Methods and means for genetically modifying genomes using designer DNA recombinant enzymes

Abstract

To provide methods and means for specifically altering the DNA sequence in a genome, in particular for genome editing by deleting or replacing a sequence of interest.SOLUTION: Disclosed is a method for producing a DNA-recombining enzyme to target sites using two non-identical sequences naturally occurring in a genome as the target sites. The invention is in particular useful for medicine, in particular to repair a mutation in a genome or to delete predefined genetic material derived from cells or tissue and to cure diseases. An advantage of the invention is that it allows precise site directed altering of DNA without engaging host DNA repair pathways and thereby works without inducing random insertions and deletions (indels).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention provides methods and means for specifically modifying DNA sequences in a genome. It provides vectors and methods for producing zyner DNA recombinase. The present invention is particularly useful for pharmaceuticals that repair mutations in the genome, or delete predetermined genetic material derived from cells or tissues, and treat diseases. Furthermore, the present invention is useful for biological research and biomedical research (for example, for creating animal models). and is useful for pharmaceuticals that treat diseases. Furthermore, the present invention is useful for biological research and biomedical research (for example, for creating animal models).

Background Art

[0002] Many genetic mutations that cause human diseases have been identified in the past few decades. Recent breakthroughs in the field of genome editing are now providing a real opportunity to establish innovative approaches to repair DNA damage, replace, manipulate, or regenerate cells that have malfunctioned in vitro or in vivo.

[0003] However, most of the recently developed genome editing techniques, such as zinc finger nucleases (e.g., US20150093802 A1), TALENs (e.g., WO2014134412 A1), and CRISPR / Cas9 (e.g., US 8,697,359 B1), introduce double-stranded DNA cleavage sites at target loci as the first step of gene modification. These cleavage sites are subsequently repaired by one of the cell's endogenous DNA repair mechanisms, typically resulting in the introduction of multiple random insertions and deletions (insertion-deletions (indels)) at the target locus. However, ideally, therapeutic genome editing should be efficient and specific without introducing indels.

[0004] ​​​​​​​​​​​DNA recombinases, particularly site-directed recombinase (SSR) systems, are involved in the endogenous DNA repair process. This enables precise manipulation of DNA without inducing pathways, and allows for the inhibition of the processed DNA. It possesses the unique ability to achieve both cutting and immediate rejoining (resealing) at the point of contact. Furthermore, SSR systems, such as Cre / loxP (EP 0 2200 009 B1), are widely used in model organisms. Applications have been found, and even if these are expressed throughout life in animals, they may be safe. It has been demonstrated that SSR acts as a tetramer on its target sites, and in these sites Depending on the direction, recombination can yield various results. Novel site-specific recombinases can be obtained. It is known that the amino acid sequence of Cre can be modified for this purpose (Buchholz F (and Stewart AF's literature, 2001).

[0005] Other SSR systems known in the art include the Flp / FRT system (WO 1999025841 A1 and US 6,774,279 B2), Dre / rox systems (US 7,422,889 B2 and US 7,915,037 B2), VCre / Vlo xP system, and sCre / SloxP system (WO 2010 / 143606 A1), as well as Vika / vox, Nigri / n There are ox and Panto / pox systems (WO 2014 / 016248 A1).

[0006] Natural DNA recombinases, especially site-directed recombinase (SSR) systems (e.g., Tyro Syn-type SSRs generally consist of four identical monomers. Generally, DNA recombinases consist of two monomers. They are separated by identical and symmetrical spacers, each often 8 nucleotides long. It recognizes a palindromic target site consisting of two 13-nucleotide subregions. Depending on the number and relative orientation of target sites, DNA recombinases can delete, insert, or reverse genetic content. Causes a position change or substitution.

[0007] The bottleneck for using recombinant DNA enzymes in therapeutic genome editing is the recombination process. The number of arrays that can be processed is limited.

[0008] In WO2008083931A1, the recognition site for the HIV long-chain terminal repeat (LTR) sequence is (loxLTR Tre 1 .0). Tailored recombinases (Tre 1.0) were used as the basis. Directed molecular evolution addresses this limitation to some extent. By using asymmetric target sites... Further development of this approach is described in WO2011147590 A2(Tre 3.0) and WO2016034553 A1 (Tre 3.1 and uTre / Brec1) and as described in the publications of Karpinski J et al., 2016 (Brec1) However, for the system to function in all cases, there must be 34 salts in the genome. There are not two exact recognition sites for the length of the base pair (for example, in the LTR of the incorporated HI provirus) Because this was not permitted, the usefulness of this approach was limited.

[0009] WO2009007982A1 is located in the LTR of HIV-1 as a potential target site for DNA recombinase. The nucleic acid sequence is described.

[0010] US2009 / 0217400 A1 describes enzymes and methods for recombination at the asymmetric loxP-M7 target site. It has been described. Further, in the literature of Zhang, C et al., (2015) and US 20170058297 A1, a heterotetrameric complex of two Cre mutants essential for recognizing asymmetric loxP-M7 target sites is described. However, these recognition sites must be artificially introduced into the genome.

[0011] US 20040003435 A1 describes a method for targeted insertion of a target sequence into a plant, comprising introducing an insertion cassette comprising the target nucleotide sequence adjacent to, or comprising, a non-identical recognition site which is a modified FRT site still recognizable by wild-type FLP recombinase.

[0012] In summary, the application of the latest known recombinase systems is limited by the fact that two recognition sites must be artificially introduced into the genome, or the target sequence in the genome must be adjacent to two identical sequences of 34 base pairs in length.

[0013] Therefore, the currently used direct genome editing approaches rely on programmable nucleases, such as RNA-guided nucleases like CRISPR / Cas9 (e.g., US 8,657,359 B1 ) because the specificity of these enzymes to target sites is defined by guide RNAs that can be adapted to target sequences and thus widely used.

Summary of the Invention

[0014] However, these nuclease systems require the cell's endogenous DNA repair mechanism, so typically a large number of random insertions and deletions (indels) are induced at the target locus. ​​​​​​​​​​

[0015] In an ideal genome editing or gene therapy, the missing parts of the genome can be removed without further modification. The defective gene is replaced with a normal locus, or the defective gene is removed from its natural position. To delete. Therefore, the ideal genome editing is efficient and does not introduce insertions or deletions. It should be specific.

[0016] Therefore, the objective of the present invention is to overcome the shortcomings of the nuclease genome editing approach and simultaneously address the following: A method for modifying nucleotide sequences in a genome that is versatile enough to be adapted to a target sequence. It is about providing.

[0017] This invention provides a genome with a length of 34 base pairs (the length of a typical target site for DNA recombinase). This is based on the discovery that two identical sequences are rarely found. [Modes for carrying out the invention]

[0018] The sole limitation of this invention for achieving excision recombination of designer DNA recombinant enzymes is that The present invention provides for the conservation of spacer arrays of a constant length of 8 bp (see, for example, Figure 17). It starts with what those people understand. For recombination to occur, these sequences must be identical. Therefore, restrictions are placed on the sequences that can be rearranged. From bioinformatics analysis of various genomes, including viruses, prokaryotes, and the human genome. It has been revealed that 8 bp long serial repeat sequences appear frequently in eukaryotic genomes. This happened (see, for example, Figures 18 and 19).

[0019] Based on this discovery, the inventors evolved to target different target sites in the genome. By combining different monomers of DNA recombinase, the cell repair pathway is activated. We understood that precise genome editing could be performed without any problems. Therefore, directed progress A combination of monomers of DNA recombinant enzymes produced by chemical, rational design, or a combination thereof. By creating a combination, it is possible to recombinate virtually any target sequence in the genome. When a sequence is deleted due to recombination, two identical sequences, each approximately 8 base pairs long, are lost to the genome. It is necessary as an internal spacer arrangement. However, if the arrangement is replaced by rearrangement... In addition, the spacer sequences in the genome are preferably not identical—in this case, the same spacer sequence The column provides the desired sequence (donor DNA) to be replaced with the target sequence on the artificial sequence.

[0020] This invention combines monomers of different DNA recombinases to create known DNA recombinase enzymes. Overcoming the limitations regarding the target sites to which these original molecules can be recombined, and naturally occurring molecules within the genome Designer DNA recombinant enzymes useful for genome editing that can recognize the target sequence being expressed provide.

[0021] In a first aspect of the present invention, the present invention induces site-directed DNA recombination and introduces ( Preferably, by rearranging two naturally occurring but non-identical target sequences, the geno Designer DNA recombinant enzymes that can modify the nucleotide sequence in DNA, and designer This invention provides a method for preparing a DNA recombinase.

[0022] The designer DNA recombinase of the present invention artificially introduces a target site into the genome. It is not necessary, and furthermore, long chain terminal repeat sequences (LTRs) that are longer than 8 nucleotides (especially 34 It has the advantage of not requiring the presence of identical sequences (that are not nucleotides).

[0023] In the context of this invention, non-identical target sequences are those in which the target site has at least two nucleotides. It means something different. The target site can differ by even more, especially by at least three nucleotides. This is advantageous in that it has the following characteristics. In embodiments of the present invention, the target site has at least 4 nucleotides, Or even 8 nucleotides different. At least 20% or more preferably 30% of the target site % of nucleotides and up to 70% or even up to 90% of nucleotides may differ. This means that the target site may have sequence identity of only 10-80%, preferably 30-90%. This is advantageous in that it means that each target site has a spacer arrangement (preferably 5 to 12 nuclei). A first partial region (preferably 10-20 nucleotides) separated by an ocide and a second It includes a partial site (preferably 10 to 20 nucleotides). One or both of the target sites are This is advantageous because it can be asymmetric, and the parts of the target site are inversely complementary to each other. This means that at least 20% or more preferably 30% of the nucleotypic portion of the part This is advantageous because up to 70% or even up to 90% of the nucleotides can be different, and this This means that all parts may have sequence identity of only 10-80%, preferably 30-90%. This means that, in one embodiment, the difference in target sites is only in the sub-sites, and the space The sequence is identical and induces deletion of the target sequence. In another embodiment, The two spacer sequences are different, and as a result, they will be replaced by the target sequence. A synthetic sequence is proposed that includes two sequences identical to the target sites appearing in the genome bordering the donor sequence. By providing this, it becomes possible to replace the target sequence.

[0024] In a preferred embodiment, the designer DNA recombinant enzyme of the present invention is one or two, Or it contains up to four monomers. In one embodiment, the designer DNA recombinant enzyme contains four monomers It contains different monomers, and each monomer recognizes different sub-parts: however, Inner DNA recombinase contains two or three different monomers, where one or two monomers It can recognize asymmetric target sites. Designer DNA recombinant enzymes can recognize four identical single sites. It contains a mer, where the designer DNA recombinase recognizes two different asymmetric target sites. It is even possible to do so. The different monomers of the present invention are those in which the monomer is at least 2%, and in particular at least This means that 4%, preferably at least 8%, of amino acid residues are different. Up to 10%, or up to 3%. It is advantageous in that even 0% of the amino acid residues can differ, and this means that the monomer is only 70- This means that it may have sequence identity of 98%, preferably 90-96%, or up to 92%.

[0025] Therefore, the DNA recombinant enzyme of the present invention is a homotetramer containing two or up to four different monomers. Alternatively, it is a heterotetramer.

[0026] In a preferred embodiment of the heterotetramer, the monomer-monomer interface is only between different monomers. Genetic modification is performed in a manner that allows for the formation of tetramers (e.g., Zhang et al., publications, 2015 and US 20 (As described in 170058297 A1). This modification removes tetramers containing only one monomer. Essential heterotetramers are formed.

[0027] The target sequence is the nucleotide sequence to be modified. This is a sequence that includes mutations. It is preferable that there be such mutations. The term "mutation" in this invention refers to all kinds of genetic or This refers to chromosomal abnormalities, particularly point mutations, frameshift mutations, translocations, duplications, deletions, or insertions. Includes.

[0028] Alternatively, the activity may cause disease, or the inhibition of gene activity may reduce symptoms. It is publicly known that the target sequence is the sequence to be inactivated, especially that of pathogens (bacteria, viruses). It is a sequence of a microorganism (such as Russ, or a parasite), or an oncogene or another gene. One example is the gene encoding PCSK9 (proprotein-converting enzyme subtilisin / kexin type 9). Therefore, inhibiting this process increases cholesterol levels and low-density lipoprotein particles (LDL). It is known to lower the blood concentration of [the substance].

[0029] If the target sequence is a sequence that you want to insert, duplicate, or inactivate (for example, a viral sequence) In some cases, the insertion is preferably achieved by recombination induced by the DNA recombinant enzyme of the present invention. The deleted or inactivated sequence is removed. This deletion leads to further designer DNA recombination. Also called enzyme-induced gene deletion (DRiGD), this involves two identical genes in two target sites. This is achieved by the spacer arrangement (see also Figure 2 for an exemplary embodiment).

[0030] Alternatively, the target sequence may be recombined by the DNA recombinase induced by the DNA recombinase of the present invention. This is replaced with the desired sequence. This substitution is further induced by designer DNA recombinase. A modified gene substitution (DRiGR), also called a chromosomal replacement, involves the synthesis of a first target site and a second target site. This is achieved by providing a donor sequence, where the first and second target sites are the desired arrangement The column is bordered. By replacing the target sequence with the desired sequence, in principle, dots can be added. Natural mutations, nonsense mutations, frameshift mutations, duplications, and even deletions and insertions It is possible to treat all mutations, including (see Figure 1 for an illustrative example). (See reference).

[0031] The DNA recombinant enzyme of the present invention enables precise genome editing without inducing endogenous DNA repair pathways. This makes it possible to perform both in vivo cleavage and immediate recombination of the processed DNA. It has an advantage in that it possesses unique abilities that it can demonstrate.

[0032] The term DNA recombinase refers to all enzymes capable of inducing site-directed DNA recombination events. Enzymes, preferably recombinases or integrases, particularly topoisomerase-like reactions, are used to carry out the reaction. The enzyme to be used is selected from, for example, the serine or tyrosine recombinase family. In one embodiment, the target sequence is used in step (a), and the molecular sequence is used in step (b). Known DNA recombinases to which tropic evolution methods have been applied include Cre, Dre, Cre, VCre, sCre, FLP, and Tre. (Including Tre 1.0 to Tre 3.1 and Brec1), selected from Vika, Nigri, and Panto.

[0033] The term "designer" DNA recombinant enzyme is used in this invention to design DNA to a given target site. This refers to the fact that recombinant enzymes have been created, and that these DNA recombinant enzymes do not occur naturally. .

[0034] In one embodiment, the present invention induces site-directed DNA recombination of a target sequence in the genome. To identify sequences that are potential target sites for DNA recombinases that can be used to induce DNA recombination. This is the method: i. The genome containing the target sequence for two sequences that are potential spacer sequences or This is a process of screening that part (these potential spacer arrays are at least 5 bp, preferably at least 7 bp, more preferably 8 bp, and preferably up to 12, more preferably The maximum length is 10 bp. One of the potential spacer sequences is upstream of the target sequence. The other potential spacer sequence is located downstream of the sequence in question. The two sequences are favorably arranged. The maximum distance is 100 kb, more preferably 10 kb, and even more preferably 2 kb, and Preferably, a minimum distance of 150 bp is taken. ii. Step to identify (define) potential target sites (potential spacers obtained in step i.) The sequence and the sequence bordering it form a potential target sequence. Each potential (obtained in step i.) Regarding the spacer sequence, the adjacent nucleotides on one side, preferably 10 to 20 nucleotides The rheotide, more preferably 12-15 nucleotides, most preferably 13 nucleotides, is a potential The first partial site forms, and the adjacent nucleotides on the other side, preferably 10 to 20 nucleotides The rheotide, more preferably 12-15 nucleotides, forms a potential second subsite. Therefore, Therefore, both potential sub-regions and the spacer array between them form a potential target site. ) includes, iii. The potential target sites identified in step ii are preferably further screened, It does not appear (anywhere) in the main genome, and therefore sequence-specific recombination, especially deletion, is guaranteed. The present invention relates to a method for selecting a potential target sequence.

