Homologous recombination method

Abstract

To provide methods for conducting recombination with simpler operation, higher recombination efficiency, smaller limitation to target DNA sequences, and suppressed residual trace sequence in molecules from which DNA sequences have been removed.SOLUTION: A homologous recombination method for deleting a specific DNA sequence from a DNA molecule comprises: preparing a DNA molecule in which a pair of target sequences, which are the targets of a site-specific recombinase, are positioned in opposite directions to sandwich the specific DNA sequence, and a pair of homologous sequences, which are homologous DNA sequences, are positioned further outside of the pair of target sequences positioned to sandwich the specific DNA sequence; and treating the DNA molecule with the site-specific recombinase to delete the specific DNA sequence together with the pair of target sequences from the DNA molecule by homologous recombination between the pair of homologous sequences.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to methods of homologous recombination. [Background technology]

[0002] Conventionally, various techniques are known for recombination in DNA molecules. For example, it is known that a homologous recombination method utilizing recombination occurring between highly homologous base sequences can be used in a relatively wide range of organisms from prokaryotes to animals (see, for example, Non-Patent Documents 1 to 4). In addition, as a method for deleting a specific DNA sequence from a DNA molecule, site-specific recombination using a site-specific recombinase is known (see, for example, Non-Patent Documents 5 and 6). In addition, as yet another method for deleting a specific DNA sequence, a method is known in which a sequence to be deleted is placed between transposon-specific inverted terminal sequences, and the sequence to be deleted is removed together with the inverted terminal sequence using a transposase (see, for example, Non-Patent Document 7).

[0003] FIG. 15 is an explanatory diagram showing, as an example of homologous recombination, a state in which homologous recombination is performed between a pair of homologous sequences on the same DNA molecule, and a marker gene between the homologous sequences is deleted. FIG. 16 is an explanatory diagram showing a homologous recombination method using genome editing technology, as another example of homologous recombination. In this method, DNA near a homologous recombination target sequence (homologous sequence) is cut by genome editing technology (e.g., ZFN, TALEN, CRISPR-Cas, etc.), thereby activating homologous recombination repair and increasing the efficiency of homologous recombination via the target sequence. FIG. 17 is an explanatory diagram showing a site-specific recombination reaction. As an example, FIG. 17 shows a site-specific recombination reaction in a Cre-loxP recombination system using Cre recombinase and loxP sequences. Such a site-specific recombination reaction makes it possible to remove a DNA sequence between target sequences such as loxP sequences (a DNA sequence containing a marker gene in FIG. 17) with such high efficiency that selection by a marker gene is not necessary. FIG. 18 is an explanatory diagram showing an example of a reaction using an excision enzyme of a transposon. As an example, Fig. 18 shows an excision reaction using the transposon PiggyBac. In this type of reaction, a DNA sequence (a DNA sequence including a marker gene in Fig. 18) between specific inverted terminal sequences (repeated sequences) that can be recognized by an excision enzyme (transposase) can be deleted, and unlike the site-specific recombination reaction shown in Fig. 17, the above DNA sequence can be deleted with high efficiency without leaving any sequence that becomes a trace after the reaction. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Francesca Storici et al., Proc. Natl. Acad. Sci. USA, 100, 14994-14999(2003) [Non-Patent Document 2] Justin M. Vento et al., J. Ind. Microbiol.Biot., 46, 1327-1341(2019) [Non-Patent Document 3] Dana Carroll, Annual Review of Biochemistry, 83, 409-439(2014) [Non-Patent Document 4] Hiroshi Ochiai et al., Experimental Medicine, 31, 95-100(2013) [Non-Patent Document 5] Yueju Wang et al., Plant Cell Rep., 30, 267-285(2011) [Non-Patent Document 6] Xueying Tian et al., J. Biol. Chem., 296, 100509(2021) [Non-Patent Document 7] Sheng Ding et al., Cell, 122, 473-483(2005) Summary of the Invention [Problem to be solved by the invention]

[0005] However, for example, the homologous recombination technique using the homologous recombination repair mechanism without DNA cleavage shown in FIG. 15 had to be limited to limited species of organisms with high homologous recombination repair activity, such as yeast, chicken DT40 cells, and mouse ES cells. Even in budding yeast (Saccharomyces cerevisiae), which is considered to be particularly efficient, the recombination frequency is only 10 -6 The degree of homologous recombination was relatively low, and selection using a marker gene was required to obtain a homologous recombination strain. In addition, in the homologous recombination method using genome editing technology shown in Figure 16, non-homologous end joining is prioritized over homologous recombination repair in many organisms, and the cut sites are repaired by direct ligation, so the frequency of the desired recombination reaction is insufficient and selection using a marker gene is often required. In addition, the homologous recombination method using genome editing technology has the problem that a series of operations such as vector construction and DNA cleavage induction are relatively complicated.

[0006] In addition, in the site-specific recombination reaction using the site-specific recombinase shown in Figure 17, the target sequence is determined by the site-specific recombinase used, so the target sequence cannot be freely designed, and there is a problem that the target sequence remains in the DNA molecule from which the above DNA sequence has been removed by recombination. The remaining target sequence may cause an unintended recombination reaction when the recombined DNA molecule is subjected to a similar site-specific recombination reaction again.

[0007] Furthermore, the reaction using the transposon excision enzyme shown in Figure 18 has the problem that the site of DNA sequence excision is limited to a specific target sequence on the genome (the four bases TTAA in the example of Figure 18). Therefore, there has been a demand for a recombination technology that allows for more efficient recombination, has no restrictions on the target DNA sequence, leaves no trace sequences in the molecule after the DNA sequence has been removed, and can be performed with simpler operations. [Means for solving the problem]

