Homologous recombination method

Abstract

In a homologous recombination method in which a specific DNA sequence is deleted from a DNA molecule, the DNA molecule is used as the DNA molecule. A DNA molecule is prepared in which a pair of target sequences, which are targets for a site-specific recombinase, are disposed so as to sandwich a specific DNA sequence therebetween and so as to have different orientations from each other, and a pair of homologous sequences, which are DNA sequences having homology, are disposed on the outside of the pair of target sequences disposed so as to sandwich the specific DNA sequence. The site-specific recombinase is caused to act on a DNA molecule, and a specific DNA sequence and a pair of target sequences are deleted from the DNA molecule by homologous recombination between a pair of homologous sequences.

Description

Technical Field

[0001] The present invention relates to a homologous recombination method. Background Art

[0002] Hitherto, various techniques have been known regarding recombination in DNA molecules. For example, it is known that homologous recombination methods that utilize recombination occurring between highly homologous base sequences can be used in a wide range of biological species from prokaryotes to animals (for example, refer to 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 (for example, refer to Non-Patent Documents 5 and 6). In addition, as another method for deleting a specific DNA sequence, a method is known in which the sequence to be deleted is previously arranged between transposon-specific inverted terminal sequences and the above-mentioned sequence to be deleted is removed together with the inverted terminal sequences using a transposase (for example, refer to Non-Patent Document 7).

[0003] Figure 15 It is an explanatory diagram showing a case where homologous recombination occurs between a pair of homologous sequences located on the same DNA molecule as an example of homologous recombination, and the marker gene between the homologous sequences is deleted. Figure 16 It is an explanatory diagram showing a homologous recombination method using a genome editing technique as another example of homologous recombination. In this method, the DNA near the homologous recombination target sequence (homologous sequence) is cut using a genome editing technique (for example, ZFN, TALEN, CRISPR-Cas, etc.), thereby activating homologous recombination repair and improving the efficiency of target sequence-mediated homologous recombination. Figure 17 It is an explanatory diagram showing a site-specific recombination reaction. Figure 17 In this, as an example, a site-specific recombination reaction in a Cre-loxP recombination system using a Cre recombinase and a loxP sequence is shown. Such a site-specific recombination reaction can, for example, remove a DNA sequence between target sequences such as a loxP sequence with high efficiency to the extent that screening using a marker gene is not required ( Figure 17 a DNA sequence containing a marker gene). Figure 18 It is an explanatory diagram showing an example of a reaction using a transposon excision enzyme. Figure 18 In this, as an example, an excision reaction using the transposon PiggyBac is shown. In such a reaction, a DNA sequence between specific inverted terminal sequences (repetitive sequences) that can be recognized by an excision enzyme (transposase) can be deleted ( Figure 18 a DNA sequence containing a marker gene), and Figure 17 different from the site-specific recombination reaction shown in

[0004] Prior Art Documents

[0005] Non-patent literature

[0006] Non-patent literature 1: Francesca Storici et al., Proc. Natl. Acad. Sci. USA, 100, 14994-14999(2003)

[0007] Non-patent literature 2: Justin M. Vento et al., J. Ind. Microbiol. Biot., 46, 1327-1341(2019)

[0008] Non-patent literature 3: Dana Carroll, Annual Review of Biochemistry, 83, 409-439(2014)

[0009] Non-patent literature 4: Hiroshi Ochiai et al., Experimental Medicine, 31, 95-100(2013)

[0010] Non-patent literature 5: Yueju Wang et al., Plant Cell Rep., 30, 267-285(2011)

[0011] Non-patent literature 6: Xueying Tian et al., J. Biol. Chem., 296, 100509(2021)

[0012] Non-patent literature 7: Sheng Ding et al., Cell, 122, 473-483(2005) Summary of the invention

[0013] Problems to be solved by the invention

[0014] However, for example, the homologous recombination technique using the homologous recombination repair mechanism without DNA cleavage as shown requires the use of limited biological species such as yeast with high homologous recombination repair activity, chicken DT40 cells, and mouse ES cells. And even in the case of Saccharomyces cerevisiae with particularly good efficiency, as described in Non-patent literature 1, the recombination frequency is about 10 Figure 15 , which is low. In order to obtain a homologous recombinant strain, screening using a marker gene is required. In addition, in -6 , lower, and in order to obtain a homologous recombinant strain, screening using a marker gene is required. In addition, in Figure 16In the homologous recombination method using genome editing technology as described above, since non-homologous end joining takes precedence over homologous recombination repair in many organisms and the cleavage sites bind directly to each other and are repaired, the frequency of the desired recombination reaction becomes insufficient, and in many cases, screening using a marker gene is required. In addition, in the homologous recombination method using genome editing technology, there are problems such as a series of operations such as the production of vectors and the induction of DNA cleavage being complicated.

[0015] In addition, Figure 17 In the site-specific recombination reaction using a site-specific recombinase as described above, the target sequence is determined by the site-specific recombinase used, so the target sequence cannot be freely designed. In addition, there is a problem that the target sequence remains in the DNA molecule from which the above DNA sequence has been removed by recombination. When the recombined DNA molecule is again subjected to the same site-specific recombination reaction, the remaining target sequence may cause an undesired recombination reaction.

[0016] In addition, Figure 18 In the reaction using the excision enzyme of a transposon as described above, there is a problem that the excision position of the DNA sequence is limited to a specific target sequence on the genome ( Figure 18 in the example of TTAA for these 4 bases). Therefore, there is a need for a recombination technique that can recombine with higher efficiency, has no restriction on the target DNA sequence, does not leave a trace sequence in the molecule from which the DNA sequence has been removed, and can be carried out with simpler operations.

[0017] Method for solving the problem

[0018] The present invention can be achieved in the following manner.

[0019] (1) According to one aspect of the present invention, there is provided a homologous recombination method for deleting a specific DNA sequence from a DNA molecule. According to this homologous recombination method, as the above DNA molecule, a pair of target sequences that are targets of a site-specific recombinase are arranged so that the specific DNA sequence is sandwiched therebetween and has different orientations from each other, and a pair of homologous sequences as DNA sequences having homology are arranged on the outer sides of the pair of target sequences sandwiching the specific DNA sequence. The site-specific recombinase is allowed to act on the DNA molecule, and the specific DNA sequence together with the pair of target sequences is deleted from the DNA molecule by homologous recombination between the pair of homologous sequences.