[0035] This method involves the latent DNA recombinase (DRiGD) that induces deletion of the target sequence in the genome. It is particularly suitable for screening to identify sequences that will be the target sites. In this case, Step i screens the genome for two identical sequences that could potentially act as spacer sequences. This method is also illustrated by the example in Figure 18.

[0036] In one embodiment, the sequence to be deleted is a sequence in the host genome, for example If it is an oncogene or another gene that you want to inactivate, then in step i, screening is performed. The genome being targeted is the genome of the host itself (or the portion containing the target sequence), for example, the human genome. It is a part of the human chromosome or a part of the human chromosome. In this case, in step iii, preferably the host genome Latent target arrays that do not appear anywhere within (and therefore only appear around the target array) The column is screened to guarantee sequence-specific deletions.

[0037] In another embodiment, the sequence to be deleted is that of a pathogen, such as a virus. This is a sequence in the genome. Here, step i is performed on the genome of the pathogen, and step iii is performed on the genome. This is performed on the genome of the host to which the pathogen infects. In this case, in step iii, preferably or latent genes that do not appear in the host genome (and therefore only appear in the pathogen genome). The target sequence is screened to ensure sequence-specific deletions.

[0038] In a more preferred step iv, the target sequence is determined by the known target site of each DNA recombinase. Step ii. includes a sub-region having homology with the sub-region, the potential target sequence obtained in step iii. Select from the list. Homology refers to the property of having the same base at a certain number of nucleotide positions. This means that. Preferably, each sub-site is a sub-site of a known target site of the DNA recombinase. At least 10%, preferably at least 20%, preferably at least 30%, of identical nucleos It has a cyd position. Furthermore, the parts preferably have homology to each other. Preferably Each sub-part is less than other sub-parts of the same target area and / or sub-parts of other target areas. At least 10%, preferably at least 20%, preferably at least 30%, of identical nucleotides It has a specific location. If there is high homology between the target sub-sites, it will have activity for both sub-sites. This allows for the production of monomers, and therefore has the potential to reduce the number of monomers required for recombination. It increases.

[0039] The selection of step iii. to ensure sequence-specific deletion can be performed before or after step iv. You may do so.

[0040] Preferably, during or before step i, the sequence to be inactivated (preferably, genetic Determine the coordinates of the offspring (or exon) within the genome. The target sequence and border to be searched. Create fragments of 150 bp to 10 kb that extend to the sequence. Remove fragments that overlap with other genes. For all fragments, there is a potentially identical spacer arrangement—and therefore repeated at least once. Identify the spacer sequence.

[0041] In one embodiment, the target sequence (for example, the exon of the gene to be inactivated) Or at least a spacer sequence that borders the overlapping sequence with the target sequence (e.g., exon). This is selected before step ii. Alternatively, the target sequence (for example, the sequence to be inactivated) is selected. Outline the sequence that overlaps with the gene's exon or at least the sequence of interest (e.g., an exon). Potential target sites are identified as described in step ii, and then pairs of target sites are selected.

[0042] In another embodiment, this method induces the substitution of a target sequence in the genome using DNA. It is used to identify sequences that are potential target sites for recombinant enzymes (DRiGR). In step i, the genome was split for two sequences that were potential spacer sequences but were not identical. Clean. In this case, the potential spacer arrangement is preferably at least 30% to 100%. They differ by at least 50% of nucleotides. Therefore, in this case, the potential spacer sequence Preferably, it has 0% to 50% or at least less than 70% sequence identity.

[0043] In a more preferred embodiment, a DNA set that induces substitution of a target sequence in the genome. Sequences that are potential target sites for the reconstitution enzyme (DRiGR): i. A genome or its sequence containing the target sequence for two sequences that are potential target sites. Screening the area (these potential target sites are at least 5 bp, preferably) The bp is at least 7 bp, more preferably 8 bp, and preferably up to 12, more preferably up to Each of the 10-20 nucleotides separated by a spacer sequence that is 10 bp long is preferable. The molecule contains a first sub-region and a second sub-region having 12 to 15 nucleotides. One of the target sites is upstream of the target sequence, and the other of the potential target sites is upstream of the target sequence. It is located downstream of the two sequences, preferably 100 kb, more preferably 10 kb, and even more preferably The maximum distance is 2 kb, and preferably the minimum distance is 150 bp. Therefore, the spacer arrangements should be selected so that they are not identical. The spacer arrangements should be at least 30 Only % to 100%, preferably 50%, of the nucleotides differ. Therefore, the spacer sequence is preferable It has 0% to 50%, or at least less than 70%, sequence identity. ii. The potential target sites identified in step i are preferably further screened to determine the host It does not appear anywhere in the genome, and therefore (using donor sequences, see below) the sequence is unique. Select a potential target sequence in which heteromorphic substitution is guaranteed, and identify it by the method described above.

[0044] In a more preferred step iii, the target sequence is a known target site of the DNA recombinase. Step i. includes a sub-region having homology with the sub-region of the potential target sequence obtained in step ii. Select from the list. Homology refers to the property of having the same base at a certain number of nucleotide positions. This means that. Preferably, each sub-site is a sub-site of a known target site of the DNA recombinase. At least 10%, preferably at least 20%, preferably at least 30%, of identical nucleos It has a chid position.

[0045] The selection of step ii. to ensure sequence-specific substitution can be performed before or after step iii. You may do so.

[0046] Preferably, during or before step i, the sequence to be replaced (preferably, genetic) Determine the coordinates of the offspring (or exon) within the genome. The target sequence and border to be searched. Create fragments of 150 bp to 10 kb that extend to the sequence. Remove fragments that overlap with other genes. For all fragments, identify potential target sites.

[0047] In one embodiment, the target sequence (e.g., the exon of the gene to be substituted) or a small Even if not, a spacer sequence is selected to surround the overlapping sequence with the target sequence (e.g., exon). Choose.

[0048] Site-directed DNA recombination is achieved by rearranging two naturally occurring target sequences in the genome. The present invention provides a designer DNA recombinant yeast capable of inducing and modifying the nucleotide sequence. Methods for preparing the element (designer recombinase-inducing gene substitution or deletion) are: a) The nucleotide sequence upstream of the nucleotide sequence to be modified (the target site) is the first The target site is downstream (preferably 30-40 base pairs) of the nucleotide sequence to be modified. The process of selecting a nucleotide sequence (which is the length) as a second target site (where the target site The sequences are not identical. al) The term "target sequence" is defined as above. One or both of the target sites are asymmetric. Furthermore, it has the advantage that symmetrical regions are rarely found in the genome. Each target region is Separated by a pacer sequence (preferably 5 to 12 nucleotides, as defined earlier) A first sub-part and a second sub-part (preferably 10 to 20 nucleotides, as defined above) It includes. Preferably, the target sequence is in phase with a sub-site of a known target site of the DNA recombinase. Select a region that contains homogeneous parts. Homology refers to a certain number of nucleotide positions. This means that they have the same base. Preferably, each sub-site is known to be a DNA recombinase. A portion of the target site and at least 10%, preferably at least 20%, preferably at least They also have 30% identical nucleotide positions. Furthermore, as explained earlier, the partial sites are preferable. The parts have homology to each other. Preferably, each part is similar to the other parts of the same target area. Parts of the site and / or other target sites and at least 10%, preferably at least 20%, (They have at least 30% identical nucleotide positions.) b) Using nucleic acids containing the first and second target sites selected as substrates in a), DNA At least one library of recombinant enzymes, preferably at least two, preferably up to four. This includes the step of applying molecularly directed evolution methods to one library, (Selected in a1) by molecular-directed evolution or a combination of molecular-directed evolution and rational design. A designer having activity against the first target site and the second target site. —Continue this process until you obtain the DNA recombinant enzyme.

[0049] Designer DNA recombinases are selected to target any pair of target sequences, and their results As a result, any target sequence adjacent to these target sites can be selected. The column contains information about host (e.g., cell or organism) genomes or pathogen genomes such as viruses. This refers to sequences that occur naturally. In the context of this invention, "sequences that occur naturally in the genome" means This means that the target sequence was not artificially introduced, and that both non-identical sequences are the same. It means appearing naturally (in the native language) during a speech.

[0050] The genome is defined as all the genetic material of an organism or pathogen. The genome is generally defined as DNA (or In RNA viruses, it consists of RNA. The genome consists of genes (coding regions or exons), ( Non-coding DNA (such as chemons and regulatory sequences), as well as mitochondria and chloroplasts. It contains the genetic material of [the animal].

[0051] Preferred target sequences include sequences unique to the target genome of the host cell or host organism. Organisms have multiple copies of their chromosomes, and in this context, "unique" means that their sequence is stained This refers to an appearance occurring only once per set of body parts.

[0052] The inventors have found that, using an unexpected DNA recombinase, all sub-regions are connected to one If only 7 to 9 of the 3 positions differ, it is possible to rearrange two different target sites. We found that this is possible (see Example F9-3. In this example, both loxF9a and loxF9b are used). (The elements can be rearranged.)

[0053] Generally, all parts are separated by no more than seven positions, preferably no more than five positions, Preferably, they differ at four positions or less. Using one DNA recombination enzyme library, both We can evolve enzymes that can recombinate target sites, particularly in the DRiGR reaction. (See Figures 15 and 16, and the table on page 63 for the examples.)

[0054] In another case, the sub-parts are up to 7, preferably 5, more preferably 4, in the pair comparison. This indicates a mismatch difference, and therefore both target sites have a maximum of 7, preferably 5, between the sub-sites. It shows two, more preferably four, mismatches. In this case, two libraries of DNA recombinase We use this to evolve the enzyme so that it can recombinate both target sites.

[0055] In further cases, only one of the target sites may have up to 7, preferably 5, more preferably 4 It shows a pair of sub-sites with a mismatch, and therefore the other target site has more than seven, preferably seven. There are only five, more preferably four, mismatches, and the other target site is a sub-site of the other site. When compared to each other, it shows more than 7 mismatches, preferably 5, and more preferably 4. Alternatively, using a library of three or four DNA recombinases, both target sites can be recombined. We will evolve enzymes to enable this.

[0056] Evolve the library(s) of DNA recombinant enzymes used in step b) to create DNA recombinant enzymes Obtain a single monomer, or co-evolve it, to create a DNA recombinase that acts together on a pair of target sites. Obtain at least two monomers. Two or more live DNA recombinases used in step b) In the case of Lari, they are preferably linked by a single expression vector for functional reasons. When using two or more libraries of DNA recombinant enzymes, the libraries are preferably the same. The regulatory elements are expressed, preferably under the control of the same promoter.

[0057] Preferably, one or two to four libraries of DNA recombinant enzymes are co-evolved in step b) It is used in a particularly preferred embodiment, where two to four libraries of DNA recombinant enzymes are used. It is used in process b).

[0058] When using two or more libraries, libraries containing different DNA recombinases are considered standard. They differ in terms of site specificity.

[0059] When using two libraries of DNA recombinase, the DNA recombinase in each library is Different asymmetrical target sites—recognizing a target site composed of two different sub-parts at a specific point. It is advantageous.

[0060] When using four libraries of DNA recombinases, the DNA recombinases in each library are Preferably, each recognizes a different asymmetrical sub-region of the target site. Therefore, they are heterogeneous. The target site is recombined only when co-expressed as a tetramer.

[0061] In a preferred embodiment, the co-evolution in step b) is performed on a library of DNA recombinant enzymes or This includes a library of at least two different DNA recombinant enzymes, and first and second target sites. This is carried out using an expression vector that codes for a substance. In particular, in a preferred embodiment, The expression vector encodes two or four libraries of DNA recombinant enzymes.

[0062] In a preferred embodiment of the vector, the negative selection marker is the first and second target. It is located between the sites. Therefore, the first and second target sites are preferably negative selection markers. It borders. Therefore, if recombination occurs, the sequence with the negative selection marker is cleaved. Therefore, negative selection is not performed. However, if recombination does not occur, negative The selection marker works.

[0063] In a preferred embodiment, the negative selection marker is the recognition site of the restriction enzyme. A unique recognition site refers to the recognition site of a restriction enzyme that appears only once in the vector. By incubation with restriction enzymes, the non-recombinant expression vector can be converted. Linearization occurs. Linearized expression vectors are, for example (illustrated in Figures 4-7) non-linearized. Select a primer pair for PCR that amplifies only plasmids and / or extract linearized DNA. By digesting with sonuclease, negative selection occurs.

[0064] In a preferred embodiment, negative selection includes two or more restriction enzyme recognition sites, Or, two or three (up to five) of the same restriction enzyme recognition sites, especially two to three (or It contains up to five different restriction enzyme recognition sites.

[0065] This significantly enhances the efficiency of linearization of non-recombinant expression vectors. To further enhance the selection of enzymes that induce substitution, exonucleases are preferably added. It digests linearized DNA.

[0066] A substrate nucleic acid comprising a first target site and a second target site (preferably bordered by a negative selection mark) The manufacturer preferably has a library of DNA recombinant enzymes or at least two DNA recombinant enzymes. Place the DNA encoding different libraries into the same vector (as illustrated in Figures 4 and 7). .

[0067] In one embodiment, the method involves introducing the previously defined expression vector into cells and This involves expressing DNA recombinant enzymes, or using a cell-free expression system.

[0068] If you want to induce a deletion using the DNA recombinant enzyme of the present invention (DRiGD), preferably further The DNA substrate or vector is not used in a single recombination reaction. If recombination is successful, negative A selection marker is excised, and the plasmid is amplified—preferably by PCR (Figure 6 and Examples are given in section 7 (pDuoSLiDE-DRiGD, pQuSLiDE-DRiGD). In this case, as previously described, The first and second target sequences contain the same spacer sequence (see also Figure 2).

[0069] The method for identifying sequences that are potential target sites for DNA recombinant enzymes, as described above, is preferably In step a) of a method for preparing designer DNA recombinant enzymes for deletion or substitution This is performed when selecting or before selecting a target site.

[0070] If you wish to induce a substitution using the DNA recombinase of the present invention (DRiGR), in addition to the substrate, the first A synthetic sequence containing the target site and the second target site is used. In this example, as previously described. The first and second target sequences contain different spacer sequences to avoid deletions. Hmm. As a result, the same target sequence on the substrate (in the vector encoding the library) and By rearranging the synthetic sequence, the recombination changes with respect to substitution. Here, negative The selected marker is replaced by rearranging it in a composite sequence.

[0071] In a preferred embodiment, the synthetic sequence is a replication-deficient plasmid (for example, Figures 4 and 5). See also). However, in an alternative embodiment, linear DNA strands are used. In a preferred embodiment, the synthetic sequence is located on a high copy number plasmid (see, for example, Figure 14). (See reference).

[0072] To further select the DNA recombinant enzyme that brings about the substitution, the synthetic sequence is preferably the first and Includes a positive selection marker between the second target. If recombination occurs, positive selection A sequence containing a marker is inserted into an expression vector, and a positive selection marker is also present. The current vector can be positively selected. In one embodiment, positive selection The vector is an antibiotic resistance gene. Therefore, after introducing the vector into cells, positive selection Cells containing vectors with selective markers can be easily selected.

[0073] In one embodiment (of DRiGR), the vector is located between the first and second target sites. It has a positive selection marker, and the synthetic sequence has a positive selection marker between the first and second target sites. Includes more kers.

[0074] Other negative and positive selection markers are known to those skilled in the art. Further applicable Among the negative selection markers is URA3(5-fluoroorotic acid), which is a toxic compound. (Converts to loruracil) or viral thymidine kinase (sensing the host to ganciclovir selection) Genes that encode toxins (such as those that make something receptive) or genes that convert prodrugs into toxic substances There are genes that encode enzymes that code for genes. Further applicable positive selection markers — is a gene that codes for an enzyme that can supplement nutritional requirements.