[0008] The present disclosure can be realized in the following forms. (1) According to one embodiment of the present disclosure, a method for homologous recombination for deleting a specific DNA sequence from a DNA molecule is proposed. According to this method for homologous recombination, a DNA molecule is prepared in which a pair of target sequences targeted by a site-specific recombinase are arranged in different orientations with the specific DNA sequence sandwiched between them, and a pair of homologous sequences, which are DNA sequences having homology, are arranged outside the pair of target sequences arranged with the specific DNA sequence sandwiched between them, and the site-specific recombination is applied to the DNA molecule to delete the specific DNA sequence together with the pair of target sequences from the DNA molecule by homologous recombination between the pair of homologous sequences. According to this form of homologous recombination method, the frequency of homologous recombination, i.e., the efficiency of recombination, can be increased by utilizing a site-specific recombination reaction using a site-specific recombinase with a relatively high recombination efficiency. Since this type of recombination reaction is for carrying out homologous recombination, it is possible to reduce restrictions on the DNA sequence that is the target of the recombination reaction. In addition, since the target sequence located closer to the specific DNA sequence than between the homologous sequences can also be removed in association with the deletion of the specific DNA sequence, it is possible to prevent trace sequences from remaining in the molecule from which the DNA sequence has been removed. Furthermore, the characteristic sequence structure comprising the above-mentioned specific DNA sequence, the target sequence, and the homologous sequence can be obtained by a relatively simple process using general gene recombination technology. (2) In the above-described homologous recombination method, the site-specific recombinase may be an enzyme belonging to the integrase family, and may be configured to proceed as a reversible reaction as the recombination reaction in the target sequence. With this configuration, it is possible to improve the effect of increasing the frequency of homologous recombination. (3) In the above-described homologous recombination method, the site-specific recombinase may be an enzyme belonging to the bidirectional tyrosine recombinase subfamily, which can improve the effect of increasing the frequency of homologous recombination. (4) In the homologous recombination method of the above aspect, the site-specific recombinase may be an enzyme selected from Cre recombinase, FLP recombinase, R recombinase, and Dre recombinase. With such a configuration, the effect of increasing the frequency of homologous recombination can be improved. (5) In the above-mentioned homologous recombination method, Saccharomyces cerevisiae or Escherichia coli may be used as the cells for performing homologous recombination, and the distance between the target sequence and the homologous sequence adjacent to each other on the upstream or downstream side of the specific DNA sequence may be 1000 bp or less. With this configuration, the effect of increasing the recombination efficiency by arranging the specific DNA sequence, a pair of target sequences in different orientations, and a pair of homologous sequences as described above can be ensured by adjusting the distance. (6) In the above-described homologous recombination method, the homologous sequence may be a sequence having a length of 60 bp or less. With this configuration, even when a short homologous sequence, which has been relatively difficult to use for homologous recombination in the past, is used, the efficiency of homologous recombination can be increased. (7) In the above-described homologous recombination method, the homologous sequence may be a sequence having a length of 25 bp or more. With such a configuration, the effect of increasing the recombination efficiency by arranging a specific DNA sequence, a pair of target sequences in different orientations, and a pair of homologous sequences in the above-described arrangement can be ensured by adjusting the length of the homologous sequences. The present disclosure can be realized in various forms other than those described above, for example, in the form of a vector used for homologous recombination, or a recombinant obtained by homologous recombination. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is an explanatory diagram showing a schematic representation of the sequence structure of DNA involved in the homologous recombination method of the present embodiment. [Diagram 2] FIG. 2 is an explanatory diagram showing an example of homologous recombination by the homologous recombination method of the present embodiment. [Diagram 3] FIG. 13 is an explanatory diagram showing a destination vector for level 0. [Figure 4] FIG. 13 is an explanatory diagram showing a destination vector for level 1. [Diagram 5]FIG. 13 is an explanatory diagram showing a destination vector for level 2. [Figure 6] FIG. 2 is an explanatory diagram showing how the sequence structure of FIG. 1 is introduced into the yeast genome. [Figure 7] FIG. 1 is an explanatory diagram showing how a sequence structure of a comparative example is introduced into a yeast genome. [Figure 8] FIG. 2 is an explanatory diagram showing how the sequence structure of FIG. 1 is introduced into the yeast genome. [Figure 9] An explanatory diagram showing the structure of each of the level 1 modules used for introduction into the yeast genome. [Figure 10] FIG. 1 is an explanatory diagram showing the structure of the V3P vector set introduced into the yeast genome. [Figure 11] FIG. 1 is an explanatory diagram showing a level 2 vector for evaluating the efficiency of homologous recombination in E. coli. [Figure 12] An explanatory diagram showing the contents of the level 2 vector introduced into E. coli. [Figure 13] FIG. 1 is an explanatory diagram showing the results of an experiment on homologous recombination in yeast. [Figure 14] An explanatory diagram showing the experimental results of homologous recombination in E. coli. [Figure 15] An explanatory diagram showing how homologous recombination occurs between homologous sequences. [Figure 16] An explanatory diagram showing a homologous recombination method using genome editing technology. [Figure 17] Schematic diagram showing a site-specific recombination reaction. [Figure 18] FIG. 1 is an explanatory diagram showing a reaction using a transposon excision enzyme. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] A. First embodiment: FIG. 1 is an explanatory diagram showing a schematic diagram of the sequence structure of DNA according to the homologous recombination method of the present embodiment. The homologous recombination method of the present embodiment is a method for deleting a specific DNA sequence from a DNA molecule by combining site-specific recombination and homologous recombination. As shown in FIG. 1, in the present embodiment, in a DNA molecule 10 to be subjected to homologous recombination, a pair of target sequences 12a and 12b targeted by a site-specific recombinase (sequence-specific recombinase) are arranged in different orientations with a specific DNA sequence 15 to be deleted sandwiched therebetween. A pair of homologous sequences 14, which are DNA sequences having homology, are arranged further outside the pair of target sequences 12a and 12b arranged in opposite orientations with the specific DNA sequence 15 sandwiched therebetween. In the present embodiment, the above-mentioned site-specific recombinase is applied to such a DNA molecule 10 to advance homologous recombination between the pair of homologous sequences 14, thereby deleting the pair of target sequences 12a and 12b as well as the specific DNA sequence 15 from the DNA molecule 10.

[0011] The target sequences 12a and 12b may be appropriately set according to the site-specific recombinase used. The site-specific recombinase used here is preferably a recombinase that undergoes a reversible reaction as the recombination reaction that occurs between the pair of target sequences 12a and 12b. Here, "a reversible reaction proceeds" means that after the recombination reaction, a recombination reaction can proceed that returns to the state before the recombination reaction.

[0012] As such a site-specific recombinase that "performs a reversible reaction," for example, a recombinase belonging to the integrase family can be used. Here, site-specific recombinases belonging to the integrase family are classified into two groups, the tyrosine recombinase family and the serine recombinase family (see Non-Patent Document 5). These are classified according to whether they have a tyrosine residue or a serine residue at the active site in the catalytic domain of the enzyme. The tyrosine recombinase family is further classified into a bidirectional tyrosine recombinase subfamily (hereinafter also referred to as the "bidirectional tyrosine subfamily") and a unidirectional tyrosine recombinase subfamily (hereinafter also referred to as the "unidirectional tyrosine subfamily") based on their mechanism of action. In addition, the serine recombinase family is classified into a large serine recombinase subfamily (hereinafter also referred to as the "large serine subfamily") and a small serine recombinase family (hereinafter also referred to as the "small serine subfamily") based on their size.