[0020] According to the homologous recombination method of this method, by using site-specific recombination reaction with a site-specific recombinase having relatively high recombination efficiency, the frequency of homologous recombination, that is, the recombination efficiency, can be increased. Such a recombination reaction is to perform homologous recombination, so it is possible to suppress the restriction of the DNA sequence that is the target of the recombination reaction. In addition, with the deletion of a specific DNA sequence, it is also possible to remove the target sequence arranged closer to the specific DNA sequence side compared with the homologous sequence, so it is possible to suppress the residual sequence that becomes a trace in the molecule from which the DNA sequence has been removed. Furthermore, the characteristic sequence structure having the specific DNA sequence, the target sequence, and the homologous sequence can be obtained through a relatively simple process using conventional gene recombination techniques.

[0021] (2) In the homologous recombination method of the above method, the site-specific recombinase can be an enzyme belonging to the integrase family, and as the recombination reaction in the above target sequence, a reversible reaction can be carried out. If such a configuration is adopted, the effect of increasing the frequency of homologous recombination can be enhanced.

[0022] (3) In the homologous recombination method of the above method, the site-specific recombinase can be an enzyme belonging to the bidirectional tyrosine recombinase subfamily. If such a configuration is adopted, the effect of increasing the frequency of homologous recombination can be enhanced.

[0023] (4) In the homologous recombination method of the above method, the site-specific recombinase can be an enzyme selected from Cre recombinase, FLP recombinase, R recombinase, and Dre recombinase. If such a configuration is adopted, the effect of increasing the frequency of homologous recombination can be enhanced.

[0024] (5) In the homologous recombination method of the above method, as the cell for performing homologous recombination, Saccharomyces cerevisiae or Escherichia coli can be used, and the distance between the above target sequence and the above homologous sequence arranged adjacent to each other on the upstream side or downstream side of the above specific DNA sequence can be 1000 bp or less. If such a configuration is adopted, by setting the specific DNA sequence, a pair of target sequences facing different directions, and a pair of homologous sequences as described above, the effect of increasing the recombination efficiency can be ensured by adjusting the above distance.

[0025] (6) In the homologous recombination method of the above method, as the above homologous sequence, a sequence with a length of 60 bp or less can be used. If such a configuration is adopted, the homologous recombination efficiency can be increased even in the case of using short homologous sequences that were previously difficult to perform homologous recombination.

[0026] (7) In the homologous recombination method of the above-described manner, as the above-described homologous sequence, a sequence having a length of 25 bp or more can be used. With such a configuration, by setting a specific DNA sequence, a pair of target sequences having different orientations, and a pair of homologous sequences as described above, the effect of ensuring an improved recombination efficiency can be achieved by adjusting the length of the above-described homologous sequence.

[0027] The present invention can be implemented by various means other than the above, for example, by a vector for homologous recombination, a recombinant obtained by homologous recombination, or the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is an explanatory diagram schematically showing the sequence structure of DNA of the homologous recombination method of the present embodiment.

[0029] Figure 2 It is an explanatory diagram showing an example of homologous recombination based on the homologous recombination method of the present embodiment.

[0030] Figure 3 It is an explanatory diagram showing a target vector for zero level.

[0031] Figure 4 It is an explanatory diagram showing a target vector for first level.

[0032] Figure 5 It is an explanatory diagram showing a target vector for second level.

[0033] Figure 6 It is a diagram showing the Figure 1 sequence structure being introduced into the yeast genome.

[0034] Figure 7 It is an explanatory diagram showing the case where the sequence structure of the comparative example is introduced into the yeast genome.

[0035] Figure 8 It is a diagram showing the Figure 1 sequence structure being introduced into the yeast genome.

[0036] Figure 9 It is an explanatory diagram showing the respective configurations of the first-level modules for introducing into the yeast genome.

[0037] Figure 10 It is an explanatory diagram showing the configuration of the V3P vector group introduced into the yeast genome.

[0038] Figure 11 It is an explanatory diagram showing a second-level vector for Escherichia coli and for evaluating homologous recombination efficiency.

[0039] Figure 12It is an explanatory diagram showing the content of the secondary vector introduced into Escherichia coli.

[0040] Figure 13 It is an explanatory diagram showing the experimental results of homologous recombination in yeast.

[0041] Figure 14 It is an explanatory diagram showing the experimental results of homologous recombination in Escherichia coli.

[0042] Figure 15 It is an explanatory diagram showing the situation of homologous recombination between homologous sequences.

[0043] Figure 16 It is an explanatory diagram showing a homologous recombination method using genome editing technology.

[0044] Figure 17 It is an explanatory diagram showing a site-specific recombination reaction.

[0045] Figure 18 It is an explanatory diagram showing the reaction of the excision enzyme using a transposon. Detailed implementation

[0046] A. First implementation:

[0047] Figure 1 It is an explanatory diagram schematically showing the sequence structure of the DNA of the homologous recombination method of this implementation. The homologous recombination method of this implementation is a method of deleting a specific DNA sequence from a DNA molecule by combining site-specific recombination and homologous recombination. As Figure 1 shown, in this implementation, in the DNA molecule 10 that is the object of homologous recombination, a pair of target sequences 12a, 12b that are the targets of the site-specific recombinase (sequence-specific recombinase) are arranged in such a way that a specific DNA sequence 15 to be deleted is sandwiched therebetween and they have different orientations from each other. And, a pair of homologous sequences 14 that are DNA sequences having homology are arranged on the outer sides of the pair of target sequences 12a, 12b that sandwich the specific DNA sequence 15 and are arranged in reverse. In this implementation, by allowing the above site-specific recombinase to act on such a DNA molecule 10, homologous recombination between a pair of homologous sequences 14 is carried out, whereby the specific DNA sequence 15 together with the pair of target sequences 12a, 12b is deleted from the DNA molecule 10.

[0048] The target sequences 12a, 12b can be appropriately set according to the site-specific recombinase (recombinase) used. The site-specific recombinase used here is preferably set as a recombinase that performs a reversible reaction as the recombination reaction occurring between the pair of target sequences 12a, 12b. Here, "performing a reversible reaction" means a recombination reaction that can be restored to the state before the recombination reaction after the recombination reaction.

[0049] 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). They are classified according to whether they have a tyrosine residue or a serine residue at the active site within 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 its mechanism of action. In addition, the serine recombinase family is classified into a large serine recombinase subfamily (hereinafter referred to as the "large serine subfamily") and a small serine recombinase family (hereinafter referred to as the "small serine subfamily") based on its size.

