ).

[0110] Next, pSET152-PtipA (lacO)-gfp uvThe lacI gene was additionally introduced into pSG5 rep. The lacI gene (including the promoter region (SEQ ID NO: 35)) was amplified by PCR from pET21b(+) (Merck Millipore) using the primers in Table 9, and the resulting vector was transformed into pSET152-PtipA(lacO)-gfp. uv - pSET152-PtipA(lacO)-gfp by inserting it into the NheI site of pSG5rep uv -pSG5rep-lacI was constructed (Figure 10).

[0111]

[0112] Next, pSET152-PtipA-gfp, which contains an expression control system using the lac operator and lac repressor, was uv The pacI gene was cloned into pSG5rep-lacI. Using the chemically synthesized pacI gene as a template, the pacI gene was amplified by PCR using the primers in Table 7 to obtain a pacI gene with NdeI and BglII sites linked to both ends. The pacI gene was digested with NdeI and BglII and the pSET152-PtipA(lacO)-gfp uv The resulting plasmid was ligated with pSG5rep-lacI using T4 DNA ligase. A portion of this reaction mixture was transformed into E. coli NEB10β, and transformants were selected based on apramycin resistance. Basal expression of PacI was controlled by adding 5% (w / v) glucose to the LB liquid medium and LB agar medium used for cloning. The plasmid extracted and purified from the transformant was confirmed for construction by restriction mapping using EcoRV and NheI double digestion and DNA sequencing to construct pSET152-PtipA(lacO)-pacI-pSG5rep-lacI (SEQ ID NO: 38). This was used as the pacI expression vector (pELIM) in the subsequent proof-of-principle experiments.

[0113] Example 3: Scarless deletion of the actinorhodin biosynthetic gene cluster using the PacI genome editing system (proof of principle) (1) Introduction of a PacI recognition sequence into the actinorhodin biosynthetic gene cluster Using the genome extracted from Streptomyces violaceoruber NBRC15146 (strain name: Streptomyces coelicolor A3(2)) as a template, approximately 1 kbp of the 5' and 3' outer regions of the actinorhodin biosynthetic gene cluster (SEQ ID NO: 39) (regions surrounding the sco5071 and sco5092 genes (SEQ ID NO: 40 and SEQ ID NO: 41, respectively)) were amplified by PCR using the primers shown in Table 10.

[0114]

[0115] pPacI, which had been previously digested and opened with EcoRI and PstI int The two 1-kbp fragments amplified by PCR were mixed and ligated by the Gibson assembly method. A portion of this reaction mixture was used to transform E. coli NEB10β competent cells, and clones showing resistance to kanamycin (Fujifilm Wako Pure Chemical Industries) were selected. Plasmids extracted and purified by a method known to those skilled in the art were subjected to restriction mapping with NdeI and NheI to identify the actinorhodin biosynthetic gene cluster-specific targeting vector (pPacI int The construction of Δact (SEQ ID NO: 46) was confirmed (FIG. 11).

[0116] pPacI int Δact was introduced into Streptomyces violaceoruber NBRC15146 by conjugation with E. coli S17-1 (ATCC). Genomic DNA extracted from a candidate strain for an editing intermediate that exhibited resistance to neomycin (Fujifilm Wako Pure Chemical Industries) was used as a template. PCR verification using the primers shown in Table 11 confirmed the creation of an editing intermediate in which the PacI recognition sequence shown in the lower panel of Figure 12 had been inserted into the genome.

[0117]

[0118] (2) Scarless deletion of the actinorhodin biosynthetic gene cluster by PacI expression. The constructed PacI expression plasmid pELIM was introduced into the edited intermediate strain by conjugation with E. coli S17-1. PacI expression from pELIM was designed to be induced by thiostrepton addition. However, when pELIM-introduced intermediate strains were selected for apramycin resistance, we confirmed that blue pigment-producing mutants were generated without the need for thiostrepton induction (Figure 13; the circled colonies are blue pigment-producing mutants). This result suggests that PacI expression was basal immediately after vector introduction, even without the need for thiostrepton induction, leading to the introduction of DSBs into the genome, which in turn induced homologous recombination repair and resulted in the formation of edited mutants. Therefore, two clones each of blue and brown colonies were picked and cultured in TSB (10% sucrose) liquid medium for 3 days to confirm the presence or absence of pigment production. As a result, the blue clones showed vigorous actinorhodin production, while the brown clones did not produce actinorhodin, strongly suggesting the possibility that simply introducing pELIM could result in the generation of edited strains in which the actinorhodin biosynthetic gene cluster was deleted (Figure 14; the two samples on the left are actinorhodin-producing strains, and the two samples on the right are actinorhodin-nonproducing strains).

