Site-specific recombinases for efficient and specific genome editing

Abstract

The present invention relates generally to the field of genome editing, provides methods for producing DNA recombinases that efficiently and specifically recombine genomic target sequences with obligate DNA recombinase enzymes, and provides DNA recombinases. More specifically, the present invention provides an engineered DNA recombinase for efficient and specific genome editing, comprising an obligate complex of recombinase, said complex comprising at least a first recombinase enzyme and at least a second recombinase enzyme, said first recombinase enzyme and said second recombinase enzyme specifically recognizing a first half-site and a second half-site of an upstream target site and / or a downstream target site of a DNA recombinase, said first recombinase enzyme and said second recombinase enzyme each comprising at least one mutation in a catalytic site, said first recombinase enzyme and said second recombinase enzyme carrying said at least one mutation in a catalytic site do not exhibit catalytic activity of a DNA recombinase when expressed alone, and said catalytic activity as a DNA recombinase is complemented in the obligate complex of recombinase. The present invention further relates to nucleic acid molecules encoding said engineered DNA recombinases and obligate complexes, and to the use of said engineered DNA recombinases and obligate complexes and nucleic acid molecules in pharmaceutical compositions.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates generally to the field of genome editing and provides DNA recombinases that efficiently and specifically recombine genomic target sequences with obligate DNA recombinase enzymes. More specifically, the present invention provides an engineered DNA recombinase for efficient and specific genome editing, comprising an obligate complex of recombinase, said complex comprising at least a first recombinase enzyme and at least a second recombinase enzyme, said first recombinase enzyme and said second recombinase enzyme specifically recognizing a first half-site and a second half-site of an upstream target site and / or a downstream target site of a DNA recognition site of a DNA recombinase, said first recombinase enzyme and said second recombinase enzyme each comprising at least one mutation in a catalytic site, said first recombinase enzyme and said second recombinase enzyme carrying said at least one mutation in a catalytic site do not exhibit the same catalytic activity of a DNA recombinase when expressed alone, and in the obligate complex of recombinase the catalytic activity as a DNA recombinase is complemented. The present invention also discloses an obligate complex of recombinases that catalyzes the recombination of DNA sequences present in the int1h region on the human X chromosome. The present invention further relates to nucleic acid molecules encoding said engineered DNA recombinases and obligate complexes, and the use of said engineered DNA recombinases and obligate complexes and nucleic acid molecules in genome editing. Furthermore, the present invention provides methods for producing said obligate DNA recombinases.

[0002] The project leading to this application has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 742133). [Background technology]

[0003] BACKGROUND OF THEINVENTION Genome engineering is becoming an increasingly important technique in biomedical research. Today, the main approach in the field of gene editing is the introduction of nuclease-mediated double-strand breaks (DSBs) at loci of interest that are subsequently corrected by cellular repair pathways. There are four types of programmable nucleases, which can be divided into two groups based on the mode of target DNA sequence recognition. Meganucleases, zinc finger nucleases (ZFNs) and transcription activator-like nucleases (TALENs) use protein-DNA interactions to guide the nuclease to specific loci, whereas clustered, regularly interspaced, short-palindromic repeat-associated (CRISPR) endonucleases direct it using RNA-DNA interactions (34, 35). Programmable nucleases are candidates for therapeutic applications, and some of them are already in clinical trials (36, 37, 38).

[0004] However, one of the main challenges of programmable nucleases is the risk of unpredictable sequence rearrangements. Introduced DSBs are mainly repaired by cells using non-homologous end joining (NHEJ) or homology-directed repair (HDR). Repair by HDR is accurate and maintains genome stability, since the sequence is copied from a second allele or donor sequence that matches the target. However, HDR is mainly active during DNA replication, and NHEJ events outnumber HDR in most cells. NHEJ is an error-prone repair mechanism that leads to insertions and deletions (indels) in the repaired DNA fragments. This can result in adverse events due to alterations in gene sequences (34; 39, 40).

