DNA editing method and cell manufacturing method using the same, and DNA editing vector and DNA editing kit for use in these methods.

Abstract

Provided is a DNA editing method in which a desired nucleotide sequence is inserted into a target site in target DNA in a cell, or the target site is removed. The method includes an introduction step for introducing a vector and a site-specific nuclease system into the cell so as to come into contact with the target DNA, wherein the vector includes a first promoter P1 and a structure (1): 5'-M1-Hv1-D-Hv2-M2-3' wherein M1 represents a 5'-side fragment of a nucleotide sequence encoding a selection marker gene and is operably linked to P1, Hv1 represents a nucleotide sequence homologous to a first nucleotide sequence Ht1 located on the 5'-side of the target site in the target DNA, D represents the desired nucleotide sequence and may be absent, Hv2 represent a nucleotide sequence homologous to a second nucleotide sequence Ht2 located on the 3'-side of the target site in the target DNA, and M2 represents a remaining 3'-side fragment of the nucleotide sequence encoding the selection marker gene. The method also includes: a cleavage step in which a fragment represented by a structure (2): 5'-Hv1-D-Hv2-3' and a fragment represented by a structure (3): 5'-M2-P1-M1-3' are generated from the vector using the site-specific nuclease system, and the target site or a portion surrounding the target site is cleaved in the target DNA; an editing step in which the target DNA and the structure (2) are bound to each other depending on the homology between Ht1 and Hv1 and are bound to each other depending on the homology between Ht2 and Hv2, so that the desired nucleotide sequence D is inserted into the target site or the target site is removed; and a selection step in which a 3'-end of M1 and a 5'-end of M2 are bound to each other in the fragment represented by the structure (3) to produce a selection vector containing a selection marker gene that is operably linked to P1 and is capable of functioning.

Description

Technical Field

[0001] The present invention relates to a DNA editing method, a cell manufacturing method using the same, a DNA editing vector for use in these methods, and a DNA editing kit.

Background Art

[0002] In recent years, the development of DNA editing techniques in useful organisms has been actively carried out. For example, site-specific nuclease systems such as the CRISPR-Cas system, a system in which a nuclease is fused to a TAL effector (TALE) (TALEN), and a system in which a nuclease is fused to a zinc finger (ZF) are used to specifically cleave a target site or its vicinity of a target DNA in a cell, and a method for editing the nucleotide sequence of the target site has been developed.

[0003] In these DNA editing techniques, a method of introducing into a cell a nucleotide sequence encoding a selection marker gene for confirming the editing is mainly adopted. For example, Non-Patent Document 1 (Yan et al., Molecular Therapy: Nucleic Acids, Vol. 19 March, 2020, p. 775-789) describes a method of introducing into a cell a vector containing the CRISPR-Cas system and an inactive puromycin resistance gene, and selecting cells expressing the CRISPR-Cas system by activating the inactive puromycin resistance gene by the cleavage activity of the CRISPR-Cas system. Further, for example, Non-Patent Document 2 (Mashiko et al., Develop.Growth Differ. 2014, 56, p. 122-129) describes a method of introducing into a cell a vector in which a sequence cleaved by the CRISPR-Cas system is sandwiched between two nucleotide sequences encoding GFP, a fluorescent protein, and generating fluorescence in cells expressing the CRISPR-Cas system to select them.

[0004] However, techniques for using these methods in DNA insertion methods to insert nucleotide sequences into target DNA have not yet been developed. Furthermore, in DNA insertion methods, the orientation and position of the inserted nucleotide sequence must be precise. Known DNA insertion methods include, for example, adding sequences homologous to 500-2000 bases of the nucleotide sequences at both ends of the target sequence to both ends of the desired nucleotide sequence, and then exchanging the desired nucleotide sequence with the target sequence by homologous recombination (HR), or inserting the desired nucleotide sequence into the target sequence. However, this method has drawbacks such as the need for long-chain vectors, making production difficult, low homologous recombination efficiency, and limited applicability to certain cells. To overcome these drawbacks, other DNA insertion methods are known that utilize microhomology-mediated end joining (MMEJ), which uses short homologous sequences of approximately 20 bases. For example, Patent Document 1 (International Publication No. 2015 / 068785) describes a method in which a site-specific nuclease system and a vector containing a desired nucleotide sequence are introduced into cells to insert the desired nucleotide sequence into a predetermined site.

[0005] However, conventional DNA insertion techniques require the introduction of sequences encoding the aforementioned selection marker genes to confirm the insertion, resulting in the presence of foreign genes other than the target nucleotide sequence in the resulting cells. Cells containing such foreign genes face various limitations in their use as genetically modified cells, thus reducing their usefulness.

[0006] As a method for removing foreign genes other than the target nucleotide sequence, for example, Non-Patent Document 3 (Kurita et al., Scientific reports, 2022, 12:2480, https: / / doi.org / 10.1038 / s41598-022-06495-y) describes a method in which an all-in-one PtTALEN-ARS vector encoding nucleotide sequences encoding CEN / ARS and TALEN is used, TALEN is introduced into cells to achieve the desired cleavage, and then the cells are cultured to remove the all-in-one PtTALEN-ARS vector from the cells. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2015 / 068785 [Non-patent literature]

[0008] [Non-Patent Document 1] Yan et al.,Molecular Therapy:Nucleic Acids,Vol.19 March,2020,p.775~789 [Non-Patent Document 2] Mashiko et al.,Develop.Growth Differ.2014,56,p.122~129 [Non-Patent Document 3] Kurita et al.,Scientific reports,2022,12:2480,https: / / doi.org / 10.1038 / s41598-022-06495-y [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The present invention has been made in view of the problems of the prior art described above, and aims to provide a DNA editing method that can accurately and efficiently insert a desired nucleotide sequence into a target site of target DNA in a cell using a vector, or remove a target site, a cell manufacturing method using the same, and a DNA editing vector and a DNA editing kit for use in these methods. [Means for solving the problem]

[0010] To achieve the above objective, the inventors diligently conducted research and first attempted to insert an endogenous promoter (ProLDSP) into the target site immediately before the start codon of the target gene (LPAT1) in genomic DNA. The DNA to be inserted was a nucleotide fragment (knock-in fragment) in which homologous sequences (homorogy arms) were added to both ends of the nucleotide sequence of ProLDSP, one being the sequence immediately before the start codon (first nucleotide sequence) and the other being the sequence after the start codon (second nucleotide sequence) of LPAT1, respectively. Furthermore, a site-specific nuclease, TALEN, was designed to cleave the target site or its vicinity. It was thought that by introducing these into cells, the genomic DNA would be cleaved by TALEN, and the ends of the nucleotide fragment would bind to the first and second nucleotide sequences of the genomic DNA in a homology-dependent manner, thereby inserting ProLDSP into the target site and overexpressing LPAT1, a gene related to membrane lipid synthesis. Furthermore, by introducing and expressing an expression vector (all-in-one PtTALEN-ARS vector) described in Non-Patent Literature 3, which includes CEN / ARS as an autonomous replication sequence, it is possible to remove it by culture after DNA insertion, thus making it possible to obtain DNA-edited cells that do not contain foreign genes other than ProLDSP (Figure 4).

[0011] However, the above method made it difficult to obtain the desired DNA-edited cells into which the nucleotide fragment was inserted. Therefore, the inventors conducted further investigations and concluded that one of the reasons for this was that, in the above method, the selection method for cells into which both the nucleotide fragment and the site-specific nuclease expression vector (all-in-one PtTALEN-ARS vector) were simultaneously introduced depended solely on the selection marker derived from the site-specific nuclease expression vector, resulting in low efficiency in obtaining cells into which both were introduced. Thus, a new method was constructed. Specifically, in the above method, the nucleotide fragment to be introduced was designed on a vector containing a selection marker gene (a selection marker gene different from the site-specific nuclease expression vector), and the selection marker gene was divided into a 5' and a 3' fragment, with the nucleotide fragment sandwiched between them, so that it could be cleaved by the site-specific nuclease. By introducing this and a site-specific nuclease expression vector into cells, the genomic DNA is cleaved by the site-specific nuclease, and the nucleotide fragments are also excised. The ends of these nucleotide fragments then bind to the first and second nucleotide sequences of the genomic DNA in a homology-dependent manner. Simultaneously, in cells where the cleavage and insertion have occurred, the 5' and 3' ends of the remaining vector fragments from which the nucleotide fragments were excised bind, and the selection marker gene becomes functional. Therefore, by performing selection according to the selection marker gene, it becomes possible to efficiently obtain cells in which the vector containing the nucleotide fragments and the site-specific nuclease expression vector have been simultaneously introduced. Furthermore, cell growth is difficult with each introduced vector alone, and if the introduced vector contains CEN / ARS, the vector in which the selection marker gene has functioned will also contain CEN / ARS. Therefore, along with the site-specific nuclease expression vector, it is possible to remove them by culture after DNA insertion (Figures 2A and 2B).

[0012] Based on the newly constructed method described above, the inventors developed and tested various vectors. They successfully inserted the desired nucleotide fragments precisely into the target sites in cells. Furthermore, when the introduced vectors contained CEN / ARS, they also succeeded in removing the vectors from the cells after DNA insertion. The inventors named this novel DNA editing method the Non-Integrative Cleavage-dependent system (NICS system). They further named the vector used in this method, which divides the selection marker gene into a 5' and a 3' fragment with the nucleotide fragments sandwiched between them, the NICS vector. Using these vectors, the inventors found that the desired nucleotide sequence can be accurately and efficiently inserted into the target site of target DNA within cells. Moreover, by including CEN / ARS in the vectors, it is possible to remove the vectors afterward and obtain genome DNA-edited cells with highly efficient removal of foreign genes other than the inserted nucleotide sequence. Furthermore, this method also allows for the precise and highly efficient removal of the target site from the target DNA by setting the nucleotide sequence to be inserted to 0 bases and the target site to several bases or more (Figure 1B), thus completing the present invention. In other words, the present invention encompasses the following aspects.

[0013] [1] A DNA editing method for inserting a desired nucleotide sequence into a target site of target DNA within a cell or removing the target site, The process includes an introduction step in which a vector and a site-specific nuclease system are introduced into cells and brought into contact with target DNA, wherein, The aforementioned vector is The first promoter P1, and The following structure (1): 5'-M1-Hv1-D-Hv2-M2-3'…(1) [(1) In the figure, M1 represents a 5'-fragment of the nucleotide sequence encoding a selectable marker gene and is operably linked to a first promoter P1. Hv1 represents a nucleotide sequence homologous to a first nucleotide sequence Ht1 on the 5'-side of the target site of the target DNA. D may or may not represent the desired nucleotide sequence. Hv2 represents a nucleotide sequence homologous to a second nucleotide sequence Ht2 on the 3'-side of the target site of the target DNA. M2 represents the remaining 3'-fragment of the nucleotide sequence encoding the selectable marker gene. In M1, Hv1, D, Hv2, and M2, adjacent sequences may partially overlap with each other.] comprising by the site-specific nuclease system, from the vector, the following structure (2): 5'-Hv1-D-Hv2-3'…(2) a fragment represented by and the following structure (3): 5'-M2-P1-M1-3'…(3) generating a fragment represented by and a cleavage step of cleaving the target site or its vicinity in the target DNA, the target DNA and the fragment represented by structure (2) bind depending on the homology between Ht1 and Hv1 and bind depending on the homology between Ht2 and Hv2, and an editing step in which the desired nucleotide sequence D is inserted into the target site or the target site is removed, In the fragment represented by structure (3), the 3'-end of M1 and the 5'-end of M2 bind to be operably linked to P1 and obtain a selection vector containing a functional selectable marker gene, a selection step, A DNA editing method comprising.

[0014] [2] the vector further contains CEN / ARS which is an autonomous replication sequence, the structure (3) generated in the cleavage step further contains CEN / ARS, The DNA editing method according to [1], wherein the selection step is a step of obtaining a selection vector in which the 3'-end of M1 and the 5'-end of M2 are ligated in the fragment represented by structure (3), which is operably linked to P1 and contains a functional selectable marker gene and CEN / ARS.

[0015] [3] The DNA editing method according to [1] or [2], wherein the length of Hv1 and the length of Hv2 are each independently 30 to 600 bases.

[0016] [4] The vector has the following structure (11) as structure (1): 5’-M1-T1-Hv1-D-Hv2-T2-M2-3’…(11) [In (11), T1 represents a TALEN_1 binding region containing the first DNA binding domain recognition sequence or its complementary sequence L1 and the second DNA binding domain recognition sequence or its complementary sequence R1, T2 represents a TALEN_2 binding region containing the third DNA binding domain recognition sequence or its complementary sequence L2 and the fourth DNA binding domain recognition sequence or its complementary sequence R2, M1, Hv1, D, Hv2, and M2 are each synonymous with those in structure (1), and in M1, T1, Hv1, D, Hv2, T2, M2, adjacent sequences may partially overlap with each other.] and includes The site-specific nuclease system is a TALEN containing a DNA binding domain and a nuclease domain, TALEN_1 containing the first DNA binding domain and the second DNA binding domain, which generates the 5'-end of structure (2), TALEN_2 containing the third DNA binding domain and the fourth DNA binding domain, which generates the 3'-end of structure (2), A TALEN_3 comprises a fifth DNA-binding domain that recognizes the nucleotide sequence on the 5' side of region T3 containing the target site of the target DNA as a fifth DNA-binding domain recognition sequence or its complementary sequence L3, and a sixth DNA-binding domain that recognizes the nucleotide sequence on the 3' side as a sixth DNA-binding domain recognition sequence or its complementary sequence R3, and cleaves the target site or its vicinity in the target DNA. A DNA editing method described in any one of [1] to [3], which includes [the specified element].

[0017] [5] The vector comprises in structure (1) a first guide RNA recognition sequence or its complementary sequence G1 and a second guide RNA recognition sequence or its complementary sequence G2, wherein G1 may be located between M1 and Hv1 and overlap with at least one of M1 and Hv1, and G2 may be located between M2 and Hv2 and overlap with at least one of M2 and Hv2. The site-specific nuclease system is a CRISPR-Cas system comprising a Cas protein and its guide RNA, The CRISPR-Cas system 1 includes a first guide RNA, and the Cas protein generates the 5' end of structure (2), The CRISPR-Cas system 2 includes a second guide RNA, and the Cas protein generates the 3' end of structure (2). A CRISPR-Cas system 3 includes a third guide RNA that recognizes a nucleotide sequence present in the target DNA containing the target site as a third guide RNA recognition sequence or its complementary sequence G3, wherein a Cas protein cleaves the target site or its vicinity in the target DNA. A DNA editing method described in any one of [1] to [3], which includes [the specified element].

[0018] [6] The introduction of the aforementioned site-specific nuclease system into cells CEN / ARS, an autonomous replicating array, The second promoter P2, and The nucleotide sequence Nuc encoding the site-specific nuclease system is operably linked to the second promoter P2. A DNA editing method described in any one of [1] to [5], which involves introducing a nuclease system expression vector containing [the specified substance].

[0019] [7] The introduction of the aforementioned vector and site-specific nuclease system into cells is First promoter P1, Structure (1), Second promoter P2, The nucleotide sequence Nuc encoding the site-specific nuclease system is operably linked to the second promoter P2. This involves the introduction of an integrated vector, The structure (3) formed in the cutting process further includes P2 and Nuc which is operably connected to P2, The DNA editing method according to any one of [1] to [6], wherein the selection step is a step of obtaining a selection vector in which the 3' end of M1 and the 5' end of M2 are bound in the fragment shown in structure (3) to operably link with P1 and a functional selection marker gene and Nuc operably linked with P2.

[0020] [8] The aforementioned integrated vector further includes CEN / ARS, which is an autonomous replicating sequence. The structure (3) formed in the cutting process further includes CEN / ARS, The DNA editing method according to [7], wherein the selection step is a step of obtaining a selection vector comprising a functional selection marker gene, CEN / ARS, and Nuc which is operably linked to P1 by binding the 3' end of M1 and the 5' end of M2 in the fragment shown in structure (3), and CEN / ARS and P2.

[0021] [9] The DNA editing method according to any one of [6] to [8], wherein the nucleotide sequence Nuc encoding the site-specific nuclease system is a nucleotide sequence encoding a fusion protein of a DNA-binding domain and a nuclease domain.

[0022]

[10] The DNA editing method according to any one of [6] to [8], wherein the nucleotide sequence Nuc encoding the site-specific nuclease system is a nucleotide sequence encoding a Cas protein and a nucleotide sequence encoding its guide RNA.

[0023]

[11] A method for producing DNA-edited cells in which a desired nucleotide sequence is inserted into a target site of target DNA or the target site is removed, A step of obtaining a cell containing the selection vector, wherein the desired nucleotide sequence D is inserted into or removed from the target site by a DNA editing method described in any one of [1] to

[10] , and The process of selecting DNA-edited cells in which the desired nucleotide sequence D is inserted into the target site or the target site is removed, using the selection marker gene contained in the selection vector as an indicator. A method for producing cells, including the method described above.

[0024]

[12] The cell manufacturing method according to

[11] , wherein the vector further comprises a vector which is an autonomous replication sequence CEN / ARS, and further comprises the step of culturing the DNA-edited cells to remove the selected vector from the cells.

[0025]

[13] A vector for inserting a desired nucleotide sequence into a target site of target DNA or removing the target site using a site-specific nuclease system. The first promoter P1, and The following structure (1'): 5'-M1-Hv1'-D'-Hv2'-M2-3'…(1') [(1') represents the 5' end fragment of the nucleotide sequence encoding the selection marker gene and is operably linked to the first promoter P1; Hv1' represents a nucleotide sequence homologous to the first nucleotide sequence Ht1 on the 5' end of the target site of the target DNA or its insertion site; D' represents the desired nucleotide sequence or its insertion site, but is optional; Hv2' represents a nucleotide sequence homologous to the second nucleotide sequence Ht2 on the 3' end of the target site of the target DNA or its insertion site; M2 represents the remaining 3' end fragment of the nucleotide sequence encoding the selection marker gene; adjacent sequences in M1, Hv1', D', Hv2', and M2 may partially overlap with each other.] Includes, The site-specific nuclease system described above produces the following structure (2'): 5'-Hv1'-D'-Hv2'-3'…(2') The fragment shown and the following structure (3'): 5'-M2-P1-M1-3'…(3') A DNA editing vector that produces the fragment shown.

[0026]

[14] The DNA editing vector described in

[13] further comprises an autonomous replication sequence CEN / ARS, wherein the site-specific nuclease system generates a fragment further containing CEN / ARS as structure (3').

[0027]

[15] Structure (1') is the following structure (11'): 5'-M1-T1'-Hv1'-D'-Hv2'-T2'-M2-3'…(11') [In (11'), T1' indicates a TALEN_1 binding region or insertion site containing a first DNA-binding domain recognition sequence or its complementary sequence L1 and a second DNA-binding domain recognition sequence or its complementary sequence R1; T2' indicates a TALEN_2 binding region or insertion site containing a third DNA-binding domain recognition sequence or its complementary sequence L2 and a fourth DNA-binding domain recognition sequence or its complementary sequence R2; M1, Hv1', D', Hv2', and M2 are the same as in structure (1'), and adjacent sequences in M1, T1', Hv1', D', Hv2', T2', and M2 may partially overlap.] Includes, The site-specific nuclease system is a TALEN comprising a DNA-binding domain and a nuclease domain, TALEN_1, which includes a first DNA-binding domain and a second DNA-binding domain, and which gives rise to the 5' end of structure (2'), TALEN_2, which includes a third DNA-binding domain and a fourth DNA-binding domain, and which gives rise to the 3' end of structure (2'), A TALEN_3 comprises a fifth DNA-binding domain that recognizes the nucleotide sequence on the 5' side of region T3 containing the target site of the target DNA as a fifth DNA-binding domain recognition sequence or its complementary sequence L3, and a sixth DNA-binding domain that recognizes the nucleotide sequence on the 3' side as a sixth DNA-binding domain recognition sequence or its complementary sequence R3, and cleaves the target site or its vicinity in the target DNA. A DNA editing vector as described in

[13] or

[14] , comprising the above.

[0028]

[16] Structure (1') includes a first guide RNA recognition sequence or its complementary sequence or insertion site G1', and a second guide RNA recognition sequence or its complementary sequence or insertion site G2', wherein G1' is located between M1 and Hv1' and may overlap with at least one of M1 and Hv1', and G2' is located between M2 and Hv2' and may overlap with at least one of M2 and Hv2'. The site-specific nuclease system is a CRISPR-Cas system comprising a Cas protein and its guide RNA, The CRISPR-Cas system 1 includes a first guide RNA, and the Cas protein generates the 5' end of the (2') structure, The CRISPR-Cas system 2 includes a second guide RNA, and the Cas protein generates the 3' end of the (2') structure. A CRISPR-Cas system 3 includes a third guide RNA that recognizes a nucleotide sequence present in the target DNA containing the target site as a third guide RNA recognition sequence or its complementary sequence G3, wherein a Cas protein cleaves the target site or its vicinity in the target DNA. A DNA editing vector as described in

[13] or

[14] , comprising the above.