[0119] Next, genomic DNA was extracted from these fungal cells, and verification (genotype analysis) was performed by PCR using primers (Table 12) designed to amplify the central region of the actinorhodin biosynthetic gene cluster (sequence number 49).

[0120]

[0121] We also reconfirmed pigment production (phenotypic analysis) in R5 medium, a medium for secondary metabolite production. Genotypic analysis confirmed PCR amplification in only blue colonies, a result completely consistent with the phenotypic analysis in R5 medium (Figure 15). These results confirmed that the blue clones, accounting for approximately 92% of all transformants, were revertants, while the remaining 8% of brown clones were fully edited. Furthermore, all blue and brown clones were neomycin-sensitive, confirming that no editing intermediates remained after pELIM introduction.

[0122] Next, to confirm whether the target region was accurately deleted, sequence analysis of the region surrounding the edited region was performed using the primers shown in Table 13.

[0123]

[0124] The wild-type revertant strain was analyzed for the 5'- and 3'-end regions of the actinorhodin biosynthetic gene cluster, while the edited mutants were analyzed for the junction regions of the 5'- and 3'-end regions. The blue clones were confirmed to have completely reverted to the wild-type sequence, while the brown clones confirmed that the target region had been precisely deleted as designed (Figure 16). Furthermore, by culturing the edited mutant strains at 40°C for 5 days, we were able to obtain apramycin-sensitive clones in which pELIM had been lost. These validation experiments demonstrated that this method, using the type II restriction enzyme PacI, can specifically introduce DSBs into the actinomycete genome, enabling precise genome editing without leaving any foreign genes behind.

[0125] Next, to confirm the effect of deletion length on genome editing efficiency, we deleted actVB (560 bp), which is located at the end of the biosynthetic gene cluster. Using the genome extracted from Streptomyces violaceoruber NBRC15146 as a template, approximately 1 kbp of the 5' and 3' outer regions of actVB (SEQ ID NO: 56) (SEQ ID NO: 57 and SEQ ID NO: 58, respectively) were amplified by PCR using the primers shown in Table 14.

[0126]

[0127] The two PCR fragments obtained were inserted into the Mobilization vector (pPacI) which had been previously digested and opened with EcoRI and PstI. int ) and ligated by the Gibson assembly method. A portion of this reaction mixture was used to transform E. coli NEB10β competent cells, and transformants were selected based on kanamycin resistance. Restriction mapping with NdeI and NheI was performed using plasmids extracted and purified from the transformants, and the actVB gene cluster-specific targeting vector (pPacI int The construction of ΔactVB (SEQ ID NO: 63) was confirmed ( FIG. 17 ).

[0128] This pPacI int ΔactVB was introduced into Streptomyces violaceoruber NBRC15146 by conjugation with E. coli S17-1. Genomic DNA extracted from a candidate editing intermediate strain exhibiting neomycin resistance was used as a template, and PCR was performed using the primers shown in Table 15 to verify the identity of the editing intermediate Streptomyces violaceoruber NBRC15146 / pPacI, in which the PacI recognition sequence had been inserted into the genome. int The construction of ΔactVB was confirmed.

[0129]

[0130] Next, to induce genome editing by PacI expression, pELIM was transfected into Streptomyces violaceoruber NBRC15146 / pPacI by conjugation with E. coli S17-1. int The pELIM-introduced strains were introduced into ΔactVB. Because the pigment-producing ability is not lost by deleting only the actVB gene located at the end of the biosynthetic gene cluster, the success of genome editing cannot be confirmed based on the phenotype. Therefore, 20 clones of pELIM-introduced strains exhibiting apramycin resistance were randomly selected and verified by PCR using the primers shown in Table 16.

[0131]

[0132] As a result, 12 clones were confirmed to be revertants to the wild type and 8 clones to be actVB gene deletion strains (Figure 18). Furthermore, as with the full-length deletion of the actinorhodin biosynthetic gene cluster, we confirmed that no editing intermediates remained after pELIM introduction. Furthermore, as shown in Figure 19, sequence analysis of the area surrounding the edited region confirmed that all blue clones were not editing intermediates but had completely reverted to the original wild-type sequence, and brown clones were confirmed to have the actVB gene region precisely deleted without a single base error.