[0005] Alternative tools widely used for genome engineering include site-specific recombinases (SSRs) from the tyrosine recombinase family. Tyrosine SSRs have considerable advantages over programmable nucleases, as they perform the complete recombination reaction without cofactors and therefore do not depend on cellular DNA repair pathways (2). This leads to highly specific, predictable and precise genome editing events, making them attractive for therapeutic applications.

[0006] One of the most commonly used SSRs is Cre, a tyrosine site-specific recombinase (SSR) that forms a homotetramer that precisely catalyzes the recombination of DNA between loxP target sites (1). The loxP sequence consists of two 13-bp palindromic half-sites flanking an 8-bp spacer where recombination occurs (Figure 1A). The Cre / loxP system is commonly used for genome modification due to its precision and robustness in orthologous hosts (2). However, application of Cre / loxP across organisms requires prior introduction of loxP target sequences into the host genome, which can be time-consuming and laborious (3, 4). To overcome this limitation and broaden the use of SSRs, directed molecular evolution has been used to generate Cre variants with altered DNA specificity for predetermined (pseudo)symmetric DNA target sites naturally occurring within the host genome (5–10). The utility of this approach was first demonstrated when an evolved Cre-like enzyme, Tre, was generated that specifically recognized and excised HIV-1 proviral DNA from the human genome (7). Tre and other evolved SSRs represent the potential for site-specific recombinases to be adapted for a wide range of therapeutic applications ( 8 , 11 , 12 ).

[0007] Substantial progress has been made in engineering novel SSRs capable of recombining at a range of DNA substrates, including asymmetric sites (5, 9, 10) (13-15). Recombination at asymmetric target sites was first demonstrated by co-expression of wild-type and mutant Cre molecules that together catalyze recombination between artificial asymmetric loxP-loxM7 sites, proving proof of concept that engineered heterospecific SSRs could be generated (5). This general principle has recently been extended to achieve recombination between naturally occurring asymmetric target sequences in the human genome by combining two evolved Cre variants (9). Cre variants, each with unique half-site specificity, were first generated via directed evolution at their respective symmetric sites. The separate variants were then expressed together to form functional heterotetramers capable of specifically excising DNA fragments adjacent to the desired asymmetric human target site (9). More recently, this technique has been demonstrated to be applicable to correct chromosomal inversions that cause genetic human disorders (10). By combining two designer recombinases targeting an asymmetric loxF8 sequence located on the human X chromosome, Lansing et al. showed that a heterotetramer (D7) could efficiently correct a genomic int1h inversion to re-establish factor VIII expression in patient-derived cells. (10) The D7 SSR is composed of two unique Cre-type subunits, D7L and D7R, which have been evolved to bind to their corresponding half-sites, loxF8L and loxF8R, respectively.

[0008] However, using two or more recombinases with different target specificities inherently carries risks. By using several Cre-derived recombinases with different specificities, the chances of subunit assembly into undesired functional complexes, including homotetramers that can trigger recombination events at non-target sites, are increased. To mitigate these potential off-target effects, approaches that ensure only the formation of heterotetramers are important to increase their safety in therapeutic applications (10). Previously, prevention of homotetramer formation was achieved by structure-guided redesign of several residues involved in the protein-protein interaction interface between different recombinase monomers (16). This approach to generate obligate SSR systems is therefore limited to enzymes with available crystal structures and is therefore not easily adaptable to engineered or distantly related recombinases.

[0009] WO 2021 / 110846 discloses a fusion protein for efficient and specific genome editing comprising a complex of recombinases comprising at least a first recombinase enzyme and a second recombinase enzyme, the first recombinase enzyme and the second recombinase enzyme being interconnected via an oligopeptide linker. The efficiency of these fusion proteins depends on the properties of the linker.