[0029]

[17] First promoter P1, Structure (1'), Second promoter P2, The nucleotide sequence Nuc encoding the site-specific nuclease system is operably linked to the second promoter P2. A DNA editing vector according to any one of

[13] to

[16] , comprising an integrated vector, wherein the site-specific nuclease system further generates a fragment containing P2 and Nuc operably linked to P2 as structure (3').

[0030]

[18] M1 and M2 are nucleotide sequences in which the selected marker gene is fragmented so that the 3' end of M1 and the 5' end of M2 of the fragment shown in structure (3') can bind together to function, as described in any one of

[13] to

[17] , for a DNA editing vector.

[0031]

[19] The DNA editing vector according to

[18] , wherein the 3' end of M1 and the 5' end of M2 are mutually overlapping nucleotide sequences, and the 3' end of M1 and the 5' end of M2 are bound via the overlapping nucleotide sequences to enable the selection marker gene to function.

[0032]

[20] The DNA editing vector described in

[19] , wherein the length of the overlapping nucleotide sequence is 40 to 500 bases.

[0033] [twenty one] A DNA editing vector according to

[20] , wherein a stop codon is added to the 3' end of M1 and / or the 5' end of M2, and the stop codon is removed from M1 and / or M2 by binding via the overlapping nucleotide sequences.

[0034] [twenty two] A DNA editing kit comprising a DNA editing vector described in any one of

[13] to

[21] and the site-specific nuclease system.

[0035] [twenty three] The aforementioned site-specific nuclease system CEN / ARS, an autonomous replicating array, The second promoter P2, and The nucleotide sequence Nuc encoding the site-specific nuclease system is operably linked to the second promoter P2. A DNA editing kit as described in

[22] , which is a nuclease system expression vector containing [the specified substance]. [Effects of the Invention]

[0036] According to the present invention, it is possible to provide a DNA editing method that can accurately and efficiently insert a desired nucleotide sequence into a target site of target DNA in a cell using a vector, or to remove the target site, a cell manufacturing method using the same, and a DNA editing vector and a DNA editing kit for use in these methods.

[0037] Furthermore, if the vector contains a CEN / ARS which is an autonomous replication sequence, it is also possible to provide a DNA editing method and a cell manufacturing method using the same, which allows for highly efficient production of genome DNA edited cells in which foreign genes other than the inserted nucleotide sequence have been removed, after inserting a desired nucleotide sequence into a target site of target DNA or removing the target site from the target DNA using the vector, by removing the vector. Additionally, DNA editing vectors and DNA editing kits for use in these methods can also be provided. [Brief explanation of the drawing]

[0038] [Figure 1A] This is a schematic diagram showing one aspect of the insertion of a nucleotide sequence into target DNA by a vector in the DNA editing method of the present invention. (a) is a schematic diagram showing the structure of a DNA editing vector and target DNA related to the DNA editing method of the present invention, (b) is a schematic diagram showing the structure of a selection vector formed in the DNA editing method of the present invention, and (c) is a schematic diagram showing the structure of edited DNA into which the nucleotide sequence has been inserted by the DNA editing method of the present invention. [Figure 1B] This is a schematic diagram showing one aspect of the removal of a target site from target DNA by a vector in the DNA editing method of the present invention. (a) is a schematic diagram showing the structure of a DNA editing vector and target DNA related to the DNA editing method of the present invention, (b) is a schematic diagram showing the structure of a selection vector formed in the DNA editing method of the present invention, and (c) is a schematic diagram showing the structure of edited DNA from which the target site has been removed by the DNA editing method of the present invention. [Figure 1C]This is a schematic diagram showing one embodiment of the configuration of a DNA editing vector and target DNA according to the DNA editing method of the present invention. (a) shows a preferred configuration of the DNA editing vector and target DNA when TALEN is used as the site-specific nuclease system, and (b) shows one embodiment of a preferred configuration of the DNA editing vector and target DNA when the CRISPR-Cas system is used as the site-specific nuclease system. [Figure 2A] This schematic diagram illustrates one aspect of the removal of a selection vector in the DNA editing method of the present invention, specifically showing the combination of a DNA editing vector and a nuclease system expression vector introduced into a cell, and the resulting state of cell survival or death. [Figure 2B] This schematic diagram illustrates one aspect of the removal of the selection vector in the DNA editing method of the present invention, showing the intracellular state (left) when a DNA editing vector and a nuclease system expression vector are introduced into a cell and editing of the target site occurs, and the state in which the selection vector is removed from the cell (right). [Figure 3] This is a schematic diagram showing a preferred embodiment when the DNA editing vector for the DNA editing method of the present invention is an all-in-one NICS vector. [Figure 4] This is a schematic diagram showing the DNA editing method constructed in Test Example 1. [Figure 5] (a) is a schematic diagram showing the positions where the primers were designed in the PCR reaction performed in Test Example 1, and (b) is an electrophoretic image of the amplified product after the PCR reaction. [Figure 6A] This is a schematic diagram showing the structure of the plasmid "pMD20-ARS-aphVIII-sep-BamHI" used to construct each vector in Test Example 2. [Figure 6B](a) is a schematic diagram showing the structure of the validation vector _NoNR (validation vector) prepared in Validation Test 1 of Test Example 2, and (b) is a schematic diagram showing the structure of the vector when the validation vector _NoNR is cleaved and paromomycin resistance is expressed. [Figure 7A] This is a graph showing the number of colonies measured in (1) Verification Test 1 of Test Example 2. [Figure 7B] (a) is a schematic diagram showing the positions where the primers were designed in the PCR reaction performed in Verification Test 1 of Test Example 2, and (b) is an electrophoretic image of the amplified product after the PCR reaction. [Figure 8] This graph shows the number of colonies measured in (2) Verification Test 2 of Test Example 2. [Figure 9] (a) is a schematic diagram showing the structure of the pNICS36 vector (or pNICS210 vector) and the TALEN vector_PDAT1 prepared in Test Example 2 (3) Construction of NICS vectors, and (b) is a schematic diagram showing the structure of the all-in-one NICS36 vector (or all-in-one NICS210 vector). [Figure 10] This graph shows the number of colonies measured in the cell introduction test of NICS vector in Test Example 2 (4). [Figure 11] This is a schematic diagram showing the positions where the primers were designed in the PCR reaction performed in (5) PCR analysis and sequencing analysis of Test Example 2. [Figure 12] This is an electrophoretic image of the amplified product after the PCR reaction performed on paromomycin-resistant colonies formed by introducing a combination of the pNICS210 vector and the TALEN vector _PDAT1 in the PCR and sequencing analysis of Test Example 2 (5). [Figure 13] This is an electrophoretic image of the amplified product after the PCR reaction performed on paromomycin-resistant colonies formed by introducing the all-in-one NICS210 vector in the PCR and sequencing analysis of Test Example 2 (5). [Figure 14] The images show the appearance of the growth status of each strain (6 strains each) that grew on the F2N plate (F2N) but did not grow on the paromomycin-containing plate (F2N+Paro) in the CEN / ARS vector shedding test (6) of Test Example 2, using colonies No. 5 and No. 8 of paromomycin-resistant colonies formed by introducing the all-in-one NICS210 vector. [Figure 15] These are electrophoretic images of the amplified products after a PCR reaction using primers to detect FokI, with DNA extracted using a simple DNA extraction kit from paromomycin-resistant colonies No. 5 and No. 8 (pre-vector elimination strains) formed by introducing the all-in-one NICS210 vector, and from six strains derived from these colonies (vector elimination strains) as confirmed in Figure 14. [Figure 16] These are electrophoresis images of the amplified products after PCR reactions performed using phenol / chloroform-based DNA templates, with the following methods used: DNA extracted from paromomycin-resistant colonies No. 5 and No. 8 (pre-vector elimination strains) formed by introducing the all-in-one NICS210 vector, and from six strains derived from these colonies (vector elimination strains) as confirmed in Figure 14. The PCR reactions were then carried out using primers to detect FokI, the N-terminal domain of TALEN (TALEN-N), the paromomycin resistance gene (ParoR), the kanamycin resistance gene (KanR), and PDAT1, respectively. [Figure 17] This is a schematic diagram illustrating one aspect of the application of the DNA editing method (NICS system) of the present invention. [Figure 18] This is a schematic diagram illustrating one embodiment of the application of the DNA editing method (NICS system) of the present invention, where two types of nucleotide sequences (X, Y) are to be inserted. [Figure 19] This is a schematic diagram showing the detailed configuration of the "all-in-one NICS210 vector" prepared in Test Example 2 (3) Construction of the NICS vector. [Figure 20]This is an electrophoretic image of the amplification product after the PCR reaction performed on paromomycin-resistant colonies formed by introducing the all-in-one NICS210-GPAT1 vector in the cell introduction test of Test Example 3 (2) NICS vector. [Figure 21] These images show the appearance of the growth status of each strain (6 strains each) that grew on the F2N plate (F2N) but did not grow on the paromomycin-containing plate (F2N+Paro) in the CEN / ARS vector shedding test (3) of Test Example 3, using colonies of paromomycin-resistant colonies No. 3 and No. 4 formed by introducing the all-in-one NICS210-GPAT1 vector. [Figure 22] This is an electrophoretic image of the amplification product after the PCR reaction performed on paromomycin-resistant colonies formed by introducing the all-in-one NICS400 vector in the cell introduction test of Test Example 4 (2) NICS vector. [Figure 23] The diagrams above show the structure of the NICS vector and the CRISPR / Cas9 vector prepared in Test Example 5 (1) Construction of the NICS vector (top); the structure of the vector when the NICS vector is cleaved and puromycin resistance is expressed (bottom left); and the configuration when a knock-in fragment (3× Flag tag sequence) is inserted into the target site (bottom right). [Figure 24] This graph shows the viability obtained in the verification trial described in (2) of Example 5. [Figure 25] This graph shows the viability at various puromycin concentrations for each combination of NICS vectors (pNICS60-PARP1 vector, pNICS192-PARP1 vector) and a PARP1-targeting CRISPR / Cas9 vector in the cell introduction study of Test Example 5 (3). [Figure 26]This graph shows the viability at various puromycin concentrations for each combination of the NICS vector (pNICS192-ATP5B vector) and the ATP5B-targeting CRISPR / Cas9 vector in the cell introduction test of Test Example 5 (3). [Figure 27A] In the PCR analysis described in Test Example 5 (2), a NICS vector (pNICS60-PARP1 vector) and a CRISPR / Cas9 vector targeting PARP1 were introduced, and the electrophoretic images of the amplified products after the PCR reaction were performed on the colonies formed under each puromycin concentration. [Figure 27B] In the PCR analysis described in Test Example 5 (2), a NICS vector (pNICS192-PARP1 vector) and a PARP1-targeting CRISPR / Cas9 vector were introduced, and the electrophoretic images of the amplified products after the PCR reaction were performed on the colonies formed under each puromycin concentration. [Figure 28A] In the PCR analysis described in Test Example 5 (2), a NICS vector (pNICS60-ATP5B vector) and a CRISPR / Cas9 vector targeting ATP5B were introduced, and the electrophoretic images of the amplified products after the PCR reaction were performed on the colonies formed under each puromycin concentration. [Figure 28B] In the PCR analysis described in Test Example 5 (2), a NICS vector (pNICS192-ATP5B vector) and a CRISPR / Cas9 vector targeting ATP5B were introduced, and the electrophoretic images of the amplified products after the PCR reaction were performed on the colonies formed under each puromycin concentration. [Figure 29] This graph shows the HDR efficiency in colonies formed at various puromycin concentrations for each combination of the NICS vector (pNICS192-PARP1 vector) and the PARP1-targeting CRISPR / Cas9 vector in the TIDER analysis of Test Example 5 (3). [Modes for carrying out the invention]

[0039] Hereinafter, the present invention will be described in detail in accordance with its preferred embodiments. Although the preferred embodiments of the present invention will be described by way of examples with reference to the drawings where appropriate, the present invention is not limited thereto. In the following description and drawings, the same or corresponding elements are denoted by the same reference numerals, and redundant descriptions are omitted.

[0040] <DNA editing method / NICS> The DNA editing method of the present invention is a method of inserting a desired nucleotide sequence into a target site of a target DNA in a cell or removing the target site from the target DNA. Here, the "insertion" of the desired nucleotide sequence includes the replacement of a sequence in which the target site is removed and the desired nucleotide sequence is inserted therein. The DNA editing method of the present invention includes an introduction step of introducing a vector and a site-specific nuclease system into a cell and bringing them into contact with the target DNA. Here, the vector includes a first promoter P1, and the following structure (1): 5’-M1-Hv1-D-Hv2-M2-3’…(1) [In (1), M1 represents a 5'-terminal fragment of a nucleotide sequence encoding a selectable marker gene and is operably linked to the first promoter P1. Hv1 represents a nucleotide sequence homologous to the first nucleotide sequence Ht1 on the 5'-side of the target site of the target DNA. D may not represent the desired nucleotide sequence. Hv2 represents a nucleotide sequence homologous to the second nucleotide sequence Ht2 on the 3'-side of the target site of the target DNA. M2 represents the remaining 3'-terminal fragment of the nucleotide sequence encoding the selectable marker gene. In M1, Hv1, D, Hv2, and M2, adjacent sequences may partially overlap with each other.] and includes a fragment represented by the following structure (2): 5’-Hv1-D-Hv2-3’…(2) and a fragment represented by the following structure (3): 5’-M2-P1-M1-3’…(3) A cutting step that produces a fragment shown by and cuts the target site or its vicinity in the target DNA, An editing step in which the target DNA and the fragment shown in structure (2) are bound in a manner dependent on the homology between Ht1 and Hv1, and in a manner dependent on the homology between Ht2 and Hv2, thereby inserting a desired nucleotide sequence D into the target site or removing the target site; A selection step to obtain a selection vector containing a functional selection marker gene, wherein the 3' end of M1 and the 5' end of M2 are joined in the fragment shown in structure (3) to operably link with P1, and the fragment is functionally linked. Includes.

[0041] (Target DNA) In the present invention, the DNA containing a target site is defined as the site where a desired nucleotide sequence is to be inserted or removed. The target DNA according to the present invention is double-stranded DNA, and for convenience, in order to show the correspondence with the DNA editing vector and site-specific nuclease system described below, the 5' end of the target site on at least one strand is designated as the first nucleotide sequence (Ht1), and the 3' end of the target site is designated as the second nucleotide sequence (Ht2), and the DNA has a structure that includes these.

[0042] As one embodiment of the structure of the target DNA, a schematic diagram of the target site where the desired nucleotide sequence is to be inserted is shown in the lower part of Figure 1A(a). In this case, it is preferable that the first nucleotide sequence (Ht1) and the second nucleotide sequence (Ht2) are adjacent to each other in the target DNA, but as shown in Figure 1A(a), they may be separated by a region of a few bases, preferably 1 to 10,000 bases, more preferably 1 to 3,000 bases, and even more preferably 1 to 1,000 bases.

[0043] Furthermore, as one embodiment of the structure of the target DNA, a schematic diagram of a case where the nucleotide sequence to be removed is used as the target site is shown in the lower part of Figure 1B(a). In this case, the length of the target site (target in Figure 1B(a)) is 1 to 10,000 bases, more preferably 1 to 3,000 bases, and even more preferably 1 to 1,000 bases. In this case, Ht1 is adjacent to the 5' end of the target site, and Ht2 is adjacent to the 3' end. In this specification, "base" indicating the length of a nucleotide sequence is synonymous with "nucleotide," or "bp" when it is a double-stranded sequence.

[0044] In Figures 1A(a) and 1B(a), the target DNA according to the present invention has the target site cleaved by a site-specific nuclease (arrow). However, the cleavage site does not have to be the target site itself, but may be in the vicinity, and may be on the first nucleotide sequence (Ht1) or the second nucleotide sequence (Ht2). In the present invention, by setting Ht1 and Ht2 as described above and designing a DNA editing vector containing the corresponding Hv1 and Hv2 described below, binding depends on the homology between Ht1 and Hv1 and the homology between Ht2 and Ht2, so a sequence in which the desired nucleotide sequence is inserted between Ht1 and Ht2, or a sequence in which the target site is removed from between Ht1 and Ht2 can be obtained. In the target DNA according to the present invention, the length between the cleavage site and the target site depends on the lengths of Ht1 and Ht2, but is preferably within 400 bases, and more preferably between 0 and 200 bases.

[0045] Furthermore, if the site-specific nuclease according to the present invention is a TALEN as described below, for convenience, in order to show the correspondence with such a TALEN, the target DNA according to the present invention shall be defined as having a structure in which the strand containing Ht1 and Ht2 further includes a TALEN_3 binding region T3 as a region that contains the target site or at least partially overlaps with the target site. A schematic diagram showing one aspect of the structure of the target DNA when the site-specific nuclease system according to the present invention is a TALEN (when the target site is a site for inserting a desired nucleotide sequence) is shown in the lower part of Figure 1C(a). In Figure 1C(a), T3 is sandwiched between Ht1 and Ht2, but as described above, Ht1 and Ht2 may be adjacent to each other, and part or all of T3 may overlap with a part of the 3' end of Ht1 and / or a part of the 5' end of Ht2. When the nucleotide sequence to be removed is the target site, the length of the target site is as described above.

[0046] For convenience, T3 is assumed to contain, from the 5' end, the fifth DNA-binding domain recognition sequence or its complementary sequence L3, spacer S3, and the sixth DNA-binding domain recognition sequence or its complementary sequence R3 in that order adjacently. By designing the fifth DNA-binding domain of TALEN_3 (TALE of Left-TALEN) to bind to L3 and the sixth DNA-binding domain (TALE of Right-TALEN) to bind to R3, the inside of S3 is cleaved (arrow) by the nuclease activity of TALEN_3. However, as mentioned above, the cleavage site does not have to be the target site, and may be on the first nucleotide sequence (Ht1) or the second nucleotide sequence (Ht2). Each DNA-binding domain recognition sequence is the sequence recognized by each DNA-binding domain of TALEN_3 (the fifth DNA-binding domain and the sixth DNA-binding domain) described below. Typically, Left-TALEN and Right-TALEN bind to opposite strands of DNA. Therefore, L3 and R3 are related such that if one is a DNA-binding domain recognition sequence, the other is the complementary sequence to the DNA-binding domain recognition sequence. The lengths of L3 and R3 are independently 10 to 30 bases, preferably 13 to 25 bases, and more preferably 15 to 20 bases. The length of S3 is 10 to 20 bases, preferably 12 to 19 bases, and more preferably 15 to 16 bases.

[0047] Furthermore, if the site-specific nuclease according to the present invention is the CRISPR-Cas system described below, for convenience in order to show the correspondence with such a CRISPR-Cas system, the target DNA according to the present invention shall, for convenience, have a structure in which at least one strand further includes a third guide RNA recognition sequence or its complementary sequence G3. A schematic diagram showing one aspect of the structure of the target DNA when the site-specific nuclease system according to the present invention is a CRISPR-Cas system (when the target site is the site for which a desired nucleotide sequence is to be inserted) is shown in the lower part of Figure 1C(b). In Figure 1C(b), Ht1 and Ht2 are separated, but as described above, Ht1 and Ht2 may be adjacent to each other. Also, even if G3 is sandwiched between Ht1 and Ht2, part or all of G3 may overlap with part of the 3' end of Ht1 and / or part of the 5' end of Ht2. When the nucleotide sequence to be removed is the target site, the length of the target site is as described above. The CRISPR-Cas system preferably cleaves between Ht1 and Ht2 (arrow), and in the CRISPR-Cas system, the site included in the guide RNA recognition sequence is cleaved by the Cas protein, so G3 is positioned to include the target site or its complementary sequence.

[0048] In the CRISPR-Cas system, for example, in the CRISPR-Cas9 system, the target site is cleaved by the Cas9 protein by designing the third guide RNA to recognize a third guide RNA recognition sequence that is G3 or its complementary sequence, and the Cas9 protein to recognize a sequence located 3' to the complementary sequence of the third guide RNA recognition sequence as the PAM sequence. The length of the guide RNA recognition sequence or its complementary sequence is 12 to 50 bases, preferably 17 to 30 bases, and more preferably 17 to 25 bases. The PAM sequence varies depending on the type of Cas protein, and typical PAM sequences are known, but it is possible to modify PAM recognition by modifying the Cas protein (e.g., by introducing mutations) to expand the range of target site options.

[0049] The base sequence of such target DNA is not particularly limited, and by designing the DNA editing vector and site-specific nuclease system described below to match the sequence, it can be targeted by the DNA editing method of the present invention.

[0050] Furthermore, the target DNA according to the present invention may be DNA present in the nucleus or DNA present outside the nucleus, depending on the purpose, and may be endogenous DNA or exogenous DNA. Examples of endogenous DNA include genomic DNA, and examples of exogenous DNA include DNA introduced into cells.

[0051] (cell) The cells containing the target DNA, that is, the cells to be treated by the DNA editing method of the present invention, are not particularly limited and may be eukaryotic or prokaryotic cells, but eukaryotic cells are preferred. Examples of eukaryotic cells include animal cells (mammalian, fish, bird, reptile, amphibian, insect cells, etc.), plant cells, algal cells, slime molds, and fungi (yeast, etc.), while examples of prokaryotic cells include Escherichia coli, Salmonella, Bacillus subtilis, Lactobacillus, hyperthermophiles, actinomycetes, and archaea.