[0133] In the deletion of the 3'-terminal gene actVB (560 bp) of the actinorhodin biosynthetic gene cluster, 8 of 20 clones were fully edited, whereas in the deletion of the entire actinorhodin biosynthetic gene cluster, which can be visually determined using a dye, only 4 of 48 clones were fully edited and showed a brown color. These results confirmed that long-distance gene deletions tend to increase the frequency of revertants.

[0134] This invention successfully established a new genome editing technology platform that avoids off-target effects, the biggest challenge in genome editing in actinomycetes. Because this invention combines promoters and temperature-sensitive replication origins that function in a wide range of actinomycete species, it can be applied to the breeding of a wide range of industrial actinomycete strains, not just the actinomycete strains exemplified in the Examples. Therefore, further improvements to this method are expected to pave the way for the molecular breeding of a wide range of industrial actinomycete strains.

[0135] Example 4: Construction of a single-vector PacI genome editing system and scarless deletion of the actinorhodin biosynthetic gene cluster using the system pPacI int Although we were able to demonstrate the effectiveness of two-step genome editing using two types of plasmids, the pELIM plasmid and the pELIM plasmid, it takes approximately 40 days to introduce the plasmids twice into the difficult-to-transform actinomycete, followed by selection of fully edited cells and the loss of pELIM. Therefore, we attempted to speed up the entire process by performing one-step genome editing in which only the elements essential for editing are integrated into a single vector.

[0136] For pELIM, pPacI int By adding the actinorhodin biosynthetic gene group targeting sequence (regions surrounding the sco5071 and sco5092 genes (sequence numbers 40 and 41, respectively)) and a PacI recognition sequence to Δact (sequence number 46), we decided to construct an actinorhodin biosynthetic gene group-specific editing vector that can manipulate, with a single vector, the insertion of the PacI recognition sequence into the genome, the induction of double-strand breaks by PacI expression, and the editing of the desired region by homologous recombination repair.

[0137] Using the primers shown in Table 17, approximately 1 kbp each of the 5' and 3' outer regions of the actinorhodin biosynthetic gene cluster (SEQ ID NO: 39) (regions surrounding the sco5071 and sco5092 genes (SEQ ID NO: 74 and SEQ ID NO: 75, respectively)) were amplified by PCR.

[0138]

[0139] pELIM, which had been previously digested and opened with XbaI and SphI, was mixed with two 1-kbp fragments amplified by PCR and ligated by Gibson assembly. A portion of this reaction mixture was used to transform E. coli NEB10β competent cells, and apramycin-resistant clones were selected. Plasmids extracted and purified by methods known to those skilled in the art were subjected to restriction mapping with XbaI and SphI to confirm the construction of a single actinorhodin biosynthetic gene cluster-specific editing vector (pELIM-Δact1 (SEQ ID NO: 78)) (Figure 20).

[0140] pELIM-Δact1 was introduced into Streptomyces violaceoruber NBRC15146 by conjugation with E. coli S17-1 (ATCC). A single-crossover homologous recombinant strain exhibiting apramycin resistance was selected as an editing intermediate in which the full-length pELIM-Δact1 had been inserted into the genome.

[0141] The selected colonies were scratched several times with the tip of a sterile toothpick and then streaked onto agar medium containing 2 mM IPTG. After culturing at 30°C for 3 days, it was confirmed that a strain lacking blue pigment production had emerged, as expected.

[0142] Two clones each from blue and brown colonies were picked and cultured in TSB (10% sucrose) liquid medium for 3 days to confirm pigment production. The blue clones showed vigorous actinorhodin production, while the brown clones did not. This strongly suggested that a single editing vector had led to the deletion of the actinorhodin biosynthetic gene cluster (Figure 21).

[0143] Genomic DNA was extracted from these fungi and subjected to PCR verification (genotype analysis) using primers designed to amplify the central region of the actinorhodin biosynthetic gene cluster (SEQ ID NO: 49) (primers listed in Table 12 above). This region was not detected. Furthermore, sequence analysis of the edited region using the primers listed in Table 13 above confirmed that the target region had been accurately deleted, as in the case of two-step editing.