[0075] A step in molecular evolution preferably involves at least one selection step and at least one mutation. This includes a heterogeneous induction step. The selected molecules are "evolved" during the selection process, and the selected candidates are... Preferably, a mutation is induced, and the resulting mutation is introduced into the sequence for testing in the next round of selection. To test. Preferred methods for mutagenesis are known to those skilled in the art, and are particularly prone to errors in PCs. R, chemical mutagenesis, use of specific mutant host species, recursive ensemble mutagenesis Mutagenesis, combinatorial cassette mutagenesis, and DNA shuffling, as well as methods thereof. This includes combinations of the above.

[0076] A co-evolution of 1 or more cycles, preferably 5 to 50, preferably 8 to 30 cycles, is preferably carried out. This process continues until a DNA recombinase with activity targeting the desired site evolves.

[0077] In some embodiments of the present invention, step b) co-evolution steps using existing live DNA recombinant enzymes It can be directly implemented in Lari.

[0078] In a preferred embodiment, the method of the present invention uses two DNA recombinases in step b) The following further steps are involved in obtaining the library. Here, step a) is further It is called a1). a2) A step to identify four sequences that will be sub-regions of known target sites of DNA recombinase (here The first sequence is homologous to the first sub-region of the first target site (selected according to (a1)), The second sequence is homologous to the second sub-region of the first target site (selected according to (a1)), and the third The sequence is homologous to the first sub-region of the second target site (selected according to (a1)), and the fourth The column is selected according to (a1) (which is homologous to the second sub-region of the second target region).

[0079] The selection step a1 and the identification step a2 are preferably performed in one step a), for example, using the SeLOX algorithm. This is achieved through computer programs like Zum (Surendranath, V. et al. (2010)). This is performed occasionally. Preferably, the upstream and downstream sequences of the target site are known to be processed by DNA recombinase enzymes. It is compared with the target site. Each sub-site is compared with a sub-site of a known target site of the DNA recombinase. Target sequences with specific homology are selected as described in a1). Identified by this method. The known sub-sites of the target site are the sites mentioned in a2). The degree of homology is preferably a As mentioned in point 1.

[0080] (Step a3):) In this process, between the partial site identified in step a2) and the target site selected in step a1) A mixture is preferably used to create an intermediate target site.

[0081] Nucleotides in the 1st to 4th sequences (as defined in (a2)) that are different from the target site selected in (a1). Determine the following: and identify one or more different nucleotides appearing in the corresponding sub-site of the target site. Preferably, an intermediate target site is created by substituting up to three nucleotides. do.

[0082] In one embodiment, regarding the isolated molecularly directed evolution of DNA recombinases at each intermediate target site: In this context, the intermediate target regions are symmetrical and therefore include identical or inversely complementary subregions. In this embodiment, preferably a set of four intermediate target sites is created. For each sub-region, select one intermediate target region in a1).

[0083] Another embodiment of the co-evolution of DNA recombinant enzymes, preferably two asymmetric intermediates A set of target sites is prepared. The first intermediate first target site is the first target site selected in (step a1). A nucleotide is substituted by a nucleotide appearing in the first sub-region of the target site (a 2) A sequence corresponding to the first sequence defined in (a1), and the first target site selected in (a1) The nucleotide is substituted by a nucleotide appearing in the second sub-site (a2) (Includes a sequence corresponding to the second sequence. The second intermediate target site is selected in (step a1).) A nucleotide is substituted by a nucleotide appearing in the first sub-region of the second target site. The sequence corresponding to the third sequence (defined in (a2)), and the second target (selected in (step a1)) A nucleotide is substituted by a nucleotide appearing in the second sub-region of the site (a2). It includes an array corresponding to the fourth array (as defined by...).

[0084] (Step a4):) A DNA recombinant enzyme that recognizes the sequence specified in step a2) is subjected to a first intermediate target site and / or A nucleic acid containing a second intermediate target site is used as a substrate, and molecularly directed evolution is applied.

[0085] In one embodiment, the nucleic acid includes both a first intermediate target site and a second intermediate target, and DNA Direct evolution of recombinant enzymes is performed on both intermediate target sites (direct co-evolution). Co-evolution Preferably, step b) is carried out as described above.

[0086] In a preferred embodiment, the first round of molecularly directed evolution is performed on each intermediate target site. Separated molecular directed evolution of DNA recombinases This will be implemented as a (separation evolution method) (see, for example, Figure 8). Further embodiments will be... Then, following these steps, one or more co-evolutionary processes take place.

[0087] The molecularly directed evolution process preferably involves positive selection of recombination at an intermediate target site. to perform and / or negative selection for recombination to other target sites include.

[0088] To "evolve" the selected molecules during the selection process, the selected candidates are preferably suddenly changed. Mutation is induced, and the resulting mutation is introduced into the sequence and tested in the next round of selection. Preferred induction methods are known to those skilled in the art, particularly PCR and chemical mutations, which are prone to errors. Heterogenization, use of specific mutant host species, recursive ensemble mutagenesis, combinatorial Alkaset mutagenesis, DNA shuffling, and combinations thereof. .

[0089] A molecularly directed evolution method is preferably carried out for 1 or more cycles, preferably 8 to 40, and preferably 15 cycles. This process continues until a DNA recombinase with activity against the intermediate target site evolves.

[0090] The molecularly targeted evolution method used is preferably the evolution of substrate-related proteins, particularly WO2. As stated in 002044409 A2. However, other directional evolutionary strategies, such as continuation, The evolutionary theory (WO2012088381 A2) can also be applied.

[0091] Next, preferably a further set of intermediate target sites (selected in (a1)) of the target sites By further adapting to specific body parts, nuclei appear in the corresponding parts of the target area. This is produced by substituting a maximum of 3 nucleotides, preferably one or more, that differ by the amount of ocidide. To manufacture.

[0092] Shuffling and further setting of intermediate target sites, which are sub-steps of molecularly directed evolution. The preparation is preferably a partial of the target site (selected in (a1)) Continue the process until a difference of 1 to 3, preferably 1 or 2 nucleotides, is achieved per site.

[0093] In one preferred embodiment for obtaining a DNA recombinase (DRiGR) that induces substitution Step b) is carried out in the three sub-steps of the molecular evolution method.

[0094] In the first step, the library(s) is created without using artificial sequences (therefore, Donabe (Without using a criterion) Evolve DRiGD as previously described, with minimal impact on the target site. Recombinases that exhibit at least some activity, and therefore DNA recombination that exhibit at least some excision activity. Select the enzyme. Preferably, this first step involves first selecting a first set of intermediate target sites, and Next, optionally, a second set of intermediate target sites, and finally, the final target site, as described above. We will proceed as planned.

[0095] In the second step, a library (multiple libraries are possible) of DNA recombinant enzymes obtained as a result of step i) ) using artificial sequences that have positive selection markers such as antibiotic resistance genes To evolve. The artificial sequence is preferably a replication-deficient donor vector. Therefore, the host cell In this case, use a plasmid or linear sequence that does not have an active origin of replication (see Figure 9 for an example). (As shown). The second step is preferably performed on the final target site. Furthermore, the sequence can be replaced by DRiGR (a more complex reaction than simple excision). Select a DNA recombinase enzyme. Preferably 5 to 20 cycles, more preferably 8 to 15 cycles. We performed the substrate-linked molecular evolution method until we obtained an enzyme that has activity against the final target site. This is done. By using replication-deficient donor vectors in combination with antibiotic selection markers, the The first recombination reaction (integration) at target site 1 is followed by the second recombination reaction at target site 2. This allows for separation from the excision, which is necessary for the success of the DRiGR reaction.

[0096] In the third step, molecular evolution methods are used with artificial sequences that do not contain positive selection markers. Furthermore, the procedure is carried out using the library(s) of DNA recombinases obtained as a result of step ii). The artificial sequence preferably carries a functional origin of replication that is active in the host cell used. The donor vector is (illustrated in Figure 14). The third step is preferably the final target The procedure is performed on the site. Preferably, 5 to 20 cycles, more preferably 8 to 15 cycles of substrate. We will perform linked molecular evolution until we obtain an enzyme that has activity against the final target site. In this third step, equivalent recombination kinetics are obtained for target site 1 and target site 2. We select DNA recombinases that exhibit high substitution activity and do not require a positive selection marker. This means that the presence of a positive selection marker (e.g., an antibiotic resistance gene) is unfavorable for selection. Many uses are available (especially medical use, or the creation of multicellular organisms with modified sequences). This is an important point regarding the case.

[0097] This three-step method involves the use of the same enzyme, or especially two to three (or even up to five) enzymes, between target sites. When combined with a vector containing one or more recognition sites of different restriction enzymes, substitution occurs. It has been shown to be highly efficient in selecting the enzyme (DRiGR) to induce.

[0098] In general, throughout this specification, the term "vector" refers to the concatenated vectors. This refers to nucleic acid molecules that can transport nucleic acids. Vectors are not limited to these. However, a nucleic acid molecule that is single-stranded, double-stranded, or partially double-stranded; containing one or more free ends. Nucleic acid molecules without ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; And there are various other polynucleotides known in the art. One type of vector is "plus There is a "mid." This is, for example, obtained by standard molecular cloning techniques to obtain additional DNA segments. It refers to a circular double-stranded DNA loop that can be inserted into another type of vector. There are virus vectors. These are vectors that contain viral DNA or RNA sequences, and These are viruses that undergo cloning (for example, retroviruses, replication-deficient retroviruses). Viruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated viruses (AAVs) Furthermore, viral vectors carry the virus for transfection into host cells. It contains polynucleotides. Some vectors autonomously replicate in the host cells into which they are introduced. It can be produced (for example, bacterial vectors and episome-type vectors having a bacterial origin of replication). Mammalian vectors). Other vectors (e.g., non-episome mammalian vectors) Upon introduction into chief cells, it is integrated into the host cell's genome, thereby multiplying with the host genome. To be manufactured.

[0099] Furthermore, certain vectors can induce the expression of genes to which they are functionally linked. Such vectors are referred to as "expression vectors" in this specification. Recombinant DNA technology Commonly useful expression vectors often take the form of plasmids.

[0100] The expression vector is preferably a plasmid, virus, or artificial chromosome.

[0101] Another object of the present invention is a vector or a vector used in a method.

[0102] Preferred expression vectors are: i) One, two, three, or four genes, each encoding a different library of DNA recombinases. (Here, the genes encoding the library are under the control of the same promoter.) ii) Preferably, a restriction enzyme recognition site that appears only once in the expression vector (unique recognition site) The identification site includes a first target site and a second target site that border the negative selection marker. It contains substrate nucleic acids.

[0103] To select a DNA recombinase (DRiGD) that induces deletion at the target site, further steps are required. No substrate DNA is required. The first intermediate target site and the second intermediate target site of the expression vector This is because the rearrangement between them is triggered independently.

[0104] Based on the desired sequence, a DNA recombinase (DRiGR) that induces substitution at the target site is selected. To that end, a synthetic nucleic acid comprising a first intermediate target site and / or a second intermediate target site is used. Use an additional (donor) vector (preferably a replication-deficient plasmid). Furthermore, the positive selection marker is located between the first and second intermediate target sites. Therefore, the first The second intermediate target site is preferably a positive selection mark such as an antibiotic resistance gene. To frame the car.

[0105] The present invention relates to a method for preparing DNA recombinant enzymes: c) Isolate the nucleic acid of at least one DNA recombinant enzyme obtained in step b) from the library. The process of doing; and preferably d) further comprising the step of cloning the nucleic acid obtained in step c) into a suitable delivery vector. obtain.

[0106] Furthermore, the present invention includes a designer DNA recombinase obtained by the method of the present invention.

[0107] The presented invention enables flexible and flawless modification of genetic information. This provides a recombinant-based genome editing solution. This method is particularly useful for treating human diseases. DNA recombinant enzymes that replace the genetic mutations that cause them, or erase specific genetic material from cells. It becomes possible to produce the element. Therefore, using the DNA recombinase of the present invention, nucleos Modify the cytoplasmic sequence, especially by repairing mutations, or by using genome-derived sequences or viruses. The introduced sequence is then erased. Mutations that can be repaired by this invention are particularly dot mutations. This includes natural mutations, frameshift mutations, deletions, and insertions.

[0108] DNA recombinases are enzymes that modify the genome of a host cell, a host organ, or a host organism, or a virus. It is possible to use naturally occurring target sequences within the genome, including the genome of pathogens. This is advantageous in that respect. The advantage of the present invention is that it can repair the precise parts of DNA without involving the host DNA repair pathway. This enables location-specific modification, thereby inducing random insertions and deletions (insertions and deletions). It functions without any problems.

[0109] Therefore, the present invention also provides site-specific recombination for non-identical naturally occurring target sites. This includes methods for modifying the genome by induction. The method involves delivering the protein itself. Or, introduce a nucleotide sequence that codes for it, and use this from an mRNA or DNA template. The present invention includes delivering the DNA recombinant enzyme to a host by expression.

[0110] The previously defined "naturally occurring" means that the target site was not artificially introduced—within the same genome. It means that it is native to [the country / region].

[0111] Suitable host cells include hematopoietic stem cells, neural stem cells, adipose tissue-derived stem cells, and embryonic stem cells. Eukaryotic cells, including stem cells such as umbilical cord stem cells, induced pluripotent stem cells, and embryonic stem cells. In the case of human embryonic stems, it is preferable that there are cells that do not originate from the destroyed embryo. Furthermore, modification of human germline cells and human gametes in host cells is preferably excluded. .

[0112] Suitable host organs for the delivery of the DNA recombinant enzyme of the present invention include bone marrow, skin, muscle, liver, lung, and liver. This includes any cells in the body, including the spleen and pleural nodes.

[0113] The method of the present invention can be performed in vitro (outside a living organism) or in vivo. Yes, it is possible. In vitro procedures include manipulating isolated cells and / or organs.

[0114] In one aspect, as described above, the present invention replaces or deletes a target sequence in the genome. Provide a method to make it disappear.

[0115] In one embodiment, the present invention involves knocking out a target sequence (e.g., a gene or exon). Cells containing or modified sequences (e.g., transgenes) or (preferably non-human) multiple For the (preferably non-therapeutic) use of the DNA recombination enzyme of the present invention for the production of cell organisms. To relate to.

[0116] In this method, the DNA recombinant enzyme of the present invention or the gene encoding the enzyme of the present invention (single One gene per mer is introduced into cells or organisms by a delivery method.

[0117] The designer DNA recombinant enzyme of the present invention borders a target sequence in the genome with two identical genes. It induces site-specific recombination at target sites where it is not present.

[0118] Site-specific deletion for target sequences (e.g., insertion sequences or viral sequences). To modify the genome by inducing recombination (DRiGD), additional DNA or RNs are required. A is not required.

[0119] Site-directed recombination (DRiGR) to replace a target sequence (e.g., a point mutation). In order to modify the genome by inducing the DNA recombinant enzyme of the present invention, A synthetic sequence containing the desired sequence (donor DNA) is further introduced into the host. The synthetic sequence is the target and The DNA includes a sequence to be replaced and a desired sequence to be replaced (donor DNA), where the desired sequence is the DNA set of the present invention. It is adjacent to the first and second target sites recognized by the reconjugate enzyme.

[0120] In another embodiment, the present invention relates to biochemical signaling pathway-related genes and polynucleotides. Mutant or modified disease-related genes or polynucleos selected from Create eukaryotic cell models containing cydos or mutated or modified sequences. The method is provided. Examples of these genes and polynucleotides are listed in Tables A to C below. .

[0121] Another application of the present invention is to introduce mutations, or preferably to address human diseases. By introducing a nucleotide sequence that has a specific mutation and / or a humanized sequence Alternatively, by substitution, a specific modification of a target sequence in the genome, for example. For example, this involves creating eukaryotic cell models or animal models. Using this method, Inducing unwanted insertions and deletions (insertions and deletions) or other uncontrolled alterations of genetic material. It has the advantage of making it possible to create animal models without guidance. In some embodiments, the introduced mutation carries a risk of having or developing a disease. This is associated with an increase in [something].