[0052] "Animal cells" include, for example, cells that make up an animal, cells that make up organs and tissues extracted from an animal, and cultured cells derived from animal tissue. Specifically, examples include germ cells such as oocytes and sperm; embryonic cells of each stage of the embryo (e.g., 1-cell stage embryo, 2-cell stage embryo, 4-cell stage embryo, 8-cell stage embryo, 16-cell stage embryo, morula stage embryo, etc.); stem cells such as induced pluripotent stem (iPS) cells and embryonic stem (ES) cells; and somatic cells such as fibroblasts, hematopoietic cells, neurons, muscle cells, osteocytes, hepatocytes, pancreatic cells, brain cells, and kidney cells. Oocytes that can be used as targets for DNA editing methods can be pre-fertilized and post-fertilized oocytes, but post-fertilized oocytes, i.e., fertilized eggs, are preferred. Particularly preferred are fertilized eggs that are in the pronuclear stage. Oocytes can be used after being frozen and thawed.

[0053] "Plant cells" include, for example, cells that make up an entire plant, cells that make up organs and tissues separated from a plant, and cultured cells derived from plant tissues. Examples of plant organs and tissues include leaves, stems, shoot apex (growth point), roots, tubers, and callus.

[0054] Among these, the cells according to the present invention are preferably at least one selected from the group consisting of algal cells and animal cells, and the algal cells are more preferably single-celled algae such as Nannochloropsis, Chlamydomonas, Euglena, Chlorella, Botryococcus, diatoms, coccolithophores, cyanobacteria, Cyanidioschizomon, Pseudococomyxa, Spirulina, and Haematococcus.

[0055] (Site-specific nuclease system) In the present invention, a "site-specific nuclease system" refers to a system that includes a nuclease for cleaving DNA double strands and is controllable so that the cleavage by the nuclease is specifically performed at the desired site. Examples of such site-specific nuclease systems include a system in which a nuclease is fused to a TAL effector (TALE) (TALEN), a system in which a nuclease is fused to a zinc finger (ZF), and a CRISPR-Cas system. In the present invention, the TALEN and / or CRISPR-Cas system are preferred.

[0056] [TALEN] TALEN (Transcription activator-like effector nuclease) is a fusion protein in which the type IIS restriction enzyme FokI is used as the nuclease domain and TALE is used as the DNA-binding domain. Each DNA-binding domain of a pair of TALENs (Left-TALEN and Right-TALEN) binds to the opposite strand of the target DNA, and the FokIs dimerize with each other, thereby exhibiting site-directed double-strand cleavage activity (nuclease activity) of DNA, cleaving within the region between the pair of DNA-binding domains. The typical nucleotide sequence encoding FokI is publicly known, but it may be modified as appropriate, and any known or similar TALEN may be used as such. Furthermore, the method for preparing the amino acid sequence of each DNA-binding domain is publicly known, and those skilled in the art can design a DNA-binding domain that can recognize and bind to the DNA-binding domain recognition sequence (DNA-binding domain recognition sequence) on the target DNA, and the nucleotide sequence encoding it.

[0057] In the present invention, when a TALEN is used, such a TALEN includes TALEN_1, which includes a first DNA-binding domain and a second DNA-binding domain; TALEN_2, which includes a third DNA-binding domain and a fourth DNA-binding domain; and TALEN_3, which includes a fifth DNA-binding domain and a sixth DNA-binding domain.

[0058] TALEN_1 and TALEN_2 recognize T1 and T2 on the DNA editing vector corresponding to their respective DNA-binding domains, and cleave the DNA editing vector at S1 between L1 and R1, and at S2 between L2 and R2, respectively. TALEN_3 recognizes T3 on the target DNA and cleaves the target DNA at S3 between L3 and R3. The cleavage site of the target DNA is either the target site or its vicinity.

[0059] The first, third, and fifth DNA-binding domains may be the same as each other, and the second, fourth, and sixth DNA-binding domains may also be the same as each other. TALEN_1, TALEN_2, and TALEN_3 may also be the same as each other. By using a common single TALEN for the three types, DNA can be cleaved more simply and efficiently. For example, a fifth DNA-binding domain that recognizes L3 on the 5' side and a sixth DNA-binding domain that recognizes R3 on the 3' side of the region (T3) containing the target site of the target DNA can be designed, and TALEN_3 can be designed to cleave the target DNA at S3 between L3 and R3 (Figure 1C(a)). In conjunction with this, by designing the DNA editing vector so that the sequences of T1 and T2 are the same as the sequence of T3, only one type of TALEN_3 can be used as TALEN_1 and TALEN_2.

[0060] [CRISPR-Cas system] The CRISPR-Cas system according to the present invention comprises at least a Cas protein and its guide RNA as components. The guide RNA consists of a crRNA (CRISPR RNA) and a tracrRNA (trans-activating crRNA) having a base sequence complementary to the target base sequence (guide RNA recognition sequence). The crRNA further includes a base sequence on its 3' side that can interact (hybridize) with the tracrRNA. On the other hand, the tracrRNA includes a base sequence on its 5' side that can interact (hybridize) with a portion of the crRNA's base sequence. Therefore, the guide RNA forms a double-stranded RNA that interacts with the Cas protein through the interaction of these base sequences. For this reason, by introducing the Cas protein and its guide RNA into a cell, the guide RNA binds to the guide RNA recognition sequence of the DNA, inducing the Cas protein to form a complex with the guide RNA. The induced Cas protein recognizes the PAM sequence located on the 3' side of the complementary sequence of the guide RNA recognition sequence, and cleaves the target site of the DNA through its nuclease activity.

[0061] The CRISPR-Cas system may be of either Class 1, which forms a complex with a guide RNA and one Cas protein to cleave nucleic acids, or Class 2, which forms a complex (cascade complex) with a guide RNA and multiple Cas proteins to cleave nucleic acids. It may also be of any of the types I, II, III, IV, V, and VI. Among these, from the viewpoint of ease of use, Class 2 is preferred, and the CRISPR-Cas9 system in which the Cas protein is the Cas9 protein is more preferred. The configuration of each CRISPR-Cas system and the typical nucleotide sequences encoding each Cas protein are all publicly known, but they may be modified as appropriate, or known or similar ones may be used as appropriate. For example, the Cas protein may be modified as appropriate, such as by introducing mutations to alter the recognition of the PAM sequence.

[0062] Various methods are known for designing the PAM sequence and guide RNA recognition sequence of such Cas proteins. For example, these can be designed using E-CRISP (http: / / www.e-crisp.org / E-CRISP / ), CRISPRdirect (http: / / crispr.dbcls.jp / ) (University of Tokyo), and Guide RNA Target Design Tool (https: / / wwws.blueheronbio.com / external / tools / gRNASrc.jsp) (Blue Heron Biotech).

[0063] In the present invention, when a CRISPR-Cas system is used, such a CRISPR-Cas system includes a CRISPR-Cas system_1 containing a first guide RNA, a CRISPR-Cas system_2 containing a second guide RNA, and a CRISPR-Cas system_3 containing a third guide RNA.

[0064] CRISPR-Cas system_1 and CRISPR-Cas system_2 recognize G1 and G2 on the DNA editing vector corresponding to each guide RNA, respectively, and cleave the DNA editing vector at G1 and G2, respectively. CRISPR-Cas system_3 recognizes G3 on the target DNA and cleaves the target DNA at G3.

[0065] The first, second, and third guide RNAs may be the same as each other, and CRISPR-Cas systems 1, 2, and 3 may also be the same as each other. By using a common one system for the three CRISPR-Cas systems, it becomes possible to cleave DNA more simply and efficiently. For example, a CRISPR-Cas system 3 can be designed that cleaves the target DNA at G3 (Figure 1C (b)) by designing a third guide RNA that recognizes the region (G3) containing the target site of the target DNA and a Cas9 protein that binds to it and recognizes a PAM sequence located on the 3' side of the complementary sequence of the third guide RNA recognition sequence. Accordingly, by designing the DNA editing vector such that the sequences of G1 and G2 are the same as or complementary to G3, and include the PAM sequence or its complementary sequence, only one type of CRISPR-Cas system 3 can be used as CRISPR-Cas system 1 and CRISPR-Cas system 2.

[0066] (DNA editing vector / NICS vector) The vector used in the DNA editing method of the present invention (hereinafter referred to as "DNA editing vector" or "NICS vector" as may be used herein) comprises a first promoter P1 and the following structure (1): 5'-M1-Hv1-D-Hv2-M2-3'…(1) The vector contains the following. As preferred embodiments of the structure of the DNA editing vector according to the DNA editing method of the present invention, the embodiment when the target site is the site for inserting a desired nucleotide sequence, that is, when the DNA editing method of the present invention is a DNA insertion method (knock-in method), is shown in Figure 1A(a), and the embodiment when the target site is the nucleotide sequence to be removed, that is, when the DNA editing method of the present invention is a DNA removal method (knock-out method), is shown in Figure 1B(a).

[0067] The vector used in the DNA editing method of the present invention preferably further contains a CEN / ARS, which is an autonomous replication sequence. CEN / ARS stands for "Centromere and autonomous replication sequence" and is a sequence involved in the chromosomal stability of budding yeast. It is commonly used as the autonomous replication sequence of a vector in vectors for budding yeast. Vectors containing this CEN / ARS have been reported to be maintained intracellularly in diatoms and unicellular algae (e.g., Nannochloropsis). Such a CEN / ARS-containing vector can be removed from host cells by repeatedly culturing the host cells (Non-Patent Literature 3, etc.). The nucleotide sequence of such a CEN / ARS is typically shown in Sequence ID No. 1, but the sequence may be modified as appropriate as long as the function of the CEN / ARS (such as the function of vector replication in cells) is maintained.

[0068] The structure (1) included in the DNA editing vector according to the DNA editing method of the present invention contains M1, Hv1, D, Hv2, and M2 in that order from the 5' end.

[0069] In structure (1), M1 represents the 5' end fragment of the nucleotide sequence encoding the selection marker gene, and M2 represents the 3' end fragment of the nucleotide sequence encoding the selection marker gene. M1 and M2 are fragments of the selection marker gene that have been divided so that they can be functional after being cleaved by site-specific nucleotides and then joined together. Here, "divided in a functional manner" means that in the nucleotide sequence encoding the two divided selection marker genes, the 3' end of the 5' end fragment and the 5' end of the 3' end fragment can join together so that the original selection marker gene can function (the 5' end fragment alone or the 3' end fragment alone (before joining) cannot function). Those skilled in the art can design this appropriately depending on the type of selection marker gene. For example, preferably, the 5' end fragment of the nucleotide sequence encoding the selection marker gene is divided into two, and M1 is formed by adding a nucleotide sequence to the remaining 3' end fragment such that the sequence overlaps with a few bases at the 3' end of M1. In other words, M1 and M2 are designed such that the 3' end of M1 and the 5' end of M2 have overlapping nucleotide sequences (overlapping sequences). This allows the 3' end of M1 and the 5' end of M2 to bind via these overlapping sequences; that is, the protruding end at the 3' end of M1, created by cleavage within the cell, and the complementary protruding end at the 5' end of M2 bind complementarily, enabling the selection marker gene to function. The length of the overlapping nucleotide sequence (overlapping sequence) between the 3' end of M1 and the 5' end of M2 is preferably 40 bases or more, more preferably 40 to 500 bases, even more preferably 50 to 400 bases, even more preferably 70 to 300 bases, and particularly preferably 150 to 300 bases.

[0070] As the selection marker genes, those conventionally used in DNA editing methods can be used as appropriate, for example, drug resistance genes and reporter genes. Examples of drug resistance genes include ampicillin resistance genes, zeosin resistance genes, bleomycin resistance genes, paromomycin resistance genes, kanamycin resistance genes, neomycin resistance genes, emesin resistance genes, spectinomycin resistance genes, hygromycin resistance genes, chloramphenicol resistance genes, erythromycin resistance genes, and puromycin resistance genes. Examples of reporter genes include green fluorescent protein (GFP), DsRed, mCherry, mOrange, mBanana, mStrawberry, mRaspberry, and mPlum. The nucleotide sequences encoding each selection marker gene are publicly known and may be modified as appropriate, as long as their function is maintained.

[0071] Among the selectable marker genes according to the present invention, drug resistance genes are preferred from the viewpoint of being able to more accurately confirm the removal of the vector from cells using a culture medium containing the drug, and paromomycin resistance genes, hygromycin resistance genes, neomycin resistance genes, or puromycin resistance genes are more preferred. For example, in the case of paromomycin resistance genes and puromycin resistance genes, by making the nucleotide sequence that overlaps between the 3' end of M1 and the 5' end of M2 (the overlapping sequence) 50 to 300 bases (for example, 207 bases in Figure 6B(a), and 60 or 192 bases in Figure 23), the 3' end of M1 and the 5' end of M2, which are formed by cleavage in the cell, join via the overlapping sequence, and sufficient paromomycin resistance or puromycin resistance is expressed. The nucleotide sequences of M1 and M2 in this case are typically shown in SEQ ID NO: 3 and SEQ ID NO: 8, respectively, for the paromomycin resistance gene, and in SEQ ID NO: 43 and SEQ ID NO: 44, or SEQ ID NO: 43 and SEQ ID NO: 53, respectively, for the puromycin resistance gene. However, they are not particularly limited as long as they bind and function as selection marker genes, and may be modified as appropriate.

[0072] The lengths of M1 and M2 in structure (1) (including the length of any overlapping sequences) may be the same or different, and each is preferably 50 to 10,000 bases, more preferably 100 to 5,000 bases, even more preferably 200 to 3,000 bases, even more preferably 200 to 2,000 bases, and particularly preferably 300 to 2,000 bases.

[0073] In structure (1), it is preferable that stop codons are added to the 3' end of M1 and / or the 5' end of M2, more preferably to both the 3' end of M1 and the 5' end of M2. This makes it possible to more reliably suppress the expression of selected marker genes that were not cleaved by the site-directed nuclease system. These stop codons are usually removed from M1 and / or M2 by the intracellular repair system when the 3' end of M1 and the 5' end of M2 are cleaved in the cell. In particular, it is preferable that the 3' end of M1 and the 5' end of M2 are duplicate sequences, as this allows for removal by binding via the duplicate sequences.

[0074] In structure (1), Hv1 represents a nucleotide sequence homologous to the first nucleotide sequence Ht1 on the 5' side of the target site of the target DNA, and Hv2 represents a nucleotide sequence homologous to the second nucleotide sequence Ht2 on the 3' side of the target site of the target DNA. In the present invention, "homologous" preferably means that the nucleotide sequences are identical to each other (100% sequence identity), but also includes sequences with 85% or more, preferably 90% or more, 95% or more (for example, 96% or more, 97% or more, 98% or more, 99% or more). From the viewpoint of DNA editing accuracy, the sequence identity between Hv1 and Hv2 is preferably 40% or less, and more preferably 20% or less.

[0075] The lengths of Hv1 and Hv2 may be the same or different, and each is preferably 30 to 600 bases, more preferably 40 to 600 bases, even more preferably 100 to 500 bases, even more preferably 140 to 500 bases, even more preferably 150 to 450 bases, 150 to 400 bases, or 200 to 450 bases, and even more preferably 150 to 250 bases or 200 to 300 bases. The inventors have found that the DNA editing method of the present invention (preferably the DNA insertion method) particularly improves DNA editing efficiency (preferably DNA insertion efficiency) in homologous sequences of different lengths than conventional homologous recombination (HR) and microhomology-mediated end joining (MMEJ), for example, 100 to 500 bases, preferably 200 to 450 bases, and even more preferably 200 to 400 bases.

[0076] When the DNA editing method of the present invention is a method for inserting a desired nucleotide sequence into a target site of target DNA (DNA insertion method), in structure (1), D represents the desired nucleotide sequence for insertion into the target site. Such a sequence is not particularly limited and can include, for example, promoters, terminators, enhancers, insulators, and binding sequences for expression regulatory genes to control the expression of a target gene; fluorescent proteins, luminescent proteins, epitope tags, etc., for observing gene expression and localization; stop codons, etc., for gene disruption; mutant sequences, etc., for modifying gene function; and expression cassettes for endogenous or exogenous gene expression. In this case, the length of D is preferably 1 to 10,000 bases, more preferably 5,000 bases or less, even more preferably 2,000 bases or less, and still more preferably 1,000 bases or less. When the DNA editing method of the present invention is a method for removing a target site from target DNA (DNA removal method), D is not included in structure (1) (i.e., the length of D is 0 bases). That is, Hv1 and Hv2 are adjacent to each other in structure (1).

[0077] In structure (1), adjacent sequences may partially overlap with each other. In this case, the length of the overlapping nucleotide sequences between M1 and Hv1, between Hv1 and D (in the case of DNA insertion), between D and Hv2 (in the case of DNA insertion), and between Hv2 and M2 is preferably 150 bases or less, more preferably 1 to 50 bases, and even more preferably 1 to 30 bases, independently for each of these. If the 3' end of M1 and the 5' end of M2 are the overlapping sequences, the number of bases obtained by dividing the sum of the lengths of the overlapping nucleotide sequences between M1 and Hv1 and the lengths of the overlapping nucleotide sequences between Hv2 and M2 from the length of the overlapping sequences is preferably 40 bases or more, more preferably 50 bases or more, and even more preferably 150 bases or more.

[0078] Furthermore, in structure (1), adjacent sequences between M1 and Hv1, and between Hv2 and M2, may each be independently separated by a region of a few bases, preferably 1 to 500 bases, more preferably 1 to 100 bases.

[0079] The DNA editing vector according to the DNA editing method of the present invention further includes a first promoter P1, and M1 in structure (1) is operably linked to the first promoter P1. Here, "operably linked" means that the first promoter P1 and M1 in structure (1) are linked so that a selection marker gene formed by the binding of M1 and M2 can be expressed, and usually P1 is located upstream of M1. The first promoter P1 and other regulatory sequences are not particularly limited, but constitutive promoters, tissue-specific promoters, time-specific promoters, inducible promoters, CMV promoters, etc., can be appropriately selected depending on the type of cells to be introduced. For example, if the cells according to the present invention are Nannochloropsis, P1 can be the Nannochloropsis LDSP promoter, Nannochloropsis LHC promoter, Nannochloropsis VCP1 promoter, Nannochloropsis TUB promoter, Nannochloropsis NR promoter, etc. Also, for example, if the cells according to the present invention are animal cells, P1 can be the CMV promoter, etc. Furthermore, the DNA editing vector according to the DNA editing method of the present invention may further appropriately include other regulatory elements (for example, terminators, enhancers, insulators, transcription factor expression cassettes, transcription factor binding sequences, etc.).

[0080] The DNA editing vector according to the DNA editing method of the present invention has the following structure when processed by the site-specific nuclease system according to the present invention (2): 5'-Hv1-D-Hv2-3'…(2) The fragment shown and the following structure (3): 5'-M2-P1-M1-3'…(3) The site-specific nuclease system is designed to produce the fragments shown, or the cleavage site is designed to match the site-specific nuclease system.

[0081] For example, one embodiment of the DNA editing vector according to the DNA editing method of the present invention, where the site-specific nuclease according to the present invention is a TALEN, is shown in the upper part of Figure 1C (a). When the site-specific nuclease according to the present invention is a TALEN, the DNA editing vector according to the DNA editing method of the present invention has the following structure (1): 5'-M1-T1-Hv1-D-Hv2-T2-M2-3'…(11) This includes the following. In structure (11), M1, Hv1, D, Hv2, and M2 are as described in structure (1) above. In structure (11), T1 is a TALEN_1 binding region containing, from the 5' end, a first DNA binding domain recognition sequence or its complementary sequence L1, a spacer S1, and a second DNA binding domain recognition sequence or its complementary sequence R1 adjacent to each other in that order, and T2 is a TALEN_2 binding region containing, from the 5' end, a third DNA binding domain recognition sequence or its complementary sequence L2, a spacer S2, and a fourth DNA binding domain recognition sequence or its complementary sequence R2 adjacent to each other in that order. However, L1 and L2 may be the same as each other and may also be the same as L3 on the target DNA, and R1 and R2 may be the same as each other and may also be the same as R3 on the target DNA, and the TALEN_1 binding region, TALEN_2 binding region, and TALEN_3 binding region may be the same as each other. Furthermore, from the viewpoint of DNA editing accuracy, the sequence identity between L1 and R1, and between L2 and R2, is preferably 40% or less, and more preferably 20% or less.

[0082] In TALEN_1, the first DNA-binding domain (TALE of Left-TALEN) is designed to bind to L1, and the second DNA-binding domain (TALE of Right-TALEN) is designed to bind to R1. Similarly, in TALEN_2, the third DNA-binding domain (TALE of Left-TALEN) is designed to bind to L2, and the fourth DNA-binding domain (TALE of Right-TALEN) is designed to bind to R2. As a result, the S1 and S2 regions are cleaved by the nuclease activity of each TALEN (arrows in Figure 1C(a)). Typically, Left-TALEN and Right-TALEN bind to opposite strands, so the relationship between L1 and R1, and between L2 and R2, is such that if one is a DNA-binding domain recognition sequence, the other is a complementary sequence to the DNA-binding domain recognition sequence. The lengths of L1, R1, L2, and R2 are independently 10 to 30 bases, preferably 13 to 25 bases, and more preferably 15 to 20 bases. Furthermore, the lengths of S1 and S2 are independently 10 to 20 bases, preferably 12 to 19 bases, and more preferably 15 to 16 bases.