[0144] This single-vector, one-step genome editing allows all operations up to editing to be performed with a single plasmid introduction, and since the introduced editing plasmid does not remain in the cells after editing, there is no need for plasmid curing. While two-step genome editing using two types of plasmids required approximately 40 days from the construction of an editing vector specific to the editing target region to the completion of editing, one-step genome editing can achieve complete editing in about 20 days.

[0145] The editing efficiency of one-step genome editing was comparable to that of two-step genome editing. This example demonstrates the scarless deletion (knockout) of a specific actinomycete genome using the type II restriction enzyme PacI. However, it is also possible to insert any sequence into a desired region (knock-in) by pre-inserting the sequence between the homologous arms of the editing vector.

Claims

1. A method for deleting, substituting or inserting a targeted base sequence in a region to be edited in an organism or cell that is the target of genome editing, comprising: (1) a step of introducing a type II restriction endonuclease recognition sequence into the region to be edited in the genome; and (2) a step of generating a double-stranded 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, wherein after steps (1) and (2) are performed, the target region is edited in the organism or cell via homologous recombination repair with a gene sequence for genome editing, and wherein the GC content in the genome of the organism or cell is 65% or more.

2. The method according to claim 1, wherein the type II restriction endonuclease recognizes a base sequence consisting of only A and T.

3. The method of claim 1, wherein the type II restriction endonuclease is PacI.

4. The method of claim 1, wherein said organism is a eukaryote or prokaryote that does not have said Type II restriction endonuclease recognition sequence in its genome.

5. The method of claim 4, wherein the organism is an actinomycete.

6. The method according to claim 5, wherein the actinomycete does not have a recognition sequence for PacI in its genome.

7. The method of claim 5, wherein the actinomycete is selected from the group consisting of the genera Streptomyces, Saccharopolyspora and Amycolaptosis.

8. The method of claim 5, wherein the actinomycete is selected from the group consisting of Streptomyces fradiae, Streptomyces venezuelae, Streptomyces griseus, Streptomyces albulus, Streptomyces coelicolor, Streptomyces rimosus, Streptomyces spectabilis, Streptomyces avermitilis, Amycolatopsis orientalis, and Saccharopolyspora spinosa.

9. The method according to any one of claims 1 to 8, wherein the introduction in step (1) is carried out using a vector for introducing a type II restriction endonuclease recognition sequence, and the vector for introduction is a vector comprising (i) a gene sequence for genome editing, (ii) a type II restriction endonuclease recognition sequence, (iii) a replication origin sequence that operates in a host organism used for cloning the vector for introduction and that does not operate in an organism or cell that is a target of genome editing, and (iv) a drug resistance gene sequence.

10. The method of claim 9, wherein the replication origin sequence is a p15A ori sequence.

11. The method according to claim 9, wherein the introduction vector further comprises a constitutive expression promoter sequence in the genome editing target organism or cell and a negative selection gene sequence operably positioned downstream thereof, and optionally, the negative selection gene sequence is codon optimized.

12. The method according to claim 11, wherein the constitutive expression promoter sequence is an ermE promoter (PermE) sequence and / or the negative selection gene sequence is a codA gene sequence.

13. The method according to claim 11, further comprising the step of (3) selecting cells into which a type II restriction endonuclease recognition sequence has been introduced using the negative selection gene.

14. The method according to any one of claims 1 to 8, wherein the step (2) is carried out using an expression vector for a type II restriction endonuclease that recognizes the introduced type II restriction endonuclease recognition sequence, the expression vector comprising a promoter sequence that operates in the organism or cell to be genome-edited and a type II restriction endonuclease sequence operably positioned downstream thereof, and a replication origin sequence that operates in a host organism used for cloning of the expression vector and does not operate in the organism or cell to be genome-edited, a drug resistance gene sequence, and a temperature-sensitive replication origin sequence that operates in the organism or cell to be genome-edited.

15. The method according to claim 14, wherein the promoter sequence is an inducible promoter sequence, the replication origin sequence that operates in a host organism used for cloning the expression vector and does not operate in the organism or cell to be subjected to genome editing is a pUC ori sequence, and / or the temperature-sensitive replication origin sequence that operates in the organism or cell to be subjected to genome editing is a pSG5rep sequence.

16. The method of claim 15, wherein the inducible promoter sequence is the tipA promoter (PtipA) sequence.

17. The method of claim 14, wherein the expression vector comprises 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 is codon-optimized.

18. The method of claim 17, wherein the operator sequence is a lacO sequence and / or the repressor sequence is a lacI sequence.