[0050] As a combination of the bidirectional tyrosine subfamily and a target sequence, for example, the Cre-loxP recombination system using the Cre recombinase and the loxP sequence, the FLP-FRT recombination system using the FLP recombinase and the FRT sequence, the R-RS recombination system using the R recombinase and the RS sequence, and the Dre-rox recombination system using the Dre recombinase and the rox sequence can be cited. Such genetic crossing mediated by the bidirectional tyrosine subfamily occurs between two identical target sequences (recognition sites). Since the recognition sites are the same, the recombination reaction is completely reversible.

[0051] As the unidirectional tyrosine subfamily, for example, λ recombinase, HK101 recombinase, and pSAM2 recombinase can be cited. In addition, as the large serine subfamily, for example, PhiC31 recombinase, TP901-1 recombinase, Bxb1 recombinase, and R4 recombinase can be cited. These unidirectional tyrosine subfamily and large serine subfamily act on two different recognition sites known as the attB and attP sequences to generate hybrid recombination sites known as attL and attR (ACS Synth. Biol. 7, 299-310 (2018)). Thus, the sequences attB and attP as the recognition sites are changed to the sequences attL and attR. Therefore, the reverse reaction does not occur in the case of only the recombinase, but by adding RDF (recombination directionality factor), the directionality of recombination is switched, and a reversible reaction can be carried out. At this time, by adjusting the concentrations of the recombinase and RDF, the reaction rates in each direction can be adjusted (Nucleic Acids Res. 44(15), 7360-7372 (2016)), and a reversible reaction can be carried out continuously.

[0052] As described above, as the site-specific recombinase that sustains the reversible reaction, a recombinase belonging to the integrase family can be preferably used. In particular, when using the bidirectional tyrosine subfamily, the reversible reaction can be more easily sustained through a simple reaction system without using further components such as RDF. It should be noted that the direction of the reaction of the small serine subfamily described above is unidirectional, and the method for reversing the reaction is still unknown. Therefore, in the present embodiment, this enzyme is not used for the reversible reaction.

[0053] The degree of identity between a pair of homologous sequences 14 is not particularly limited as long as homologous recombination can occur. Whether it is a degree of identity that allows homologous recombination is affected by factors such as the length of the homologous sequences. The degree of identity can be set to, for example, at least 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and most preferably 100%. The length of the homologous sequences 14 is not particularly limited as long as homologous recombination can occur. From the perspective of suppressing unwanted recombination reactions, the length of the homologous sequences 14 can be set to, for example, 20 bp or more, desirably 30 bp or more, and more desirably 40 bp or more. From the perspective of improving the homologous recombination efficiency in the homologous sequences 14, the length of the homologous sequences 14 is desirably 25 bp or more, and more desirably 30 bp or more. In addition, from the perspective of easily preparing vectors with a desired sequence structure, the length of the homologous sequences 14 can be set to, for example, 500 bp or less, desirably 400 bp or less, and more desirably 300 bp or less. Generally, if the homologous sequences 14 are too short, homologous recombination becomes difficult to perform and the recombination efficiency decreases. However, in the homologous recombination method of the present embodiment, even if the length of the homologous sequences 14 is set to, for example, 60 bp or less, and further 50 bp or less, high homologous recombination efficiency can be achieved.

[0054] As Figure 1 shown, between the 5'-end of the target sequence 12a located upstream (5'-side) of the specific DNA sequence 15 in the target sequences 12a, 12b and the 3'-end of the homologous sequence 14 closer to the target sequence 12a among the pair of homologous sequences 14, a first spacer sequence Spu can be provided. In addition, between the 3'-end of the target sequence 12b located downstream (3'-side) of the specific DNA sequence 15 in the target sequences 12a, 12b and the 5'-end of the homologous sequence 14 closer to the target sequence 12b among the pair of homologous sequences 14, a second spacer sequence Spd can be provided. The lengths of the first spacer sequence Spu and the second spacer sequence Spd, that is, the distances between the target sequences and the homologous sequences adjacent to each other on the upstream or downstream side of the specific DNA sequence 15, can be set to 0 bp or more. The above distances can be set to, for example, 50 bp or more, and can be set to 1500 bp or less.

[0055] For example, in the case of using Cre recombinase and loxP sequences as a combination of recombinase and target sequence and performing homologous recombination in Saccharomyces cerevisiae or Escherichia coli, the above distance can be set to 1500 bp or less, can be set to 1200 bp or less, and is desirably set to 1000 bp or less. Thereby, the lengths of the above-mentioned first spacer sequence Spu and second spacer sequence Spd can be appropriately set according to the site-specific recombinase and its target sequence, the type of cells in which recombination occurs, the locus in the cells where recombination occurs, etc., so that the recombination efficiency based on homologous recombination can be improved.

[0056] Figure 2 This is an example of homologous recombination occurring by the homologous recombination method of the present embodiment, and is an explanatory diagram showing an example of using Cre recombinase and loxP sequences as a combination of recombinase and target sequence. In the same Figure 1 as Figure 2 (A), the site-specific recombinase ( Figure 2 in the case of being Cre recombinase) is allowed to act, causing homologous recombination between homologous sequences, and deleting the specific DNA sequence 15 together with the target sequence (loxP sequence) from the DNA molecule 10 ( Figure 2 (E)). The mechanism of performing such homologous recombination is considered as follows.

[0057] That is, when the Cre recombinase is allowed to act as described above, within the same DNA molecule 10 where the distance between the target sequences ( Figure 2 in this case is the loxP sequence) is relatively close, a reversible recombination reaction occurs between a pair of target sequences, and the inversion of the specific DNA sequence 15 continues. The destabilization of DNA caused by the continuous reversible recombination reaction between the target sequences may activate the DNA repair mechanism based on homologous recombination, and it is considered that homologous recombination can be induced between a pair of homologous sequences arranged close to a pair of target sequences within the DNA molecule 10. In addition, the fact that the distance between the homologous sequences repeatedly approaches itself may also be one of the factors that increase the frequency of homologous recombination. Therefore, it is considered that during the process of continuously performing the reversible recombination reaction between the target sequences, the frequency of homologous recombination increases, that is, homologous recombination occurs at a frequency much higher than the frequency of natural homologous recombination in a state where no reversible reaction occurs between the target sequences. Figure 2 The example of Figure 2 (A) to Figure 2 (D) shows the situation where the reaction of repeatedly flipping the "specific DNA sequence 15" occurs between the loxP sequences in opposite directions due to the change between the states shown. And Figure 2(A) Different, when a pair of loxP sequences are arranged in the same orientation, a reaction in which a specific DNA sequence 15 is excised by site-specific recombination between the pair of loxP sequences occurs. Such an excision reaction proceeds at a overwhelmingly high frequency compared to the reverse reaction in which the excised specific DNA sequence 15 is integrated into the position before excision in the DNA molecule 10. Therefore, most of the DNA molecules 10 are stable in a state where 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 opposite directions as in the present embodiment, by repeatedly performing the flipping reaction of the specific DNA sequence 15, the DNA-unstable state persists, and the state where the distance between a pair of homologous sequences 14 is close can be maintained for a longer period. Therefore, the homologous recombination efficiency in which the specific DNA sequence 15 between the homologous sequences and the target sequences are excised together can be improved.