[0083] It is preferable that the sequences between M1 and L1, R1 and Hv1, Hv2 and L2, and R2 and M2 do not overlap with each other, but they may overlap to some extent. In this case, the length of each overlapping nucleotide sequence is preferably 15 bases or less, more preferably 15 to 10 bases, and even more preferably 1 to 10 bases. If the 3' end of M1 and the 5' end of M2 are the overlapping sequences, the number of bases obtained by dividing the total length of the overlapping nucleotide sequence between M1 and T1 and the length of the overlapping nucleotide sequence between T2 and M2 by the length of the overlapping sequence is preferably 40 bases or more, more preferably 50 bases or more, and even more preferably 150 bases or more.

[0084] Furthermore, in structure (11), the spaces between M1 and L1, between R1 and Hv1, between Hv2 and L2, and between R2 and M2 may each be independently separated by a region of several bases, preferably 1 to 500 bases, more preferably 1 to 100 bases.

[0085] Furthermore, for example, one embodiment in which the site-specific nuclea of ​​the present invention is a CRISPR-Cas system as the DNA editing vector for the DNA editing method of the present invention is shown in the upper part of Figure 1C (b). When the site-specific nuclease of the present invention is a CRISPR-Cas system, the DNA editing vector for the DNA editing method of the present invention has a structure (1) that includes a first guide RNA recognition sequence or its complementary sequence G1 and a second guide RNA recognition sequence or its complementary sequence G2, preferably the following structure (12): 5'-M1-G1-Hv1-D-Hv2-G2-M2-3'…(12) Includes.

[0086] In the CRISPR-Cas system, the region included in the guide RNA recognition sequence is cleaved by the Cas protein. Therefore, in structure (12), G1 is located between M1 and Hv1 and may overlap with at least one of M1 and Hv1, and G2 is located between M2 and Hv2 and may overlap with at least one of M2 and Hv2. When the 3' end of M1 and the 5' end of M2 are the overlapping sequences, the number of bases obtained by dividing the length of the overlapping sequence by the sum of the lengths of the overlapping nucleotide sequences between M1 and G1 and the lengths of the overlapping nucleotide sequences between G2 and M2 is preferably 40 bases or more, more preferably 50 bases or more, and even more preferably 150 bases or more.

[0087] Furthermore, if the site-specific nuclease according to the present invention is a CRISPR-Cas system, the DNA editing vector according to the DNA editing method of the present invention also includes a PAM sequence recognized by the Cas protein. For example, in the case of a CRISPR-Cas9 system, the PAM sequence is located a few bases ahead of the 3' end of the complementary strand of the G1 and G2 guide RNA recognition sequences.

[0088] G1 and G2 may be the same as each other, and may also be the same as G3 on the target DNA. The lengths of G1 and G2 are independently 12 to 50 bases, preferably 17 to 30 bases, and more preferably 17 to 25 bases.

[0089] The DNA editing vector according to the DNA editing method of the present invention may further include other components, for example, a nucleotide sequence encoding a selection marker gene for confirming the introduction of the vector. In this case, the selection marker gene is the same as that mentioned above, but it must be different from the selection marker genes related to M1 and M2.

[0090] The DNA editing vector according to the DNA editing method of the present invention is a double-stranded, circular vector. Examples of such vectors include plasmid vectors, cosmid vectors, viral vectors, and artificial chromosome vectors, and may consist solely of DNA, or of RNA, GNA, LNA, BNA, PNA, TNA, etc., or a mixture thereof. It may also be modified with components other than nucleic acids, such as sugars. The DNA editing vector according to the DNA editing method of the present invention can be prepared by known methods or similar methods as appropriate, for example, by artificial synthesis or by introducing the above-mentioned components based on an existing vector.

[0091] (Introduction process) In the DNA editing method of the present invention, the DNA editing vector and the site-specific nuclease system are introduced into the cell and brought into contact with the target DNA. The site-specific nuclease system may be introduced into the cell in the form of a protein or RNA, introduced into the form of DNA encoding the protein or RNA and expressed in the cell, or introduced into the form of a vector that expresses the protein or RNA (expression vector) and expressed in the cell. Furthermore, if the site-specific nuclease system is a TALEN, the Left-TALEN and Right-TALEN may be introduced together or separately, and if there are multiple TALENs, they may be introduced together or separately. In addition, if the site-specific nuclease system is a CRISPR-Cas system, the Cas protein and guide RNA may be introduced together or separately, and if there are multiple CRISPR-Cas systems, they may be introduced together or separately. Among these, from the viewpoint of easy introduction together with the DNA editing vector, it is preferable to introduce the site-specific nuclease system in the form of a vector that expresses the site-specific nuclease system (nuclease system expression vector) and express it in the cell.

[0092] Such nuclease system expression vectors are not particularly limited, but from the viewpoint of being able to detach from cells, it is preferable that they contain CEN / ARS as an autonomous replication sequence. Examples of such vectors include the ARS+ all-in-one vector described in Non-Patent Document 3, and more specifically in the present invention, for example, CEN / ARS, an autonomous replicating array, The second promoter P2, and The nucleotide sequence Nuc encoding the site-specific nuclease system is operably linked to the second promoter P2. Examples of nuclease system expression vectors include those containing the above. The CEN / ARS is as described above. The second promoter P2 and other regulatory sequences are not particularly limited, but are similar to those listed as the first promoter P1 above, and P1 and P2 may be the same. Here, "operably linked" means that the second promoter P2 and Nuc are linked so that the site-specific nuclease system encoded by Nuc can be expressed, and usually P2 is located upstream of Nuc. Note that if there are multiple nucleotide sequences that are independent of Nuc (for example, a TALEN containing a nucleotide sequence encoding a Left-TALEN and a nucleotide sequence encoding a Right-TALEN, as described below), there may be multiple P2s corresponding to each nucleotide sequence, and the multiple P2s may be the same or different from each other.

[0093] Nuc is a nucleotide sequence encoding the site-specific nuclease system. If the site-specific nuclease system is a TALEN, examples include a nucleotide sequence encoding a fusion protein of a DNA-binding domain and a nuclease domain as the Left-TALEN, and a nucleotide sequence encoding a fusion protein of a DNA-binding domain and a nuclease domain as the Right-TALEN. These may be on different vectors, but it is preferable that they be on the same vector. Furthermore, if there are multiple TALENs, they may be on different vectors, but it is preferable that they be on the same vector.

[0094] Furthermore, in the case of a CRISPR-Cas system, Nuc can be a combination of the nucleotide sequence encoding the Cas protein and the nucleotide sequence encoding the guide RNA. These may be on different vectors, but it is preferable that they be on the same vector. Moreover, if there are multiple CRISPR-Cas systems, they may be on different vectors, but it is preferable that they be on the same vector.

[0095] As described above, the nuclease domain (FokI) and the nucleotide sequences encoding the Cas protein are publicly known, and the nucleotide sequences encoding the DNA-binding domain and guide RNA can be appropriately designed according to the sequence near the target site of the target DNA.

[0096] Furthermore, the nuclease system expression vector may further appropriately include other regulatory elements (e.g., terminators, inducible promoters, transcription factor expression cassettes, transcription factor binding sequences), and may further include a nucleotide sequence encoding a selection marker gene for confirming the introduction of the vector. The selection marker gene is the same as that listed for the DNA editing vector related to the DNA editing method of the present invention, but it is necessary that it is at least different from the selection marker genes M1 and M2 of the DNA editing vector. Also, if the DNA editing vector further includes a nucleotide sequence encoding a selection marker gene different from the selection marker genes M1 and M2, this selection marker gene may be the same as necessary, but it is preferable that it be different. Such a nuclease system expression vector can be prepared by appropriately known methods or similar methods, for example, by artificial synthesis or by introducing the above components based on an existing vector.

[0097] Furthermore, from the viewpoint of improving DNA editing efficiency, it is more preferable to introduce the DNA editing vector and the nuclease system expression vector into cells in the form of an integrated vector (integrated NICS vector) that combines them. Examples of such integrated vectors include: First promoter P1, Structure (1), Second promoter P2, The nucleotide sequence Nuc encoding the site-specific nuclease system is operably linked to the second promoter P2. An integrated vector containing the above is an example. Preferably, the integrated vector further contains the autonomous replication sequence CEN / ARS. A preferred embodiment of the configuration of the integrated vector of the present invention is shown in Figure 3. The CEN / ARS, P1, structure (1), P2, and Nuc operably linked to P2 are as described above. Furthermore, when designing T1 and T2, or G1 and G2, corresponding to each site-specific nuclease system, the above-described DNA editing vectors are used, respectively.

[0098] The integrated vector may further appropriately include other regulatory elements (e.g., terminators, enhancers, insulators, transcription factor expression cassettes, transcription factor binding sequences), etc., in accordance with the DNA editing vector described above, and may further include a nucleotide sequence encoding a selection marker gene for confirming the introduction of the vector. However, the selection marker gene is the same as that listed above, but it must be different from the selection marker genes related to M1 and M2. Such an integrated vector can be prepared by appropriately known methods or similar methods, for example, by artificial synthesis or by introducing the above components based on an existing vector.

[0099] Various known methods can be used to introduce the DNA editing vector (including the integrated vector) and site-specific nuclease system into the cells, such as the polyethylene glycol method, electroporation, Agrobacterium-mediated method, particle gun method, laser ablation method, and whisker method.

[0100] (cutting process) In the DNA editing method of the present invention, the DNA editing vector and the site-specific nuclease system are introduced into cells in the introduction step and brought into contact with the target DNA, thereby the site-specific nuclease system extracts the following structure from the vector (2): 5'-Hv1-D-Hv2-3'…(2) The fragment shown and the following structure (3): 5'-M2-P1-M1-3'…(3) (If the vector further includes CEN / ARS, then 5'-M2-CEN / ARS-P1-M1-3'…(3)) This produces a fragment represented by and can cleave the target site or its vicinity in the target DNA.

[0101] Structure (2) contains Hv1, D, and Hv2 in that order from the 5' end, and its composition is as described in structure (1). Structure (3) is the remaining structure after structure (2) has been excised from the DNA editing vector, and contains M2, P1, and M1 in that order from the 5' end, preferably M2, CEN / ARS, P1, and M1 in that order. Each component of structure (3) is as described in the DNA editing vector, and other components may be included as appropriate. Furthermore, the cleavage sites in the DNA editing vector and the target DNA are as described above.

[0102] For example, when S1 and S2 are cleaved by TALEN, in some cases, parts of T1 and T2 may remain attached to the 5' end of Hv1 and the 3' end of Hv2 in structure (2), respectively. However, these are usually removed by the intracellular repair system during binding of Hv1 and Hv2 to Ht1 and Ht2 in the cell, which depends on homology. Similarly, when S1 and S2 are cleaved by TALEN, in some cases, parts of T1 and T2 may remain attached to the 3' end of M1 and the 5' end of M2 in structure (3), respectively. These are removed by the intracellular repair system or by binding of M1 and M2 in the cell (preferably binding that depends on complementarity via the aforementioned duplicate sequence). Likewise, when G1 and G2 are cleaved by the CRISPR-Cas system, if parts of bases remain attached to each end, they are usually removed by the intracellular repair system during cleavage and / or binding, similar to the case of cleavage by TALEN.

[0103] (Editing process) The target DNA, which has been cut at or around the target site in the above cleavage step, and the fragment shown in structure (2) excised from the DNA editing vector, bind in a homology-dependent manner between Ht1 and Hv1, and in a homology-dependent manner between Ht2 and Hv2. In Ht1 and Hv1, and Ht2 and Hv2, "binding in a homology-dependent manner" means binding by homologous sequence-dependent repair (HDR), in which each homologous sequence aligns according to its sequence and undergoes annealing and ligation, as well as removal of overhanging ends, by the intracellular repair system. This makes it possible to obtain DNA-edited cells in which the desired nucleotide sequence D is precisely inserted between the target site, i.e., between Ht1 and Ht2, or in which the target site is removed from between Ht1 and Ht2.

[0104] Confirmation of whether or not the desired nucleotide sequence D is inserted or whether or not the target site is removed can be performed based on conventionally known methods, such as PCR, sequencing, or Southern blotting, and the DNA editing method of the present invention may further include confirmation steps using these methods.

[0105] (Selection process) In the fragment shown in structure (3) obtained by cutting out the fragment shown in structure (2) in the above cutting process, M1 and M2 are functionally separated, so that the 3' end of M1 and the 5' end of M2 are joined, and a selection vector containing P1 and a functionally selectable marker gene that is operably linked to P1 is obtained, preferably a selection vector containing CEN / ARS, P1, and a functionally selectable marker gene that is operably linked to P1 is obtained. When the integrated vector is used as the DNA editing vector, a selection vector containing P1, a functionally selectable marker gene that is operably linked to P1, P2, and Nuc that is operably linked to P2 is obtained, preferably a selection vector containing CEN / ARS, P1, a functionally selectable marker gene that is operably linked to P1, P2, and Nuc that is operably linked to P2 is obtained.

[0106] Since the selection marker gene functions in cells from which the selection vector has been obtained, these cells can be selected and obtained using the selection marker gene as an indicator. For example, if the selection marker gene is a drug resistance gene, cells can be selected based on their survival in an environment containing the drug, and if the selection marker gene is a reporter gene, cells can be selected based on their reporter activity (e.g., fluorescence).

[0107] Furthermore, since the acquisition of the selection vector strongly suggests that the above-mentioned cleavage and editing steps were also performed within the same cell, cells selected using the selection marker gene as an indicator can be selected as cells in which the desired nucleotide sequence D has been inserted into the target site or the target site has been removed, thereby enabling the efficient acquisition of DNA-edited cells in which the nucleotide sequence D has been inserted into the target site or the target site has been removed.

[0108] (Removal of selected vectors) The DNA-edited cells obtained by the DNA editing method of the present invention also include the selection vector, and it is preferable if the vector has CEN / ARS because it can be removed from the cells by culturing them. Furthermore, if an expression vector is used to introduce the nuclease system, a vector having CEN / ARS, such as the nuclease system expression vector described above, can similarly be removed from the cells.

[0109] For example, if the selection marker gene is a drug resistance gene, the selection process described above is carried out under conditions (selective medium) where a corresponding drug is added to apply selective pressure corresponding to the selection marker gene, thereby obtaining DNA-edited cells in which the selection marker gene functions and the desired nucleotide sequence D is inserted into the target site or the target site is removed. Next, the cells whose survival in the selective medium has been confirmed are cultured under conditions without selective pressure, i.e., in a medium without the drug, thereby removing the selection vector, including CEN / ARS. The culture period under the conditions without selective pressure is usually preferably 10 days or more, and more preferably 10 to 14 days. The removal of the selection vector from the cells can be confirmed, for example, by the fact that a portion of the cell line cultured under the conditions without selective pressure cannot survive when transferred back to conditions with selective pressure (selective medium), or by the fact that nucleotide sequences derived from the selection vector cannot be detected by PCR, etc.

[0110] For example, if the selection marker gene is a reporter gene, cells are selected using the detection of fluorescence originating from the selection marker gene as an indicator up to the selection step described above, thereby obtaining DNA-edited cells in which the selection marker gene is functional and the desired nucleotide sequence D is inserted into the target site or the target site is removed. These cells are then cultured for at least 10 days, preferably 10 to 14 days, to remove the selection vector containing CEN / ARS, and cells in which the fluorescence is no longer detected are obtained as cells from which the selection marker has been removed. The removal of the selection vector from the cells can also be confirmed, for example, by the absence of detection of nucleotide sequences originating from the selection vector by PCR.

[0111] In the DNA editing method of the present invention, the cleavage step, editing step, and selection step may occur in this order after the introduction step, but they may also occur simultaneously and in parallel with each other. A preferred embodiment of the DNA editing method of the present invention, up to the removal of the selection vector, will be described below with reference to Figures 2A and 2B, using the nuclease system expression vector as an example for the introduction of the site-specific nuclease system, but the present invention is not limited thereto.

[0112] In the DNA editing method of the present invention, first, the above-mentioned DNA editing vector and nuclease system expression vector are introduced into cells, causing the nuclease system (Nuc) to be expressed from the nuclease system expression vector, and the DNA editing vector and target DNA are cleaved (three arrows on the left of Figure 2A: introduction step and cleavage step). As a result, a cell is obtained in which the desired nucleotide sequence D is inserted into the target site or the target site is removed (D is inserted into the target site in Figures 2A and 2B), and which also contains the selection vector (upper left of Figure 2B: editing step and selection step). These cells can be selected using a selection marker gene contained in the selection vector. For example, if the selection marker gene is a drug resistance gene, cells into which only the DNA editing vector or the nuclease system expression vector has been introduced, or cells into which both have been introduced but cleavage has not occurred, cannot survive by culturing under selective pressure conditions with the corresponding drug added (right of Figure 2A). These cells can then be selected and obtained using this as an indicator. Furthermore, by culturing these cells (for example, in a medium without added drugs if the selection marker gene is a drug resistance gene), the selection vector containing CEN / ARS and the nuclease system expression vector are removed (Figure 2B right), thus obtaining cells that do not contain foreign genes other than the desired nucleotide sequence D (Figure 2B bottom left).

[0113] <Cell manufacturing method> The present invention is a method for producing DNA-edited cells in which a desired nucleotide sequence is inserted into or removed from a target site of target DNA. The steps of obtaining a cell containing the selection vector, wherein the desired nucleotide sequence D is inserted into the target site or the target site is removed by the DNA editing method of the present invention described above, and The process of selecting DNA-edited cells in which the desired nucleotide sequence D is inserted into the target site or the target site is removed, using a selection marker gene contained in the selection vector as an indicator. The invention also provides a method for producing DNA-edited cells, including the method described above.

[0114] The steps for obtaining cells containing the selection vector, in which the desired nucleotide sequence D is inserted into or removed from the target site, by the DNA editing method of the present invention, are as described in the introduction step to the selection step above. Furthermore, the steps for selecting DNA-edited cells in which the desired nucleotide sequence D is inserted into or removed from the target site, using a selection marker gene contained in the selection vector as an indicator, are as described in the selection step above.

[0115] The cell production method of the present invention preferably further includes the step of obtaining cells containing a selection vector containing CEN / ARS using a vector containing CEN / ARS as the DNA editing vector, and further culturing the DNA-edited cells to remove the selection vector from the cells. Such a step is as described in the section on the removal of the selection vector. This makes it possible to obtain cells that do not contain foreign genes other than the inserted desired nucleotide sequence.

[0116] Therefore, the present invention also provides a method for producing a non-human organism, which includes cells in which a desired nucleotide sequence has been inserted into target DNA or the target site has been removed, and preferably further includes a non-human organism, which includes cells that do not contain foreign genes other than the desired nucleotide sequence. This method includes the step of producing a non-human organism from cells obtained by the cell production method of the present invention described above.

[0117] Examples of non-human organisms include non-human animals, plants, and algae. Examples of non-human animals include mammals (mice, rats, guinea pigs, hamsters, rabbits, humans, monkeys, pigs, cows, goats, sheep, etc.), fish, birds, reptiles, amphibians, and insects. Examples of plants include grains, oil crops, fodder crops, fruits, and vegetables. Examples of algae include Nannochloropsis, Chlamydomonas, Euglena, Chlorella, Botryococcus, diatoms, coccolithophores, cyanobacteria, Cyanidioschizophyllum, Pseudococomyxa, Spirulina, and Haematococcus.

[0118] As a method for producing a non-human individual from cells, known methods can be appropriately used. When producing a non-human individual from cells in an animal, usually, germ cells or pluripotent stem cells are used. For example, the above DNA editing vector and site-specific nuclease system are microinjected into an oocyte, and the obtained oocyte is then transplanted into the uterus of a pseudo-pregnant female non-human mammal, and offspring are obtained thereafter. The transplantation can be performed on a fertilized egg at the 1-cell stage embryo, 2-cell stage embryo, 4-cell stage embryo, 8-cell stage embryo, 16-cell stage embryo, or morula stage embryo. The microinjected oocyte can be cultured under appropriate conditions until transplantation if necessary. The transplantation and culture of the oocyte can be performed based on conventionally known techniques.

[0119] In plants, it has long been known that their somatic cells have totipotency, and methods for regenerating plant bodies from plant cells have been established in various plants. Therefore, for example, by microinjecting the above DNA editing vector and site-specific nuclease system into a plant cell and regenerating a plant body from the obtained plant cell, a DNA-edited plant body in which a desired nucleotide sequence is inserted or the target site is removed can be obtained.

[0120] The confirmation of the presence or absence of DNA editing can be performed based on conventionally known techniques, and for example, the PCR method, sequencing method, Southern blotting method, etc. can be appropriately used. From the obtained non-human individual, offspring and clones in which the desired DNA is edited can also be obtained.