[0122] In a preferred embodiment, the method of the present invention is used to produce animal models useful for biomedical research. For example, to create a model of a human disease. When implementing the present invention in animals, preferably This is performed for non-therapeutic use in animals.

[0123] Suitable host organisms include invertebrates and vertebrates, especially Bovidae and yellow staghorn Fruit fly (Drosophila melanogaster), nematode (Caenorhabditis elegans), African African clawed frog (Xenopus laevis), medaka, zebrafish, house mouse (Mus musc) This includes *Anoplophora ulus*, *Ratus norvegicus*, or embryos of these organisms.

[0124] However, plants and fungi, as well as their cells, can also be used as hosts. .

[0125] In some embodiments, one or more vectors described herein are used to non-human Produces transgenic animals or transgenic plants. In some embodiments the transgenic animal is a mammal such as a mouse, rat, or rabbit. In certain embodiments, the organism or subject is a plant. In certain embodiments, the organism or subject or plant is an alga. Methods for producing transgenic plants and animals are known in the art and generally begin using methods of cell transfection. Transgenic animals, as well as transgenic plants, particularly grains and algae, are provided. Transgenic animals or plants can be useful in applications other than providing disease models. These can include, for example, the production of food or diet through higher protein expression, sugars, nutrients, or vitamin levels than are normally found in the wild type. In this regard transgenic plants, particularly legumes and potatoes, and animals, particularly mammalian animals such as livestock (cattle, sheep, goats, and pigs), in addition to poultry and edible insects, are preferred.

[0126] Delivery vectors can be used for the introduction and expression of DNA recombinant enzymes into a host, or other delivery methods such as DNA, mRNA, or proteins encapsulated in liposomes or nanoparticles (e.g., as described in Wang M et al., (2016)), or in vivo electroporation, or cell extracts are also applicable.

[0127] The delivery method used is determined according to the host, host cell, or target tissue in the host to which the sequence to be modified is desired.

[0128] Also, the choice of delivery vector used is determined according to the host, host cell, or determined according to the target tissue in the host. Suitable delivery vectors are known to those skilled in the art, such as plasmids, artificial chromosomes, retroviral vectors, lentiviral vectors, spumavirus vectors, and in addition to adenoviral vectors, there are Agrobacterium carrying shuttle vectors terium. Also, lentivirus and adeno-associated virus, and delivery vectors including these vectors types (especially adeno-associated virus vectors) are selected to be advantageous in that they can be targeted to specific types of cells or tissues.

[0129] The delivery vector preferably includes regulatory elements. The term "regulatory element" refers to promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and polyU sequences) are intended to be included. In some embodiments, the regulatory element is a cellular, bacterial, viral, or hybrid promoter, where the promoter is preferably a constitutive or inducible promoter. Also, the promoter is preferably selected according to the host, host cell, or the target tissue in the host. Also, suitable promoters are known to those skilled in the art . Preferred constitutive promoters are selected from the promoters of cytomegalovirus, Rous sarcoma virus, murine leukemia virus-related retrovirus, phosphoglycerokinase gene, murine spleen focus-forming virus, or human elongation factor 1α.

[0130] Also, for substitution of the target sequence (DRiGR), the delivery vector preferably contains a donor sequence with two non-identical target sites recognized by the designer DNA recombinase of the present invention ​​This includes, where the target site borders the desired sequence (donor DNA) to be replaced with the target sequence. Here, the desired sequence is adjacent to the first target site and the second target site.

[0131] Alternatively, the donor sequence may be an isolated nucleic acid molecule or vector (e.g., a replication-deficient plasmid). ) Provided on top.

[0132] Furthermore, the present invention includes nucleic acids or vectors that encode the designer DNA recombinant enzyme of the present invention. In a preferred embodiment, the monomer of the designer DNA recombinant enzyme of the present invention is encoded. A nucleic acid is either a single nucleic acid molecule or a single vector. Preferably, a designer DNA recombinant enzyme. The monomer is expressed under the control of one promoter or other regulatory element.

[0133] Furthermore, the present invention: 1. The expression vector or delivery vector described above that encodes the designer DNA recombinant enzyme of the present invention (Here, the designer DNA recombinase of the present invention borders the target sequence in the genome) (Inducing site-directed recombination at two non-identical target sites) 2. Two identical but non-identical marks border the target sequence and the desired replacement sequence (donor DNA). A donor sequence containing the target region, a nucleotide sequence (where the desired sequence is the first target region) Transduction into cells having the expression of DNA recombinase at the second target site (and adjacent to the second target site). This includes methods for doing so.

[0134] The donor sequence can be placed in an expression vector or delivery vector, or isolated. The nucleic acid, preferably as a replication-deficient plasmid, is provided. Preferably, the donor sequence is provided per cell. The target sequence is provided in multiple copies to increase the substitution rate (see also Figure 3).

[0135] In a further aspect, the invention relates to: i) a designer DNA recombinase of the invention, or a nucleic acid or vector encoding the designer DNA recombinase of the invention, wherein the designer DNA recombinase of the invention induces site-specific recombination at two non-identical target sites flanking the target sequence in the genome ii) a nucleotide sequence comprising a donor sequence comprising the same two non-identical target sites flanking the target sequence and the desired sequence (donor DNA) to be substituted, wherein the desired sequence is adjacent to the first target site and the second target site), in a set or kit. Preferably, the donor sequence is provided in multiple copies per cell to increase the replacement rate of the target sequence (see also Figure 3).

[0136] Here again, the donor sequence is arranged on the same vector (encoding the designer DNA recombinase of the invention) or provided as a separate nucleic acid, preferably a replication-deficient plasmid.

[0137]

[0138]

[0139]

[0140]

[0141] A further important part of the invention is the medical applicability of the DNA recombinase of the invention, the nucleic acid or vector of the invention, or the set / kit of the invention. Regarding medical applicability, the target sequence has been modified to treat diseases, or has been modified to treat diseases. Even if it reduces symptoms or extends lifespan, "disease-related" genes or polynucleotides It is Do.

[0140] In one embodiment, the present invention relates to an in vitro method. In this method, the present invention first Applies to the isolated cells or organs described.

[0141] In another embodiment, the present invention relates to a method for treating a subject, preferably a human or an animal, and DNA recombinant enzyme of the invention, nucleic acid or vector encoding the designer DNA recombinant enzyme of the invention The method comprises the step of applying a tar or a set of the present invention.

[0142] The delivery method and vector are preferably selected as described above.

[0143] In one embodiment, the target to be treated is a nucleotide sequence that causes the target to develop a disease ( Target sequences), particularly mutations that cause a decrease or loss of function of the gene product. For example, it possesses a mutation in the factor IX gene that causes hemophilia. DNA recombination according to the present invention Enzymes, nucleic acids or vectors that encode them, and wild-type or other functional varians of genes. By delivering a donor sequence (or a portion thereof, such as an exon) that carries the desired sequence, This involves replacing the mutated portion of a gene with a desired (functional) sequence. To enable substitution, the desired sequence in the donor sequence is targeted by a DNA recombinase enzyme. Place them adjacent to each other.

[0144] In another embodiment, the target to be treated is the nucleotide sequence that causes the disease (target) It possesses a sequence that reduces the symptoms of this condition. Here, the target sequence is this Applying the method of the invention, the DNA recombinant enzyme of the present invention, the nucleic acid encoding it, or the vector The deletion occurs upon delivery. Here, the target sequence is particularly pathogenic (bacteria, viruses). A sequence of a microorganism (such as a parasite) or an oncogene or another gene, where the The activity causes disease, or the gene (such as the gene encoding PCSK9) Inhibiting its activity is known to reduce symptoms.

[0145] Furthermore, the present invention encodes the DNA recombinant enzyme of the present invention and the designer DNA recombinant enzyme of the present invention. A pharmaceutical composition comprising nucleic acids or vectors, or the set of the present invention and a suitable carrier. .

[0146] The target sequence targeted by the DNA recombinase according to the present invention is in eukaryotic cells. This can be any polynucleotide, either endogenous or extrinsic. For example, the target is The sequence is integrated into the nucleus, or the mitochondria of a eukaryotic cell, or the genome of a host cell. Regardless of whether it is or not, it can be considered a polynucleotide within the virus. The sequence may be a sequence that codes for a gene product (e.g., a protein) or a non-coding sequence (e.g., These can be introns or regulatory polynucleotides.

[0147] The target sequences targeted by the DNA recombinant enzyme of the present invention are numerous disease-related genes. Genes and polynucleotides, as well as genes and polynucleotides related to biochemical signaling pathways. It may include.

[0148] "Disease-related" genes or polynucleotides are genes derived from cells of diseased tissue. Compared to non-disease control tissues or cells, the transcript or translation product is at an abnormal level, or This refers to any gene or polynucleotide produced in an abnormal form. Disease-related genes are abnormal. Genes can always be expressed at high levels; disease-related genes may be expressed at abnormally low levels. It may be a gene that becomes a certain state. Here, the change in expression correlates with the onset and / or progression of the disease. Furthermore, disease-related genes are those resulting from mutations (multiple mutations are possible) or genes (multiple mutations are possible) that cause the disease. This refers to genes that directly contribute to or have genetic variations that are in linkage disequilibrium with it. (Transcript) Alternatively, the translated product may or may not be publicly known, and its level may be normal or abnormal. This may also be the case. Examples of disease-related genes and polynucleotides are provided by McKusick-Nathans Genetics. Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and The National Center for Biotechnology Information, It is available from the National Library of Medicine (Bethesda, Md.) These are available on the World Wide Web.

[0149] The target sequences targeted by the DNA recombinant enzyme of the present invention are numerous disease-related genes. The entirety of the offspring and polynucleotides, as well as the genes and their contents related to biochemical signaling pathways Polynuclear, as listed in U.S. Patent US8697359 B1 incorporated herein by reference May contain leotide.

[0150] Examples of disease-related genes and polynucleotides are listed in Tables A and B. Specific diseases For specific information, please contact the McKusick-Nathans Institute of Genetics and Medicine, Johns Hopkins University (Baltimore, Md.). It is also available from the National Biotechnology Information Center and the National Library of Medicine (Bethesda, Md.). These are available on the World Wide Web. Biochemical signaling pathway-related genes Examples of polynucleotides are listed in Table C.

[0151] These gene mutations and pathways result in abnormal proteins or abnormalities that affect function. This can result in the production of large amounts of protein. In further examples of genes, disease and protein The material is incorporated herein by reference from a U.S. provisional application. Such genes, The protein and pathway may be the target sequence for the DNA recombinant enzyme of the present invention. .

[0152] [Table 1]

[0153] [Table 2] TIFF0007842465000003.tif234170TIFF0007842465000004.tif153170

[0154] [Table 3] TIFF0007842465000006.tif243170TIFF0007842465000007.tif246170TIFF000 7842465000008.tif245170TIFF0007842465000009.tif245170TIFF00078424650 00010.tif245170TIFF0007842465000011.tif246170TIFF0007842465000012.t if244170TIFF0007842465000013.tif247170TIFF0007842465000014.tif150170

[0155] Furthermore, embodiments of the present invention include knocking out genes, amplifying genes, and repair of specific mutations, including point mutations, frameshift mutations, deletions, or insertions. This relates to methods and compositions for doing so.

[0156] In yet another aspect of the present invention, the present invention is used to obtain from several genetic mutations The resulting visual defects can be corrected.

[0157] Some further aspects of the present invention relate to the National Institutes of Health. h) The website further describes a wide variety of genetic disorders in the topic subcategory. Regarding correcting defects associated with various genetic disorders (website: health.nih.gov) (See / topic / GeneticDisorders). Genetic brain disorders include, but are not limited to, these. Renal leukodystrophy, agenesis of the corpus callosum, Aicardi syndrome, Alpers disease, Alzheimer's disease Marr's disease, Barth syndrome, Batten disease, CADASIL, cerebellar degeneration, Fabry disease, Gerstoma N'Streusler-Scheinker disease, Huntington's disease, and other triplet repeats Triplet Repeat Disorders, Leigh syndrome, Lesch-Nyhan syndrome, This may include Menkes disease, mitochondrial myopathy, and NINDS corpus callosum agenesis. These diseases are, Further details are available on the National Institutes of Health website under the subcategories of genetic brain disorders.

[0158] In some embodiments, the state can be a neoplasm. In this embodiment, the gene to be targeted is one of the genes listed in Table A. (In this case, it is PTEN, etc.). In some embodiments, the condition is age-related macular degeneration. It is possible. In some embodiments, the condition may be schizophrenia. In terms of the treatment method, the condition is Trinucleotide Repeat Disorder. It is possible. In some embodiments, the condition may be fragile X syndrome. In that embodiment, the condition may be secretase-related dysfunction. In some embodiments In this context, the condition may be a prion-related disorder. In some embodiments, the condition is ALS. It is possible. In some embodiments, the condition may be drug poisoning. In some embodiments, the condition may be autism. In some embodiments, the condition may be Alzheimer's. It could be Immer's disease. In some embodiments, the condition can be inflammatory. In this embodiment, the condition may be Parkinson's disease.

[0159] Examples of proteins associated with Parkinson's disease include, but are not limited to, α-synucleoproteins. Crane, DJ-1, LRRK2, PINK1, Parkin, UCHL1, Synphyrin-1, and NURR1 are available. ru.

[0160] An example of a toxicity-related protein might be ABAT.

[0161] Examples of inflammation-related proteins include, for example, monocyte chemotactic proteins encoded by the Ccr2 gene. Protein-1 (MCP1), CC chemokine receptor type 5 (CCR5), encoded by the Ccr5 gene, The IgG receptor IIB (FCGR2b, also known as CD32), encoded by the Fcgr2b gene, is transmitted through Fcer1g. There may be an FcεR1g (FCER1g) protein encoded by this.

[0162] Examples of cardiovascular disease-related proteins include, for example, IL1B (interleukin-1, β), XDH( Xanthine dehydrogenase, TP53 (tumor protein p53), PTGIS (prostaglandin 12 (prostacyclin synthase), MB (myoglobin), IL-4 (interleukinase) 4), ANGPT1 (angioprotein 1), ABCG8 (ATP-binding cassette, subfamily G) Possible candidates include WHITE), member 8), or CTSK (cathepsin K).

[0163] Examples of Alzheimer's disease-related proteins include, for example, those encoded by the VLDLR gene. Very low-density lipoprotein receptor protein (VLDLR), encoded by the UBA1 gene. NEDD8 encoded by the ubiquitin-like modifier activator 1 (UBA1) or UBA3 gene. - An activating enzyme E1 catalytic subunit protein (UBE1C) may be involved.

[0164] Examples of proteins associated with autism spectrum disorder include, for example, the BZRAP1 gene. The benzodiazepine receptor (peripheral)-related protein (BZRAP1) encoded in the AFF2 gene Therefore, the encoded AF4 / FMR2 family member 2 protein (AFF2) (also known as MFR2), FXR Fragile X intellectual disability autosomal homolog 1 protein (FXR1), encoded by a single gene, or the fragile X mental retardation autosomal homolog 2 protein (FXR2 gene) encoded by the FXR2 gene R2) is possible.

[0165] Examples of proteins associated with macular degeneration include, for example, AT, encoded by the ABCR gene. P-binding cassette, subfamily A (ABC1) member 4 protein (ABCA4), APOE gene Therefore, the apolipoprotein E protein (APOE), or the CCL2 gene, encodes There may be an encoded chemokine (CC motif) ligand 2 protein (CCL2).

[0166] Examples of proteins associated with schizophrenia include NRG1, ErbB4, CPLX1, TPH1, TPH2, and NRX Possible configurations include N1, GSK3A, BDNF, DISC1, GSK3B, and combinations thereof.

[0167] Examples of proteins involved in tumor suppression include ATM (ataxia telangiectasia mutation). Type), ATR (ataxia telangiectasia and Rad3-related), EGFR (epidermal growth factor receptor), E RBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2 erythroid ERBB4 (v-erb-b2 erythroblastic leukemia virus oncogene homolog 3), ERBB4 (v-erb-b2 erythroblastic leukemia virus oncogene Gene homologs4) may include Notch1, Notch2, Notch3, or Notch4.