[0058] At this time, for example, when the specific DNA sequence 15 contains a marker gene such as drug resistance, cells containing the DNA molecule 10 in which homologous recombination has occurred can be selected using the expression of the marker gene as an index. In addition, when the specific DNA sequence 15 contains a gene for a site-specific recombinase, during the period when a reversible recombination reaction continuously occurs between the target sequences, when the gene for the site-specific recombinase becomes in an expressible state, the site-specific recombinase is generated, and the site-specific recombination reaction can be continuously performed. And after homologous recombination occurs, the specific DNA sequence 15 containing the gene for the site-specific recombinase is deleted, and the influence of the gene for the unnecessary enzyme can be suppressed.

[0059] 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 and 12b are arranged so as to sandwich a specific DNA sequence 15 and have different orientations from each other, and a pair of homologous sequences 14 are arranged outside the pair of target sequences 12a and 12b that sandwich the specific DNA sequence 15. And a site-specific recombinase is allowed to act on the DNA molecule 10, and the specific DNA sequence 15 is deleted from the DNA molecule 10 by homologous recombination between the pair of homologous sequences 14. Generally, compared to the homologous recombination that occurs between the homologous sequences 14, the frequency of the recombination reaction of the site-specific recombination that occurs between the target sequences 12a and 12b is extremely high. When a site-specific recombination reaction occurs between the target sequences 12a and 12b within the same molecule, as in Figure 2 (B) and Figure 2As shown in (C), in the same DNA molecule 10, the distance between a pair of homologous sequences 14 disposed outside the target sequences 12a and 12b is shortened. As a result, the frequency of homologous recombination between the homologous sequences 14 is increased, and it becomes easy to delete a specific DNA sequence 15 from the DNA molecule 10 by homologous recombination. In particular, by using an enzyme belonging to the integrase family and performing a reversible reaction as the enzyme for the recombination reaction in the target sequences 12a and 12b as a site-specific recombinase, the distance between the pair of homologous sequences 14 can be shortened, and the effect of increasing the frequency of homologous recombination can be enhanced.

[0060] In addition, according to the present embodiment, by utilizing the reaction of the site-specific recombinase and performing homologous recombination between a pair of homologous sequences 14 disposed outside the target sequences 12a and 12b to delete the specific DNA sequence 15, the target sequences 12a and 12b can be deleted from the DNA molecule 10 together with the specific DNA sequence 15. Thus, the target sequences 12a and 12b do not remain in the DNA molecule 10 after the deletion of the specific DNA sequence 15. Therefore, even when the same site-specific recombination technique is applied to the DNA molecule 10 after the deletion of the specific DNA sequence 15, an unwanted recombination reaction due to the remaining target sequences can be suppressed.

[0061] Furthermore, since the excision of the specific DNA sequence 15 is performed by homologous recombination, the degree of freedom in designing the sequences (homologous sequences 14) for excision can be ensured, unlike the case of excising the specific DNA sequence 15 by site-specific recombination using a site-specific recombinase. In addition, according to the homologous recombination method of the present embodiment, an operation (e.g., a series of operations such as the production of a vector and the induction of DNA cleavage) for obtaining a sequence structure in which genes are stacked as shown in Figure 1 can be performed by a relatively simple process using conventional genetic recombination techniques. As a method for gene stacking for obtaining the sequence structure shown in Figure 1 , various known methods capable of connecting a plurality of DNA fragments regardless of the shape of the DNA ends can be adopted. Specifically, the cloning process for obtaining the sequence structure shown in Figure 1 can be performed, for example, by a classical method using restriction enzymes, the Golden gate method, the Gibson Assembly system (Nature Methods 6 (5), 343-345 (2009)), or a method using the NEBuilder HiFi DNA Assembly Cloning Kit (manufactured by NEB) and the In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.) (IN-FUSION is a registered trademark). Therefore, as described above Figures 15 to 18Different from the previously known DNA recombination methods shown, it is possible to achieve recombination with higher efficiency, suppress the restriction on the DNA sequence that is the target of the recombination reaction, not leave a trace sequence in the molecule from which the DNA sequence has been removed, and perform DNA recombination with simpler operations.

[0062] Furthermore, according to the homologous recombination method of the present embodiment, high recombination efficiency can also be achieved in systems using a wider range of cells including prokaryotes, eukaryotes, and animal cultured cells, as well as in vitro systems. For example, in previously known homologous recombination, the recombination efficiency varies significantly depending on the biological species and cell type. Even in Saccharomyces cerevisiae, which is considered to have a relatively high homologous recombination efficiency, as described in Non-Patent Document 1, the recombination frequency is about 10 -6 . In addition, as the most efficient genomic homologous recombination method in Escherichia coli, a method of performing transformation while introducing / inducing a homologous recombinase from λ phage is known. However, in this case, the efficiency is 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 recombinase from λ phage in Escherichia coli, the cells and enzymes to be recombined do not need to be limited to a specific combination, and homologous recombination can be performed with a higher efficiency of, for example, about 10 -0 to about 10 -2 by using a conventional site-specific recombinase that can be applied to a wider range of cell types, etc.