[0013] Examples of combinations of bidirectional tyrosine subfamily and target sequences include the Cre-loxP recombination system using Cre recombinase and loxP sequence, the FLP-FRT recombination system using FLP recombinase and FRT sequence, the R-RS recombination system using R recombinase and RS sequence, and the Dre-rox recombination system using Dre recombinase and rox sequence. Such genetic crossover via the bidirectional tyrosine subfamily occurs between two identical target sequences (recognition sites), and since the recognition sites are identical, the recombination reaction is completely reversible.

[0014] Examples of the unidirectional tyrosine subfamily include λ recombinase, HK101 recombinase, and pSAM2 recombinase. Examples of the large serine subfamily include PhiC31 recombinase, TP901-1 recombinase, Bxb1 recombinase, and R4 recombinase. These unidirectional tyrosine and large serine subfamilies act on two recognition sites with different sequences, known as attB and attP, to generate hybrid recombination sites known as attL and attR (ACS Synth. Biol. 7, 299-310(2018)). In this way, the recognition sites attB and attP are changed to attL and attR, so the recombinase alone does not cause a reverse reaction, but the addition of RDF (recombination directionality factor) switches the direction of recombination, making the reaction reversible. By adjusting the concentrations of recombinase and RDF, it is possible to control the reaction rates in each direction (Nucleic Acids Res. 44(15), 7360-7372(2016)), allowing the reversible reaction to proceed continuously.

[0015] As described above, a recombinase belonging to the integrase family can be suitably used as a site-specific recombinase that continues the reversible reaction. In particular, when the bidirectional tyrosine subfamily is used, the reversible reaction can be more easily continued in a simple reaction system without using additional components such as RDF. Note that the small serine subfamily described above is not used in this embodiment to advance the reversible reaction because the reaction direction is unidirectional and there is no known method for reversing the reaction.

[0016] The degree of identity between a pair of homologous sequences 14 is not particularly limited as long as homologous recombination is possible. Whether or not the identity is sufficient to enable homologous recombination is affected by the length of the homologous sequences, etc., but the degree of identity may be, for example, at least 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 100%. The length of the homologous sequence 14 is not particularly limited as long as homologous recombination is possible. From the viewpoint of suppressing unintended recombination reaction, the length of the homologous sequence 14 may be, for example, 20 bp or more, preferably 30 bp or more, and more preferably 40 bp or more. From the viewpoint of increasing the efficiency of homologous recombination in the homologous sequence 14, the length of the homologous sequence 14 is preferably 25 bp or more, and more preferably 30 bp or more. In addition, from the viewpoint of facilitating the production of a vector having a desired sequence structure, the length of the homologous sequence 14 may be, for example, 500 bp or less, preferably 400 bp or less, and more preferably 300 bp or less. Generally, if the homologous sequence 14 is too short, homologous recombination is difficult to proceed and the recombination efficiency decreases. However, in the homologous recombination method of this embodiment, even if the length of the homologous sequence 14 is, for example, 60 bp or less, or even 50 bp or less, it is possible to achieve high homologous recombination efficiency.

[0017] As shown in FIG. 1, a first spacer sequence Spu may be provided between the 5' end of the target sequence 12a, which is located on the upstream side (5' side) of the specific DNA sequence 15 among the target sequences 12a and 12b, and the 3' end of the homologous sequence 14 adjacent to the target sequence 12a among the pair of homologous sequences 14. Also, a second spacer sequence Spd may be provided between the 3' end of the target sequence 12b, which is located on the downstream side (3' side) of the specific DNA sequence 15 among the target sequences 12a and 12b, and the 5' end of the homologous sequence 14 adjacent to the target sequence 12b among the pair of homologous sequences 14. The length of the first spacer sequence Spu and the second spacer sequence Spd, i.e., the distance between the target sequence and the homologous sequence adjacent to each other on the upstream or downstream side of the specific DNA sequence 15, may be 0 bp or more. The distance may be, for example, 50 bp or more, or 1500 bp or less.

[0018] For example, when homologous recombination is performed in Saccharomyces cerevisiae or Escherichia coli using Cre recombinase and loxP sequence as a combination of recombinase and target sequence, the above distance can be 1500 bp or less, may be 1200 bp or less, and is preferably 1000 bp or less. In this way, by appropriately setting the lengths of the first spacer sequence Spu and the second spacer sequence Spd according to the site-specific recombinase and its target sequence, the type of cell in which recombination is performed, the gene locus in which recombination is performed in the cell, etc., it is possible to increase the efficiency of recombination by homologous recombination.

[0019] Fig. 2 is an explanatory diagram showing an example of homologous recombination caused by the homologous recombination method of this embodiment, in which Cre recombinase and loxP sequence are used as a combination of recombinase and target sequence. When a site-specific recombinase (Cre recombinase in the case of Fig. 2) is allowed to act in the state of Fig. 2(A) similar to Fig. 1, homologous recombination between homologous sequences occurs, and a specific DNA sequence 15 is deleted together with the target sequence (loxP sequence) from the DNA molecule 10 (Fig. 2(E)). The mechanism by which such homologous recombination proceeds is considered to be as follows.

[0020] That is, when Cre recombinase is allowed to act as described above, a reversible recombination reaction proceeds between a pair of target sequences (loxP sequences in FIG. 2) in the same DNA molecule 10 in which the distance between the target sequences is relatively close, and the inversion of the specific DNA sequence 15 continues. In this way, the destabilization of DNA caused by the continuous reversible recombination reaction between the target sequences may activate the DNA repair mechanism by homologous recombination, and it is considered that homologous recombination is induced between a pair of homologous sequences located close to a pair of target sequences in the DNA molecule 10. In addition, the repeated closeness between the homologous sequences may also be a factor in increasing the frequency of homologous recombination. Therefore, it is considered that the frequency of homologous recombination increases in the process in which the reversible recombination reaction continues between the target sequences, that is, homologous recombination proceeds at a frequency much higher than the frequency at which homologous recombination naturally occurs in a state in which the reversible reaction does not progress between the target sequences. In the example of FIG. 2, the state where the reaction of inversion of the "specific DNA sequence 15" is repeated between loxP sequences in mutually opposite directions due to the change between the states shown in FIG. 2(A) to FIG. 2(D) is shown. Unlike FIG. 2(A), when a pair of loxP sequences are arranged in the same direction, a reaction of excising the specific DNA sequence 15 by site-specific recombination between the pair of loxP sequences proceeds, and such an excision reaction proceeds overwhelmingly more frequently than a reverse reaction in which the excised specific DNA sequence 15 is incorporated into the DNA molecule 10 at the position before excision. Therefore, most of the DNA molecules 10 are stable in a state in which the specific DNA sequence 15 is excised between a pair of target sequences (loxP sequences). In contrast, when a pair of loxP sequences are arranged in mutually opposite directions as in this embodiment, the reversal reaction of the specific DNA sequence 15 is repeated, so that the DNA remains unstable, and the distance between the pair of homologous sequences 14 can be made longer and closer. Therefore, the efficiency of homologous recombination in which the specific DNA sequence 15 is excised together with the target sequence between the homologous sequences can be improved.