[0168] Examples of proteins associated with secretase disorders include, for example, PSENEN (presenilin enhancer). Cathepsin B homolog (C. elegans), CTSB (Cathepsin B), PSEN1 (Presenilin) 1) APP (Amyloid-beta (A4) precursor protein), APH1B (prepharyngeal defect 1 homolog B (line Parasite), PSEN2 (Presenilin 2 (Alzheimer's disease 4)), or BACE1 (β-site APP-cleavage) Enzyme 1) is possible.

[0169] Examples of proteins associated with amyotrophic lateral sclerosis (ALS) include SOD1 (superoxide dismutase). - 1), ALS2 (Amyotrophic Lateral Sclerosis 2), FUS (Intrasarcoma Fusion), TARDBP (TAR DNA-bound tannins) Proteins, VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B), and VAGFC (blood) Endothelial growth factor C), and any combination thereof, are possible.

[0170] Examples of proteins associated with prion diseases include SOD1 (superoxide dismutase 1). ), ALS2 (Amyotrophic Lateral Sclerosis 2), FUS (Intrasarcoma Fusion), TARDBP (TAR DNA Binding Protein) VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B), and VAGFC (vascular endothelial growth factor B) Skin growth factor C), and any combination thereof, are possible.

[0171] Examples of proteins involved in neurodegenerative states in prion disorders include, for example, A2M(α-2-ma Chloglobulin), AATF (anti-apoptotic transcription regulator), ACPP (acid phosphatase) (Prostate type), ACTA2 (Actin α2 vena cava smooth muscle type), ADAM22 (ADAM metallopeptidase) Main receptors, ADORA3 (adenosine A3 receptor), or ADRA1D (alpha-1D adrenergic receptor) There may be α-1D adrenergic receptors that perform this function.

[0172] Examples of proteins associated with immune deficiencies include, for example, A2M [α-2-macroglobulin]; AAN AT [arylalkylamine N-acetyltransferase]; ABCA1 [ATP-binding cassette] [subfamily A(ABC1), member 1]; ABCA2 [ATP-binding cassette, subfamily A] (ABC1), member 2]; or ABCA3 [ATP-binding cassette, subfamily A(ABC1), member 2]; [Bar 3] is possible.

[0173] Examples of proteins associated with triplet repeat diseases include, for example, AR (androgen receptor). Body), FMR1 (fragility x intellectual disability 1), HTT (huntingtin), or DMPK (myotonic dystrophy) These include protein kinases (Frataxin), FXN, and ATXN2.

[0174] Examples of proteins associated with neurotransmission disorders include, for example, SST (somatostatin) and NOS1 ( Nitric oxide synthase 1 (neuronal type), ADRA2A (adrenergic α-2A receptor), ADRA 2C (adrenergic α-2C receptor), TACR1 (tachykinin receptor 1), or HTR2c (5-H) There is a droxytryptamine (serotonin) receptor 2C.

[0175] Examples of neurodevelopment-related sequences include, for example, A2BP1 [ataxin 2-binding protein 1], AADAT [ataxin 2-binding protein 1]. Minoadipine aminotransferase, AANAT [arylalkylamine N-acetyl [Transferase], ABAT [4-aminobutyrate aminotransferase], ABCA1 [ATP-bonding [ATP-binding cassette, subfamily A (ABC1), member 1], or ABCA13 [ATP-binding cassette, sa There is a family A (ABC1), with 13 members.

[0176] Further examples of desirable conditions treatable by the present invention include: Eicardi-Gutierre syndrome. Syndrome; Alexander disease; Alan Herndon-Dudley syndrome; POLG-related disorder; Alpha - Mannosidosis (types II and III); Alström syndrome; Angelman syndrome; Ataxia with telangiectasia; Neuronal ceroid lipofuscinosis; β-thalassemia; Bilateral optic nerve Atrophy and (childhood) neurotrophy type 1; retinoblastoma (bilateral); Canavan disease; brain, eye, and face • Skeletal syndrome 1 [COFS1]; Cerebral tendon xanthomatous syndrome; Cornelia de Lange syndrome; MAPT-related disorders Hereditary prion diseases; Drabé syndrome; Early-onset familial Alzheimer's disease; Freetra Ihi ataxia [FRDA]; Flins syndrome; Fucosidosis; Fukuyama type congenital muscular dystrophy Galactosialidosis; Gaucher disease; Organic acidemia; Hemophagocytic lymphohistiocytosis; Hutchinson-Gilford progeria syndrome; mucolipidosis II; childhood free sialic acid storage Disease; PLA2G6-associated neurodegeneration; Jörbel-Langenilsen syndrome; Junctional epidermolysis bullosa Diseases; Huntington's disease; Krabbe disease (childhood); Mitochondrial DNA-associated Lie syndrome and NA RP; Lesch-Nyhan syndrome; LIS 1-associated gyrus defect; Lowe syndrome; Maple syrup Urinary urosis; MECP2 duplication syndrome; ATP7A-related copper transport disorder; LAMA2-related muscular dystrophy; Ally Lusulfatase A deficiency; Mucopolysaccharidosis type I, II, or III; Peroxisome formation dysplasia Common symptoms, Zellweger syndrome spectrum; neurodegenerative diseases with impaired iron storage in the brain; acidic Fingomyelinase deficiency; Niemann-Pick disease type C; Glycine encephalopathy; ARX-related disorders; Urea cycle disorders; COL1A1 / 2-associated osteogenesis imperfecta; Mitochondrial DNA deletion syndrome; PLP1 - Related disorders; Perry syndrome; Phelan-McDermid syndrome; Glycogen storage disease type II (Pompe disease) (minor Childhood-related; MAPT-related disorder; MECP2-related disorder; radicular chondrodysplasia type 1; Robert's syndrome; Sandhoff disease; Schindler disease type 1; Adenosine deaminase deficiency; Smith & Lemley disease Opitz syndrome; spinal muscular atrophy; childhood-onset spinocerebellar ataxia; hexosaminidase A deficiency Disorders; Thanatophoric dysplasia type 1; Collagen type VI-related disorder; Usher syndrome type I Type; Congenital muscular dystrophy; Wolff-Hirschorn syndrome; Lysosomal acid lipase deficiency Disorders; and xeroderma pigmentosum may be selected.

[0177] This system can be used to target any desired polynucleotide sequence. It will become clear that this is a possibility. Treatment can be beneficially provided using this system. Some examples of conditions or diseases are listed above. These conditions are currently associated with Examples of genes are also provided there. However, the genes exemplified are not exhaustive. do not have.

[0178] (Example F-9:) In a preferred embodiment of DRiGR, the designer DNA recombinant enzyme of the present invention is used in blood The mutation in exon 8 of the F9 gene, which causes Tomopathy B, is replaced. Exon 8 is a severe form Mutations occur frequently in hemophilia B, and the mutations that appear in this population are known. (www.factorix.org)

[0179] In one embodiment, the F9 designer DNA recombinant enzyme is composed of two different monomers: SEQ ID NO: Sequence 1 (Rec F9-1) or sequence number 1 has at least 90% sequence identity, more preferably 95% Monomers having sequences with sequence identity, and sequences of sequence number 2 (Rec F9-2), or Column number: At least 80% sequence identity with 2, preferably 90% sequence identity, more preferably 9 A heterotetramer containing monomers having sequence identity of 5% or even 98% ru.

[0180] Furthermore, the object of the present invention is the sequence number 1 (Rec F9-1) or sequence number 2 (Rec F9-2) Sequence, or sequence number: at least 80% sequence identity with 1 or 2, preferably 90% sequence identity. A designer including a sequence having uniformity, more preferably 95% or even 98% sequence identity. —It is a DNA recombination enzyme.

[0181] Sequence ID: 1 (Rec F9-1) [ka]

[0182] In a monomer having the sequence sequence number 1 (Rec F9-1), preferably at the following positions It has the following amino acid residues: X17 is any amino acid, preferably D or G. X19 is any amino acid, preferably T or A. X22 is any amino acid, preferably G or E. X30 is a small nonpolar amino acid, preferably V, L, I, A, or G, more preferably. The selection will be made from either V or A. X43 is a positively charged amino acid, preferably K or R. X60 is any amino acid, preferably N or D. X68 is any amino acid, preferably P or S. X69 is a positively or negatively charged amino acid, preferably D, E, K, or R, more preferably The option is selected from E or K. X81 is a polar amino acid, preferably consisting of S, T, C, K, or R, more preferably C or R. Selected. X84 is any amino acid, preferably A or T. X89 is any amino acid, preferably L or Q. X93 is a small amino acid, preferably selected from C, S, and A, more preferably from C or A. It will be done. X140 is any amino acid, preferably I or T. X166 is a nonpolar amino acid, preferably selected from I, L, or V, more preferably from I or V. It will be selected. X177 is a nonpolar amino acid, preferably selected from I, L, or V, more preferably from I or V. It will be selected. X182 is a nonpolar amino acid, preferably selected from I, L, or V, more preferably from I or V. It will be selected. X183 is a positively charged amino acid, preferably K or R. X186 is a polar amino acid, preferably S or T. X206 is any amino acid, preferably A or T. X222 is any amino acid, preferably V or E. X225 is a nonpolar amino acid, preferably selected from I, L, or V, more preferably from I or V. It will be selected. X237 is a polar amino acid, preferably selected from S, T, C, or Y, more preferably from Y or C. It will be selected. X239 is a nonpolar amino acid, preferably I, L, V, or F, more preferably F or I. Selected. X249 is a small nonpolar amino acid, preferably V, L, I, A, or G, more preferably V Alternatively, it will be selected from A. X259 is any amino acid, preferably F or P. X260 is any amino acid, preferably T or A. X264 is any amino acid, preferably A or D. X278 is any amino acid, preferably L or Q. X284 is a nonpolar amino acid, preferably selected from I, L, or V, more preferably from I or V. It will be selected. X318 is a nonpolar amino acid, preferably selected from I, L, or V, more preferably from I or L. It will be selected. X342 is any amino acid, preferably G or D.

[0183] Uppercase letters represent single-character codes in the lUPAC naming convention. The preferred array for array index 1 is array number Sequence of code: 28, sequence number: 3, and sequence number: 5, and sequence number: 28, sequence number: 3, or Sequence ID: 5 has at least 80% sequence identity, preferably 90% sequence identity, more preferably The sequences are selected from sequences that have 95% or even 98% sequence identity.

[0184] Sequence ID: 2 (Rec F9-2) [ka]

[0185] In a monomer having the sequence sequence number 2 (Rec F9-2), preferably at the following positions It has the following amino acid residues: X3 is any amino acid, preferably K or N. X5 is any amino acid, preferably P or Q. X8 is any amino acid, preferably H or T. X14 is a nonpolar amino acid, preferably I, L, or V, more preferably I or L. Selected. X16 is a small nonpolar amino acid, preferably V, L, I, A, or G, more preferably The selection will be made from either V or A. X18 is a small amino acid, preferably A or G. X19 is any amino acid, preferably T or A. X20 is a polar amino acid, preferably selected from S, T, N, or Q, more preferably S or N. It will be selected. X21 is any amino acid, preferably D or G. X31 is a nonpolar amino acid, preferably I, L, V, and F, more preferably L or F. It will be selected. X39 is a positively or negatively charged amino acid, preferably D, E, K, or R, more preferably This is selected from K or E. X80 is any amino acid, preferably T or A. X84 is any amino acid, preferably T or A. X93 is any amino acid, preferably C or A. X97 is a nonpolar amino acid, preferably I, L, V, and M, more preferably L or M. Selected. X125 is a nonpolar amino acid, preferably selected from I, L, and V, more preferably from I or V. It will be selected. X132 is a positively charged amino acid, preferably K or R. X182 is a nonpolar amino acid, preferably selected from I, L, and V, more preferably from I or V. It will be selected. X183 is a positively charged amino acid, preferably K or R. X186 is a polar amino acid, preferably T or S. X195 is a nonpolar amino acid, preferably selected from I, L, and V, more preferably from I or V. It will be selected. X206 is any amino acid, preferably A or T. X216 is any amino acid, preferably G or R. X249 is a small nonpolar amino acid, preferably V, L, I, A, or G, more preferably It is selected from V or A. X253 is any amino acid, preferably I or T. X259 is any amino acid, preferably P or S. X260 is any amino acid, preferably A or T. X264 is a nonpolar amino acid, preferably I, L, and V, more preferably V or I (and It is selected from L). X266 is a small amino acid, preferably A or G. X273 is an aromatic or heteroaromatic amino acid, preferably H or Y. X276 is any amino acid, preferably K or Q. X278 is any amino acid, preferably A or D. X306 is a nonpolar amino acid, preferably selected from I, L, and V, more preferably from L or I. It will be selected. X325 is a nonpolar amino acid, preferably selected from I, L, and V, more preferably from L or I. It will be selected.

[0186] Here again, uppercase letters represent single-letter codes in the lUPAC naming convention.

[0187] A preferred sequence for sequence number 2 is the sequence for sequence number 29, sequence number 4, and sequence number 6. , and sequence identity of at least 85% with sequence number: 29, sequence number: 4, or sequence number: 6, good Sequences having 90% sequence identity, more preferably 95% or even 98% sequence identity. Selected from.

[0188] A preferred pair of monomers that form the F9 designer DNA recombinant enzyme of the present invention is Sequence ID: 28 It has the amino acid sequences of 29 and, in particular, SEQ ID NOs: 3 and 4 and SEQ ID NOs: 5 and 6.

[0189] In another embodiment, the F9 designer DNA recombinant enzyme is sequence number 36 (Rec F9- 3) At least 90% sequence identity with sequence number 36, preferably 95%, more preferably 98%. It contains a sequence that has sequence identity. In this case, the F9 designer DNA recombinant enzyme is preferred It is a homotetramer.

[0190] The F9 designer DNA recombinant enzyme of the present invention is obtained using the method of the present invention as described herein. This is obtained, and recognizes the asymmetric loxF9-A and loxF9-A target sites of sequence numbers 7 and 8. : [Table 4]

[0191] These sequences are present in the human genome and are adjacent to exon 8 of the F9 gene. This invention relates to Nucleic acid selected from sequence number: 7 (loxF9a) and sequence number: 8 (loxF9b), or sequence number: Sequence identity of at least 90% with sequence number 7 or sequence number 8, preferably at least 95% sequence identity. Sequences having identity, more preferably at least 99% sequence identity, or one of these sequences A reverse complementary nucleic acid sequence, and a DNA recombinase thereof, preferably the F9 designer of the present invention. - Including use as a target site for DNA recombinant enzymes. In one embodiment, these of the present invention The nucleic acid has a length of 30 to 40 nucleotides, preferably 32 to 36, more preferably 34 nucleotides. It is.

[0192] Furthermore, the present invention relates to nucleic acids and vectors encoding the F9 designer DNA recombinant enzyme of the present invention. include.

[0193] Furthermore, the present invention: 1. A delivery vector comprising a nucleic acid encoding the F9 designer DNA recombinant enzyme of the present invention, 2. loxF9a and the sequence that borders the sequence encoding exon 8 of the F9 gene or its functional mutant. Transduction into cells is performed using a donor sequence containing the LoxF9b region and the expression of a DNA recombinase. This includes methods for achieving this.

[0194] The donor sequence can be placed in an expression vector or delivery vector, or isolated. The nucleic acid, preferably as a replication-deficient plasmid, is provided. Preferably, the donor sequence is provided per cell. Provided in multiple copies to increase the substitution rate of the target sequence (as illustrated in Figures 3 and 11). (See also).