[0010] US Patent No. 10,017,832 B2 discloses DNA recombinases produced by introducing several mutations into the protein-protein interface between DNA recombinase monomers to form a so-called obligate heterotetrameric complex. However, this approach has the drawback of requiring extensive efforts to introduce several mutations into each of the recombinase enzymes, and also leads to a reduction in their recombinase activity compared to wild-type enzymes that do not contain mutations to allow the formation of obligate heterotetrameric complexes. In addition, it is not straightforward to create obligate versions of other naturally occurring enzymes, since the target residues are not well conserved in related recombinases. Summary of the Invention [Problem to be solved by the invention]

[0011] (Summary of the invention) It is therefore an object of the present invention to overcome the drawbacks of the prior art and to provide a complex of recombinases having improved properties, in particular a catalytic activity allowing efficient recombination events, increased specificity and / or reduced activity at non-target or off-target sites.

[0012] This problem is solved by providing an engineered DNA recombinase for efficient and specific genome editing, comprising an obligate complex of recombinase, said complex comprising at least a first recombinase enzyme and at least a second recombinase enzyme, said first recombinase enzyme and said second recombinase enzyme specifically recognizing a first half-site and a second half-site of an upstream target site and / or a downstream target site of a DNA recombinase, said first recombinase enzyme and said second recombinase enzyme respectively comprising at least one mutation in their catalytic domain, said first recombinase enzyme and said second recombinase enzyme carrying said at least one mutation do not exhibit catalytic activity of a DNA recombinase when expressed alone, and wherein the catalytic activity as a DNA recombinase is complemented in the obligate complex of recombinase. [Means for solving the problem]

[0013] The present invention is not based on the redesign of the protein-protein interface between DNA recombinase monomers, as disclosed, for example, in US Patent No. 10,017,832 B2. In contrast to the prior art, at least one mutation in the catalytic region of the first recombinase enzyme and in the catalytic region of the second recombinase enzyme is not located in a region involved in protein-protein interactions between the recombinase enzymes or in the DNA binding region, thereby avoiding undesired interference with protein-protein complex formation and target recognition. Advantageously, the recombinase complex of the present invention is active at the desired target site only when the correct subunits come together and complement each other's catalytic activities, thereby forming an active complex.

[0014] Preferably, said at least one mutation is a point mutation in the form of an amino acid substitution leading to a substitution of an amino acid in the protein sequence of the first recombinase enzyme and a substitution of an amino acid in the amino acid sequence of the second recombinase enzyme. Most preferably, the at least one point mutation is a single amino acid substitution in the catalytic domain of each recombinase. According to a preferred embodiment, the first recombinase enzyme and the second recombinase enzyme do not have point mutations at the same position or of the same type. By not having point mutations of the same type, it is meant that if the mutation is at the same position in the first and second recombinases, the first amino acid substituted for the amino acid at this position in the first recombinase is different from the second amino acid substituted for the amino acid at this position in the second recombinase.

[0015] Tyrosine site-specific recombinases represent versatile genome editing tools with considerable therapeutic potential. The recent development of engineering and evolving SSRs into heterotetramers to improve target site flexibility has represented an important step towards their broad application in genome editing. However, tyrosine site-specific recombinase monomers tend to form different homo- and heterotetramer combinations in cells, increasing their off-target potential. Thus, the present invention provides two pairs of mutations that target residues involved in catalysis and lead to a simple obligate system of tyrosine site-specific recombinase. Only when the pair of mutations are applied as single mutations on each recombinase subunit (e.g., the first and second recombinase enzymes), the engineered tyrosine site-specific recombinase can efficiently recombine the intended target sequence, whereas the monomers carrying the point mutations are inactive when expressed alone. The utility of the obligate system of tyrosine site-specific recombinase to improve the recombination specificity of designer recombinases against therapeutic targets in human cells is demonstrated herein. Moreover, the mutation has been shown to make certain naturally occurring tyrosine site-specific recombinases, such as Cre, Vika, Panto, and Dre, obligate, which provides a simple method to improve their application properties.The present invention contributes to the development of safe and effective therapeutic designer recombinases and to the promotion of mechanistic understanding of catalysis by tyrosine site-specific recombinases.Therefore, undesirable side reactions, i.e., off-target recombination events, can be effectively avoided.The target specificity of engineered DNA recombinases is further increased compared to the methods known in the prior art.