[0121] <DNA editing vector> The present invention provides a DNA editing vector for use in the above DNA editing method of the present invention. The DNA editing vector of the present invention a first promoter P1, and the following structure (1'): 5'-M1-Hv1'-D'-Hv2'-M2-3'...(1') contains, by the site-specific nuclease system, the following structure (2'): 5'-Hv1'-D'-Hv2'-3'…(2') The fragment shown and the following structure (3'): 5'-M2-P1-M1-3'…(3') The vector produces the fragment shown by . The DNA editing vector of the present invention preferably further contains an autonomous replication sequence CEN / ARS, in which case the fragment shown as 5'-M2-CEN / ARS-P1-M1-3' is produced as structure (3').

[0122] The DNA editing vector of the present invention is the same as the DNA editing vector relating to the DNA editing method of the present invention described above, including its preferred embodiments, except that in the above structure (1), D is replaced by the desired nucleotide sequence or its insertion site D' (however, this may be omitted in the case of a DNA removal method), and Hv1 and Hv2 are replaced by a nucleotide sequence homologous to the first nucleotide sequence Ht1 or its insertion site Hv1' and a nucleotide sequence homologous to the second nucleotide sequence Ht2 or its insertion site Hv2', respectively. In the DNA editing vector provided by the present invention, these components may be used as insertion sites so that users can design them according to their desired target site. In this case, Hv1'-D'-Hv2' may together be a single insertion site. Furthermore, if Hv1', D', or Hv2' is the insertion site, structure (2') is the structure obtained by cutting structure (1') after inserting each component, i.e., 5'-Hv1-D-Hv2-3'…(2) (structure (2) is as described above).

[0123] In addition, when the site-specific nuclease system is TALEN, the DNA editing vector of the present invention, in the above structure (11), in addition to the above structure (1'), instead of T1 and T2, respectively, the TALEN_1 binding region or its insertion site T1' and the TALEN_2 binding region or its insertion site T2', is the same as the DNA editing vector according to the DNA editing method of the present invention when the above site-specific nuclease system is TALEN, including its preferred embodiments. In such a DNA editing vector, these components may also be used as their insertion sites so that the user can design them according to the target site of interest. Also in this case, T1'-Hv1'-D'-Hv2'-T2' may be a single insertion site together.

[0124] Furthermore, when the site-specific nuclease system is the CRISPR-Cas system, the DNA editing vector of the present invention, in the above structure (12), in addition to the above structure (1'), instead of G1 and G2, respectively, the first guide RNA recognition sequence or its complementary sequence or their insertion site G1', and the second guide RNA recognition sequence or its complementary sequence or their insertion site G2', is the same as the DNA editing vector according to the DNA editing method of the present invention when the above site-specific nuclease system is the CRISPR-Cas system, including its preferred embodiments. In such a DNA editing vector, these components may also be used as their insertion sites so that the user can design them according to the target site of interest. Also in this case, G1'-Hv1'-D'-Hv2'-G2' may be a single insertion site together.

[0125] Examples of the insertion site in each of the above components include, but are not limited to, a multiple cloning site.

[0126] <DNA Editing Kit> The present invention also provides a kit for use in the DNA editing method of the present invention. The kit of the present invention includes the DNA editing vector of the present invention and the site-specific nuclease system. The DNA editing vector and the site-specific nuclease system are as described above, including their preferred embodiments. Further, the DNA editing vector and the site-specific nuclease system may be in the form of the above-described integrated vector.

[0127] The DNA editing kit of the present invention may further include one or more reagents. Examples of the reagents include, but are not limited to, drugs and substrates corresponding to the selection marker gene, restriction enzymes and their reaction solutions for inserting a target sequence into the insertion site, dilution buffers, reconstitution solutions, washing buffers, control reagents (e.g., control DNA targeting nuclease systems), and reagents for introducing vectors into cells.

[0128] Each element included in the DNA editing kit of the present invention may be contained in separate containers or in the same container. Further, it may be contained in a container for each single use amount, or an amount for multiple uses may be contained in one container (the user can take out and use the amount required for a single use). Each element may be contained in a container in a dry form or in a form dissolved in an appropriate solvent.

[0129] <Application Modes of NICS System> In the DNA editing method of the present invention, as described above, a site-specific nuclease system is used to cleave the target DNA and excavate a fragment (structure (2)) containing nucleotide sequences homologous to the nucleotide sequences before and after the target site of the target DNA, and a selection vector is obtained to which a selection marker gene, divided into a 5' fragment M1 and a 3' fragment M2, binds and functions. Therefore, the presence of such a selection vector makes it possible to obtain DNA-edited cells in which the desired nucleotide sequence is inserted into the target site of the target DNA or the target site is removed with high efficiency. The knowledge of the NICS system discovered by the present inventors is applicable, and one example of an application of such a NICS system is to perform multi-step excision and binding by combining two or more nuclease systems that cleave different sites, respectively. An example of such an example is shown in Figures 17 and 18.

[0130] For example, if the site-specific nuclease system is a combination of two types, nuclease system A (NucA) and nuclease system B (NucB), the configuration of the vector introduced into the cell can be applied as an integrated vector according to the present invention. Promoter Pa, The following structure (a): 5'-M1-Y-M2-3'…(a), promoter Pb, The following structure (b): 5'-Nb1-X-Nb2-3'…(b), The following structure (c): 5'-Hv1-D-Hv2-3'…(c) Examples of such embodiments include the above. Preferably, the integrated vector further includes an autonomous replicating sequence called CEN / ARS.

[0131] Here, in structure (a), M1 and M2 are the same as in structure (1), except that M1 is operably linked to promoter Pa (whereas Pa is also synonymous with P1), and Y represents an arbitrary nucleotide sequence. In structure (b), Nb1 represents the 5' end of the nucleotide sequence NucB encoding nuclease system B and is operably linked to promoter Pb (Pb is synonymous with P2), X represents an arbitrary nucleotide sequence different from Y, and Nb2 represents the remaining 3' end of the nucleotide sequence encoding nuclease system B. Furthermore, structure (c) is synonymous with structure (2) above, but may consist of one or two types. Structure (c) is also included in the vector as X and / or Y. If there is only one type of structure (c) and X or Y is other than structure (c), the nucleotide sequence of X or Y other than structure (c) is not particularly limited and can be any sequence, as long as it can be fragmented in such a way that the selection marker gene or nuclease system B does not function (Figure 17).

[0132] Furthermore, Nb1 and Nb2 are nucleotide sequences in which nuclease system B (NucB) is fragmented so that it can function by binding the 3' end of Nb1 to the 5' end of Nb2, similar to M1 and M2 described above. Therefore, by designing nuclease system A (NucA, for example, the nuclease system expression vector described above) to excise X from structure (b), and introducing the vector and NucA into cells, X is excised, and the 3' end of Nb1 and the 5' end of Nb2 bind to form NucB, allowing it to function. The functional NucB can then be further designed to excise Y from structure (a), allowing Y to be excised, and the 3' end of M1 and the 5' end of M2 bind to form a selection marker gene, allowing the selection vector containing the selection marker gene (and NucB) to be obtained, similar to the selection vector described above. Structure (c), consisting of X and / or Y, is excised, and, similar to the NICS system described above, nucleotide sequence D is inserted into the target site of the target DNA (Figures 17-18) or the target site is removed from the target DNA (not shown), depending on the homology between Ht1 and Hv1 and between Ht2 and Hv2. If there is only one structure (c), the site-specific nuclease system that cleaves the target DNA may be NucA or NucB. If there are two structures (c), it is preferable that the nuclease that excises the structure and the nuclease that cleaves the site or surrounding area where the structure is inserted are the same (for example, in Figure 18, NucA excises X and cleaves the target site or surrounding area where X is inserted, and NucB excises Y and cleaves the target site or surrounding area where Y is inserted). [Examples]

[0133] The present invention will be described more specifically below based on examples and comparative examples, but the present invention is not limited to the following examples. Each of the following tests was carried out under the conditions and methods described below.

[0134] 1. Target cells and their culture conditions 1 As one form of target cell, we used the single-celled alga Nannochloropsis oceanica strain (NIES Collection, National Institute for Environmental Studies, No. 2145). The culture medium used was F2N liquid medium as described in section 2 below. We used an LP-200P incubator (NK system) in a filtered Erlenmeyer flask at a temperature of 25°C and a light intensity of 57 μmol photons·m. -2 ·s -1 The cultures were incubated at a shaking speed of 102 rpm. For normal plate culture, the F2N plate shown in section 3 below was used. For culture with added antibiotics (culture using a selective plate), the amount of Daigo artificial seawater SP in (B) was halved (18 g), and an F2N 50% seawater medium plate prepared in the same manner as the F2N plate shown in section 3 below was used, except that the antibiotic was aseptically added when mixing (A) to (D). The final antibiotic concentrations were zeosin: 2 μg / mL and paromomycin: 150 μg / mL.

[0135] 2. Preparation of F2N liquid culture medium The F2N liquid medium was prepared by mixing (A), (B), and (C) below. The composition for preparing 1 L of F2N liquid medium is shown below.

[0136] (A) NaNO3 (75 mg / mL) 1 mL NaH2PO4·2H2O (30 mg / mL) 1 mL Na2SiO3·9H2O (10 mg / mL) 1 mL f / 2 metal 5mL 1M Tris-HCl (pH7.6) (12.11g / 100mL) 10mL 500mM NH4Cl (2.67g / 100mL) 10mL The above composition was diluted to 500 mL with ultrapure water and sterilized by autoclaving.

[0137] For the f / 2 metal, a solution with the following composition was mixed, diluted to 100 mL with ultrapure water, autoclaved, and stored at 4°C.

[0138] Na2·EDTA 440mg FeCl3·6H2O 316mg CoSO4·7H2O (1.2 mg / mL) 1 mL ZnSO4·7H2O (2.1 mg / mL) 1 mL MnCl2·4H2O (18 mg / mL) 1 mL CuSO4·7H2O (0.7 mg / mL) 1 mL Na2MoO4·7H2O (0.7mg / mL) 1mL.

[0139] (B) Daigo Artificial Seawater SP (Fujifilm Wako Pure Chemical Industries) 36g The solution was diluted to 500 mL with ultrapure water and then autoclaved.

[0140] (C) Thiamine HCl (1 mg / mL) 500 μL Vitamin B12 (50 μg / mL) 50 μL Biotin (50 μL / mL) 50 μL Each was sterilized by filter and stored at -20°C, then aseptically added to a mixture of (A) and (B) that had been autoclaved.

[0141] 3. Fabrication of F2N plate The F2N plate (solid culture medium) was prepared by mixing (A), (B), (C), and (D) below, pouring the mixture into a 9mm diameter plastic plate, and allowing it to cool and solidify. The composition for preparing 1L of F2N solid culture medium is shown below.

[0142] (A) NaNO3 (75 mg / mL) 1 mL NaH2PO4·2H2O (30 mg / mL) 1 mL Na2SiO3·9H2O (10 mg / mL) 1 mL f / 2 metal 5mL 1M Tris-HCl (pH7.6) (12.11g / 100mL) 10mL 500mM NH4Cl (2.67g / 100mL) 10mL The above composition was diluted to 250 mL with ultrapure water and sterilized by autoclaving. The f / 2 metal is as shown in section 2 above.

[0143] (B) Daigo Artificial Seawater SP (Fujifilm Wako Pure Chemical Industries) 36g The solution was diluted to 450 mL with ultrapure water and then autoclaved.

[0144] (C) Thiamine HCl (1 mg / mL) 500 μL Vitamin B12 (50 μg / mL) 50 μL Biotin (50 μL / mL) 50 μL Each was sterilized by filter and stored at -20°C, then aseptically added to the autoclaved mixtures of (A), (B), and (D).

[0145] (D) Agar 8g The material was suspended in 300 mL of ultrapure water and sterilized by autoclaving.

[0146] 4. Cell count of Nannochloropsis The Nannochloropsis culture medium was collected, and 2 μL was added to a bacterial counter chamber (SLGC) using a pipette. The cells were then observed using an ECLIPSE E100 microscope (Nikon) and the cell count was measured. The count was performed twice, and the average value was calculated as the cell count.

[0147] 5. Electroporation Method For electroporation, we used the conventional method described in 5-2 and the novel method described in 5-3. Unlike the conventional method, method 5-3 does not require carrier DNA, which is an exogenous gene derived from salmon sperm DNA. Pre-culture 5-1 and plating 5-4 are common to both methods.

[0148] 5-1. Preculture The Nannochloropsis strain was cultured for approximately 7-10 days under the normal culture conditions described in 1. above, until the cell concentration reached 2.5 × 10⁶. 6 The cells were subcultured in 250 mL of F2N liquid medium to achieve a cell / mL ratio, and then further cultured.

[0149] 5-2. Electroporation using Gene Pulser Xcell (manufactured by BIORAD) For Nannochloropsis strains cultured for approximately 4-6 days after the aforementioned pre-culture, cell concentration was measured by cell count. 200 mL of culture medium was collected in four 50 mL Falcon tubes and centrifuged at 4°C and 4,900 rpm for 7 minutes. The supernatant was removed, and 37.5 mL of ice-cold 375 mM sorbitol was added to each tube for suspension. The tubes were then centrifuged at 4°C and 4,900 rpm for 7 minutes, and the supernatant was removed. This procedure was repeated twice for washing, and then the final cell concentration was measured to 1.5 × 10⁶. 9 Cells were suspended in 375 mM sorbitol to a concentration of cells / mL. 3 μL of salmon sperm DNA (10 mg / mL) was incubated at 95°C for 1 minute as carrier DNA, and then rapidly cooled on ice. Subsequently, the DNA to be introduced (each vector, knock-in fragment, etc.) was mixed with 3 μL of carrier DNA and placed in a Gene Pulser Cuvette 0.2 cm (BIORAD). 150 μL of the cell suspension solution was added, and the mixture was cooled on ice for at least 5 minutes. The moisture from the cuvette was wiped off, and the following conditions were met: Voltage: 2200V, Capacitance: 50μF Resistance: 600Ω Cuvette length: 2mm Electroporation was performed. Immediately after electroporation, the entire volume of the solution was transferred to a 15 mL Falcon tube containing 5 mL of F2N liquid medium as soon as possible. The cap of the Falcon tube was loosened and secured with surgical tape, and the sides were wrapped with two layers of paper towels to create low-light conditions, and recovery culture was performed by shaking.

[0150] 5-3. Electroporation using ELEPO21 (manufactured by Neppageen Inc.) Similarly to 5-2 above, the Nannochloropsis strains cultured for 4-6 days after the pre-culture were harvested and washed twice. The cell suspension was then combined into two centrifuge tubes, and the cells were washed twice more with 40 mL each of 375 mM sorbitol to remove the salt from the culture medium. Subsequently, the final cell concentration was 1.0 × 10⁶. 10 Cells were suspended in 375 mM sorbitol cooled on ice to a concentration of cells / mL. 2 μL (or 1 μL) of DNA to be introduced (total amount), 38 μL of the cell suspension solution, and 40 μL of 375 mM sorbitol were combined and allowed to stand at room temperature for 10 minutes. The mixture was then transferred to a 1 mm gap cuvette and cooled on ice for at least 5 minutes. After wiping off any moisture from the ice-cooled cuvette, the corners were lightly tapped on the workbench to remove air bubbles, and then the cuvette was placed in the chamber for electroporation. Electroporation was performed under the following conditions: Poring pluse: Voltage: 1750V, Pulse width: 3.5ms, Pulse interval: 50ms, Number of pulses: 1, Polarity: + Transfer plus: Voltage: 100V, Pulse width: 50ms, Pulse interval: 50ms, Number of pulses: 3, Polarity: + / - The procedure was carried out as described above. After electroporation, recovery culture was performed using the same procedure as in 5-2.

[0151] 5-4. Plating After recovery culture for 2-4 days, the paper towel and tape were removed from the 15 mL Falcon tube used for recovery culture, and the culture was centrifuged at 2,000 rpm for 5 minutes, with the supernatant removed. The cultured DNA was suspended in 2 mL of Top Agar, which had been melted by heating in a microwave oven and then cooled to approximately 40°C. This suspension was then spread onto an F2N 50% seawater medium plate containing antibiotics appropriate to the introduced DNA, and dried for approximately 30 minutes to allow the agar to solidify. The plate was sealed with surgical tape and cultured statically in an incubator to allow colony formation. As the Top Agar, F2N liquid medium containing 0.4% agar was used.

[0152] 6. Extraction of genomic DNA To extract genomic DNA from the cells (Nannochloropsis), the method described in 6-1 or 6-2 below was used.

[0153] 6-1. Method using phenol / chloroform First, cells were cultured in a 12-well plate in 2 mL of F2N liquid medium for approximately 2 weeks. The culture medium was then centrifuged at 20°C and 4,900 rpm for 5 minutes to collect the cells. 500 μL of TEN buffer was added to suspend the pellet, and the mixture was centrifuged at 20°C and 4,900 rpm for 5 minutes. The supernatant was removed by pipette. The pellet was suspended in 150 μL of ice-cold sterile ultrapure water, and immediately afterwards, 350 μL of SDS-EB buffer was added and mixed. 500 μL of a 1:1 phenol / chloroform solution was then added and mixed vigorously. The mixture was then centrifuged at room temperature and 14,500 rpm for 10 minutes. The upper layer was transferred to a new 1.5 mL tube, another 500 μL of phenol / chloroform solution was added and mixed, and the mixture was centrifuged at room temperature and 14,500 rpm for 10 minutes. The upper layer was transferred to a new 1.5 mL tube, 50 μL of 1.82 M NaCl and 500 μL of isopropanol were added and mixed slowly, and the mixture was centrifuged at room temperature at 14,500 rpm for 15 minutes, with the supernatant removed. The pellet was slowly suspended in 200 μL of TE buffer, 20 μL of 1.82 M NaCl and 550 mL of 100% ethanol were added and mixed, and the mixture was centrifuged at 20°C at 14,500 rpm for 15 minutes, with the supernatant removed. 200 μL of 70% ethanol was added, and the mixture was centrifuged at room temperature at 14,500 rpm for 10 minutes, the supernatant was removed by pipette, and the mixture was dried under reduced pressure for 10 minutes. Then, 40 μL of TE buffer was added, the mixture was heated at 55°C for 10 minutes, air-cooled to room temperature, and allowed to stand overnight at 4°C to dissolve the genomic DNA in TE buffer. The concentration of genomic DNA was measured and diluted with sterile water to 10 ng / μL to be used as template DNA for PCR. (TEN buffer) 1M Tris-HCl 1mL 500 mM EDTA 2 mL NaCl 0.8766g The solution was diluted to 100 mL with ultrapure water and sterilized in an autoclave. (SDS-EB buffer) 1M Tris-HCl 10mL 500mM EDTA 8mL NaCl 2.3376g SDS 2.0g The solution was diluted to 100 mL with ultrapure water and sterilized in an autoclave. (1.82M NaCl) NaCl 5.3g The solution was diluted to 50 mL with ultrapure water and sterilized in an autoclave. (fish buffer) 1 mL of 1 M Tris-HCl (pH 8.0) 0.5M EDTA (pH 8.0) 0.2mL The solution was diluted to 100 mL with ultrapure water and sterilized in an autoclave.

[0154] 6-2. Method using a simple DNA extraction kit For the extraction of genomic DNA when examining a large number of samples in a simple manner, a simple DNA extraction kit (Kaneka Simple DNA Extraction Kit version 2 (Kaneka Corporation)) was used. Specifically, cell colonies were suspended in 10 μL of reagent A and incubated at 98°C for 8 minutes. After cooling to room temperature, 1.4 μL of reagent B was added and mixed by pipetting. The resulting crude extract was diluted to 1 / 10 with sterile water and used as the template DNA for PCR.

[0155] 7. PCR reaction The PCR reaction was carried out using a reaction solution with the following composition, and scaled up as needed. The PCR reaction was also performed using a thermal cycler following the reaction cycle described below. (Reaction solution) KOD FX Neo buffer (manufactured by TOYOBO) 5 μL 2 mM dNTPs 2 μL Sterile ultrapure water 0.4μL 10 mM primer solution, 0.4 μL each Template DNA 2 μL KOD FX Neo (manufactured by TOYOBO) 0.2 μL (Reaction cycle) 94℃ 2min ↓ 98℃ 10sec 68℃ 1 min / kb (1 min per 1 kb of amplified product) 30 cycles ↓ 68℃ 1 min / kb + 1 min 4℃ ∞.

[0156] 8. Agarose gel electrophoresis 50×TAE was diluted with ultrapure water to prepare 1×TAE. 1g / 100mL or 3g / 100mL of Agarose S (Nippon Gene Co., Ltd.) was added to the 1×TAE, heated in a microwave oven to dissolve, and poured into a gel maker equipped with a comb to prepare 1% or 3% agarose gels. A 3% agarose gel was used when the band size of the DNA to be electrophoresed was 1kb or less. 6×loading buffer (New England Biolabs Co., Ltd.) was added to the electrophoresis sample, applied to the well, and electrophoresis was performed at 100V until the gel reached approximately 70% capacity. 4μL of ethidium bromide was added to deionized water, and the post-electrophoresis gel was immersed and shaken for approximately 30 minutes to stain it. The electrophoresis pattern was observed using a transilluminator. (50×TAE) Tris 48.4g 0.5M EDTA 20mL Acetic acid 11.42 mL The solution was diluted to 200 mL using ultrapure water.