[0063] As described above, in the homologous recombination method of the present embodiment, various site-specific recombinases can be used for homologous recombination in the homologous sequence 14. In particular, a system using the Cre recombinase, which is widely used in various biological species including in vivo systems and is known for its broad reactivity, is highly desirable. If a system using the Cre recombinase is utilized, the homologous recombination efficiency can be improved for a wide range of biological species, whether prokaryotes or eukaryotes, and whether animal cells or plant cells, etc. In addition, in addition to the system using the Cre recombinase, for example, systems using FLP recombinase, R recombinase, and Dre recombinase, which also belong to the bidirectional tyrosine subfamily like the Cre recombinase, can also be widely used for various biological species. It is also known that genes of various recombinases such as Dre, VCre, SCre, Vika, and Nigri among the recombinases belonging to the bidirectional tyrosine subfamily are homologous genes of the Cre recombinase gene. Although the target sequences are different, their action mechanisms are basically the same as those of the Cre recombinase. When using a recombinase obtained from a homologous gene of the Cre recombinase gene like this, similar to when using the Cre recombinase, the homologous recombination efficiency can be effectively improved in a wide range of biological species, which is thus preferred.

[0064] Examples

[0065] Hereinafter, the present invention will be described more specifically by way of examples, but the present invention is not limited to the descriptions of these examples. In the examples described below, vectors having the Figure 1 shown sequence structure were prepared, and the sequence structure in the vector was integrated into the genome of the haploid of laboratory yeast, Saccharomyces cerevisiae BY4742 strain, or introduced into Escherichia coli DH5α strain or BL21(DE3) strain in the form of a plasmid, thereby investigating the homologous recombination efficiency in both eukaryotes and prokaryotes.

[0066] <Production of cloning vector set>

[0067] All the vectors used in the experiments were constructed by the Golden Gate method (PLoS ONE 6, e16765 (2011)). The Golden Gate method is a well-known gene stacking method that uses IIS-type restriction enzymes and T4 DNA ligase to insert multiple DNA fragments into a vector in a pre-designed order. As the subcloning vectors required for the Golden Gate method, vectors corresponding to the zero-level, first-level, and second-level target vectors were prepared. The production methods of the zero-level, first-level, and second-level target vectors will be described in turn below.

[0068] Figure 3 is an explanatory diagram showing the zero-level target vector. The zero-level target vector is as Figure 3Shown is a vector containing a BsaI recognition site. When producing the target vector for level 0, the target DNA fragment is amplified by PCR using pUC19 and synthetic DNA as templates. Regarding the primers used in PCR, artificial sequences are synthesized with DNA sequences added in a manner that repeats approximately 15 bp with the adjacent DNA sequence for binding each DNA fragment and used. The amplified PCR fragments are sequentially ligated using the In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.) (IN-FUSION is a registered trademark), thereby completing the target vector for level 0.

[0069] Figure 4 is an explanatory diagram showing the target vector for level 1. As Figure 4 shown, the target vector for level 1 is a group of multiple vectors, and each vector has the zFP538 gene of the fluorescent protein (Nat Biotechnol. 17, 969-973 (1999)) and BsaI recognition sites arranged in opposite directions sandwiching this gene. In addition, the presence, combination, and arrangement of the loxP sequences, sequences 1 to 4 described below, and the restriction enzyme sites and cleavage sites used in the Golden Gate reaction are different for each of the target vectors for level 1. These target vectors for level 1 are produced by amplifying the target DNA fragment using pUC19 and synthetic DNA (including the kanamycin resistance gene) as templates and sequentially ligating 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 in order to ligate the amplified fragments obtained by PCR in the desired order. Sequences 1 to 4 can be any sequences as long as the similarity of their sequences to each other is sufficiently low. Here, artificial sequences with low similarity to the yeast genome sequence are set, and sequences 1 and 2 use the sequences described in Microb Cell Fact 12, 47 (2013), and sequences 3 and 4 use the sequences described in Nucleic Acids Res. 43, 6620-6630 (2015).

[0070] Figure 5 is an explanatory diagram showing the target vector P1301 for level 2. The target vector for level 2 is a vector for research in Escherichia coli produced by repeating the Golden Gate reaction twice to produce a vector, and is produced by the same method as the above-mentioned target vectors for level 0 and level 1.

[0071] <Production of yeast vector group>

[0072] Figure 6 is an explanatory diagram showing the use of homologous recombination of the level 1 module produced using the above-mentioned target vector for level 0 and target vector for level 1 to Figure 1Explanatory diagram of the case where the shown sequence structure is introduced into the ECM38 locus of the yeast genome. Figure 7 It is an explanatory diagram showing the case where the sequence structure of the comparative example is similarly introduced into the yeast genome. Here, as target sequences 12a and 12b, loxP sequences are used, and the homologous sequence 14 is represented as homologous sequence A. As Figure 6 and Figure 7 show, homologous sequence A is set as the sequence in the range of 20 bp, 30 bp, or 50 bp starting from the 3'-terminal side of the region (CDS) encoding the ECM38 protein in the yeast genomic DNA to be introduced. As 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 is used (refer to Figure 9 , Figure 10 , Figure 13 described later). It should be noted that Figure 7 the sequence structure of the comparative example shown does not have the loxP sequence as the target sequence.

[0073] Figure 8 Similar to Figure 6 , it is an explanatory diagram showing the case where the sequence structure shown in Figure 1 is introduced into the ALD4 locus of the yeast genome. Here, similar to Figure 6 , as target sequences 12a and 12b, loxP sequences are used, and the homologous sequence 14 is represented as homologous sequence AA. As Figure 8 shows, homologous sequence AA is set as the sequence in the range of 50 bp starting from the 3'-terminal side of the region (CDS) encoding the ALD4 protein in the yeast genomic DNA to be introduced. As homologous sequence AA, a sequence with a length of 50 bp is used (refer to Figure 9 , Figure 10 , Figure 13 described later).

[0074] As Figures 6 to 8 shows, it is designed as follows: all the vectors for yeast are mixed and transformed in a group of three fragments, so that recombination occurs between the homologous regions (any one of sequences 1 to 4) of each fragment, and finally one fragment is formed and introduced into the yeast genome. Figures 6 to 8 In

[0075] Figure 9 , each fragment constituting the group of three fragments for obtaining one fragment to be introduced into the yeast genome is represented as primary module A, primary module B, and primary module C in the order from the 5'-side after binding. Figures 6 to 8 It is an explanatory diagram showing the respective constitutions of the primary modules used in the introduction into the yeast genome shown in Figure 9Among them, for each first-level module, the types of first-level target vectors used are summarized (refer to Figure 4 ), the types of zero-level modules, and the composition of zero-level module inserts. It should be noted that Figure 9 also shows the first-level modules used in the evaluation of homologous recombination efficiency in Escherichia coli described later.