[0195] This invention relates to cells: —The F9 designer DNA recombinant enzyme according to the present invention or nucleic acid that encodes and expresses it or vector, —Exon 8 of the F9 gene adjacent to the loxF9a and loxF9b target sites or its functional mutant Donor array containing the sequence that codes By introducing this, the (non-functional or dysfunctional) mutant exon 8 of the F9 gene is introduced. (Here, the F9 designer DNA recombinase performs site-directed recombination by the donor sequence) The method further includes a method for substituting the sequence containing mutant exon 8.

[0196] Furthermore, the present invention: —F9 designer DNA recombinant enzyme or nucleic acid or vector encoding it according to the present invention —Exon 8 of the F9 gene adjacent to the loxF9a and loxF9b target sites or its functional mutant Includes a kit containing a donor sequence that includes a sequence encoding [the specified character].

[0197] In a preferred embodiment, the defective F9 exon 8 sequence is replaced with an overactive Padua mutation (R3 The replacement is performed with a donor sequence carrying 38L)(PMID:23197580).

[0198] The specific preferred donor sequence for the method and kit mentioned earlier is sequence number: 26( Selected from wild-type exon 8) and Sequence ID No. 27 (exon 8 with Padua mutation). ru.

[0199] The donor sequence can be placed in a vector that encodes the F9 designer DNA recombinase. (See Figure 11 for an example), or isolated nucleic acid, preferably replication-deficient plasmid It will be offered as a gift.

[0200] Preferably, the donor sequence is the target sequence substitution rate in the method and kit mentioned above. To increase the number of copies per cell (e.g., by an AAV vector) (See also Figure 11, for example.)

[0201] (Example: Hex:) In a preferred embodiment of DRiGD, the designer DNA recombinant enzyme of the present invention is This deletes a sequence on chromosome 7. This designer DNA recombinase further deletes a sequence on chromosome 7. This is called the NER DNA recombinase, and it is the asymmetric Hex1 and Hex2 variant of SEQ ID NO: 40 and SEQ ID NO: 41. Recognize the target area: [Table 5]

[0202] In one embodiment, the designer DNA recombinant enzyme is composed of two different monomers: SEQ ID NO: 46 The sequence (Hex-R-#7) or sequence number 46 has at least 90% sequence identity, more preferably 95% Monomers having sequences with sequence identity, and the sequence of sequence number 47 (Hex-L-#7), or Column number: At least 80% sequence identity with 47, preferably 90% sequence identity, more preferably A heterotetramer containing monomers having sequence identity of 95% or even 98% ru.

[0203] In another embodiment, the designer DNA recombinant enzyme consists of two different monomers: SEQ ID NO: Sequence 48 (Hex-R-#30) or sequence number 48 has at least 90% sequence identity, more preferably 95%. A monomer having a sequence with % sequence identity, and the sequence of sequence number 49 (Hex-L-#30), This has at least 80% sequence identity with sequence number 49, preferably 90% sequence identity, more preferably Heterotetramers containing monomers having sequence identity of 95% or even 98% That is the case.

[0204] Furthermore, the present invention relates to nucleic acids and vectors encoding the Hex designer DNA recombinant enzyme of the present invention. include.

[0205] In a further embodiment, the present invention relates to medical use, particularly to the diseases mentioned above. Related genes or polynucleotides, or genes related to the biochemical signaling pathways mentioned earlier. DNA recombination of the present invention for the treatment of diseases or conditions associated with one of the polynucleotides This includes an enzyme, the nucleic acid or vector of the present invention, or the set / kit of the present invention.

[0206] In a similar embodiment, the present invention relates to the DNA recombinant enzyme, nucleic acid or vector of the present invention. The disease genes or polynucleotides mentioned above, including the ter, or the biochemical cells mentioned above. Treatment of diseases or conditions associated with one of the genes and polynucleotides related to the signaling pathway. This includes the preparation of pharmaceuticals for use in the present invention, or the use of the set / kit of the present invention, or the method of the present invention. nothing.

[0207] In this disclosure, particularly in the claims and / or paragraphs, the words "including" and "including" are used. The terms "and" and "including" have meanings that originate from this term in U.S. patent law. Please note that these terms may have the meanings of "including", "had included", and It can mean "contains," etc.; as well as "essentially consists of" and "essential." Terms such as "consisting of ~" have meanings that fall under U.S. patent law. For example, these terms allow for elements that are not explicitly stated, but which are fundamental or novel to the invention. Exclude elements found in prior art that affect the characteristics of these and others. Embodiments of the present invention are disclosed, or these will become apparent from the following detailed description and are otherwise disclosed. It is included.

[0208] Furthermore, the embodiments described in the present invention can be combined with each other. [Brief explanation of the drawing]

[0209] The present invention is illustrated by the following drawings and non-limiting embodiments: [Figure 1] This shows a general scheme for gene substitution (DRiGR) induced by the preferred DNA recombinase of the present invention. It shows different monomers R1-R4 of the DNA recombinase and their respective DNA binding sequences loxR1-R4 (sub-regions of the target site). The target site in the genome (frown) is replaced with the desired sequence (smile). [Figure 2] A general scheme for gene deletions (DRiGDs) induced by designer DNA recombinases is shown. Here, the binding of different monomers R1-R4 of the DNA recombinase to their respective DNA binding sequences loxR1-R4 is shown in the scheme above. Each target site has the same 8 bp spacer. Here, the target region (the frown) in the genome is cut out. [Figure 3] This illustrates that when a donor vector is present in excess of genomic DNA, gene substitution induced by DNA recombinases is driven toward the replacement of genomic DNA with the donor vector DNA. Preferably, the donor vector, such as an AAV vector, is present in multiple copies within the nucleus of the target cell to efficiently repair cellular mutations. [Figure 4]We demonstrate a general method for obtaining DNA recombinases for DRiGR by applying a novel directed evolutionary strategy (Duo-SLiDE DRiGR) that generates a pair of DNA recombinase monomers that function to jointly recombinate two different target sites (lox1, lox2) present in the genome, starting from a source vector with two libraries of DNA recombinases. Expression of a single recombinase in this system is also feasible (see Figure 15). R1, R2, and R3 represent three different restriction enzymes or their respective restriction sites. P1 and P2 represent two different PCR primers or their respective primer binding sites. When these monomers of DNA recombinases are expressed with a DNA template (here, a pDonor plasmid) also carrying the two target sites, DNA fragments are exchanged due to recombination at both target sites. Since the restriction site derived from the pDuoSLiDE vector is removed by the insertion of the DNA fragments, the effectively recombined recombinase can be amplified by PCR after DNA digestion with restriction enzymes. By periodically repeating this system in combination with random mutagenesis and DNA shuffling, the most efficient recombinase that facilitates the reaction can be identified. Using replication origins (R6K-ori) on inactive pDonors in the host, the processes of integration through one target site (e.g., lox1) and excision through the other target site (e.g., lox2) can be separated. Furthermore, the integrated vector can be identified by antibiotic selection on a kanamycin-containing plate. [Figure 5]We demonstrate a similar method for obtaining DNA recombinases for DRiGR by applying a novel directed evolutionary strategy (QuSLiDE DRiGR) that generates two pairs of DNA recombinase monomers that function to jointly recombinate two different target sites. Here, each monomer is guided from a source vector containing four libraries of DNA recombinases to one sub-site of one target site present in the genome (white triangles represent lox1, black triangles represent lox2) (R1, R2, and R3, as well as P1 and P2, have the same meaning as in Figure 4). If the DNA on the donor vector contains wild-type alleles and the genome contains disease-causing mutations, the gene defects are repaired in the cell (see Figure 1). [Figure 6] By applying a novel directed evolutionary strategy (Duo-SLiDE DRiGD), we demonstrate a general method for obtaining DNA recombinases for DRiGDs by generating a pair of DNA recombinase monomers that function to jointly recombinate two different target sites in the genome (white triangles represent lox1, black triangles represent lox2), starting from a source vector with two libraries of DNA recombinases (R1, R2, and R3, as well as P1 and P2, have the same meaning as in Figure 4). When these DNA recombinase monomers are expressed, the restriction site derived from the pDuoSLiDE vector is removed by recombination between two copies of the pDuoSLiDE vector. The effective recombinase that promotes the deletion can be amplified by PCR after restriction enzyme digestion of DNA. Periodically repeating this system in combination with random mutagenesis and DNA shuffling reveals the most efficient recombinase that promotes the reaction. [Figure 7]We demonstrate a similar method for obtaining DNA recombinases for DRiGDs by applying a novel directed evolutionary strategy (QuSLiDE DRiGD) that generates two pairs of DNA recombinase monomers that function to jointly recombinate two different target sites. Here, each monomer is derived from a source vector containing four libraries of DNA recombinases to one sub-site of one target site present in the genome (white triangles represent lox1, black triangles represent lox2) (R1, R2, and R3, as well as P1 and P2, have the same meaning as in Figure 4). This set of monomers makes it possible to delete the target site in the genome (see Figure 2). [Figure 8] We present a modern known method for obtaining a tailor-made recombinase that acts on one target site by substrate-associated protein evolution (SLiPE) (Buchholz F and Stewart AF, 2001). Here, the vector contains only one library and two identical target sites (dashed triangle). [Figure 9] The present invention is demonstrated for replacing mutations in exon 8 of the F9 gene using Duo-SLiDE DRiGR. Figure 9A shows a schematic representation of exon 8 and adjacent sequences of the F9 gene. Two selected target sequences (loxF9a—sequence number: 3 and loxF9b—sequence number: 4) are shown as triangles (black and white), with their nucleotide sequences and chromosome locations indicated. Figure 9B illustrates the method and substitution reaction for obtaining two recombinase monomers F9-1 ​​(sequence numbers: 1, 3, 5, and 28) and F9-2 (sequence numbers: 2, 4, 6, and 29). Confirmation that the recombination reaction occurred as predicted was confirmed by DNA sequencing. Figure 9C shows an agarose gel demonstrating the restriction patterns of pDuoF9 (source) (lane 1) and pDuoF9 (product) (lane 2) after digestion with NdeI and SacI. M = molecular marker. [Figure 10]This document describes an exemplary evolutionary process for adapting a library of DNA recombinases to a given target site—here, it was applied to the loxF9a and loxF9b target sites. Here, an active DNA recombinase on the loxF9a site (F9A) was obtained by designing two symmetric intermediate sites, AL and AR, and performing 20 or 17 cycles of SLiPE (see Figure 8). The resulting DNA recombinase libraries were combined and shuffled, and then 108 cycles of SLiPE were performed on the asymmetric loxF9a site (F9A). The agarose gels for the indicated generation cycles are shown. Lines with two triangles indicate unrecombined bands, and lines with one triangle indicate the size of the recombined bands. The gray triangles below the gel image indicate a decrease in the amount of L-arabinose added to the growth medium. To obtain an active DNA recombinase at the loxF9b site (F9B), a first set of two symmetric intermediate sites, BLS and BRS, was designed and subjected to 9 cycles of SLiPE (see Figure 8). After shuffling the DNA recombinase library, SLiPE was again performed on a second set of symmetric intermediate sites, BL (30 cycles) and BR (22 cycles). Finally, the obtained DNA recombinase libraries were combined and shuffled, and then subjected to 17 cycles of SLiPE at the asymmetric loxF9b site (F9B). Lastly, the two DNA recombinase libraries obtained at the loxF9a and loxF9b sites were cloned into pDuoF9 (source) (see Figure 9) (not shown). [Figure 11] The use of F9 designer DNA recombinase in a therapeutic setting is illustrated, illustrating the cloning of both F9-1 ​​and F9-2 coding sequences into a delivery vector (e.g., an adeno-associated virus vector) along with donor sequences containing loxF9a and loxF9b adjacent to the wild-type or Padua mutation (R338L) in exon 8 of the F9 gene. Delivery of multiple copies of such vectors to target cells replaces the inactivating mutation in the genome. [Figure 12]The results of screening for 8 bp repeat sequences in the human PCSK9 gene on chromosome 1 are shown. Exons of the PCSK9 gene are indicated by thick rectangles. The 8 bp repeat sequences are separated by at least 150 bp and no more than 2 kb, and are located in 34 bp long potential target regions (indicated by vertical lines) that do not appear anywhere else in the genome, as determined by the screening. Target sequences between potential target regions are indicated by horizontal lines. [Figure 13] Similar screening results are shown for 8 bp repeat sequences located at a 34 bp potential target site in the human papillomavirus 16 (HPV16) genome that is separated by at least 150 bp and no more than 2 kb, and does not appear in the human genome obtained through screening. Sequences encoding viral proteins are indicated by thick rectangles with arrows (larger than the symbols). [Figure 14] By applying a novel directed evolutionary strategy (SLiDE DRiGR 2.0) that generates a library of DNA recombinases that recombinate two different target sites in the genome (white triangles represent lox1, black triangles represent lox2), we demonstrate a different general method for obtaining DNA recombinases for use in Step 3 of Example 3. R1, R2, R3, R4, and R5 represent five different restriction enzymes or their respective restriction sites. P3 and P4 represent two different PCR primers or their respective primer binding sites. When this library of DNA recombinases is expressed in the presence of a DNA template (here, the pDonor-ex8 plasmid) also carrying two target sites, DNA fragments are exchanged by recombination of both target sites. Since the restriction site derived from the pF9 vector is removed by the insertion of the DNA fragments, the effectively recombined recombinase, which facilitates the substitution, can be amplified by PCR after digestion of DNA with restriction enzymes R1-3 and RecBCD. By periodically repeating this system in combination with random mutagenesis and by shuffling DNA, the most efficient recombinase that promotes the DRIGR reaction can be identified. [Figure 15]This section highlights the steps of the novel directed evolutionary strategy (SLiDE DRiGR) used in Step 3 of Example 3. In Sub-Step 1, bacteria are transformed with pF9 (source) and pDonor. Growth of antibiotic-resistant colonies can be observed only when recombinase expression is induced. In Sub-Step 2, the plasmid DNA of the obtained colonies is digested using restriction enzymes R1-R3. This enriches the colonies that successfully underwent DRIGR, yielding a pure preparation (product) of pF9. The purity of the plasmid preparation is demonstrated by restriction digestion using enzymes XhoI and XmaI, which exhibit the unique restriction pattern of pDuoF9 (product). Furthermore, the precise sequence of pF9 (product) is revealed by DNA sequencing. [Figure 16] We demonstrate a different general method for obtaining DNA recombinases for DRiGR by applying a novel directed evolutionary strategy (Duo-SLiDE DRiGR 2.0) that generates one or a pair of DNA recombinase monomers that function to jointly recombinate two different target sites present in the genome (white triangles represent lox1, black triangles represent lox2), starting from a source vector having one or two libraries of DNA recombinases. R1, R2, R3, R4, and R5 represent five different restriction enzymes or their respective restriction sites. P3 and P4 represent two different PCR primers or their respective primer binding sites. When these DNA recombinase monomers are expressed with a DNA template (here, a high copy number plasmid pDonor-ex8) also carrying the two target sites, DNA fragments are exchanged by recombination at both target sites. Since the restriction site derived from the pDuoSLiDE vector is removed by the insertion of the DNA fragments, the substitution-advanced effective recombinase can be amplified by PCR after DNA digestion with restriction enzymes. Importantly, antibiotic selection markers are not used in this assay. Periodically repeating this system in combination with random mutagenesis and DNA shuffling will reveal the most efficient recombinase that facilitates the reaction. [Figure 17]The alignment shows the latest Cre, the recombinase obtained after step 1 of Example 3 (R#1), the recombinase obtained after step 2 of Example 3 (3R#7-B5), and the recombinase obtained after step 3 of Example 3 (F9-3). The alignment shows what further mutations occurred during the later stages of evolution. [Figure 18] The general scheme of the method of the present invention, as exemplified by the DRiGD method and process of the present invention, is shown. [Figure 19] A more detailed scheme of the present invention's method, exemplified by the DRiGD method and applicable to the human genome, is shown. [Figure 20] A more detailed scheme of the present invention's method, exemplified by the DRiGD method and applicable to the human genome, is shown. [Figure 21] The DRIGG method of the present invention, applied to a target site on human chromosome 7, which will be further described in Example 5, is illustrated. A. Shows the target sites: Hex1 and Hex2 are target sites used for excision—HexL, HexR, HexR1, and HexR2 are intermediate target sites. B. Shows the evolution of specific recombinases at the Hex1 and Hex2 target sites. C. Shows the recombination efficiency of the two resulting clones (clone #7 and clone #30). [Figure 22] This demonstrates the precise excision of sequences from plasmid substrates using the recombinase obtained in Example 5. [Examples]

[0210] (Example 1:) To demonstrate the usefulness of DRiGD, the inventors have found that the data is separated by at least 150 bp. The human genome was screened for all 8 bp repeat sequences: up to 2 kb were missing from the genome. The potential target sites within the range to be eliminated do not appear anywhere else in the human genome. The left 13 bp and right 13 bp sequences of the bp repeat sequence are shown. Figure 12 shows DNA recombination in the human PCSK9 gene. The results for all 309 possible target sites of the enzyme are shown. Figure 13 shows the results for human papillomavirus (HPV). 16) The results for all 176 possible target sites of the DNA recombinases are shown.