[0021] In this case, for example, if the specific DNA sequence 15 contains a marker gene such as a drug resistance gene, it is possible to select cells containing the DNA molecule 10 in which homologous recombination has occurred, using the expression of this marker gene as an indicator. In addition, if the specific DNA sequence 15 contains a site-specific recombinase gene, the site-specific recombinase is produced when the site-specific recombinase gene is in an expressible state while the reversible recombination reaction continues between the target sequences, and the site-specific recombination reaction can be continued. After homologous recombination has occurred, the specific DNA sequence 15 containing the site-specific recombinase gene is deleted, and the influence of the unnecessary enzyme gene can be suppressed.

[0022] According to the homologous recombination method of the present embodiment configured as described above, a DNA molecule 10 is used in which a pair of target sequences 12a, 12b are arranged in different directions with a specific DNA sequence 15 sandwiched therebetween, and a pair of homologous sequences 14 are arranged further outside the pair of target sequences 12a, 12b arranged with the specific DNA sequence 15 sandwiched therebetween. Then, a site-specific recombinase is applied to this DNA molecule 10 to delete the specific DNA sequence 15 from the DNA molecule 10 by homologous recombination between the pair of homologous sequences 14. In general, the frequency of recombination reaction is much higher in site-specific recombination that proceeds between the target sequences 12a, 12b than in homologous recombination that proceeds between the homologous sequences 14. When a site-specific recombination reaction proceeds between the target sequences 12a, 12b in the same molecule, the distance between the pair of homologous sequences 14 arranged outside the target sequences 12a, 12b in the same DNA molecule 10 is reduced, as shown in Figures 2(B) and 2(C). As a result, the frequency of homologous recombination between homologous sequences 14 increases, making it easier to delete specific DNA sequence 15 from DNA molecule 10 by homologous recombination. In particular, by using, as the site-specific recombination enzyme, an enzyme that belongs to the integrase family and that proceeds with a reversible reaction as the recombination reaction in target sequences 12a and 12b, it is possible to improve the effect of shortening the distance between a pair of homologous sequences 14 and increasing the frequency of homologous recombination.

[0023] Furthermore, according to this embodiment, the specific DNA sequence 15 is deleted by performing homologous recombination between a pair of homologous sequences 14 located outside the target sequences 12a and 12b while utilizing the reaction of a site-specific recombinase, so that the target sequences 12a and 12b can be deleted from the DNA molecule 10 together with the specific DNA sequence 15. In this way, the target sequences 12a and 12b do not remain in the DNA molecule 10 after the deletion of the specific DNA sequence 15, so that, for example, even when a similar site-specific recombination technique is applied to the DNA molecule 10 after the deletion of the specific DNA sequence 15, unintended recombination reactions caused by the remaining target sequence can be suppressed.

[0024] Furthermore, because the specific DNA sequence 15 is excised by homologous recombination, the degree of freedom in designing the sequence for excision (homologous sequence 14) can be ensured, unlike the case where the specific DNA sequence 15 is excised by site-specific recombination using a site-specific recombinase. Furthermore, according to the homologous recombination method of this embodiment, operations for obtaining a sequence structure in which genes are integrated as shown in FIG. 1 (for example, a series of operations such as the construction of a vector and induction of DNA cleavage) can be performed in a relatively simple process using a general gene recombination technique. As a gene integration method for obtaining the sequence structure shown in FIG. 1, various known methods that can join multiple DNA fragments regardless of the terminal shape of the DNA can be adopted. Specifically, the cloning step for obtaining the sequence structure of FIG. 1 can be performed, for example, by a method using classical restriction enzymes, the Golden gate method, the Gibson Assembly system (Nature Methods 6 (5), 343-345 (2009)), or a method using NEBuilder HiFi DNA Assembly Cloning Kit (NEB) or In-Fusion HD Cloning Kit (Takara Bio Inc.) (IN-FUSION is a registered trademark). Therefore, unlike the conventionally known DNA recombination methods shown in FIGS. 15 to 18, it is possible to perform recombination with higher efficiency, to suppress restrictions on the DNA sequence that is the target of the recombination reaction, to leave no sequence as a trace in the molecule from which the DNA sequence has been removed, and to perform DNA recombination with simpler operations.

[0025] Furthermore, according to the homologous recombination method of this embodiment, it is possible to achieve high recombination efficiency in systems using a wider range of cells, including prokaryotes, eukaryotes, and cultured animal cells, and in in vitro systems. For example, in conventionally known homologous recombination, the recombination efficiency varies greatly depending on the organism species and cell species, and even in budding yeast (Saccharomyces cerevisiae), which is considered to have a relatively high efficiency of homologous recombination, the recombination frequency is only 10 -6In addition, in Escherichia coli, the most efficient method of genome homologous recombination is known to be transformation while introducing and inducing a homologous recombination enzyme derived from Lambda phage, but in this case, the efficiency was about 10 -3 (Front. Microbiol., 11 September 2020, https: / / doi.org / 10.3389 / fmicb.2020.548410). According to the homologous recombination method of the present embodiment, unlike the case of using a homologous recombination enzyme derived from Lambda phage in E. coli, a combination of a cell to be recombined and an enzyme is not specified, but a general site-specific recombination enzyme that can be applied to a wider range of cell types, etc., is used, and the efficiency of the recombination can be increased to, for example, 10 -0 ~10 -2 This allows homologous recombination to be carried out with a higher efficiency.