[0016] A drawback of mutations in the interface between monomers (e.g., as disclosed in U.S. Pat. No. 10,017,832 B2) is a reduced recombination activity of the engineered recombinase compared to the wild-type enzyme. In contrast, the engineered recombinases of the present invention, which contain at least a single mutation in the catalytic site, do not exhibit such a loss of recombination activity. It is demonstrated herein that the recombination activity of the engineered recombinases of the present invention with the above-mentioned greatly improved target specificity is comparable to that of the wild-type enzyme. Effect of the Invention

[0017] According to one aspect, the present invention provides a method for producing an obligate DNA recombinase for genome editing, comprising the steps of: i. providing a nucleic acid molecule encoding a first recombinase enzyme and a nucleic acid molecule encoding a second recombinase enzyme, wherein the first recombinase enzyme binds to a first half-site of an asymmetric recombinase target site and the second recombinase enzyme binds to a second half-site of the asymmetric recombinase target site, wherein the first recombinase enzyme and the second recombinase enzyme form a heterodimer capable of inducing site-specific DNA recombination of a sequence of interest at the asymmetric recombinase target site in a DNA sequence, wherein the asymmetric recombinase target site comprises a first half-site and a second half-site of an upstream target site and / or a downstream target site of a DNA recombinase, and wherein the first half-site and the second half-site are non-identical and non-palindromic; ii. performing mutagenesis to generate a library of nucleic acid molecules encoding a first mutant recombinase enzyme and nucleic acid molecules encoding a second mutant recombinase enzyme, where mutations are introduced into the first recombinase enzyme and the second recombinase enzyme; iii. generating an expression vector by cloning the library of nucleic acid molecules encoding a first mutant recombinase enzyme and the library of nucleic acid molecules encoding a second mutant recombinase enzyme into an expression vector, wherein said expression vector carries the DNA sequence of interest to be recombined; iv. transfecting a cell with the expression vector of step iii) and expressing the first mutant recombinase enzyme and the library of second mutant recombinase enzymes in the same cell, thereby forming a recombinase heterodimer comprising the first mutant recombinase enzyme and the second mutant recombinase enzyme; v. performing a positive selection screening for heterodimers obtained in step iv that are capable of inducing site-specific DNA recombination of the sequence of interest at the asymmetric recombinase target site in the DNA; vi. performing a negative selection screen for heterodimers obtained in step iv or v that are unable to induce site-specific DNA recombination of the sequence of interest at an off-target site, preferably a symmetric recombinase target site in the DNA; and vii. selecting an obligate DNA recombinase capable of recombining a DNA sequence of interest at a recombinase target site in DNA comprising a first half-site and a second half-site of an upstream target site and / or a downstream target site of the DNA recombinase, wherein the first half-site and the second half-site are not identical or palindromic; and wherein the obligate DNA recombinase is not capable of recombining a DNA sequence of interest at an off-target site, preferably a symmetric recombinase target site in DNA; wherein in the obligate DNA recombinase obtained in step vii, the first mutant recombinase enzyme and the second mutant recombinase enzyme comprise at least one mutation in a catalytic site that renders the first recombinase enzyme and the second recombinase enzyme, respectively, catalytically inactive when expressed alone.

[0018] According to a preferred embodiment, the obligate DNA recombinase is for the recombination of DNA sequences.

[0019] According to one embodiment, the first recombinase enzyme and the second recombinase enzyme according to steps ii to vi have been evolved by substrate-linked directed evolution (SLiDE) or directed evolution.

[0020] In a further embodiment, the selection according to steps v and vi is cycled between the selection of obligate heterodimers that are catalytically active at an asymmetric target site (positive selection) and heterodimers that are catalytically inactive at an off-target site, preferably a symmetric target site.