[0211] (Example 2:) To demonstrate the feasibility of the DRiGR invention, the inventors have found that when applied together, it can lead to human IX We created a recombinase that can replace exon 8 of the factor (F9) gene. Exons frequently undergo mutations in patients with hemophilia B. The results provided the expression of a suitable donor vector and two recombinases that function in conjunction. This demonstrates that this exon can be efficiently substituted with a different sequence (Figure 6). (Figures 9 and 11).

[0212] Unless otherwise specified, WO 2008 / 083931 A1, WO 2011 / 147590 A1, WO2016034553 A1, Furthermore, the materials and methods described in the publications of Buchholz F and Stewart AF, 2001, are used.

[0213] DNA recombination with pre-identified target sequences (loxF9a—SEQ ID NO: 7 and loxF9b—SEQ ID NO: 8) The enzyme has a partial site of the target site (LoxP, LoxH, LoxM7, LoxM5, loxLTR, LoxBTR). Selected by comparing sequences in the togenome: [Table 6] The first (left) and second (right) sub-regions within the target region in the table are underlined.

[0214] In the first step, two recombinase libraries are subjected to the SLiDE approach described above. They were individually evolved using (Buchholz and Stewart, 2001 and WO 2002044409 A2) (See Figure 8). In short, each library is used to target intermediate sites (AL, AR, BLS). Evolved towards recombination of BLRS, BL, BR) and ultimately two target sites, loxF9a or loxF9 We achieved one recombination of b. New mutations were detected using error-prone PCR and DNA shuffling. The enzyme was introduced using a specific method, and the selective pressure was further regulated via the expression level of the recombinase. Recombinases possessing properties are recombined at their respective target sites and the target sites derived from the vector They were selected by their ability to delete adjacent sequences (see Figure 8).

[0215] Use the following intermediate array: [Table 7]

[0216] The code sequences of the recombinases derived from these two libraries are then vectorized. It was cloned into Kuborn pDuoF9 (source) (Sequence ID: 21, see Figure 9). This vector contains two recombinase libraries, which link the transcription of both enzymes together. Expression is initiated from a common inducible promoter that adopts a Peron-like structure. The size of this library... It has exceeded 100,000 clones.

[0217] To perform DRiGR, 50 μl of electrocompetent Escherichia coli (E. coli) XL1-Blu E cells were transformed with 1 ng pDuoF9 (source) library and 15 μg / ml chloramphenicol. The cultures were grown overnight in 200 ml LB containing the oat. The next day, 1 ml of the overnight culture was used with fresh medium 1. The culture was inoculated into 00 ml and allowed to grow for 2 hours. Subsequently, the culture was divided into 2 x 50 ml portions, and L-arabinose was added. The solution was added to achieve a final concentration of 50 μg / ml, and recombinase expression was induced in one of the cultures. After incubating the cells for 2.5 hours, place them on ice and electroporate them as described above. Prepared for rations (Sambrook and Russell, 2001). Electrocompi Resuspend the tent cells in 200 μl of water, and immediately add 0.4 ng pDonor to 50 μl of the cell suspension. It was used for transformation at 00 V. Subsequently, the bacteria were allowed to recover in SOC medium for 2 hours, and then the entire suspension was used. The cells were seeded on LB agar plates containing 15 μg / ml kanamycin.

[0218] When uninduced cells were seeded, the colonies did not grow even after being left on the agar plate overnight. Therefore, the expression of recombinase is required for integration of the pDonor vector (SEQ ID NO: 22). This was proven. All growing colonies from the induced samples were pooled and 15 μg / m² was added. The cells were cultured in 220 ml LB medium containing l-kanamycin and 50 μg ml L-arabinose, and the second pair Recombination was induced through the target site.

[0219] The following day, plasmid DNA was isolated from the culture. The clones that successfully underwent DRiGR were selected. To enrich the DNA, digest it with an enzyme that cleaves any non-pDuoF9 (product) - negative Selection—re-transformation was performed. For this purpose, 500 ng of plasmid preparation was mixed with 1 μl of NdeI and AvrII. The DNA was digested with PspXI, respectively. After incubation at 37°C for 3 hours, the DNA was precipitated. Then, it was resuspended in 50 μl of water. Of this, 1 μl was added to 50 μl of electrocompetent Used for the transformation of E. coli XL1 Blue cells. Transformed cells were treated with 15 μg / ml kanamai. Propagated in 220 ml LB containing - positive selection.

[0220] The following day, plasmid DNA was isolated from the culture. The recombinase library was placed under primer P The product was amplified by PCR using 1 and P2 (SEQ ID NOs: 23 and 24), and the PCR product was purified on a column. The recombinases were prepared and subjected to digestion using restriction enzymes SacI and SbfI. A recombinase library was then isolated. Next, we ligate the pDuoF9 (source) again and start another cycle of DRiGR. Three rounds of Duo-SLiDE DRIGR (see Fig. 10) were performed to target the loxF9 site. We enriched the recombinase that allows for efficient DRiGR in a and LoxF9b.

[0221] Primers used: [Table 8]

[0222] The sequence of plasmid pDuoF9 (product) is given as sequence number 25.

[0223] This method yielded the following pairs of recombinase monomer sequences: Rec F9-1: Sequence ID: 3 and Rec F9-2a: Sequence ID: 4 Rec F9-1b: Sequence ID: 5 and Rec F9-2b: Sequence ID: 6

[0224] (Example 3) (Step 1:) In the first step, two recombinase libraries are subjected to the SLiDE approach described above. They were individually evolved using (Buchholz and Stewart, 2001 and WO 2002044409 A2) (See Figure 8). Briefly, each library described in Example 2 is used as an intermediate target unit. Evolution towards recombination of the following positions (loxF9-AL, AR, BLS, BLRS, BL, BR - see Example 2): This ultimately led to the recombination of one of two target sites, loxF9a or loxF9b. Natural mutations are introduced using error-prone PCR and DNA shuffling, and the selective pressure is recombined. Further regulation was achieved through the expression level of the enzyme. The active recombinase was then adjusted for each These abilities involve rearranging the target site and deleting sequences adjacent to the target site derived from the vector. The selection was made by force (see Figure 8).

[0225] (Step 2:) In the second step, a single recombinase library is evolved to remove replication-deficient pDonors. DRiGR was performed according to the selection scheme used (see Figure 9). This involved vectors. Library created in step 1 of backbone pF9 (source) (= pDuoF9, array index: 21) This involves cloning of a recombinase coding sequence derived from [the vector]. 1 to 4 recombinase coding sequences are found in this vector. The combinase library adopts a common operon-like structure that links the transcription of both enzymes together. It can be expressed using an inducible promoter. The size of this library is 100,000 It surpasses 0 clones.

[0226] To perform DRiGR, 50 μl of electrocompetent E. coli XL1-Blue cells were added. The g pDuoF9 (source) library was transformed and 2 μg / ml chloramphenicol was added. The cultures were grown overnight in 00 ml of LB medium. The next day, 1 ml of the overnight culture was used to transfer the cultures to 100 ml of fresh medium. The culture was inoculated and allowed to grow for 2 hours. Subsequently, the culture was divided into 2 x 50 ml portions, and L-arabinose was added. The final concentration was set to 1-200 μg / ml, and recombinase expression was induced in one of the cultures. After incubating the cells for 2.5 hours, place them on ice and electroporate as described above. Prepared for use in (Sambrook and Russell, 2001). Electrocomputation Resuspend the cell in 200 μl of water, and immediately add 80 ng pDonor 1700 to the 200 μl cell suspension. It was used for transformation in V. The bacteria were then allowed to recover in SOC medium for 2 hours, and then the whole suspension was used. Use 15 μg / ml chloramphenicol, 5 μg / ml kanamycin, and 1-200 μg / ml L - Inoculated into 200 ml LB medium containing arabinose. To evaluate library size, a small amount was used. A portion of the suspended material was spread onto an agar plate.

[0227] If uninduced cells are seeded the following day, the colonies will not grow on the agar plate. This confirmed that the expression of recombinase was integrated into the pDonor vector (SEQ ID NO: 22). It was proven to be necessary for [the process]. Plasmid DNA was isolated from liquid culture. DRiGR was applied. To enrich the clones that were successfully created, the DNA was cut into arbitrary non-pDuoF9 (products). The cells were digested with an enzyme, negatively selected, and re-transformed. For this purpose, 500 ng of plasmid was added. The prepared samples were digested with 1 μl each of NdeI, AvrII, PspXI, and FspI. The samples were then incubated at 37°C for 3 hours. After incubation, the DNA is cleaned by microdialysis on a membrane filter. It was mixed up. Of this digest, 3 μl was added to 50 μl of electrocompetent E. coli XL1 - Used for the transformation of Blue cells. Transformed cells were given chloramphenicol at a dose of 15 μg / ml. And they were grown in 100 ml LB containing 15 μg / ml kanamycin—positive selection.

[0228] The following day, plasmid DNA was isolated from the culture, and 500 ng of DNA was diluted in 1 μl each of NdeI and AvrII. Digestion completed. Recombinase library with primers P1# and P2# (SEQ ID NOs: 30 and 31) The product was amplified using the error-prone PCR method, the PCR product was purified on a column, and restriction enzymes were used. Digestion was performed using SacI and SbfI. The isolated recombinase library was then processed using pDuoF9(S We re-established the system and started another cycle of DRiGR. 3 and 8 cycles After the procedure, DNA shuffling was performed as described above (Buchholz and Stewart, 2001). 11 rounds of Duo-SLiDE DRIGR were performed to efficiently achieve DRiGR at target sites loxF9a and loxF9b. We enriched the recombinase that can be implemented in a targeted manner.

[0229] (Step 3:) In the third step, a single recombinase library is evolved to produce high copy number plasmids. Using pDonor-ex8 (see Figure 14, Sequence ID: 37), selection by antibiotic and The integration of the Nervectors is performed without separating it from the division, according to different selection schemes (Figure 14). (See reference), DRiGR 2.0 was implemented. This includes the vector backbone pF9 (source). (= pDuoF9(source) Sequence ID:21) The library created in step 2 of this process This involves cloning the Binase code sequence. Subsequently, the library already has pDonor-ex8. The cells were transformed into electrocompetent E. coli XL1-Blue cells. 15 μg of transformed cells / ml chloramphenicol, 15 μg / ml kanamycin, and 1-200 μg / ml L-arabinose The cells were grown in 100 ml LB medium containing [the specified substance]. The size of this library exceeded 100,000 clones. I did it.

[0230] The following day, plasmid DNA was isolated from the culture, and 1000 ng of DNA was divided into 1 μl each of NdeI, AvrII, and P. spXI (restriction enzymes R1, R2, and R3—see Figures 14 and 15), and exonucleases The recombinase library was digested using primers P3 and P4 (SEQ ID NO: 32 and The product was amplified by error-prone PCR using 33), and the PCR product was purified on a column. The enzymes SacI and SbfI were used for digestion. Subsequently, the isolated recombinase library was used. Re-ligated to pF9 (source), and the electro-competent large pDonor-ex8 Enterococcus XL1-Blue cells were transformed. The transformed cells were treated with 15 μg / ml chloramphenicol, 5 Grow in 100 ml LB medium containing μg / ml kanamycin and 1-200 μg / ml L-arabinose. This was done. In this way, the next cycle was started. DNA shuffling was performed every 3 cycles. The procedure was carried out as described above (Buchholz and Stewart, 2001). 11 rounds of SLiDE DRI GR (see Fig. 14) was performed, and without antibiotic selection, the donor vector Efficiently perform DRiGR at target sites loxF9a and LoxF9b without separating the ter integration from the partition. The recombinase that can be used in this process was enriched.

[0231] Primers used: [Table 9]

[0232] This method yielded the following recombinase monomer sequences: After step 1, R#1 (sequence number: 34); after step 2, R#7-B5 (sequence number: 35), and the final step 3. Following that, Rec F9-3 (Sequence number: 36).

[0233] The recombinase efficiency (DRGR efficiency) of the recombinase obtained after steps 1, 2, and 3 is as follows: Compare in the table below: [Table 10]

[0234] (Example 4) To utilize F9 recombinase in therapeutic settings, both F9-1 ​​and F9-2 codes are used. The sequences loxF9a and l adjacent to the wild-type or Padua mutation (R338L) in exon 8 of the F9 gene. A delivery vector (e.g., an adeno-associated virus vector) along with a donor sequence containing oxF9a. Cloning into such vectors. Delivery of multiple copies of such vectors to target cells is possible with the genome. Replace the inactivating mutation inside (see Figure 11).

[0235] (Example 5) To demonstrate the usefulness of DRiGD, a specific DNA sequence on chromosome 7 of the human genome will be excised. We evolved a recombinase for this purpose.

[0236] Firstly, two different recombinase libraries were described as part of a study on substrate-associated directional evolution. Tokol (See Buchholz and Stewart's literature, 2001 and WO 2002044409 A2—Figure 8) The library was evolved using the left and right subregions (He) of the final target region Hex1 and Hex2. Evolved for xL or HexR (see Figure 21A). Recombined for HexR. To evolve the ZERA library, two intermediate target sites (HexR1 and HexR2) were used (Figure 2). (See 1B). Throughout the entire evolutionary process, new mutations are made more easily than they should be. The DNA was introduced and selected by pancreatic PCR (using primers P1# and P2#) and DNA shuffling. The pressure was further regulated via the expression level of recombinase. Active recombinase By rearranging each target site, the sequence adjacent to the target site derived from the vector is deleted. The selection was based on their respective capabilities (see Figure 8—Standard SLiPE and Figure 21B).

[0237] Next, in order to recombinate the final target sites Hex1 and Hex2, both libraries (HexR and HexL) are used. (obtained about) was combined. Therefore, the library (library size is 100,000 recon Cloning a larger enzyme (than vinase) into the same expression plasmid and combining the transcriptions of both enzymes It was expressed from a common inducible promoter that adopts a binding operon-like structure (pDUO- in Figure 6). (See SLiDE DRiGD). Next, transfer the plasmid containing both libraries to XL-1 The cells were transformed into blue E. coli cells. After allowing the cells to recover in 1 ml of SOC medium for 1 hour, 10 μl was added to 15 ml of medium. The cells were seeded on LB agar plates containing μg / ml chloramphenicol. The following day, 32 clones were selected. The cells were incubated for 8 hours in 500 μl LB containing 25 μg / ml chloramphenicol. Next, 250 μl The pre-culture was used to inoculate 2 × 5 ml LBs containing 25 μg / ml chloramphenicol. In one of the 5 ml cultures, in order to induce the expression of the recombinase dimer, 1 0 μg / ml L-arabinose was added. The next day, plasmid DNA was isolated and SacI(R2) and SbfI The samples were digested using (R3), and the recombination efficiency in the induced and non-induced samples was evaluated.

[0238] This method allows for the efficient excision reaction using two recombinases. Dimers (clones #7 and #30) were obtained. Precise excision was performed using recombinant pDUO-SLiDE DR. This was confirmed by sequencing iGD—see Figure 22.