[0076] Hereinafter, first, the production method of each first-level module shown in Figure 9 will be described. Here, first, using the genome of Saccharomyces cerevisiae BY4742 strain, the genome of Escherichia coli K12 strain, or synthetic DNA as a template, a sequence of 1080 bp from the beginning of the ORF of ECM38, which is a part of the sequence encoding the ECM38 protein (CDS) for homologous recombination with the genome, i.e., sequence B (refer to Figure 6 ) and the 50-bp sequence A described above are amplified by PCR. For the target DNA fragment, the DNA fragments are successively ligated using the In-Fusion HD Cloning Kit and subcloned into a vector equivalent to the zero-level target vector for the Golden Gate method ( Figure 3 ), and the resulting module is named zero-level module V1P1042. In addition, zero-level modules similar to zero-level module V1P1042 are obtained, that is, four sequences (50 bp, 100 bp, 200 bp, 1000 bp) of the spacer sequence between the loxP sequence and sequence A ( Figure 1 , the first spacer sequence Spu) are added downstream of the 3' side of the insert of V1P1042 to form zero-level modules V1P1087, V1P1245, V1P1246, and V1P1260. Furthermore, zero-level modules similar to zero-level module V1P1246 are obtained, that is, zero-level modules V1P1265 and V1P1263 in which the length of sequence A is changed to 20 bp and 30 bp, and zero-level module V1P1248 in which sequence A is deleted from the insert of V1P1042 (refer to Figure 9 ). It should be noted that as the spacer sequence (the first spacer sequence Spu), a part of the CDS of the araB gene of Escherichia coli K12 strain is used. These zero-level modules are modules for obtaining the first-level module A shown in Figure 6 and Figure 7 .

[0077] Similarly, a DNA fragment of the homologous recombination region containing the 5'-terminal side 804 bp range of the CDS of the ALD4 gene for homologous recombination with the genome, i.e., sequence BB (refer to Figure 8By using the 50-bp sequence AA described above, the resulting module was named the zero-level module V1P1262. Additionally, a zero-level module similar to the zero-level module V1P1262 was obtained, which was the zero-level module V1P1274 of the vector formed by adding a sequence (200 bp) of the first spacer sequence Spu between the loxP sequence and the sequence AA downstream of the 3' side of the insert in V1P1262. These zero-level modules are modules for obtaining the first-level module A shown in Figure 1 . Figure 8

[0078] In addition, a fusion DNA fragment of the G418 resistance gene (G418 marker), the EGFP gene which is a modified form of the GFP gene (Gene, 173, 33 - 38 (1996)), and the Cre gene bound to the promoter of the GAL10 gene induced by galactose was subcloned into the zero-level target vector Figure 3 , and the resulting module was named the zero-level module V1P256 (see Figure 9 ). This is a module for obtaining the first-level module B shown in Figures 6 to 8 .

[0079] Furthermore, a DNA fragment of the homologous recombination region ranging from 301 bp at the 3' end side of the CDS of the ECM38 gene in the yeast genomic DNA to be imported, further 841 bp (the sequence C shown in Figure 6 and Figure 7 ) was subcloned into the zero-level target vector Figure 3 , and the resulting module was named the zero-level module V1P70 (see Figure 9 ). Furthermore, as vectors formed by adding sequences (200 bp, 50 bp) of the second spacer sequence Spd between the loxP sequence and the homologous recombination sequence A upstream of the 5' side of the insert in the zero-level module V1P70, the zero-level modules V1P1261 and V1P1249 were obtained (see Figure 1 Figure 9 Figure 6 ). It should be noted that as the spacer sequence (the second spacer sequence Spd), a partial sequence of the CDS of the araA gene of Escherichia coli K12 strain was used. These zero-level modules are modules for obtaining the first-level module C shown in Figure 6 and Figure 7 .

[0080] Similarly, a DNA fragment of the homologous recombination region ranging from the 3' end side of the CDS of the ALD4 gene in the yeast genomic DNA to be imported, further 849 bp (the sequence CC shown in Figure 8 ) was subcloned into the zero-level target vector Figure 3 ​), name the resulting module as the zero - level module V1P73 (refer to Figure 9 ). This zero - level module is used to obtain Figure 8 the first - level module C shown.

[0081] Make the carriers of each of the above - mentioned zero - level modules react with Figure 9 the corresponding target carriers for the first - level respectively (the restriction enzyme is BsaI) to produce the first - level modules. Combine the 3 types of first - level modules obtained and use them as the V3P vector group, as shown in Figures 6 to 8 for introduction into the yeast genome.

[0082] Figure 10 is an explanatory diagram showing the composition of the V3P vector group introduced into the yeast genome. Figure 10 shows the combination of the 3 types of first - level modules constituting each V3P vector group, the name of the yeast strain into which each V3P vector group has been introduced, the locus of the gene into which Figure 1 the sequence structure has been introduced, the presence or absence of the loxP sequence in each V3P vector group, 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). As shown in Figures 6 to 8 , each V3P vector group has a homologous recombination sequence (sequence B or sequence BB) in its insert that can perform homologous recombination with the 5'-side upstream of the insertion position in the yeast genomic DNA, a homologous recombination sequence (sequence C or sequence CC) that can perform homologous recombination with the 3'-side downstream of the insertion position in the yeast genomic DNA, or a sequence selected from sequences 1 - 4 that can perform homologous recombination between the inserts. When the 3 types of inserts of the V3P plasmid group shown in Figure 10 are transformed into yeast, homologous recombination occurs between the sequences and with the yeast genome, and finally all the fragments are ligated and introduced into the yeast genome in the order shown as "first - level module combination" in Figure 10 .

[0083] <Introduction of the vector into the yeast genome>

[0084] Amplify the inserts from each plasmid of each vector group prepared by PCR, use each amplified fragment to transform the Saccharomyces cerevisiae BY4742 strain, spread it on YPD agar medium containing G418, and purify the grown colonies. Then use PCR to screen the strains that have undergone homologous recombination as expected. It should be noted that the transformation is carried out according to the method of Akada et al. (BioTechniques 28, 854 (2000)). Show the transformants of the yeast prepared in Figure 10 .