[0026] As described above, in the homologous recombination method of this embodiment, various site-specific recombinases can be used to perform homologous recombination in the homologous sequence 14. In particular, a system using Cre recombinase, which is widely used in various biological species including in vivo systems and is known to exhibit a wide range of reactivity, is desirable. By utilizing a system using Cre recombinase, it becomes possible to increase the efficiency of homologous recombination for a wide range of biological species, regardless of whether the biological species is a prokaryote or eukaryote, or an animal cell or a plant cell. In addition to the system using Cre recombinase, for example, a system using FLP recombinase, R recombinase, or Dre recombinase, which belongs to the bidirectional tyrosine subfamily like Cre recombinase, can also be widely used for various biological species. In addition, among the recombinases belonging to the bidirectional tyrosine subfamily, for example, the genes of Dre, VCre, SCre, Vika, and Nigri recombinases are all known to be homologous to the Cre recombinase gene, and although their target sequences are different, their action mechanisms are almost the same as those of Cre recombinase. In the case of using such recombinases obtained from homologous genes of the Cre recombinase gene, it is desirable to be able to effectively increase the efficiency of homologous recombination in a wide range of organisms, as in the case of using Cre recombinase. EXAMPLES

[0027] The present disclosure will be described in more detail below with reference to examples, but the present disclosure is not limited to the description of these examples. In the examples described below, a vector having the sequence structure shown in Figure 1 was prepared, and the sequence structure in the vector was incorporated into the genome of Saccharomyces cerevisiae BY4742 strain, which is a haploid strain of laboratory yeast, or introduced as a plasmid into Escherichia coli DH5α strain or BL21 (DE3) strain, to examine the efficiency of homologous recombination in both eukaryotes and prokaryotes.

[0028] <Preparation of cloning vector set> All vectors used in the test were constructed by the Golden Gate method (PLoS ONE 6, e16765(2011)). The Golden Gate method is a well-known gene integration method in which multiple DNA fragments are inserted into a vector in a pre-designed order using Type IIS restriction enzymes and T4 DNA ligase. As subcloning vectors required for the Golden Gate method, vectors corresponding to destination vectors for level 0, level 1, and level 2 were constructed. Below, the methods for constructing destination vectors for level 0, level 1, and level 2 are explained in order.

[0029] FIG. 3 is an explanatory diagram showing a destination vector for level 0. As shown in FIG. 3, the destination vector for level 0 is a vector containing a BsaI recognition site. When preparing a destination vector for level 0, a target DNA fragment was amplified by PCR using pUC19 or synthetic DNA as a template. The primers used in PCR were artificially synthesized to have DNA sequences added so as to overlap with adjacent DNA sequences by about 15 bp in order to bind each DNA fragment. The amplified PCR fragments were sequentially bound using In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.) (IN-FUSION is a registered trademark) to complete a destination vector for level 0.

[0030] FIG. 4 is an explanatory diagram showing a destination vector for level 1. As shown in FIG. 4, the destination vector for level 1 is a set of multiple types of vectors, each of which has a fluorescent protein zFP538 gene (Nat Biotechnol. 17, 969-973 (1999)) and BsaI recognition sites arranged in opposite directions on either side of the gene. In addition, each of the destination vectors for level 1 differs in the presence or absence, combination, and arrangement of a loxP sequence, sequences 1 to 4 described below, and a restriction enzyme site and cutting site used in the Golden Gate reaction. These destination vectors for level 1 were prepared by amplifying the target DNA fragment using pUC19 and synthetic DNA (including a kanamycin resistance gene) as templates, and sequentially binding the DNA fragments using the In-Fusion HD Cloning Kit. Here, the above-mentioned sequences 1 to 4 are 57 bp homologous sequences provided at the ends of each amplified fragment obtained by PCR amplification in order to bind the amplified fragments obtained by PCR in the desired order. Any sequence can be used as long as the sequence similarity between the sequences is sufficiently low for sequences 1 to 4. Here, artificial sequences with low similarity to the yeast genome sequence are set, and sequences 1 and 2 are sequences described in Microb Cell Fact 12, 47 (2013), and sequences 3 and 4 are sequences described in Nucleic Acids Res. 43, 6620-6630 (2015).

[0031] 5 is an explanatory diagram showing destination vector P1301 for level 2. The destination vector for level 2 is a vector used for studies in E. coli in which the Golden Gate reaction is repeated twice to prepare a vector, and was prepared in the same manner as the destination vectors for level 0 and level 1 described above.

[0032] <Preparation of vector set for yeast> FIG. 6 is an explanatory diagram showing how the sequence structure shown in FIG. 1 is introduced into the ECM38 locus of the yeast genome by homologous recombination of the level 1 module prepared using the destination vector for level 0 and the destination vector for level 1 described above. FIG. 7 is an explanatory diagram showing how the sequence structure of the comparative example is introduced into the yeast genome in a similar manner. Here, loxP sequences are used as the target sequences 12a and 12b, and the homologous sequence 14 is shown as the homologous sequence A. As shown in FIG. 6 and FIG. 7, the homologous sequence A is a sequence ranging from 301 bp on the 3'-end side of the region (CDS) encoding the ECM38 protein in the yeast genome DNA to be introduced to 20 bp, 30 bp, or 50 bp. As the homologous sequence A, a sequence with a length of 20 bp, a sequence with a length of 30 bp, or a sequence with a length of 50 bp was used (see FIG. 9, FIG. 10, and FIG. 13 described later). Note that the sequence structure of the comparative example shown in FIG. 7 does not have the loxP sequence, which is the target sequence.

[0033] FIG. 8 is an explanatory diagram showing how the sequence structure shown in FIG. 1 is introduced into the ALD4 locus of the yeast genome in the same manner as in FIG. 6. Here, loxP sequences are used as the target sequences 12a and 12b as in FIG. 6, and the homologous sequence 14 is shown as the homologous sequence AA. As shown in FIG. 8, the homologous sequence AA is a sequence ranging from the 3' end of the region (CDS) encoding the ALD4 protein in the yeast genome DNA to be introduced to 50 bp. As the homologous sequence AA, a sequence having a length of 50 bp was used in this manner (see FIG. 9, FIG. 10, and FIG. 13 described later).

[0034] As shown in Figures 6 to 8, all vectors tested for yeast were designed so that by mixing and transforming the three fragment sets, recombination would occur between the homologous regions of each fragment (any of sequences 1 to 4), and the resulting fragment would be introduced into the yeast genome as one fragment. In Figures 6 to 8, the fragments constituting the three fragment sets for obtaining one fragment to be introduced into the yeast genome are shown as level 1 module A, level 1 module B, and level 1 module C in order from the 5' end after ligation.