[0239] The target site, intermediate target site, and primer used are listed in the table below: [Table 11]

[0240] This method yielded the following pairs of recombinase monomer sequences: Hex-R-#7: Sequence ID: 46 and Hex-L-#7: Sequence ID: 47 Hex-R-#30: Sequence ID: 48 and Hex-L-#30: Sequence ID: 49

[0241] Cited non-patent literature [ka] This application provides the invention in the following embodiments. (Aspect 1) DNA recombination enzymes can induce site-specific DNA recombination of target sequences in the genome. A method for identifying sequences that are potential target sites: i. The genome or portion thereof containing the target sequence, with a length of at least 5 bp and a maximum of 12 bp. A step of screening for two sequences that are potential spacer sequences, One of the potential spacer sequences is upstream of the sequence in question, and the potential spacer The other sequence is downstream of the sequence in question, and the two sequences are at a maximum distance of 100 kb and 1 The process of taking a minimum distance of 50 bp; ii. For each potential spacer array, the adjacent nucleotides on one side of them Preferably, 10 to 20 nucleotides form a potential first subsite, and the other Adjacent nucleotides on the side, preferably 10 to 20 nucleotides, more preferably 12 to 15 nucleotides The potential target is determined by the creotide to form a potential second sub-site. A step of identifying the target area, wherein both potential sub-parts and the space between them The process includes forming the potential target site by a siphon arrangement, iii. Further screening of the potential target sites identified in step ii. for host genotypes A potential target sequence that does not appear anywhere in the sequence, and therefore guarantees sequence-specific deletion. Select the method described above. (Aspect 2) This method induces site-directed DNA recombination to recombine two naturally occurring target sequences in the genome. This allows for the modification of the nucleotide sequence in the genome through designer DNA recombination. A method for preparing enzymes: a) The nucleotide sequence upstream of the nucleotide sequence to be modified is used as the first target site, and Furthermore, a nucleotide sequence downstream of the nucleotide sequence to be modified is selected as the second target site. This is a process in which the arrangement of these target sites is not identical, and each target site is 5 to 1 The first portion consists of 10-20 nucleotides each, separated by a 2-nucleotide spacer sequence. Including the part and the second subpart, the process described in Embodiment 1 is performed before step a), or as part thereof. The aforementioned process; b) A vector comprising the first target site and the second target site selected as substrates in a). —to apply molecularly directed evolution to at least one library of DNA recombinant enzymes This process includes, a) At least one which is active against the first target site and the second target site selected in a) The method thereof, which is carried out until the designer DNA recombinant enzyme is obtained. (Aspect 3) The DNA recombinase is suitable for deletion, and the (potential) spacer sequences are identical. A method according to one embodiment 1 or 2. (Aspect 4) The DNA recombinase is suitable for substitution, and the (potential) spacer sequences are identical. No, the method described in embodiment 1 or 2. (Aspect 5) This method induces site-directed DNA recombination to recombine two naturally occurring target sequences in the genome. This allows for the substitution of nucleotide sequences in the genome using designer DNA recombination. A method for preparing enzymes: a) The nucleotide sequence upstream of the nucleotide sequence to be modified is used as the first target site, and Furthermore, a nucleotide sequence downstream of the nucleotide sequence to be modified is selected as the second target site. This is a process in which the arrangement of these target sites is not identical, and each target site is 5 to 1 The first portion consists of 10-20 nucleotides each, separated by a 2-nucleotide spacer sequence. The process including the part and the second subpart b) A vector comprising the first target site and the second target site selected as substrates in a). —to apply molecularly directed evolution to at least one library of DNA recombinant enzymes This process includes, Here, step b is to select the first target site and the recombination between the vector and the synthetic sequence. The procedure is carried out in the presence of the synthetic sequence including the second target site, a) At least one which is active against the first target site and the second target site selected in a) The method thereof, which is carried out until the designer DNA recombinant enzyme is obtained. (Aspect 6) The nucleotide sequence to be modified is a mutation, particularly a point mutation or frameshift mutation. The method according to any one of embodiments 1 to 5, wherein the sequence includes anomalies, deletions, or insertions. (Aspect 7) The vector used in step b) contains at least one library of the DNA recombinant enzymes A negative selection marker located between the first and second target sites, more preferably k is an expression vector containing at least one specific recognition site of a restriction enzyme, embodiments 1-6 The method described in any one of the items. (Pattern 8) The synthetic sequence includes a positive selection marker located between the first and second target sites. The method according to any one of embodiments 5 to 7 for preparing a designer DNA recombinant enzyme suitable for substitution. Law. (Aspect 9) Process b) consists of three sub-processes: i) A sub-step of evolving at least one of the libraries without artificial sequencing; ii) The at least one library obtained as a result of step i) is positively selected A secondary process that uses an artificial sequence containing a maker to induce evolution; iii) As a result of step ii), the at least one library is positively selected as a marker. Designer DNA recombination suitable for substitution is performed in a sub-step using an artificial sequence that is not present in the original DNA. A method for preparing an enzyme according to any one of embodiments 5 to 8. (Aspect 10) Designer DNA recombination can be obtained by the method described in any one of the descriptions in 1 to 9. enzyme. (Aspect 11) It comprises at least two different monomers, where the enzyme naturally appears in the genome. By rearranging two target sequences, site-directed DNA recombination in the genome of the host organism is achieved. It is possible to induce the target site, and the arrangement of the target sites is not the same, preferably one of embodiments 1 to 9 Designer D, preferably according to embodiment 10, can be obtained by the method described in any one of the items. NA recombinant enzyme. (Aspect 12) The monomer is arranged according to sequence number 1 (Rec F9-1), or sequence number 1, or preferably a sequence Column number: 28, sequence number: 3, or sequence number: 5, with at least 95% sequence identity, preferred. It contains a sequence with 98% sequence identity, and the monomer is sequence number 2 (Rec F9-2) Sequence, or sequence number 2, or preferably sequence number 29, sequence number 4, or sequence Number: 6 and at least 90% sequence identity, preferably 95% sequence identity, more preferably 98% A designer DNA recombinant enzyme according to embodiment 10 or 11, comprising a sequence having sequence identity. (Aspect 13) Sequence ID: 1 (Rec F9-1), and / or Sequence ID: 2 (Rec F9-2), or sequence number Code number: 36 (Rec F9-3), or sequence number: 46 (Hex-R-#7), and / or sequence number: 47 (Hex-L-#7), or sequence number: 48 (Hex-R-#30), and / or sequence number: 49 (Hex- Sequence by L-#30), sequence number: 1 and / or 2, or sequence number: 36, or sequence Number: 46, and / or Sequence ID: 47, or Sequence ID: 48, and / or Sequence ID: :49, or preferably at least one selected from sequence numbers:28, 29, or 3-6 The sequence and at least 90% sequence identity, preferably 95% sequence identity, more preferably 98% Designer DNA recombinant enzymes containing sequences with % sequence identity. (Aspect 14) Site-directed DNA recombination to modify the target nucleotide sequence is the first target site. The second target site is induced in the genome by rearranging the second target site, and both target sites are in the genome They appear naturally in the environment, and they are the designer DNA recombinant enzyme or the DNA recombinant enzyme By introducing the encoding nucleic acid or vector, it becomes not identical, and here the target part The position borders the aforementioned target sequence, and each target site has a space of 5 to 12 nucleotides. It contains a first and second sub-region of 10-20 nucleotides separated by a siphon sequence. M, Designer DNA Recombinant Enzyme as described in any one of the embodiments 10 to 13 for use in pharmaceuticals for use in pharmaceuticals encoding DNA recombinant enzymes as described in any one of embodiments 9 to 11 Nucleic acid or vector. (Aspect 15) Both target sites contain the same spacer sequence, and the target sequence is missing due to recombination. Loss of designer DNA recombinant enzyme or nucleic acid or for use in pharmaceuticals according to embodiment 14 vector. (Aspect 16) Both target sites include spacer sequences that are not identical, and further border both desired sequences. A synthetic donor sequence containing the target site is provided, and the target sequence is recombined to obtain the desired configuration. Designer DNA recombinant yeast for use in pharmaceuticals according to embodiment 14, which is replaced by columns Elements, nucleic acids, or vectors. (Aspect 17) Nucleic acid or vector encoding a designer DNA recombinant enzyme as described in any one of embodiments 10 to 13. —. (Aspect 18) Sequence ID: 7 (loxF9a) and Sequence ID: 8 (loxF9b), and at least one of Sequence IDs: 7 or 8 Nuclei selected from sequences having 90% sequence identity, or nucleic acid sequences that are inversely complementary to them. acid. (Aspect 19) Host cells, host organs, or non-nucleic acids or vectors as described in Embodiment 17 or 18 A human host organism and / or a designer DNA recombinant enzyme according to any one of embodiments 10 to 13. (Aspect 20) By rearranging the first and second target sites, the target nucleo in the genome is created. The cydone sequence is modified, where both target sites appear naturally in the genome and they are Designer DNA recombinant enzyme as described in any one of items 10 to 13 or as described in any one of items 10 to 13 By introducing nucleic acids or vectors that encode the DNA recombinant enzyme listed, the DNA becomes identical. A method for inducing site-specific DNA recombination without causing the aforementioned site-specific DNA recombination. (Aspect 21) Selecting the first and second target sites which have different spacer arrangements, and the first The target is further provided by providing a synthetic sequence including the target site and the second target site. This induces substitution of nucleotide sequences, where the first and second target sites border the desired sequence. By selecting the first and second target sites which include the same spacer array, The method according to embodiment 20, which induces deletion of the target nucleotide sequence. (Aspect 22) Site-directed DNA recombination by rearranging two naturally occurring target sequences in the genome. A kit for replacing a target sequence in the genome: —Designer DNA recombinant enzyme described in any one of the descriptions in Actuals 10 to 13 or the description in Actuals 17 or 18 Nucleic acid or vector; —to replace the target sequence adjacent to the first target site and the second target site, The kit comprises a synthetic sequence containing a desired sequence. (Aspect 23) The method described in any one of embodiments 10 to 13, which catalyzes site-specific DNA recombination between two recognition sites. Use of designer DNA recombinant enzymes. (Aspect 24) Cells or multicellular cells having the aforementioned target sequence knocked out or modified. The use described in embodiment 23 for producing living organisms. (Aspect 25) Whether the aforementioned target site is the same as sequence number 7(loxF9a) and sequence number 8(loxF9b), The use according to embodiment 23, wherein the sequence is selected from sequences that are inversely complementary to these. (Aspect 26) Designer DNA recombinant enzyme according to any one of the embodiments 10 to 13 or nucleic acid according to embodiment 9 This is a pharmaceutical composition containing a vector.

Claims

1. A designer DNA recombinant enzyme comprising at least two different monomers, The enzyme can induce site-directed DNA recombination in the genome of a host organism by recombining two naturally occurring target sequences in the genome, wherein the target sequences are not identical, and the designer DNA recombinase can recognize target sites having sequences that are identical to or inversely complementary to sequence numbers 7 (loxF9a) and 8 (loxF9b). The first monomer contains a sequence having at least 95% sequence identity with sequence number 3, and the second monomer contains a sequence having at least 95% sequence identity with sequence number 4, or The designer DNA recombinase wherein the first monomer contains a sequence having at least 95% sequence identity with SEQ ID NO: 5, and the second monomer contains a sequence having at least 95% sequence identity with SEQ ID NO:

6.

2. The designer DNA recombinant enzyme according to claim 1, wherein the target sequence borders a target nucleotide sequence, and each of the two target sequences includes a target region comprising a first subregion and a second subregion having 10 to 20 nucleotides, separated by a spacer sequence having 5 to 12 nucleotides.

3. It is a designer DNA recombinase, (i) A sequence comprising the sequence assigned to sequence number 46 (Hex-R-#7), or a sequence having at least 95% sequence identity with sequence number 46, and a sequence assigned to sequence number 47 (Hex-L-#7), or a sequence having at least 95% sequence identity with sequence number 47, and capable of recognizing sequence number 40 (Hex1) and sequence number 41 (Hex2), or (ii) A sequence comprising the sequence assigned to sequence number 48 (Hex-R-#30), or a sequence having at least 95% sequence identity with sequence number 48, and a sequence assigned to sequence number 49 (Hex-L-#30), or a sequence having at least 95% sequence identity with sequence number 49, and capable of recognizing sequence number 40 (Hex1) and sequence number 41 (Hex2); or (iii) The designer DNA recombinant enzyme comprising the sequence of sequence number 36 (Rec F9-3), or a sequence having at least 95% sequence identity with sequence number 36, and capable of recognizing sequence number 7 (loxF9a) and sequence number 8 (loxF9b).

4. A nucleic acid or vector encoding a designer DNA recombinant enzyme according to any one of claims 1 to 3.

5. A pharmaceutical composition comprising a designer DNA recombinant enzyme according to any one of claims 1 to 3, or a nucleic acid or vector according to claim 4.

6. A pharmaceutical composition for inducing site-directed DNA recombination, which modifies a target nucleotide sequence in the genome by rearranging a first target site and a second target site, The pharmaceutical composition comprises a designer DNA recombinant enzyme according to any one of claims 1 to 3, or a nucleic acid or vector according to claim 4.

7. The pharmaceutical composition according to claim 6, wherein both the first target site and the second target site contain the same spacer sequence, and the site-specific DNA recombination results in the deletion of the target nucleotide sequence.

8. A synthetic donor sequence is provided which includes a spacer sequence in which both the first target site and the second target site are not identical, and further includes both the first target site and the second target site bordering a desired sequence, wherein the site-directed DNA recombination results in the substitution of the target nucleotide sequence by the desired sequence, according to claim 6.

9. A host cell, a non-human host organ, or a non-human host organism comprising a designer DNA recombinant enzyme according to any one of claims 1 to 3, or a nucleic acid or vector according to claim 4, The host cell, non-human host organ, or non-human host organism is one in which the host cell does not contain human gametes or human germ cells.

10. A method for inducing site-directed DNA recombination, which modifies a target nucleotide sequence in a non-human genome in vitro by rearranging a first target site and a second target site, The method comprises the step of introducing a designer DNA recombinant enzyme according to any one of claims 1 to 3 or a nucleic acid or vector according to claim 4, wherein both the first target site and the second target site are naturally occurring in the non-human genome and are not identical.

11. A method according to claim 10, for in vitro inducing the substitution of the target nucleotide sequence by selecting the first target site and the second target site such that the spacer sequences are different, and further providing a synthetic sequence including the first target site and the second target site, The method wherein the first target region and the second target region border a desired arrangement.

12. The method according to claim 10, wherein a deletion of the target nucleotide sequence is induced in vitro by selecting the first target site and the second target site to contain the same spacer sequence.

13. A pharmaceutical composition for inducing site-directed DNA recombination, which modifies a target nucleotide sequence in a non-human genome in vitro by rearranging a first target site and a second target site, The pharmaceutical composition comprises a designer DNA recombinant enzyme according to any one of claims 1 to 3 or a nucleic acid or vector according to claim 4, wherein both the first target site and the second target site are naturally occurring and not identical in the non-human genome.

14. A kit for substituting a target nucleotide sequence in a non-human genome by site-directed DNA recombination by rearranging a first target site and a second target site, wherein both the first and second target sites naturally occur in the same genome, and the kit is: A designer DNA recombinant enzyme according to any one of claims 1 to 3, or a nucleic acid or vector according to claim 4; and A synthetic sequence containing a desired sequence that replaces the target sequence adjacent to the first target site and the second target site, The kit includes the above.

15. The use of a designer DNA recombinase according to any one of claims 1 to 3, which catalyzes site-specific DNA recombination between a first target site and a second target site in vitro, The use thereof, wherein the site-specific DNA recombination is not in human gametes or human germ cells.

16. The use according to claim 15, which involves in vitro producing cells or multicellular non-human organisms having a sequence in which the target nucleotide sequence is knocked out or modified.

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