[0021] According to a further aspect, the present invention provides an engineered DNA recombinase for genome editing, comprising an obligate complex of recombinase, said complex comprising at least a first recombinase enzyme and at least a second recombinase enzyme, said first recombinase enzyme and said second recombinase enzyme specifically recognizing a first half-site and a second half-site of an upstream target site and / or a downstream target site of a DNA recombinase, said first recombinase enzyme and said second recombinase enzyme each comprising at least one mutation in a catalytic site, said first recombinase enzyme and said second recombinase enzyme carrying said at least one mutation in a catalytic site do not exhibit catalytic activity of a DNA recombinase when expressed alone, and wherein the catalytic activity as a DNA recombinase is complemented in the obligate complex of recombinase.

[0022] According to a preferred embodiment, the first half-site and the second half-site are non-identical and non-palindromic.

[0023] According to a further preferred embodiment, the engineered DNA recombinase is obtained by the method for producing an obligate DNA recombinase for genome editing according to the invention.

[0024] According to one embodiment, the at least one first recombinase and the at least one second recombinase are of the same type.Preferably, the at least one first recombinase and the at least one second recombinase are both Cre-, Dre-, VCre-, SCre-, Vika-, lambda-Int-, Flp-, R-, Kw-, Kd-, B2-, B3-, Nigri- or Panto-recombinases.

[0025] In a further embodiment, the DNA recombinase is a complex of recombinases in the form of a heterotetramer.

[0026] According to another embodiment, at least one mutation is a single amino acid substitution in the catalytic domain of the recombinase. Preferably, said single amino acid substitution in the at least one first recombinase enzyme and the at least one second recombinase enzyme is at a conserved amino acid position within the catalytic domain.

[0027] According to a preferred embodiment, the present invention provides an engineered DNA recombinase enzyme comprising a complex of at least a first DNA recombinase enzyme and at least a second DNA recombinase enzyme, wherein said first DNA recombinase enzyme and said second DNA recombinase enzyme specifically recognize a first half-site and a second half-site of an upstream target site and / or a downstream target site of a DNA recombinase, said first DNA recombinase enzyme and said second DNA recombinase enzyme each comprise a single amino acid substitution in their catalytic site, wherein said first DNA recombinase enzyme and said second DNA recombinase enzyme carrying said single amino acid substitution do not exhibit DNA recombinase catalytic activity when expressed alone, and wherein said first DNA recombinase enzyme and said second DNA recombinase enzyme carrying said single amino acid substitution exhibit DNA recombinase catalytic activity when co-expressed to form a complex.

[0028] According to one embodiment, at least one mutation or single mutation in the catalytic domain of the first DNA recombinase is different from at least one mutation or single mutation in the catalytic domain of the second DNA recombinase.

[0029] According to another embodiment, the present invention provides a DNA recombinase of the present invention, (i) the first recombinase enzyme comprises a Cre recombinase and the second recombinase enzyme comprises a Cre recombinase, each Cre recombinase comprising a single amino acid substitution within an amino acid region selected from the group consisting of SEQ ID NO:109 to SEQ ID NO:114; (ii) the first recombinase enzyme comprises a Vika recombinase and the second recombinase enzyme comprises a Vika recombinase, each Vika recombinase comprising a single amino acid substitution within an amino acid region selected from the group consisting of SEQ ID NO:115 to SEQ ID NO:120; (iii) the first recombinase enzyme comprises a Dre recombinase and the second recombinase enzyme comprises a Dre recombinase, each Dre recombinase comprising a single amino acid substitution within an amino acid region selected from the group consisting of SEQ ID NO: 121 to SEQ ID NO: 126; or (iv) the first recombinase enzyme comprises a Panto recombinase and the second recombinase enzyme comprises a Panto recombinase, each Panto recombinase comprising a single amino acid substitution within an amino acid region selected from the group consisting of SEQ ID NO: 127 to SEQ ID NO: 132; The present invention provides a DNA recombinase.

[0030] According to one embodiment, the first recombinase enzyme and the second recombinase enzyme do not comprise inter-monomer interface mutations.

[0031] In another embodiment, the engineered DNA recombinase is a mutant of a naturally occurring site-specific recombinase or a mutant of a designer DNA recombinase.