19. The method according to claim 14, further comprising the step of: (4) 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, wherein the introduction of a type II restriction endonuclease recognition sequence in step (1) and the expression of a type II restriction endonuclease that recognizes the type II restriction endonuclease recognition sequence in step (2) are carried out using a single vector, wherein the single vector comprises: (i) a gene sequence for genome editing; (ii) a type II restriction endonuclease recognition sequence; (iii) a promoter sequence that operates in the organism or cell that is the target of genome editing and a type II restriction endonuclease sequence operably arranged downstream thereof; (iv) a replication origin sequence that operates in a host organism used for cloning of the single vector and that does not operate in the organism or cell that is the target of genome editing; and (v) a drug resistance gene sequence.

21. The method according to claim 20, wherein the promoter sequence is an inducible promoter sequence, and / or the replication origin sequence that operates in a host organism used for cloning the single vector and does not operate in an organism or cell to be subjected to genome editing is a pUC ori sequence.

22. The method of claim 21, wherein the inducible promoter sequence is the tipA promoter (PtipA) sequence.

23. The method of claim 20, wherein the single vector comprises 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 is codon-optimized.

24. The method of claim 23, wherein the operator sequence is a lacO sequence and / or the repressor sequence is a lacI sequence.

25. A vector for introducing a type II restriction endonuclease recognition sequence, 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) an origin of replication sequence that functions in a host organism used for cloning the vector for introduction and that does not function in an organism or cell to be subjected to genome editing; and (iv) a drug resistance gene sequence.

26. The introduction vector of claim 25, wherein the replication origin sequence is a p15A ori sequence.

27. The introduction vector described in claim 25, further comprising a constitutive expression promoter sequence in the organism or cell to be genome edited and a negative selection gene sequence operably positioned downstream thereof, and optionally, the negative selection gene sequence is codon optimized.

28. The introduction vector according to claim 27, wherein the constitutive expression promoter sequence is an ermE promoter (PermE) sequence, and / or the negative selection gene sequence is a codA gene sequence.

29. A type II restriction endonuclease expression vector comprising a promoter sequence that operates in an organism or cell to be genome edited and a type II restriction endonuclease sequence operably positioned downstream of the promoter sequence, as well as a replication origin sequence that operates in a host organism used for cloning of the expression vector and does not operate in the organism or cell to be genome edited, a drug resistance gene sequence, and a temperature-sensitive replication origin sequence that operates in the organism or cell to be genome edited.

30. The expression vector described in claim 29, wherein the promoter sequence is a tipA promoter (PtipA) sequence, the replication origin sequence that operates in a host organism used for cloning the expression vector and does not operate in the organism or cell to be subjected to genome editing is a pUC ori sequence, and / or the temperature-sensitive replication origin sequence that operates in the organism or cell to be subjected to genome editing is a pSG5rep sequence.

31. The expression vector of claim 29, wherein the expression vector comprises an operator sequence and a repressor sequence, the operator sequence being inserted between the promoter sequence and a transcription start site, and optionally, the Type II restriction endonuclease sequence is codon optimized.

32. An expression vector according to claim 31, wherein the operator sequence is a lacO sequence and / or the repressor sequence is a lacI sequence.

33. A single vector for introducing a type II restriction endonuclease recognition sequence and 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 operates in the organism or cell to be the target of genome editing and a type II restriction endonuclease sequence operably arranged downstream of the promoter sequence; (iv) a replication origin sequence that operates in a host organism used for cloning of the vector and does not operate in the organism or cell to be the target of genome editing; and (v) a drug resistance gene sequence.

34. The single vector described in claim 33, wherein the promoter sequence is a tipA promoter (PtipA) sequence, and / or the replication origin sequence that operates in a host organism used for cloning of the single vector and does not operate in an organism or cell to be subjected to genome editing is a pUC ori sequence.

35. The single vector of claim 33, wherein the single vector comprises 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 codon optimized.

36. The single vector according to claim 35, wherein the operator sequence is a lacO sequence and / or the repressor sequence is a lacI sequence.

37. A kit for deleting, substituting or inserting a targeted base sequence in a region to be edited in a genome in an organism or cell to be edited, comprising: (i) an introduction vector according to any one of claims 25 to 28 and an expression vector according to any one of claims 29 to 32; or (ii) a single vector according to any one of claims 33 to 36.

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