[0035] Fig. 9 is an explanatory diagram showing the configuration of each of the level 1 modules used for introduction into the yeast genome shown in Fig. 6 to Fig. 8. That is, Fig. 9 shows, for each level 1 module, the type of level 1 destination vector used (see Fig. 4), the type of level 0 module, and the configuration of the level 0 module insert. Note that Fig. 9 also shows the level 1 module used for evaluation of the efficiency of homologous recombination in E. coli, which will be described later.

[0036] In the following, the method of producing each level 1 module shown in FIG. 9 will be described. First, sequence B (see FIG. 6), which is a part of the sequence of the region (CDS) encoding the protein of ECM38 for performing homologous recombination with the genome and is a 1080 bp sequence from the beginning of the ORF of ECM38, and sequence A of a length of 50 bp described above were amplified by PCR using the S. cerevisiae BY4742 strain genome, the E. coli K12 strain genome, or synthetic DNA as a template. The target DNA fragments were sequentially linked using the In-Fusion HD Cloning Kit, and subcloned into a vector corresponding to the destination vector for level 0 of the Golden Gate method (FIG. 3), and the obtained module was named V1P1042, a level 0 module. In addition, level 0 modules V1P1087, V1P1245, V1P1246, and V1P1260 were obtained as vectors in which four types of sequences (50 bp, 100 bp, 200 bp, and 1000 bp) were added to the 3' downstream of the insert of V1P1042 as spacer sequences between the loxP sequence and sequence A (first spacer sequence Spu in FIG. 1). Furthermore, level 0 modules V1P1265 and V1P1263, which are level 0 modules similar to the level 0 module V1P1246, were obtained by changing the length of sequence A to 20 bp and 30 bp, and level 0 module V1P1248 was obtained by deleting sequence A from the insert of V1P1042 (see FIG. 9). As the spacer sequence (first spacer sequence Spu), a partial sequence of the CDS of the araB gene of the E. coli K12 strain was used. These level 0 modules are used to obtain the level 1 module A shown in FIGS.

[0037] Similarly, sequence BB (see FIG. 8), which is a DNA fragment containing a homologous recombination region in the range of 804 bp on the 5'-end side of the CDS of the ALD4 gene for performing homologous recombination with the genome, and sequence AA of a length of 50 bp described above were subcloned, and the obtained module was named level 0 module V1P1262. In addition, level 0 module V1P1274 was obtained as a vector in which a sequence (200 bp) serving as a spacer sequence between the loxP sequence and sequence AA (first spacer sequence Spu in FIG. 1) was added downstream of the 3'-side insert of V1P1262, which is a level 0 module similar to level 0 module V1P1262. These level 0 modules are used to obtain level 1 module A shown in FIG. 8.

[0038] In addition, a fusion DNA fragment of a G418 resistance gene (G418 marker), an EGFP gene (Gene, 173, 33-38 (1996)), which is a modified form of the GFP gene, and a Cre gene linked to the promoter of the GAL10 gene induced by galactose, was subcloned into a destination vector for level 0 (Figure 3), and the resulting module was named level 0 module V1P256 (see Figure 9). This is used to obtain level 1 module B shown in Figures 6 to 8.

[0039] In addition, a DNA fragment (sequence C shown in Figures 6 and 7) containing a homologous recombination region ranging from 301 bp on the 3'-end side of the CDS of the ECM38 gene in the yeast genomic DNA to be introduced to an additional 841 bp was subcloned into a destination vector for level 0 (Figure 3), and the resulting module was named level 0 module V1P70 (see Figure 9). Furthermore, level 0 modules V1P1261 and V1P1249 were obtained as vectors in which a spacer sequence (second spacer sequence Spd in Figure 1) (200 bp, 50 bp) was added between the loxP sequence and the homologous recombination sequence A upstream of the 5'-end insert of the level 0 module V1P70 (see Figure 9). Note that a part of the sequence of the CDS of the araA gene of the E. coli K12 strain was used as the spacer sequence (second spacer sequence Spd). These level 0 modules are used to obtain the level 1 module C shown in Figures 6 and 7.

[0040] Similarly, a DNA fragment (sequence CC shown in FIG. 8) containing a further 849 bp of the homologous recombination region from the 3'-end side of the CDS of the ALD4 gene in the yeast genomic DNA to be introduced was subcloned into a destination vector for level 0 (FIG. 3), and the resulting module was named level 0 module V1P73 (see FIG. 9). This level 0 module is used to obtain level 1 module C shown in FIG. 8.

[0041] Level 1 modules were prepared by Golden Gate reaction (restriction enzyme: BsaI) between each of the level 0 module vectors and the corresponding level 1 destination vectors shown in Figure 9. Three types of the obtained level 1 modules were combined and used as a V3P vector set for introduction into the yeast genome as shown in Figures 6 to 8.

[0042] FIG. 10 is an explanatory diagram showing the configuration of a V3P vector set introduced into a yeast genome. FIG. 10 shows the combination of three types of level 1 modules constituting each V3P vector set, the name of the yeast strain into which each V3P vector set was introduced, the gene locus into which the sequence structure of FIG. 1 was introduced, and the presence or absence of a loxP sequence, the length of the first spacer sequence Spu, the length of the second spacer sequence Spd, and the length of the homologous sequence (A or AA) in each V3P vector set. As shown in FIG. 6 to FIG. 8, each V3P vector set has, in its insert, a homologous recombination sequence (sequence B or sequence BB) capable of homologous recombination with the 5' upstream of the introduction site in yeast genomic DNA, a homologous recombination sequence (sequence C or sequence CC) capable of homologous recombination with the 3' downstream of the introduction site in yeast genomic DNA, or a sequence selected from sequences 1 to 4 capable of homologous recombination between the respective inserts. When the three types of inserts of the V3P plasmid set shown in Figure 10 are transformed into yeast, homologous recombination occurs between each sequence and with the yeast genome, and all the fragments are eventually linked and introduced into the yeast genome in the order shown as "Level 1 module combination" in Figure 10.

[0043] <Introduction of vectors into the yeast genome> The inserts from each plasmid of each vector set were amplified by PCR, and the amplified fragments were used to transform Saccharomyces cerevisiae BY4742 strain, which was then spread onto YPD agar medium containing G418, and the grown colonies were purified. Then, strains that had undergone homologous recombination as expected were selected by PCR. The transformation was performed according to the method of Akada et al. (BioTechniques 28,854(2000)). The yeast transformants prepared are shown in FIG. 10.