[0085] <Induction of homologous recombination in yeast and determination of homologous recombination efficiency>

[0086] The prepared strains were cultured on 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 to induce the expression of the Cre gene. When homologous recombination was considered to occur with the expression of the Cre gene, the marker gene, EGFP gene, and Cre gene sandwiched by the homologous sequences (sequence A or sequence AA) were detached from the genome as a group, and the fluorescence of EGFP was quenched. Therefore, the colonies with fluorescence quenching parts were counted within the grown colonies as candidates for the colonies in which homologous recombination had occurred, and the quenching rate of the colonies was investigated with all the colonies as the denominator. As a control, for the Uz4161 strain, the efficiency was also measured under the condition of YPD medium without inducing the expression of the Cre gene. Furthermore, for some strains, for the colonies with quenching, PCR was performed using primers set in such a way as to sandwich both sides of the genomic integration site, and whether homologous recombination was correctly carried out was evaluated by the size level of the amplified DNA.

[0087] <Preparation of Escherichia coli vectors>

[0088] All the tested vectors were prepared by the two-stage Golden Gate method, and the homologous recombination efficiency was evaluated in Escherichia coli in the form of circular plasmids. The sequences for homologous recombination used sequence A used in yeast, and the vectors were also shared with the vectors for yeast ( Figure 9 ), but some modules were additionally prepared.

[0089] Here, in order to obtain the vectors for homologous recombination, gene sequences containing the spectinomycin resistance gene (SpR marker) and the mRFP1.1 gene (Nat Biotechnol 22, 1567 (2004)) were amplified by PCR using Escherichia coli K12 strain genomic DNA and synthetic DNA as templates, and subcloned into the zero-stage target vector ( Figure 3 ), and the obtained module was named zero-stage module V1P415 (refer to Figure 9 ).

[0090] Next, the vectors of each zero-stage module were subjected to Golden Gate reactions (the restriction enzyme was BsaI) with the Figure 9 described first-stage target vectors respectively to prepare first-stage modules. Each first-stage module was subjected to a second Golden Gate reaction (the restriction enzyme was Eps3I) with the second-stage target vector P1301 ( Figure 5 ), and was completed as the second-stage vector.

[0091] Figure 11 is an explanatory diagram showing the representative constitution of the second-stage vector for evaluating the homologous recombination efficiency prepared as above, which is used as an Escherichia coli vector. In addition, Figure 12It is an explanatory diagram showing the content of each secondary vector introduced into Escherichia coli.

[0092] In addition, an expression vector for the Cre gene was prepared separately from the above-mentioned secondary vector. Here, using Escherichia coli K12 strain genomic DNA and synthetic DNA as templates, a fusion DNA fragment of the tetR gene required for induction with tetracycline and the Cre gene bound to the tetA promoter was amplified by PCR, and subcloned into the zero-level target vector ( Figure 3 ), and the resulting module was named zero-level module V1P408. Then, using this zero-level module V1P408 and the first-level target vector V1P402 ( Figure 4 ), a Golden Gate reaction (restriction enzyme: BsaI) was performed to prepare the first-level module V2P591 (refer to Figure 9 ).

[0093] <Preparation of Strains for Evaluating Homologous Recombination in Escherichia coli>

[0094] When performing a homologous recombination experiment, any one of the secondary vectors shown in Figure 12 and the Cre gene expression vector V2P591 were simultaneously transformed into Escherichia coli DH5α strain or BL21(DE3) strain, and the resulting transformants were used for the homologous recombination experiment. In addition, as a control, strains obtained by transforming only the secondary vector were also evaluated.

[0095] <Induction of Homologous Recombination in Escherichia coli and Measurement of Homologous Recombination Efficiency>

[0096] It is considered that if homologous recombination occurs, the fluorescence of mRFP1.1 is quenched. In the medium in which Escherichia coli obtained by transforming two vectors was grown, most of the transformed colonies had quenched fluorescence, so it was speculated that the Cre gene had been weakly expressed and homologous recombination had occurred. Therefore, the colonies with quenched fluorescence were counted among the grown colonies as candidate colonies that had undergone homologous recombination, and the quenching rate of the colonies was investigated with all the colonies as the denominator. A part of the quenched colonies was isolated and cultured in LB medium supplemented with anhydrous tetracycline to induce the expression of the Cre gene, and then PCR was performed using primers set in such a way as to sandwich both sides of the homologous recombination region, and the homologous recombination efficiency was calculated based on the band size.

[0097] <Evaluation Results>

[0098] Figure 13 It is an explanatory diagram showing the experimental results of homologous recombination in yeast. As shown in Figure 13As shown, in yeast, under control conditions that satisfy any one of the following: "(i) no homologous sequence exists in the part outside a pair of loxP sequences (Uz4167 strain)", "(ii) no loxP sequence (Uz4163 strain)", or "(iii) all components are present, but under the condition of Cre non-expression (Uz4161 strain)", no quenched colonies were observed, and as a result, it was considered that homologous recombination did not occur at all. On the other hand, it was found that, regardless of the presence or absence of a spacer sequence (the first spacer sequence Spu or the second spacer sequence Spd), under the condition of having two loxP sequences with different directions and a pair of homologous sequences outside them and expressing the Cre gene, fluorescence quenching occurred with an efficiency of at least 100 times or more compared to the control. Depending on the conditions, fluorescence quenching also occurred in most cells. According to the results of PCR, it was considered that when the length of the homologous sequence region (length of the homologous sequence) was 30 bp or more, homologous recombination occurred correctly in more than 90% of the colonies where recombination occurred, while when the homologous sequence length was 20 bp, the ratio of correct homologous recombination decreased to about 33%. Therefore, it was considered that the homologous sequence length was desirably set to 25 bp or more. However, the survival rate of the cells grown in the Cre gene induction medium (counted by the number of colonies) was 10 - 100%, so it was considered that under any condition, the grown cells underwent homologous recombination with a probability of 10 -0 ~10 -2 . Therefore, it can be said that Figure 13 in the results shown, including the case where the homologous sequence length was 20 bp, homologous recombination occurred at an extremely high frequency compared to 10 -6 (see Non-Patent Document 1), which is known as the efficiency of genome homologous recombination based on foreign gene introduction in yeast.

[0099] It should be noted that for the Uz4161 strain, five clones that were speculated to have undergone correct recombination based on the DNA size level were sequenced, and it was also confirmed that correct homologous recombination occurred. In addition, it was confirmed that even under conditions with poor recombination efficiency, when the cells with fluorescence quenching were passaged without isolation, most cells showed fluorescence quenching and became a cell population in which homologous recombination occurred. In addition, when a pair of target sequences (loxP sequences) were arranged in the same orientation (cis orientation) (Uz4162 strain), a site-specific recombination reaction occurred in the pair of loxP sequences arranged in the cis orientation, and the proportion of fluorescence quenching colonies reached an extremely high value of 99.9% or more. It is considered that this is because when a site-specific recombination reaction occurs in a pair of loxP sequences arranged in the cis orientation, the DNA molecule (yeast genome) becomes stable in a state where a specific DNA sequence 15 is excised between the pair of loxP sequences.