[0157] 9. Sequence First, prepare the reaction mix for sequencing with the following composition: 5×Sequence Buffer (Thermo Fisher Scientific) 2 μL 10 mM primer, 0.32 μL each Template DNA 100 ng / kb The reaction was then prepared by adding sterile ultrapure water to make a volume of 9 μL. The prepared sequencing reaction mix was heated at 95°C for 5 minutes, then rapidly cooled on ice for 5 minutes, and then 1 μL of Big Dye terminator (Thermo Fisher Scientific) was added for the following reaction: 96℃ 2min ↓ 96℃ 10sec 50℃ 5sec 60℃ 4min 30 cycles ↓ 4℃ ∞ The analysis was performed using a thermal cycler. The primers used were one designed based on the nucleotide sequence upstream of PDAT1 in Nannochloropsis (SEQ ID NO: 26, showing the nucleotide sequence) and another designed based on the nucleotide sequence downstream of PDAT1 in Nannochloropsis (SEQ ID NO: 27, showing the nucleotide sequence).

[0158] After the reaction was complete, 10 μL of the reaction product was mixed with 1 μL of 1.5 M sodium acetate (pH 5.0), 1 μL of 0.25 M EDTA (pH 8.0), and 40 μL of 100% ethanol, and allowed to stand at room temperature for 15 minutes. The mixture was centrifuged at 20°C and 14,500 rpm for 15 minutes, and the supernatant was removed. 100 μL of 70% ethanol was added, and the mixture was centrifuged at 20°C and 14,500 rpm for 15 minutes, the supernatant was removed, and the mixture was dried under reduced pressure for 10 minutes. 20 μL of HiDi formamide was added to the dried sample to dissolve the pellet, and the mixture was heated at 95°C for 2 minutes and rapidly cooled on ice for 5 minutes. The sample was transferred to a sequencing plate, a plate sealing mat was set, and sequencing was performed using a SeqStudio™ Genetic Analyzer (Thermo Fisher Scientific).

[0159] 10. Dropping of CEN / ARS vectors Colonies grown on the selection plate were collected on an F2N plate (without antibiotics) and used as the master plate. From the master plate, subcultured in 30 mL of F2N liquid medium and cultured for 1-2 weeks, resulting in 1 × 10⁶ colonies. 6The cells were subcultured in fresh F2N liquid medium to a concentration of cells / mL and cultured for another two weeks. The culture medium was then diluted with F2N medium to a concentration of 3000 cells / plate and spread onto F2N plates. Candidate colonies that were eliminated from the F2N plates were seeded onto F2N plates and F2N 50% seawater plates containing antibiotics, respectively. Strains that did not grow on the antibiotic-containing plates were identified as strains that had lost the CEN / ARS vector.

[0160] 11. DNA extraction from agarose gel For DNA extraction from the agarose gel described in 8. above, the Wizard(R) SV Gel and PCR Clean-Up System (Promega) was used. From the agarose gel after electrophoresis described in 8. above, gel fragments containing the target band were cut out with a razor blade using a WSE-5400 Printgraph Classic (ATTO). 100 μL of Membrane Binding Solution (Promega) was added per 100 mg of gel fragment, followed by an additional 100 μL. The gel fragments were heated at 55°C in a block incubator to completely dissolve them and then added to the column. The column was centrifuged at room temperature at 12,000 rpm for 2 minutes, the flow-through was removed from the collection tube, and 750 μL of Membrane Wash Solution (Promega) was added. The column was then washed by centrifugation at room temperature at 12,000 rpm for 2 minutes. The column was washed again with 500 μL of Membrane Wash Solution. The column was centrifuged at room temperature at 12,000 rpm for 2 minutes to remove the Membrane Wash Solution. The column was then placed in a new 1.5 mL tube, 30 μL of Nuclease-Free Water (Promega) was added, and the mixture was allowed to stand for 1 minute. Finally, the column was centrifuged at 20°C at 12,000 rpm for 2 minutes to elute the DNA. The concentration of the extracted DNA solution was measured using Nanodrop (Thermo Fisher Scientific) and used as the template DNA for the sequence described in step 9 above.

[0161] 12. Target cells and their culture conditions 2 Another variant of the target cell used was HEK293T (human embryonic kidney cells). The following cell experiments were performed in a clean bench, and before each operation, the instruments to be used (electric pipettes, pipettes, plates, etc.) were placed in the clean bench and subjected to UV treatment for at least 15 minutes.

[0162] 12-1. Preparation of complete culture medium 500 mL of DMEM (High Glucose) with L-Glutamine and Phenol Red was mixed with 5.5 mL of 10 × Non-Essential Amino Acid (NEAA), 5.5 mL of Penicillin-Streptomycin (ST-PN), and 55 mL of Fetal Bovine Serum (FBS) inactivated at 56°C for 30 minutes. This mixture was stored at 4°C and used as the complete culture medium described below.

[0163] 12-2. Culture of HEK293T The culture medium was removed from a 100 mm plate containing HEK293T cells using an aspirator. 10 mL of the complete medium prepared in 12-1. above was added, and the cells were detached from the plate by pipetting with an electric pipette. The cell aggregates were then broken down to obtain a cell suspension. 10 mL of the cell suspension was added to a 100 mm plate that had previously contained the complete medium. The plate was shaken to ensure uniform cell distribution, and the plate was incubated at 37°C under 5% CO2 conditions. This procedure was repeated every 2-3 days to prevent excessive cell growth in the plate.

[0164] 12-3. Introduction of vectors into cultured cells (Lipofection) First, each vector solution was prepared at 200 ng or 100 ng / μL, and the vectors were mixed as needed. The total concentration of the vectors to be introduced was then prepared with sterile ultrapure water to 100 ng / 6 μL or 200 ng / 6 μL. 25 μL of D-MEM was added to each well of a 96-well plate, followed by 6 μL of the prepared vector. Furthermore, a mixture of 25 μL of D-MEM and 0.7 μL of Lipofectamine LTX (Thermo Fisher Scientific) was added to each well in a 1.5 mL tube, and the mixture was allowed to stand at room temperature for 30 minutes.

[0165] Furthermore, the culture medium was removed from a 100 mm plate containing HEK293T cells in complete medium using an aspirator, and 3 mL of TrypLE™ Express was added and incubated for 30 seconds. 7 mL of complete medium was then added, and the cells were suspended using an electric pipette. The entire volume was transferred to a 50 mL tube. The mixture was centrifuged at 20°C and 1000 rpm for 3 minutes, the supernatant was removed using an aspirator, and the cells were resuspended in 3 mL of complete medium. The cell count was measured using a Luna automated cell counter (Logos Biosystems), and the result was 1.0 × 10⁶. 4 The cells were diluted with complete medium until the cell concentration was cells / mL. 100 μL of the diluted cell suspension was added to each of the wells prepared above, and the cells were incubated at 37°C under 5% CO2 conditions for 24 hours.

[0166] 12-4. Selection of vector-transferred cells using antibiotics After 24-hour culture in 12-3 above, the cells transfected with the GFP expression vector (pMAX_GFP) were observed under a microscope. After confirming that Lipofection had been performed, cell selection was carried out. First, the medium in the culture in the 96-well plate was removed with an aspirator, and 150 μL of a complete medium with the concentration of the antibiotic (puromycin) adjusted to 0-20 μg / mL was added to each well, followed by culturing at 37 °C and 5% CO2 for 48 hours. After the culture (72 hours after Lipofection), the cells were collected and Cell Count was performed by the method described in 12-5 below. During the 48 hours from the addition of the antibiotic to the collection of the cells, the medium was not changed.

[0167] 12-5. Cell Count of HEK293T Before collecting the cells, the cells transfected with pMAX_GFP were observed under a microscope to confirm that the expression of pMAX_GFP decreased in a puromycin concentration-dependent manner. Next, the medium in the culture in the 96-well plate was transferred to a separate-type PCR tube, 50 μL of TrypLE Express (manufactured by Thermo Fisher Scientific) was added to each well, and the mixture was incubated at 37 °C for 5 minutes. The cells were suspended with a multi-pipettor using the medium in the PCR tube where it had been transferred, and the suspension was collected back into the PCR tube again. Then, 10 μL of the cell suspension collected in the PCR tube was taken out, and the cell count was measured using a Luna automatic cell counter (manufactured by Logos Biosystems), and the Viability (survival rate: number of cells after 48-hour culture in 12-4 (after antibiotic addition) / number of cells after 24-hour culture in 12-3 (before antibiotic addition) × 100 [%]) was calculated.

[0168] 13. Genotyping 13-1. Genome Extraction and Purification After the above step 12-4, the cell suspension recovered in step 12-5 was centrifuged at 13,000 rpm for 3 minutes to remove the medium (supernatant). Then, 150 μL of PBS(-) was added, centrifuged at 13,000 rpm for 3 minutes to remove the supernatant, another 15 μL of PBS(-) was added, and centrifuged at 13,000 rpm for 3 minutes again to remove the supernatant. A mixture of 20 μL of Lysis Buffer and 0.8 μL of Protein Degrader was added to each PCR tube, reacted at 68°C for 15 minutes, then at 95°C for 10 minutes, and stored at ④. After that, it was transferred to -20°C and used as the following PCR template.

[0169] 13-2. PCR Amplification of the Inserted Sequence After the genomic extraction in step 13-1 above, in order to confirm whether the desired knock-in fragment (3×Flag sequence) was inserted into the target locus, forward and reverse primers were designed outside the nucleotide sequences (Ht1, Ht2) at both ends of the target site, and out-out PCR was performed. The primers were designed using Primer 3plus (https: / / www.primer3plus.com) and NCBI BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) based on the nucleotide sequences encoding each target gene and its surrounding regions. As the PCR enzyme, PrimeSTAR MAX DNA Polymerase (manufactured by Takara Bio Inc.) was used for ATP5B, and PrimeSTAR GXL DNA Polymerase (manufactured by Takara Bio Inc.) was used for PARP1, respectively.

[0170] [Method Using PrimeSTAR MAX DNA Polymerase] 0.2 μL of the PCR template prepared in 13-1 above, 5.0 μL of 2×PrimeSTAR MAX Premix (Takara Bio Inc.), 0.3 μL of 10 μM forward primer, 0.3 μL of 10 μM reverse primer, and 4.2 μL of sterile ultrapure water were mixed. Using a thermal cycler, the mixture was preheated at 94°C for 2 minutes, followed by thermal denaturation at 98°C for 10 seconds, annealing at 66°C for 5 seconds, and extension at 72°C for 5 seconds, repeated for 35-40 cycles, and then stored at 4°C. The PCR product was subjected to agarose gel electrophoresis using the method in 8 above, and a band of the desired length containing the knock-in sequence was confirmed.

[0171] [Method using PrimeSTAR GXL DNA Polymerase] 1.25 μL of the PCR template prepared in 13-1 above, 0.5 μL of PrimeSTAR GXL DNA Polymerase (Takara Bio Inc.), 5.0 μL of 5× PrimeSTAR GXL Buffer (Takara Bio Inc.), 2.0 μL of 2.5 mM each dNTP mixture, 0.75 μL of 10 μM forward primer, 0.75 μL of 10 μM reverse primer, and 14.75 μL of sterile ultrapure water were mixed. Using a thermal cycler, the mixture was preheated at 94°C for 2 minutes, followed by thermal denaturation at 98°C for 10 seconds, annealing at 70°C for 15 seconds, and extension at 68°C for 5 seconds, for 40 cycles, after which it was stored at 4°C. The PCR product was subjected to agarose gel electrophoresis using the method in 8 above, and a band of the desired length containing the knock-in sequence was confirmed.

[0172] 14.TIDER analysis For the TIDER (Tracking of Insertion, Deleteions, and Recombination Events) analysis, a TA vector was created in which a desired knock-in fragment (3×Flag sequence) containing homologous sequences (Hv1, Hv2) was inserted.

[0173] 14-1. TA cloning Mix 9 μL of the PCR product from 13-2 above with 1 μL of 10×A-attachment mix (TArget Clone -Plus-, TOYOBO) and incubate at 60°C for 30 minutes using a thermal cycler. Mix 3 μL of the incubated solution with 5 μL of 2×Ligation Buffer (TArget Clone -Plus-, TOYOBO), 1 μL of 50 ng / μL pTA2 vector (TArget Clone -Plus-, TOYOBO), and 1 μL of T4 DNA Ligase (TArget Clone -Plus-, TOYOBO) in a PCR tube. After incubating at 16°C for 30 minutes using a thermal cycler, mix 3 μL of this reaction mixture with 30 μL of XL10-Gold (Agilent) and let stand on ice for 10 minutes. Incubate at 42°C for 30 seconds using a constant temperature bath, then let stand on ice for at least 2 minutes. This mixture was spread onto an LB+Amp plate coated with 20 μL of 5% X-gal, 10 μL of 0.1 M IPTG, and 70 μL of SOC. After incubation at 37°C for approximately 16 hours, white colonies that could no longer degrade X-gal were selected, and vector introduction was confirmed by colony PCR. After small culture of the colonies in which vector introduction was confirmed, each plasmid (TA cloning vector) was extracted using miniprep.

[0174] 14-2. Sequencing and TIDER analysis Using the TA cloning vector obtained in 14-1 above as a template, sequencing analysis was performed using vector primers in the method described in 9 above. HDR efficiency (HDR efficiency[%]) was then calculated using TIDER (https: / / tide.nki.nl / ) with the following parameter settings: Alignment Window (bp): 69, Decomposition Window (bp): 70~94, Indel size range: 5 (for PARP1).

[0175] <Test Example 1> Insertion of knock-in fragment (Comparative Example) First, we designed the method shown in Figure 4 and attempted to insert a knock-in fragment (nucleotide fragment) into the upstream of LPAT1, a gene (target DNA) related to membrane lipid synthesis in the target cell Nannochloropsis. This knock-in fragment consisted of an LDSP promoter (ProLDSP) with homologous sequences (homology arms) attached to the nucleotide sequences at both ends of the desired target site upstream of LPAT1. In the method shown in Figure 4, the knock-in fragment containing the LDSP promoter (ProLDSP) is inserted upstream of LPAT1, while the CEN / ARS vector, the all-in-one PtTALEN-ARS vector, is not inserted into the genomic DNA, the target DNA. Therefore, it was thought that by removing the CEN / ARS vector after the desired insertion was achieved, it would be possible to insert DNA without any foreign genes other than the knock-in fragment remaining in the cell.

[0176] The knock-in fragment used was a 1304bp (total, approximately 790ng) fragment in which the nucleotide sequence shown in SEQ ID NO: 20, which encodes Nannochloropsis ProLDSP, had the same sequence as the 217 bases before the start codon of Nannochloropsis LPAT1 (SEQ ID NO: 21) added to the 5' end, and the same sequence as the 320 bases after the start codon of LPAT1 added to the 3' end. However, in the nucleotide sequence shown in SEQ ID NO: 20, a partial mutation was introduced in the sequence corresponding to the DNA binding domain recognition sequence of the R-TALEN of PtTALEN, as described below, to prevent the knock-in fragment from being cleaved by TALEN.

[0177] Furthermore, in the all-in-one PtTALEN-ARS plasmid described in Non-Patent Literature 3, an all-in-one PtTALEN-ARS vector (520 ng) was constructed by designing the amino acid sequence of the PtTALEN DNA-binding domain to cleave between 111 and 125 bases from the start codon of Nannochloropsis LPAT1.

[0178] The knock-in fragment and the all-in-one PtTALEN-ARS vector were electroporated into Nannochloropsis cells using the method described in 5-3. Colonies were formed on a plate of F2N 50% seawater medium containing zeosin (selection plate) using the zeosin resistance gene (ShBle) present in the all-in-one PtTALEN-ARS vector. Genomic DNA was extracted from each colony (12 cells) using the method described in 6-1. PCR was performed using the method described in 7. above, with primer combinations designed to be placed inside the knock-in fragment (ProLDSP) and on the target DNA (upstream and downstream of LPAT1), as shown in Figure 5(a). The nucleotide sequences of each primer are shown in the order of Out-out PCR forward primer (5' Junction PCR forward primer), reverse primer (3' Junction PCR reverse primer), 5' Junction PCR reverse primer, and 3' Junction PCR forward primer, as shown by Sequence IDs: 22-25.

[0179] Figure 5(b) shows the results of agarose gel electrophoresis of the amplified product after the PCR reaction using the method described in section 8 above. As shown in Figure 5(a), if the desired insertion is achieved, bands of 3151 bp, 1369 bp, and 1517 bp are all observed. However, no such combination of bands was observed in any of the colonies, confirming that the DNA insertion efficiency is insufficient using the method shown in Figure 4.

[0180] <Example Test 2> Construction of Non-Integration-Type Tear-Dependent Selection (NICS) Vectors One reason for the low DNA insertion efficiency in Test Example 1 was thought to be that few of the colonies formed on the selection plate had both the knock-in fragment and the all-in-one PtTALEN-ARS vector simultaneously introduced. Therefore, a new method shown in Figure 1A was designed. In the method shown in Figure 1A, the selection marker gene (paromomycin resistance gene in this test) was divided into a 5' side (M1) and a 3' side (M2). Between these two sides, homologous sequences to the nucleotide sequences at both ends of the target site (Hv1, Hv2) and the target DNA (D, LDSP promoter in this test) were inserted as a knock-in fragment in a manner that could be removed by a site-specific nuclease system (TALEN in this test) (Figure 1A(a)). This vector is cleaved in cells into which a site-directed nuclease system has been introduced, i.e., in cells where the target DNA is likely to have also been cleaved and the knock-in fragment inserted (Figure 1A(c)), and the remaining fragment binds to enable the function of the selection marker gene (Figure 1A(b)), thus improving DNA insertion efficiency. In this example, the upstream site of PDAT1, a gene in Nannochloropsis that transfers an acyl group to diacylglycerol to synthesize lipids and has been reported to increase lipid production when overexpressed in other algae, was selected as the target site.

[0181] (1) Verification Test 1 First, to confirm that this system functions in nannochloropsis, a validation vector was prepared and verified. First, in a vector containing CEN / ARS (nucleotide sequence shown SEQ ID NO: 1), the paromomycin resistance gene (aphVIII), and the nannochloropsis promoter (ProLHC, nucleotide sequence shown SEQ ID NO: 2) and terminator (terFCP, nucleotide sequence shown SEQ ID NO: 9) for the paromomycin resistance gene, the paromomycin resistance gene (aphVIII) was split into a 5' fragment (aphVIII-5', nucleotide sequence shown SEQ ID NO: 3) and a 3' fragment (aphVIII-3', nucleotide sequence shown SEQ ID NO: 8) with a 207 bp overlap (pMD20-ARS-aphVIII-sep-BamHI, Figure 6A). Next, the PtTALEN binding region for nitrate reductase NoNR (Kurita et al., Genes Cells., 2020; 00: p.1~8, DOI: 10.1111 / gtc.12805 (Reference I)) was inserted between these fragments to create the validation vector _NoNR. A conceptual diagram of the constructed validation vector _NoNR is shown in Figure 6B(a). When this vector is cleaved by the aforementioned PtTALEN for NoNR, the paromomycin resistance gene becomes functional, and paromomycin-resistant colonies are formed on the selection plate (Figure 6B(b)).

[0182] Furthermore, in the all-in-one PtTALEN-ARS plasmid described in Non-Patent Literature 3, we created an all-in-one PtTALEN vector (TALEN vector_NoNR) in which the amino acid sequence of the DNA-binding domain of PtTALEN was configured to bind to the binding region of PtTALEN for NoNR, and an all-in-one PtTALEN vector (TALEN vector_LPAT1) in which the sequence was configured to bind to the binding region of PtTALEN near the target site of LPAT1 (Reference I).

[0183] The tests were conducted using the validation vector NoNR alone (No. 2), a combination of the validation vector NoNR and the TALEN vector NoNR (No. 3), and a combination of the validation vector NoNR and the TALEN vector LPAT1 (No. 4). The test was also performed using the pMD20-ARS-aphVIII-sep-BamHI (a control vector in which the BamHI site is inserted between the 5' and 3' fragments of the paromomycin resistance gene, replacing the PtTALEN binding region, so that the 5' and 3' fragments bind in-frame) (No. 1). Each vector was administered in 200 ng doses, and nannochloropsis cells were electroporated using the method described in 5-3. The number of paromomycin-resistant colonies formed on F2N 50% seawater plates containing paromomycin was measured. The results are shown in Figure 7A. Furthermore, genomic DNA was extracted from representative colonies of each vector combination using the method described in 6-1. PCR was performed using the primer combinations designed on the 5' and 3' fragments, as shown in Figure 7B(a), using the method described in 7. above. The results of agarose gel electrophoresis of the amplified products after the PCR reaction using the method described in 8. above are shown in Figure 7B(b).