[0044] <Induction of homologous recombination in yeast and measurement of homologous recombination efficiency> The strains thus prepared were cultured in YPGa (10 g / L yeast extract, 20 g / L peptone, 20 g / L galactose) agar medium at a concentration of 100 or 1000 cells / plate, and Cre gene expression was induced. When homologous recombination occurs with the expression of the Cre gene, the marker gene, EGFP gene, and Cre gene, which are sandwiched between the homologous sequences (sequence A or sequence AA), are dropped from the genome as a set, and the fluorescence of EGFP is quenched. Therefore, among the grown colonies, colonies with quenched fluorescence were counted as candidates for colonies that have undergone homologous recombination, and the quenching rate of colonies was examined using the total number of colonies as the denominator. As a control, the efficiency of the Uz4161 ​​strain was also measured under YPD medium conditions, in which Cre gene expression is not induced. Furthermore, for some strains, PCR was performed on quenched colonies using primers set to sandwich both sides of the genome introduction site, and whether or not homologous recombination had occurred accurately was evaluated at the amplified DNA size level.

[0045] <Preparation of vector for E. coli> All tested vectors were constructed using the two-step Golden Gate method, and the efficiency of homologous recombination was evaluated in E. coli as circular plasmids. The sequence for homologous recombination was sequence A used in yeast, and the vector was also shared with the yeast vector (Figure 9), but some modules were constructed additionally.

[0046] Here, to obtain a vector for homologous recombination, a gene sequence containing a spectinomycin resistance gene (SpR marker) and the mRFP1.1 gene (Nat Biotechnol 22, 1567 (2004)) was amplified by PCR using Escherichia coli K12 genomic DNA and synthetic DNA as templates, and subcloned into a level 0 destination vector (Figure 3). The resulting module was named level 0 module V1P415 (see Figure 9).

[0047] Next, each level 0 module vector was subjected to Golden Gate reaction (restriction enzyme BsaI) with the level 1 destination vector shown in Figure 9 to generate a level 1 module. Each level 1 module was subjected to a second Golden Gate reaction (restriction enzyme Eps3I) with the level 2 destination vector P1301 (Figure 5) to complete the level 2 vector.

[0048] Fig. 11 is an explanatory diagram showing a typical structure of a level 2 vector for E. coli for evaluating efficiency of homologous recombination prepared as described above, and Fig. 12 is an explanatory diagram showing the contents of each level 2 vector introduced into E. coli.

[0049] In addition to the level 2 vector, an expression vector for the Cre gene was also prepared. Here, the tetR gene required for the Teton system induced by tetracycline and the fusion DNA fragment of the Cre gene linked to the tetA promoter were amplified by PCR using the genomic DNA of E. coli K12 strain and synthetic DNA as templates, and subcloned into a destination vector for level 0 (Figure 3). The resulting module was named level 0 module V1P408. Then, this level 0 module V1P408 and the destination vector for level 1 V1P402 (Figure 4) were used to carry out the Golden Gate reaction (restriction enzyme BsaI) to prepare the level 1 module V2P591 (see Figure 9).

[0050] <Creation of strains for evaluating homologous recombination in E. coli> When carrying out a homologous recombination experiment, one of the level 2 vectors shown in Figure 12 and V2P591, an expression vector for the Cre gene, were simultaneously transformed into the E. coli DH5α or BL21(DE3) strain, and the resulting transformants were subjected to the homologous recombination experiment. As a control, a strain transformed only with the level 2 vector was also evaluated.

[0051] <Induction of homologous recombination in E. coli and measurement of homologous recombination efficiency> It is believed that the fluorescence of mRFP1.1 is quenched when homologous recombination occurs. In the medium in which E. coli transformed with the two vectors was grown, the fluorescence of most of the transformed colonies was already quenched, so it was assumed that the Cre gene was already weakly expressed and homologous recombination had already occurred. Therefore, among the grown colonies, colonies with parts where the fluorescence was quenched were counted as colonies that were candidates for homologous recombination, and the quenching rate of colonies was examined using the total number of colonies as the denominator. Some of the quenched colonies were isolated and cultured in LB medium containing anhydrotetracycline, and the expression of the Cre gene was induced. PCR was performed using primers set to flank both sides of the homologous recombination region, and the efficiency of homologous recombination was calculated from the band size.

[0052] <Evaluation Results> FIG. 13 is an explanatory diagram showing the results of an experiment on homologous recombination in yeast. As shown in FIG. 13, in yeast, under control conditions that fulfill any of the following conditions: "(i) no homologous sequence in the part outside the pair of loxP sequences (Uz4167 strain)", "(ii) no loxP sequence (Uz4163 strain)", and "(iii) all components are present but Cre is not expressed (Uz4161 ​​strain)", no quenched colonies were observed, and it was considered that no homologous recombination occurred at all. On the other hand, regardless of the presence or absence of a spacer sequence (the first spacer sequence Spu or the second spacer sequence Spd), under conditions in which the Cre gene was expressed with two loxP sequences of different orientations and a pair of homologous sequences outside them, fluorescence was found to be quenched at least 100 times more efficiently than the control, and in some cases, the fluorescence of most of the cells was quenched under certain conditions. From the PCR results, it is believed that when the region of homologous recombination (homologous sequence length) is 30 bp or more, homologous recombination has occurred correctly in over 90% of the colonies in which recombination occurred, but when the homologous sequence length is 20 bp, the rate of correct homologous recombination dropped to approximately 33%. Therefore, it is considered desirable for the homologous sequence length to be 25 bp or more. However, since the survival rate (counted from the number of colonies) of cells grown in Cre gene induction medium was 10-100%, it is believed that under all conditions, the cells grown were more than 10%. -0 ~10 -2 Therefore, in the results shown in Figure 13, including the case where the homologous sequence length is 20 bp, the efficiency of homologous recombination in yeast genomes due to the introduction of foreign genes is 10 -6 (See Non-Patent Document 1), it can be said that homologous recombination occurs at an extremely high frequency.

[0053] For the Uz4161 ​​strain, we sequenced five clones that were presumed to have undergone correct recombination at the DNA size level, and confirmed that correct homologous recombination had occurred. We also confirmed that even under conditions of poor recombination efficiency, most cells lost their fluorescence and changed into a cell population that had undergone homologous recombination by simply subculturing the cells without isolating the cells that had lost their fluorescence. When a pair of target sequences (loxP sequences) was arranged in the same direction (forward orientation) (Uz4162 strain), site-specific recombination reaction occurred at the pair of loxP sequences arranged in the forward orientation, and the percentage of colonies that lost fluorescence was extremely high, at over 99.9%. This is thought to be because when site-specific recombination reaction occurs at a pair of loxP sequences arranged in the forward orientation, the DNA molecule (yeast genome) becomes stable in a state in which a specific DNA sequence15 is excised between the pair of loxP sequences.