[0100] Figure 14 is an explanatory diagram showing the experimental results of homologous recombination in Escherichia coli. AsFigure 14 As shown, in the case of Escherichia coli and yeast, no fluorescence-quenched colonies were observed under control conditions. On the other hand, regardless of the presence or absence of the spacer sequence (the first spacer sequence Spu), under the condition of having two loxP sequences with different directions and a pair of homologous sequences outside them and expressing the Cre gene, the fluorescence of almost all colonies was quenched. However, if the length between the loxP sequence and homologous sequence A (the length of the first spacer sequence Spu) is long, there is a tendency for the probability of correct homologous recombination to slightly decrease. It has been previously known that in the case of genomic homologous recombination based on transformation in Escherichia coli, when the homologous recombination enzyme from λ phage is not used, homologous recombination does not occur in the case of a relatively short homologous sequence of about 50 bp, and even when the homologous recombination enzyme from λ phage is used, the homologous recombination efficiency when the homologous sequence length is 50 bp is about 10 -3 (Front. Microbiol., 11 September 2020, https: / / doi.org / 10.3389 / fmicb.2020.548410). In contrast, Figure 14 In the results shown, under any condition, the grown cells underwent homologous recombination with a probability of 10 -0 ~10 -1 in the case of a 50-bp homologous sequence, so it can be said that a high-frequency homologous recombination that could not be conceived in the prior art occurred.

[0101] As described above, by using a homologous recombination method utilizing the Figure 1 sequence structure shown, not only in the case of using yeast, but also in a biological species where homologous recombination is not likely to occur, such as in the case of using a homologous sequence of about 50 bp length in the above-mentioned Escherichia coli, a high homologous recombination efficiency was also obtained. Therefore, it is considered that in the homologous recombination method using the Figure 1 sequence structure shown, regardless of whether the recombination target is a prokaryote or a eukaryote, and regardless of the host's original homologous recombination efficiency, as a reaction with universality in a wider range of biological species, the homologous recombination efficiency can be improved.

[0102] The present invention is not limited to the above-described embodiments and the like, and can be implemented by various configurations without departing from its gist. For example, the technical features in the embodiments corresponding to the technical features in each mode described in the summary of the invention can be appropriately replaced and combined in order to solve part or all of the above problems or to achieve part or all of the above effects. In addition, if the technical feature is not described as an essential feature in this specification, it can be appropriately deleted.

[0103] The present invention can also be implemented in the following manner.

[0104] [Application Example 1]

[0105] A homologous recombination method, which is a homologous recombination method for deleting a specific DNA sequence from a DNA molecule, wherein,

[0106] As the above DNA molecule, a pair of target sequences that are to be targets of a site-specific recombinase are arranged in such a way that the above specific DNA sequence is sandwiched therebetween and they have different orientations, and a pair of homologous sequences that are DNA sequences with homology are arranged on the outermost sides of the pair of above target sequences sandwiching the above specific DNA sequence.

[0107] The above site-specific recombinase is allowed to act on the above DNA molecule, and the above specific DNA sequence together with the above pair of target sequences is deleted from the above DNA molecule by homologous recombination between the above pair of homologous sequences.

[0108] [Application Example 2]

[0109] The homologous recombination method according to Application Example 1, wherein the above site-specific recombinase is an enzyme belonging to the integrase family, and as the recombination reaction in the above target sequence, a reversible reaction is carried out.

[0110] [Application Example 3]

[0111] The homologous recombination method according to Application Example 2, wherein the above site-specific recombinase is an enzyme belonging to the bidirectional tyrosine recombinase subfamily.

[0112] [Application Example 4]

[0113] The homologous recombination method according to Application Example 3, wherein the above site-specific recombinase is an enzyme selected from Cre recombinase, FLP recombinase, R recombinase, and Dre recombinase.

[0114] [Application Example 5]

[0115] The homologous recombination method according to any one of Application Examples 1 to 4, wherein,

[0116] As the cell for carrying out homologous recombination, Saccharomyces cerevisiae or Escherichia coli is used,

[0117] On the upstream side or downstream side of the above specific DNA sequence, the distance between the above adjacent target sequence and the above homologous sequence is 1000 bp or less.

[0118] [Application Example 6]

[0119] The homologous recombination method according to any one of Application Examples 1 to 5, wherein a sequence having a length of 60 bp or less is used as the homologous sequence described above.

[0120] [Application Example 7]

[0121] The homologous recombination method according to any one of Application Examples 1 to 6, wherein a sequence having a length of 25 bp or more is used as the homologous sequence described above.

[0122] Symbol Explanation

[0123] 10…DNA molecule

[0124] 12a, 12b…target sequence

[0125] 14…homologous sequence

[0126] 15…specific DNA sequence

Claims

1. A homologous recombination method, which is a homologous recombination method for deleting a specific DNA sequence from a DNA molecule, wherein, as the DNA molecule, a pair of target sequences that are to be targets of a site-specific recombinase are arranged in such a way that the specific DNA sequence is sandwiched therebetween and they have different orientations, and a pair of homologous sequences that are DNA sequences having homology are arranged on the outer sides of the pair of target sequences sandwiching the specific DNA sequence, the site-specific recombinase is allowed to act on the DNA molecule, and the specific DNA sequence together with the pair of target sequences is deleted from the DNA molecule by homologous recombination between the pair of homologous sequences.

2. The homologous recombination method according to claim 1, wherein, the site-specific recombinase is an enzyme belonging to the integrase family, and a reversible reaction is carried out as the recombination reaction in the target sequence.

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

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

5. The homologous recombination method according to any one of claims 1 to 4, wherein, as the cell for carrying out homologous recombination, Saccharomyces cerevisiae or Escherichia coli is used, and the distance between the adjacent target sequence and the homologous sequence on the upstream side or downstream side of the specific DNA sequence is 1000 bp or less.

6. The homologous recombination method according to any one of claims 1 to 5, wherein, as the homologous sequence, a sequence having a length of 60 bp or less is used.

7. The homologous recombination method according to any one of claims 1 to 6, wherein, as the homologous sequence, a sequence having a length of 25 bp or more is used.

Images