[0184] As shown in Figure 7A, paromomycin-resistant colonies were sufficiently formed in cells introduced with the combination of validation vector _NoNR and TALEN vector _NoNR (No. 3). Furthermore, as shown in Figure 7B(a), a 529 bp band was observed in cells introduced with a control vector in which the 5' and 3' fragments of the paromomycin resistance gene were linked in frame, a 558 bp band was observed in cells where the validation vector _NoNR was not cleaved by PtTALEN, and a 301 bp band was observed in cells where the validation vector _NoNR was cleaved by PtTALEN and rejoined via a duplicate sequence. As shown in Figure 7B(b), a 301 bp band was observed in the combination of validation vector _NoNR and TALEN vector _NoNR (No. 3), confirming that the validation vector _NoNR was cleaved by PtTALEN and rejoined via a duplicate sequence, and confirming that the paromomycin resistance described above was due to the paromomycin resistance gene after this rejoining. However, as shown in Figure 7A, many paromomycin-resistant colonies were also formed in cells into which a control vector (No. 1) was introduced, in which the 5' and 3' fragments of the paromomycin resistance gene were connected in frame.

[0185] (2) Verification Test 2 From (1) above, even when recombination occurred in-frame between the 5'-side fragment and the 3'-side fragment of the paromomycin resistance gene, a large number of paromomycin-resistant colonies were formed as background. Therefore, as each vector, a vector (verification vector_NoNR_stop, control vector_stop) with a stop codon (TAG) inserted immediately after the 5'-side fragment of the paromomycin resistance gene was prepared, and the same test as (1) was conducted. Also, a vector (verification vector_LPAT1_stop) with the binding region of PtTALEN for LPAT1 inserted between the 5'-side fragment and the 3'-side fragment of the paromomycin resistance gene instead of the binding region of PtTALEN for NoNR was similarly prepared. Only the control vector_stop (No. 1), only the verification vector_NoNR_stop (No. 2), the combination of the verification vector_NoNR_stop and the TALEN vector_NoNR (No. 3), the combination of the verification vector_NoNR_stop and the TALEN vector_LPAT1 (No. 4), only the verification vector_LPAT1_stop (No. 5), the combination of the verification vector_LPAT1_stop and the TALEN vector_LPAT1 (No. 6), and the combination of the verification vector_LPAT1_stop and the TALEN vector_NoNR (No. 7) were each tested. The results are shown in Figure 8.

[0186] As shown in Figure 8, in the cells into which the combination of the verification vector_NoNR_stop and the TALEN vector_NoNR (No. 3) and the combination of the verification vector_LPAT1_stop and the TALEN vector_LPAT1 (No. 6) were each introduced, paromomycin-resistant colonies were sufficiently formed in the same manner as in (1) above. On the other hand, in the strain into which the control vector_stop (No. 1) was introduced, the amount of paromomycin-resistant colonies formed decreased compared to (1), and successful reduction of the background was achieved.

[0187] (3) Construction of NICS vector (Example) Based on the verification results of (1) and (2) above, a vector (NICS vector) to be used in the method shown in Figure 1A was constructed using pMD20-ARS-aphVIII-sep-BamHI shown in Figure 6A. A conceptual diagram showing the structure of the vector constructed as the NICS vector shown in Figure 1A(a) and Figure 1C(a) is shown in Figure 9(a) on the left. The NICS vector shown in Figure 9(a) on the left has the following structure: CEN / ARS (Sequence ID: 1), Promoter P1 (ProLHC, SEQ ID NO: 2) and terminator (terFCP, SEQ ID NO: 9) for the paromomycin resistance gene, Between the promoter and the terminator, the paromomycin resistance gene (aphVIII) is divided into a 5' fragment M1 (aphVIII-5', SEQ ID NO: 3) and a 3' fragment M2 (aphVIII-3', SEQ ID NO: 8) with a 207 bp duplicate sequence. Between M1 and M2 are the PtTALEN binding regions T1 and T2 for PDAT1 (both sequence numbers: 4, 5'-L1 (Left-TALEN DNA binding domain recognition sequence, sequence number: 10)-S1 (spacer sequence)-R1 (complementary sequence of the Right-TALEN DNA binding domain recognition sequence, sequence number: 11)-3'), Between T1 and T2, homologous sequences Hv1 and Hv2, Between Hv1 and Hv2, ProLDSP (SEQ ID NO: 6) is used as the knock-in fragment D. Stop codons (TAGs) were inserted between the 5' fragment M1 and T1, and between the 3' fragment M2 and T2, respectively, of the paromomycin resistance gene (the latter partially overlapping with M2). Hv1 and Hv2 are the same sequences as the sequence immediately before the start codon and the sequence after the start codon, respectively, of PDAT1 in Nannochloropsis. Two vectors were prepared: one with the homologous sequences Hv1 and Hv2 lengths of 36 bp each (Hv1: SEQ ID NO: 5, same sequence as the nucleotide sequence immediately before the start codon of PDAT1 / Hv2: SEQ ID NO: 7, same sequence as the nucleotide sequence after the start codon of PDAT1) (pNICS36 vector), and another with lengths of 210 bp each (Hv1: SEQ ID NO: 12, same sequence as the nucleotide sequence immediately before the start codon of PDAT1 / Hv2: SEQ ID NO: 13, same sequence as the nucleotide sequence after the start codon of PDAT1) (pNICS210 vector). The nucleotide sequence of the pNICS36 vector is shown as sequence number 14, and the nucleotide sequence of the pNICS210 vector is shown as sequence number 15.

[0188] Furthermore, as a site-directed nuclease system expression vector to be combined with the above-mentioned NICS vector (pNICS36 vector or pNICS210 vector), an all-in-one PtTALEN-ARS vector (TALEN vector_PDAT1) was constructed in which the amino acid sequence of the DNA-binding domain of PtTALEN in the all-in-one PtTALEN-ARS plasmid described in Non-Patent Literature 3 was modified to bind to sequence T3 (the same sequence as T1 and T2) around the start codon of PDAT1. A conceptual diagram showing the structure of the constructed vector is shown in accordance with Figure 9(a) on the right.

[0189] Furthermore, a vector was constructed by fusing the above NICS vector and the TALEN vector_PDAT1 as an integrated vector (integrated NICS vector) as shown in Figure 3. A conceptual diagram showing the structure of the constructed integrated NICS vector in more detail is shown in Figure 9(b). The integrated NICS vector shown in Figure 9(b) has the following configuration: CEN / ARS(*), Promoter P1 (ProLHC(*)) and terminator (terFCP(*)) for the paromomycin resistance gene, Between the promoter and the terminator, the 5' fragment M1(*) and the 3' fragment M2(*) of the paromomycin resistance gene are located. Between M1 and M2 are the PtTALEN binding regions T1 and T2 (*) for PDAT1, Between T1 and T2, homologous sequences Hv1 and Hv2(*) Between Hv1 and Hv2, ProLDSP(*) is present as knock-in fragment D. Nucleotide sequences encoding TALEN-L (TALEN-L, SEQ ID NO: 16) containing a DNA-binding domain that recognizes L1 of T1, T2, and T3, Nucleotide sequences encoding TALEN-R (TALEN-R, SEQ ID NO: 17) containing a DNA-binding domain that recognizes R1 of T1, T2, and T3, Promoter P2 (ProLHC(*)) and terminator (terFCP(*)) for TALEN-L and TALEN-R The components indicated by "*" above are common to the above NICS vectors. The nucleotide sequence of the all-in-one NICS36 vector, in which the homologous sequences Hv1 and Hv2 are 36 bp long, is shown as SEQ ID NO: 18, and the nucleotide sequence of the all-in-one NICS210 vector, in which the homologous sequences Hv1 and Hv2 are 210 bp long, is shown as SEQ ID NO: 19. A schematic diagram showing the detailed structure of the all-in-one NICS210 vector is shown in Figure 19. In Nannochloropsis electroporation, it is thought that only about 20% of cells are simultaneously inserted into a single cell by both types of vectors (Reference I), so a higher knock-in efficiency was expected with an integrated NICS vector.

[0190] (4) Tests on introducing NICS vectors into cells The tests were conducted using either the pNICS36 vector or the pNICS210 vector alone, a combination of the pNICS36 vector or the pNICS210 vector with the TALEN vector PDAT1, the all-in-one NICS36 vector, and the all-in-one NICS210 vector. 200 ng of each vector was used, and nannochloropsis cells were electroporated using the method described in section 5-3 above. The number of paromomycin-resistant colonies formed on F2N 50% seawater plates containing paromomycin was measured. The results are shown in Figure 10. As shown in Figure 10, sufficient paromomycin-resistant colonies were confirmed to form in cells introduced with the pNICS36 vector or the pNICS210 vector in combination with the TALEN vector PDAT1, the all-in-one NICS36 vector, and the all-in-one NICS210 vector. Furthermore, since no colonies formed with the pNICS36 vector or the pNICS210 vector alone, it was confirmed that background suppression was more highly effective.

[0191] (5) PCR analysis and sequencing analysis Genomic DNA was extracted from 16 randomly selected colonies from those formed in (4) above using the method described in 6-2. PCR reactions were performed using the method described in 7. above, with primer combinations designed to be placed inside the knock-in fragment (ProLDSP) and on the target DNA (upstream and downstream of PDAT1), as shown in Figure 11. The nucleotide sequences of each primer are shown in the order of forward primer for Out-out PCR (forward primer for 5' Junction PCR), reverse primer (reverse primer for 3' Junction PCR), reverse primer for 5' Junction PCR, and forward primer for 3' Junction PCR, as shown by Sequence IDs: 26-29. Figure 12 shows the results of agarose gel electrophoresis of the amplification products after PCR reaction of colonies formed by introducing the pNICS210 vector and TALEN vector PDAT1 using the method described in 8. above, and Figure 13 shows the results of agarose gel electrophoresis of the amplification products after PCR reaction of colonies formed by introducing the all-in-one NICS210 vector using the method described in 8. above.

[0192] As shown in Figure 11, when the desired insertion is achieved, bands of 1601 bp, 443 bp, and 461 bp are all observed. However, as shown in Figures 12 and 13, the desired bands were observed in one strain (colony No. 9 in Figure 12) when the combination of the pNICS210 vector and the TALEN vector _PDAT1 was introduced, and in five strains (colonies No. 5, 8, 11, 13, and 14 in Figure 13) when the all-in-one NICS210 vector was introduced.

[0193] Furthermore, for the colonies from which the target band was obtained as described above, each band was excised, DNA was extracted, and sequence analysis was performed using the method described in section 9 above to determine whether the desired knock-in fragment ProLDSP sequence had been introduced. Ultimately, when the combination of the pNICS210 vector and the TALEN vector _PDAT1 was introduced, the correct sequence was detected in 1 / 16 (DNA insertion efficiency: 6.25%), and when the all-in-one NICS210 vector was introduced, the correct sequence was detected in 4 / 16 (DNA insertion efficiency: 25%), confirming that the desired insertion was possible in both cases. In particular, it was confirmed that DNA introduction was possible with higher efficiency when the all-in-one NICS210 vector was introduced.

[0194] (6) Deletion test of CEN / ARS vector In the strains in which the insertion of the knock-in fragment was confirmed in (5) above, a selection vector containing CEN / ARS was formed from the NICS vector. Therefore, from some of the strains in which the insertion of the knock-in fragment was confirmed in (5) above (colonies No. 5 and 8 in Figure 13), the selection vector, which is the CEN / ARS vector, was removed using the method described in 10. above, with paromomycin as the antibiotic. Specifically, candidate colonies for removal (parent plants before vector removal) obtained by culturing in F2N liquid medium for 14 days and then spreading them onto F2N plates were seeded onto F2N plates and F2N 50% seawater plates containing paromomycin (selection plates), respectively, and six strains that grew on the F2N plates but not on the selection plates containing paromomycin were obtained from each. Figure 14 shows the appearance of the plates indicating the growth status of each strain.

[0195] Next, each of the six strains obtained above, along with the parent strains before vector elimination (colonies No. 5 and 8 in Figure 13), were cultured in a 12-well plate using conventional methods. Genomic DNA was extracted using the method described in 6-2 above, and the elimination of the selected vector was confirmed by PCR (method described in 7 above) using a primer that detects FokI, the nuclease domain of TALEN, as this was used as a template. The results of agarose gel electrophoresis of the amplified products after the PCR reaction using the method described in 8 above are shown in Figure 15. As shown in Figure 15, FokI was not detected in any of the six strains.

[0196] Furthermore, instead of the method described in 6-2 above, highly purified genomic DNA was extracted using the method described in 6-1. Using this as a template, the DNA was examined again in detail by PCR using primers to detect the N-terminal domain of TALEN (TALEN-N), the paromomycin resistance gene (ParoR), and the kanamycin resistance gene (KanR), in addition to FokI, as well as primers for out-out PCR of the knock-in fragment (donor) PDAT1 (Figure 11). The results of agarose gel electrophoresis of the amplified products after the PCR reaction are shown in Figure 16.

[0197] As shown in Figure 16, strains in which the PDAT1 band was confirmed and none of the bands of genes derived from the selection vector (FokI, TALEN-N, ParoR, KanR) were confirmed were obtained with high probability, with two strains each from colony No. 5 and colony No. 8 (colony No. 5 strains: #1, #2; colony No. 8 strains: #4, #5). These results confirm that, using the NICS vector of the present invention, it is possible to accurately insert the desired nucleotide sequence (LDSP promoter in this example) into the target site of the target DNA (upstream of PDAT1 in this example), and furthermore, it is possible to construct a genome DNA edited strain in which the vector is subsequently removed and foreign genes other than the desired nucleotide sequence are removed.

[0198] <Test Example 3> Verification of DNA editing targeting GPAT1 We confirmed that DNA can be introduced into different target sites using the NICS vector of the present invention. In this test example, the upstream site of GPAT1 (glycerol triphosphate acyltransferase) of Nannochloropsis was selected as the target site.

[0199] (1) Construction of NICS vectors (Example) First, using the same configuration as the all-in-one NICS210 vector in Test Example 2(3), the following configuration is used: CEN / ARS(*), Promoter P1 (ProLHC(*)) and terminator (terFCP(*)) for the paromomycin resistance gene, Between the promoter and the terminator, the 5' fragment M1(*) and the 3' fragment M2(*) of the paromomycin resistance gene are located. Between M1 and M2 are the PtTALEN binding regions T1 and T2 for GPAT1 (both sequence numbers: 30, 5'-L1 (Left-TALEN DNA binding domain recognition sequence, sequence number: 31)-S1 (spacer sequence)-R1 (complementary sequence of the Right-TALEN DNA binding domain recognition sequence, sequence number: 32)-3'), Between T1 and T2, homologous sequences Hv1 and Hv2, Between Hv1 and Hv2, ProLDSP(*) is present as knock-in fragment D. Nucleotide sequences encoding TALEN-L containing a DNA-binding domain that recognizes L1 of T1, T2, and T3 (TALEN-L, SEQ ID NO: 35), Nucleotide sequences encoding TALEN-R (TALEN-R, SEQ ID NO: 36) containing a DNA-binding domain that recognizes R1 of T1, T2, and T3, Promoter P2 (ProLHC(*)) and terminator (terFCP(*)) for TALEN-L and TALEN-R The components indicated by "*" above, including the above-mentioned NICS vector and integrated NICS vector, are common to each of them. In addition, T1 and T2 are the same sequences as T3, the sequence around the start codon of GPAT1 in Nannochloropsis. The lengths of homologous sequences Hv1 and Hv2 were set to 200 bp (Hv1; SEQ ID NO: 33, the same sequence as the nucleotide sequence immediately before the start codon of GPAT1) and 208 bp (Hv2; SEQ ID NO: 34, the same sequence as the nucleotide sequence after the start codon of GPAT1) (all-in-one NICS210-GPAT1 vector). The nucleotide sequence of the all-in-one NICS210-GPAT1 vector is shown in SEQ ID NO: 37.

[0200] (2) Test of introducing NICS vectors into cells The cell introduction test was performed in the same manner as in Test Example 2(4), except that the all-in-one NICS210-GPAT1 vector was used instead of the all-in-one NICS210 vector. Genomic DNA was extracted from 12 randomly selected colonies from among the colonies formed on a plate of F2N 50% seawater containing paromomycin, in the same manner as in Test Example 2(5), using the method described in 6-2 above, and PCR was performed. The results of agarose gel electrophoresis of the amplified products after the PCR reaction are shown in Figure 20. In Figure 20, the position of the band observed when the desired insertion (knock-in) is achieved is indicated by a black triangle, and the position of the band observed when insertion did not occur (WT) is indicated by a white triangle.

[0201] As shown in Figure 20, the desired band was observed in 5 strains (colonies No. 1-4 and 7 in Figure 20) after introduction of the all-in-one NICS210-GPAT1 vector. Furthermore, when the colonies from which the desired band was obtained were sequenced in the same manner as in Test Example 2(5), accurate sequences were detected in 2 out of 12 cases (DNA insertion efficiency: 17%) even when the all-in-one NICS210-GPAT1 vector was introduced, confirming that DNA can be introduced to the target site, i.e., the upstream site of GPAT1, with high efficiency.

[0202] (3) Deletion test of CEN / ARS vector From some of the strains in which accurate insertion of the knock-in fragment was confirmed in (2) above (colonies No. 3 and 4 in Figure 20), the selection vector, which is the CEN / ARS vector, was removed using paromomycin as the antibiotic, in the same manner as in Test Example 2(6). As a result, six colonies that grew on the F2N plate but not on the selection plate containing paromomycin were obtained from colony No. 3 (#3, #4, #6~9) and six colonies from colony No. 4 (#1~4, #6, #7). This confirmed that in this test example as well, the CEN / ARS vector was removed, and a genome DNA editing strain with foreign genes other than the desired nucleotide sequence removed could be constructed. Figure 21 shows the appearance of the plates indicating the growth status of each strain.

[0203] <Test Example 4> Verification of homologous sequence lengths in integrated NICS vectors (1) Construction of NICS vectors (Example) An integrated NICS vector (all-in-one NICS400 vector) was constructed in the same manner as the all-in-one NICS210 vector in Test Example 2(3), except that the lengths of homologous sequences Hv1 and Hv2 in the integrated NICS vector were both set to 414 bp (Hv1: SEQ ID NO: 38, the same sequence as the nucleotide sequence immediately before the start codon of PDAT1 / Hv2: SEQ ID NO: 39, the same sequence as the nucleotide sequence after the start codon of PDAT1). The nucleotide sequence of the all-in-one NICS400 vector is shown as SEQ ID NO: 40.

[0204] (2) Test of introducing NICS vectors into cells The cell introduction test was performed in the same manner as in Test Example 2(4), except that the all-in-one NICS400 vector was used instead of the all-in-one NICS210 vector. Genomic DNA was extracted from 12 randomly selected colonies from the colonies formed on a plate of F2N 50% seawater containing paromomycin, in the same manner as in Test Example 2(5), using the method described in 6-2 above, and PCR was performed. The results of agarose gel electrophoresis of the amplified products after the PCR reaction are shown in Figure 22. In Figure 22, the position of the band observed when the desired insertion was achieved is indicated by a black triangle, and the position of the band observed when insertion did not occur (WT) is indicated by a white triangle.

[0205] As shown in Figure 22, the desired bands were observed in three strains (colonies No. 4, 6, and 9 in Figure 22) after introducing the all-in-one NICS400 vector. Furthermore, when the colonies from which the desired bands were obtained were sequenced in the same manner as in Test Example 2(5), accurate sequences were detected in 3 out of 12 cases (DNA insertion efficiency: 25%) even when the all-in-one NICS400 vector was introduced, confirming that DNA can be introduced with high efficiency.

[0206] <Test Example 5> Application of the NICS system to human cultured cells As an alternative to the method shown in Figure 1A, the method shown in Figure 23 was designed to confirm that the vector of the present invention is also applicable to human cultured cells. In the method shown in Figure 23, the selected marker gene (puromycin resistance gene in this example) was divided into a 5' side (M1) and a 3' side (M2), and a vector was constructed in which homologous sequences to the nucleotide sequences at both ends of the target site (Hv1, Hv2) and the target DNA (D, a 3× Flag tag sequence in this example) were inserted as a knock-in fragment between them, so that it could be removed by a site-specific nuclease system (CRISPR-Cas system in this example) (upper panel of Figure 23). This vector is cleaved in cells into which the site-specific nuclease system has been introduced, i.e., in cells where the target DNA is also likely to have been cleaved and the knock-in fragment inserted (lower right panel of Figure 23), and the remaining fragment binds, allowing the selected marker gene to function (lower left panel of Figure 23). In this study, the downstream regions of PARP1 and ATP5B, which are endogenous genes on the human genome, were selected as target sites.