[0054] FIG. 14 is an explanatory diagram showing the experimental results of homologous recombination in E. coli. As shown in FIG. 14, in E. coli, no fluorescence quenching colonies were observed under the control conditions, as in the case of yeast. On the other hand, regardless of the presence or absence of a spacer sequence (first spacer sequence Spu), under the conditions in which two loxP sequences with different orientations and a pair of homologous sequences outside them were provided and the Cre gene was expressed, the fluorescence of almost all colonies was quenched. However, when the distance between the loxP sequence and the homologous sequence A (the length of the first spacer sequence Spu) became relatively long, the probability of accurate homologous recombination tended to decrease slightly. In E. coli, when a homologous recombination enzyme derived from lambda phage is not used in genome homologous recombination by transformation, homologous recombination does not occur with a relatively short homologous sequence of about 50 bp, and even when a homologous recombination enzyme derived from lambda phage is used, the efficiency of homologous recombination when the homologous sequence length is 50 bp is 10 -3It has been known that the number of copies of the 50-bp homologous sequence is about 10 (Front. Microbiol., 11 September 2020, https: / / doi.org / 10.3389 / fmicb.2020.548410). In contrast, the results shown in Figure 14 show that under all conditions, the cells grown had a number of copies of the 50-bp homologous sequence of 10 -0 ~10 -1 Since homologous recombination occurs with a probability of , it can be said that homologous recombination occurs at a high frequency that cannot be predicted by conventional techniques.

[0055] As described above, by using the homologous recombination method using the sequence structure shown in Figure 1, high efficiency of homologous recombination was obtained not only in the case of using yeast, but also in organisms in which homologous recombination is difficult to occur, such as the above-mentioned case of using a homologous sequence of about 50 bp in length in E. coli. Therefore, the homologous recombination method using the sequence structure shown in Figure 1 is considered to be able to increase the efficiency of homologous recombination as a universal reaction in a wider range of organisms, regardless of whether the recombination target is a prokaryote or a eukaryote, and regardless of the original homologous recombination efficiency of the host.

[0056] The present disclosure is not limited to the above-mentioned embodiments, etc., and can be realized in various configurations without departing from the spirit of the present disclosure. For example, the technical features in the embodiments corresponding to the technical features in each form described in the Summary of the Invention column can be appropriately replaced or combined in order to solve some or all of the above-mentioned problems or to achieve some or all of the above-mentioned effects. Furthermore, if a technical feature is not described as essential in this specification, it can be appropriately deleted.

[0057] The present disclosure can also be realized in the following forms. [Application example 1] A method of homologous recombination for deleting a specific DNA sequence from a DNA molecule, comprising: As the DNA molecule, a pair of target sequences targeted by a site-specific recombinase are arranged in different orientations with the specific DNA sequence sandwiched between them, and a pair of homologous sequences, which are DNA sequences having homology, are arranged outside the pair of target sequences arranged with the specific DNA sequence sandwiched between them, The site-specific recombinase is allowed to act on the DNA molecule, and the specific DNA sequence is deleted from the DNA molecule together with the pair of target sequences by homologous recombination between the pair of homologous sequences. Homologous recombination methods. [Application example 2] The method for homologous recombination according to Application Example 1, The site-specific recombinase is an enzyme belonging to the integrase family, and proceeds with a reversible reaction as a recombination reaction in the target sequence. Homologous recombination methods. [Application example 3] The method for homologous recombination according to Application Example 2, The site-specific recombinase is an enzyme belonging to the bidirectional tyrosine recombinase subfamily. Homologous recombination methods. [Application example 4] The method for homologous recombination according to Application Example 3, The site-specific recombinase is an enzyme selected from Cre recombinase, FLP recombinase, R recombinase, and Dre recombinase. Homologous recombination methods. [Application example 5] The method for homologous recombination according to any one of Application Examples 1 to 4, As cells for carrying out homologous recombination, budding yeast (Saccharomyces cerevisiae) or Escherichia coli (Escherichia coli) are used. The distance between the target sequence and the homologous sequence adjacent to each other on the upstream or downstream side of the specific DNA sequence is 1000 bp or less. Homologous recombination methods. [Application Example 6] The method for homologous recombination according to any one of Application Examples 1 to 5, The homologous sequence is a sequence of 60 bp or less in length. Homologous recombination methods. [Application Example 7] The method for homologous recombination according to any one of Application Examples 1 to 6, The homologous sequence is a sequence having a length of 25 bp or more. Homologous recombination methods. [Explanation of symbols]

[0058] 10...DNA molecule 12a, 12b...Target sequence 14…Homologous sequence 15...Specific DNA sequence

Claims

1. A method of homologous recombination for deleting a specific DNA sequence from a DNA molecule, comprising: As the DNA molecule, a pair of target sequences targeted by a site-specific recombinase are arranged in different orientations with the specific DNA sequence sandwiched between them, and a pair of homologous sequences, which are DNA sequences having homology, are arranged outside the pair of target sequences arranged with the specific DNA sequence sandwiched between them, The site-specific recombinase is allowed to act on the DNA molecule, and the specific DNA sequence is deleted from the DNA molecule together with the pair of target sequences by homologous recombination between the pair of homologous sequences. Homologous recombination methods.

2. The method for homologous recombination according to claim 1, The site-specific recombinase is an enzyme belonging to the integrase family, and proceeds with a reversible reaction as a recombination reaction in the target sequence. Homologous recombination methods.

3. The method for homologous recombination according to claim 2, The site-specific recombinase is an enzyme belonging to the bidirectional tyrosine recombinase subfamily. Homologous recombination methods.

4. The method for homologous recombination according to claim 3, The site-specific recombinase is an enzyme selected from Cre recombinase, FLP recombinase, R recombinase, and Dre recombinase. Homologous recombination methods.

5. The method for homologous recombination according to claim 2, As cells for carrying out homologous recombination, budding yeast (Saccharomyces cerevisiae) or Escherichia coli (Escherichia coli) are used. The distance between the target sequence and the homologous sequence adjacent to each other on the upstream or downstream side of the specific DNA sequence is 1000 bp or less. Homologous recombination methods.

6. The method for homologous recombination according to claim 2, The homologous sequence is a sequence having a length of 60 bp or less. Homologous recombination methods.

7. The method for homologous recombination according to claim 2 or 6, The homologous sequence is a sequence having a length of 25 bp or more. Homologous recombination methods.

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