[0207] (1) Construction of NICS vectors (Example) The vector (NICS vector) used in the method shown in Figure 1A was constructed in the same manner as in Test Example 2 (3). A conceptual diagram showing the structure of the vector constructed as the NICS vector shown in Figure 1A (a) and Figure 1C (b) is shown in the upper part of Figure 23. The NICS vector shown in Figure 23 has the following structure: Promoter P1 (pCMV, SEQ ID NO: 41) and polyA (bGH polyA signal, SEQ ID NO: 42) for the puromycin resistance gene, Between the promoter and polyA, there is a sequence in which the puromycin resistance gene (PuroR) is divided into a 5' fragment M1 (PuroR-5', SEQ ID NO: 43) and a 3' fragment M2 (PuroR-3', SEQ ID NO: 44) with a 60 bp overlapping sequence, or a sequence in which the puromycin resistance gene (PuroR) is divided into a 5' fragment M1 (PuroR-5', SEQ ID NO: 51 (same as SEQ ID NO: 43)) and a 3' fragment M2 (PuroR-3', SEQ ID NO: 52) with a 192 bp overlapping sequence. Between M1 and M2 are gRNA recognition sequence G1 (sequence number: 45, common to both PARP1 and ATP5B) and gRNA recognition sequence G2 (complementary sequence of G1, sequence number: 46, common to both PARP1 and ATP5B), Between G1 and G2, homologous sequences Hv1 and Hv2, Between Hv1 and Hv2 is a sequence (sequence number: 47) that encodes a 3× Flag tag as the knock-in fragment D. Stop codons (TAG or TAA) were inserted between the 5' fragment M1 and G1, and between the 3' fragment M2 and G2, respectively, of the puromycin resistance gene. Hv1 and Hv2 are the same sequences as the sequence immediately before the stop codon and the sequence after the stop codon, respectively, of PARP1 and ATP5B. The length of the homologous sequences Hv1 and Hv2 was set to 40 bp each ([PARP1]Hv1: SEQ ID NO: 48, same sequence as the nucleotide sequence immediately before the stop codon of PARP1 / Hv2: SEQ ID NO: 49, same sequence as the nucleotide sequence after the stop codon of PARP1, [ATP5B]Hv1: SEQ ID NO: 54, same sequence as the nucleotide sequence immediately before the stop codon of ATP5B / Hv2: SEQ ID NO: 55, same sequence as the nucleotide sequence after the stop codon of ATP5B). The nucleotide sequence of the NICS vector for PARP1 with a 60bp duplicate marker gene (pNICS60-PARP1 vector) is shown as SEQ ID NO: 50, the nucleotide sequence of the NICS vector for PARP1 with a 192bp duplicate marker gene (pNICS192-PARP1 vector) is shown as SEQ ID NO: 53, the nucleotide sequence of the NICS vector for ATP5B with a 60bp duplicate marker gene (pNICS60-ATP5B vector) is shown as SEQ ID NO: 56, and the nucleotide sequence of the NICS vector for ATP5B with a 192bp duplicate marker gene (pNICS192-ATP5B vector) is shown as SEQ ID NO: 57.

[0208] Furthermore, as a site-directed nuclease system expression vector to be combined with the above-mentioned NICS vector, we constructed a CRISPR / Cas9 vector in which the guide RNA (gRNA) sequences were configured to bind to G1, G2, and G3 (common to G1-G3, and sequences around the stop codon of PARP1 or ATP5B), respectively, in the CRISPR-Cas9 vector described by Sakuma et al., NATURE PROTOCOLS, Vol.11, No.1, 2016, pp.118-133.

[0209] (2) Verification Test We confirmed that cells introduced with vectors other than the non-divisible PuroR expression vector did not exhibit puromycin resistance. Specifically, HEK293T cells were introduced with the following vectors using the method described in 12-3 above: untransfected cells, pTA2-PuroR (a vector containing non-divisible PuroR, Positive Control), CRISPR / Cas9 vector (Negative Control), and pTA2-PuroR-Mut-T162A vector (a vector in which PuroR was inactivated by introducing a mutation). The cells were cultured and harvested using the method described in 12-4 above with puromycin concentrations of 0 μg / mL (-) or 20 μg / mL (+). The number of cells was measured using the method described in 12-5 above, and viability was calculated. The results are shown in Figure 24.

[0210] As shown in Figure 24, in all groups except the Positive Control, puromycin treatment resulted in the death of most cells, and the cell viability decreased to less than 20%, indicating that the cells could not grow in the presence of puromycin.

[0211] (3) Test of introducing NICS vectors into cells The tests were conducted for the following conditions: no vector (Untransfected:WT), NICS vector only (pNICS60-PARP1 vector, pNICS192-PARP1 vector, pNICS60-ATP5B vector, or pNICS192-ATP5B vector), and combinations of each NICS vector with a CRISPR / Cas9 vector encoding the gRNA corresponding to the guide RNA recognition sequence (G3) on the gene targeted by that NICS vector (i.e., a CRISPR / Cas9 vector targeting the aforementioned gene).

[0212] Each of these was introduced into HEK293T in the same manner as in (2) above, and viability was calculated at puromycin concentrations of 0 μg / mL (-) or 20 μg / mL (+). For PARP1, the survival rates at each puromycin concentration for each combination of NICS vectors (pNICS60-PARP1 vector, pNICS192-PARP1 vector) and a PARP1-targeting CRISPR / Cas9 vector are shown in Figure 25. For ATP5B, the survival rates at each puromycin concentration for each combination of NICS vectors (pNICS192-ATP5B vector) and a ATP5B-targeting CRISPR / Cas9 vector are shown in Figure 26.

[0213] As shown in Figures 25 and 26, cells introduced with only the NICS vector had a survival rate of less than 20% at a puromycin concentration of 20 μg / mL, with most cells dying. However, cells co-introduced with the CRISPR / Cas9 vector all showed high survival rates. This confirmed that the NICS vector, from which the 3× Flag tag sequence was excised by the CRISPR / Cas9 vector, rejoined due to the duplication sequence, resulting in the expression of the puromycin resistance gene (PuroR) (Figure 23, lower left).

[0214] (4) PCR analysis For the untransfected (WT) and each NICS vector paired with a CRISPR / Cas9 vector, out-out PCR reactions were performed using the methods described in 13-1 to 13-2 above, following the procedure in (3) above (n=4 for each). The results of agarose gel electrophoresis of the amplified products after the PCR reaction using the method described in 8 above are shown in Figures 27A and 27B (PARP1) and 28A and 28B (ATP5B), respectively. When the desired insertion was achieved, the observed band length changed from 349 bp (before insertion) to 425 bp (after insertion) for PARP1, and from 347 bp (before insertion) to 424 bp (after insertion) for ATP5B.

[0215] As shown in Figures 27A to 28B, each NICS vector was co-introduced with a CRISPR / Cas9 vector, and the expected post-insertion band length was confirmed in the colonies formed at a puromycin concentration of 20 μg / mL(+).

[0216] (5)TIDER analysis From the PCR products described in (4) above, TIDER analysis was performed using the methods described in 14-1 to 14-3 above to determine the HDR efficiency (HDR efficiency [%], n=4). As a result, it was confirmed that insertion of the 3× Flag tag sequence occurred under all conditions. As an example of comparing HDR efficiency (insertion efficiency) with and without puromycin selection, Figure 29 shows the results when the pNICS192-PARP1 vector and the CRISPR / Cas9 vector were introduced in combination (Figure 27B). As shown in Figure 29, it was confirmed that the insertion efficiency significantly increased with the addition of the antibiotic corresponding to the marker gene (puromycin) in this combination.

[0217] Based on the above, it has been confirmed that the NICS system of the present invention makes it possible to obtain DNA-edited cells (e.g., genome-edited cells) with edited target DNA, not only in algae but also in cells derived from other species (e.g., humans), and that the obtained DNA-edited cells can be easily selected and isolated while appropriately selecting the target gene locus or marker gene according to the purpose. [Industrial applicability]

[0218] As described above, the present invention provides a DNA editing method and a cell manufacturing method using the same, which can accurately and efficiently insert a desired nucleotide sequence into a target site of target DNA in a cell using a vector, or remove the target site. It also provides a DNA editing vector and a DNA editing kit for use in these methods.

[0219] Furthermore, if the vector contains a CEN / ARS which is an autonomous replication sequence, it is also possible to provide a DNA editing method and a cell manufacturing method using the same, which allows for highly efficient production of genome DNA edited cells in which foreign genes other than the inserted nucleotide sequence have been removed, after inserting a desired nucleotide sequence into a target site of target DNA or removing the target site from the target DNA using the vector, by removing the vector. Additionally, DNA editing vectors and DNA editing kits for use in these methods can also be provided.

[0220] For example, by placing the target site immediately before the start codon of the target gene, inserting only a strong endogenous promoter (e.g., the ProLDSP mentioned above) at this site, and removing all other vectors, it becomes possible to overexpress the target gene.

Claims

1. A DNA editing method for inserting a desired nucleotide sequence into a target site of target DNA within a cell or removing the target site, The process includes an introduction step in which a vector and a site-specific nuclease system are introduced into cells (excluding cells within a human organism, human germ cells, human embryonic cells, and human fertilized eggs) and brought into contact with target DNA, wherein, The aforementioned vector is The first promoter P1, and The following structure (1): 5'-M1-Hv1-D-Hv2-M2-3'...(1) [(1) M1 represents the 5' end fragment of the nucleotide sequence encoding the selection marker gene and is operably linked to the first promoter P1; Hv1 represents a nucleotide sequence homologous to the first nucleotide sequence Ht1 on the 5' end of the target site of the target DNA; D represents the desired nucleotide sequence, but may be omitted; Hv2 represents a nucleotide sequence homologous to the second nucleotide sequence Ht2 on the 3' end of the target site of the target DNA; M2 represents the remaining 3' end fragment of the nucleotide sequence encoding the selection marker gene; adjacent sequences in M1, Hv1, D, Hv2, and M2 may partially overlap with each other.] Includes, The site-specific nuclease system extracts the following structure (2) from the vector: 5'-Hv1-D-Hv2-3'...(2) The fragment shown and the following structure (3): 5'-M2-P1-M1-3'...(3) A cutting step that produces a fragment shown by and cuts the target site or its vicinity in the target DNA, An editing step in which the target DNA and the fragment shown in structure (2) are bound in a manner dependent on the homology between Ht1 and Hv1, and in a manner dependent on the homology between Ht2 and Hv2, thereby inserting a desired nucleotide sequence D into the target site or removing the target site; A selection step to obtain a selection vector containing a functional selection marker gene, wherein the 3' end of M1 and the 5' end of M2 are joined to the fragment shown in structure (3), thereby operably linking to P1, and the fragment is functionally linked. DNA editing methods, including those mentioned above.

2. The vector further comprises CEN / ARS, which is an autonomous replication sequence. The structure (3) formed in the cutting process further includes CEN / ARS, The DNA editing method according to claim 1, wherein the selection step is a step of obtaining a selection vector in which the 3' end of M1 and the 5' end of M2 are bound together in the fragment shown in structure (3) to operably link with P1 and a functional selection marker gene and CEN / ARS.

3. The DNA editing method according to claim 1, wherein the length of Hv1 and the length of Hv2 are independently 30 to 600 base pairs.

4. The aforementioned vector has the following structure (11): 5'-M1-T1-Hv1-D-Hv2-T2-M2-3'...(11) [In (11), T1 represents a TALEN_1 binding region including a first DNA-binding domain recognition sequence or its complementary sequence L1 and a second DNA-binding domain recognition sequence or its complementary sequence R1; T2 represents a TALEN_2 binding region including a third DNA-binding domain recognition sequence or its complementary sequence L2 and a fourth DNA-binding domain recognition sequence or its complementary sequence R2; M1, Hv1, D, Hv2, and M2 are the same as in structure (1), and adjacent sequences in M1, T1, Hv1, D, Hv2, T2, and M2 may partially overlap.] Includes, The site-specific nuclease system is TALEN, which comprises a DNA-binding domain and a nuclease domain. TALEN_1, which includes a first DNA-binding domain and a second DNA-binding domain, and which gives rise to the 5' end of structure (2), TALEN_2, which includes a third DNA-binding domain and a fourth DNA-binding domain, and which gives rise to the 3' end of structure (2), TALEN_3 includes a fifth DNA-binding domain that recognizes the nucleotide sequence on the 5' side of region T3 containing the target site of the target DNA as a fifth DNA-binding domain recognition sequence or its complementary sequence L3, and a sixth DNA-binding domain that recognizes the nucleotide sequence on the 3' side as a sixth DNA-binding domain recognition sequence or its complementary sequence R3, and cleaves the target site or its vicinity in the target DNA, The DNA editing method according to claim 1, comprising:

5. The vector comprises in structure (1) a first guide RNA recognition sequence or its complementary sequence G1 and a second guide RNA recognition sequence or its complementary sequence G2, wherein G1 may be located between M1 and Hv1 and overlap with at least one of M1 and Hv1, and G2 may be located between M2 and Hv2 and overlap with at least one of M2 and Hv2. The site-specific nuclease system is a CRISPR-Cas system comprising a Cas protein and its guide RNA, The CRISPR-Cas system_1 includes a first guide RNA, and the Cas protein generates the 5' end of structure (2), The CRISPR-Cas system_2 includes a second guide RNA, and the Cas protein generates the 3' end of structure (2), The CRISPR-Cas system _3 includes a third guide RNA that recognizes a nucleotide sequence present in the target DNA containing the target site as a third guide RNA recognition sequence or its complementary sequence G3, wherein the Cas protein cleaves the target site or its vicinity in the target DNA, The DNA editing method according to claim 1, comprising:

6. The introduction of the site-specific nuclease system into the cells is CEN / ARS, an autonomous replication sequence, The second promoter P2, and Nucleotide sequence Nuc encoding the site-specific nuclease system operably linked to the second promoter P2 The DNA editing method according to claim 1, comprising the introduction of a nuclease system expression vector containing [the specified substance].

7. The introduction of the vector and site-specific nuclease system into the cells is First promoter P1, Structure (1), The second promoter P2, Nucleotide sequence Nuc encoding the site-specific nuclease system operably linked to the second promoter P2 This involves the introduction of an integrated vector, The structure (3) formed in the cutting process further includes P2 and Nuc which is operably connected to P2, The DNA editing method according to claim 1, wherein the selection step is a step of obtaining a selection vector comprising a functional selection marker gene in which the 3' end of M1 and the 5' end of M2 are bound together in the fragment shown in structure (3) to operably link with P1, and Nuc which is operably linked with P2.

8. The aforementioned integrated vector further comprises CEN / ARS, which is an autonomous replication sequence. The structure (3) formed in the cutting process further includes CEN / ARS, The DNA editing method according to claim 7, wherein the selection step is a step of obtaining a selection vector comprising a functional selection marker gene, CEN / ARS, and Nuc which is operably linked to P1 by the 3' end of M1 and the 5' end of M2 in the fragment shown in structure (3), and CEN / ARS.

9. The DNA editing method according to any one of claims 6 to 8, wherein the nucleotide sequence Nuc encoding the site-specific nuclease system is a nucleotide sequence encoding a fusion protein of a DNA-binding domain and a nuclease domain.

10. The DNA editing method according to any one of claims 6 to 8, wherein the nucleotide sequence Nuc encoding the site-specific nuclease system is a nucleotide sequence encoding a Cas protein and a nucleotide sequence encoding its guide RNA.

11. A method for producing DNA-edited cells in which a desired nucleotide sequence is inserted into a target site of target DNA or the target site is removed, A step of obtaining a cell (excluding cells within a human organism, human germ cells, human embryonic cells, and human fertilized eggs) containing the selection vector, wherein the desired nucleotide sequence D is inserted into the target site or the target site is removed by a DNA editing method according to any one of claims 1 to 8, and The process of selecting DNA-edited cells in which the desired nucleotide sequence D is inserted into or removed from the target site, using the selection marker gene contained in the selection vector as an indicator. A method for producing cells, including the method described above.

12. The cell production method according to claim 11, further comprising the step of culturing the DNA-edited cells and removing the selected vector from the cells, wherein the vector further comprises a vector containing CEN / ARS which is an autonomous replication sequence.

13. A vector for inserting a desired nucleotide sequence into a target site of target DNA or removing the target site using a site-specific nuclease system. The first promoter P1, and The following structure (1'): 5'-M1-Hv1'-D'-Hv2'-M2-3'...(1') [In (1'), M1 represents the 5' end fragment of the nucleotide sequence encoding the selection marker gene and is operably linked to the first promoter P1; Hv1' represents a nucleotide sequence homologous to the first nucleotide sequence Ht1 on the 5' end of the target site of the target DNA or its insertion site; D' represents the desired nucleotide sequence or its insertion site, but is optional; Hv2' represents a nucleotide sequence homologous to the second nucleotide sequence Ht2 on the 3' end of the target site of the target DNA or its insertion site; M2 represents the remaining 3' end fragment of the nucleotide sequence encoding the selection marker gene; adjacent sequences in M1, Hv1', D', Hv2', and M2 may partially overlap with each other.] Includes, The site-specific nuclease system described above produces the following structure (2'): 5'-Hv1'-D'-Hv2'-3'...(2') The fragment shown and the following structure (3'): 5'-M2-P1-M1-3'...(3') A DNA editing vector that produces the fragment shown.

14. The DNA editing vector according to claim 13, further comprising an autonomous replication sequence CEN / ARS, wherein the site-specific nuclease system generates a fragment further comprising CEN / ARS as structure (3').

15. Structure (1') is as follows: Structure (11'): 5'-M1-T1'-Hv1'-D'-Hv2'-T2'-M2-3'...(11') [In (11'), T1' indicates a TALEN_1 binding region or insertion site including a first DNA-binding domain recognition sequence or its complementary sequence L1 and a second DNA-binding domain recognition sequence or its complementary sequence R1; T2' indicates a TALEN_2 binding region or insertion site including a third DNA-binding domain recognition sequence or its complementary sequence L2 and a fourth DNA-binding domain recognition sequence or its complementary sequence R2; M1, Hv1', D', Hv2', and M2 are the same as in structure (1'), and adjacent sequences in M1, T1', Hv1', D', Hv2', T2', and M2 may partially overlap.] Includes, The site-specific nuclease system is TALEN, which comprises a DNA-binding domain and a nuclease domain. TALEN_1 includes a first DNA-binding domain and a second DNA-binding domain, which give rise to the 5' end of structure (2'), TALEN_2 includes a third DNA-binding domain and a fourth DNA-binding domain, which give rise to the 3' end of structure (2'), TALEN_3 includes a fifth DNA-binding domain that recognizes the nucleotide sequence on the 5' side of region T3 containing the target site of the target DNA as a fifth DNA-binding domain recognition sequence or its complementary sequence L3, and a sixth DNA-binding domain that recognizes the nucleotide sequence on the 3' side as a sixth DNA-binding domain recognition sequence or its complementary sequence R3, and cleaves the target site or its vicinity in the target DNA, The DNA editing vector according to claim 13, comprising

16. Structure (1') includes a first guide RNA recognition sequence or its complementary sequence or insertion site G1', and a second guide RNA recognition sequence or its complementary sequence or insertion site G2', wherein G1' is located between M1 and Hv1' and may overlap with at least one of M1 and Hv1', and G2' is located between M2 and Hv2' and may overlap with at least one of M2 and Hv2'. The site-specific nuclease system is a CRISPR-Cas system comprising a Cas protein and its guide RNA, The CRISPR-Cas system_1 includes a first guide RNA, and the Cas protein generates the 5' end of the (2') structure, The CRISPR-Cas system_2 includes a second guide RNA, and the Cas protein generates the 3' end of the (2') structure, The CRISPR-Cas system _3 includes a third guide RNA that recognizes a nucleotide sequence present in the target DNA containing the target site as a third guide RNA recognition sequence or its complementary sequence G3, wherein the Cas protein cleaves the target site or its vicinity in the target DNA, The DNA editing vector according to claim 13, comprising

17. First promoter P1, Structure (1'), The second promoter P2, Nucleotide sequence Nuc encoding the site-specific nuclease system operably linked to the second promoter P2 The DNA editing vector according to claim 13, comprising an integrated vector containing, wherein the site-specific nuclease system generates a fragment further comprising P2 and Nuc operably linked to P2 as structure (3').

18. The DNA editing vector according to claim 17, further comprising an autonomous replication sequence CEN / ARS, wherein a fragment further comprising CEN / ARS as structure (3') is generated by the site-specific nuclease system.

19. The DNA editing vector according to claim 13, wherein M1 and M2 are nucleotide sequences in which the selection marker gene is fragmented such that the 3' end of M1 and the 5' end of M2 of the fragment shown in structure (3') are bound together to enable function.

20. The DNA editing vector according to claim 19, wherein the 3' end of M1 and the 5' end of M2 are mutually overlapping nucleotide sequences, and the 3' end of M1 and the 5' end of M2 are bound together via the overlapping nucleotide sequences, thereby enabling the selection marker gene to function.

21. The DNA editing vector according to claim 20, wherein the length of the overlapping nucleotide sequence is 40 to 500 bases.

22. The DNA editing vector according to claim 21, wherein a stop codon is added to the 3' end of M1 and / or the 5' end of M2, and the stop codon is removed from M1 and / or M2 by binding via the overlapping nucleotide sequences.

23. A DNA editing kit comprising a DNA editing vector according to any one of claims 13 to 22 and the site-specific nuclease system.

24. The aforementioned site-specific nuclease system CEN / ARS, an autonomous replication sequence, The second promoter P2, and Nucleotide sequence Nuc encoding the site-specific nuclease system operably linked to the second promoter P2 The DNA editing kit according to claim 23, comprising a nuclease system expression vector containing the above.

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