[0032] According to a preferred embodiment, the at least one mutation or single amino acid substitution in the at least one first recombinase enzyme and the at least one second recombinase enzyme is selected from the group consisting of E129R, Q133H, R173A, R173C, R173D, R173E, R173F, R173G, R173I, R173K, R173L, R173M, R173N, R173P, R173Q, R173S, R173T, R173V, R173W, R173Y, E176H, E176I, E176L, E176M, E176V, E176W, E176Y, K201A, K201C, K201C, K201D, K201F, K201 G, K201H, K201I, K201L, K201M, K201N, K201P, K201Q, K201R, K201S, K201T, K201V, K201W, K201Y, H289D, H289E, H28 9I, H289K, H289R, H289W, R292A, R292C, R292E, R292F, R292G, R292H, R292I, R292L, R292M, R292N, R292P, R292Q, R 292S, R292T, R292V, R292W, R292Y, Q311R, W315C, W315E, W315G, W315I, W315K, W315L, W315M, W315N, W315Q, W315R, or at the corresponding amino acid position of another recombinase, preferably at the corresponding position of SEQ ID NO:14, SEQ ID NO:17 or SEQ ID NO:20.

[0033] According to a further preferred embodiment, the at least one mutation or single amino acid substitution in the at least one first recombinase enzyme and the at least one second recombinase enzyme is (i) E129, Q133, R173, E176, K201, H289, R292, Q311, W315, and Y324 of SEQ ID NO: 1; (ii) E146, Q151, R191, N194, K219, H308, R311, Q330, W334, and Y343 of SEQ ID NO: 14; (iii) E130, Q134, R174, E177, K202, H290, R293, Q312, W316, and Y325 of SEQ ID NO: 17; (iv) E131, Q135, R175, E178, K202, H290, R293, Q312, W316, and Y325 of SEQ ID NO: 20; or (v) Amino acid position in another recombinase wherein said amino acid positions in other recombinases correspond to positions E129, Q133, R173, E176, K201, H289, R292, Q311, W315, or Y324 of SEQ ID NO:1.

[0034] According to one embodiment, the first recombinase enzyme and the second recombinase enzyme do not comprise inter-monomer interface mutations.

[0035] In another embodiment, the engineered DNA recombinase is a mutant of a naturally occurring site-specific recombinase or a mutant of a designer DNA recombinase.

[0036] According to a preferred embodiment, the first recombinase enzyme comprises a mutation selected from the group consisting of the mutation K201R of SEQ ID NO: 1, 2 or 7, the mutation K202R of SEQ ID NO: 18 or 21, the mutation K219R of SEQ ID NO: 15 and the mutation K221R of SEQ ID NO: 24, and the second recombinase enzyme comprises a mutation selected from the group consisting of the mutation Q311K of SEQ ID NO: 4 or 11, the mutation Q311R of SEQ ID NO: 5 or 12, the mutation Q312R of SEQ ID NO: 19 or 22, the mutation Q330R of SEQ ID NO: 16 and the mutation Q336R of SEQ ID NO: 25.

[0037] In one embodiment, the recombinase target site is a target site for a tyrosine site-specific recombinase, such as Cre-, Dre-, VCre-, SCre-, Vika-, lambda-Int-, Flp-, R-, Kw-, Kd-, B2-, B3-, Nigri- and Panto-recombinase, and the engineered DNA recombinase is a genetically engineered DNA recombinase enzyme comprising a first recombinase enzyme and a second recombinase enzyme, wherein the first recombinase enzyme is a polypeptide having an amino acid sequence with at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO:7 and comprising a single mutation K201R in the catalytic site; and the second recombinase enzyme is a polypeptide having an amino acid sequence with at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO:11 and comprising a single mutation at position Q311R in the catalytic site; a genetically engineered DNA recombinase enzyme comprising a first recombinase enzyme and a second recombinase enzyme, wherein the first recombinase enzyme is a polypeptide having an amino acid sequence with at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO:7 and comprising a single mutation K201R in the catalytic site; and the second recombinase enzyme is a polypeptide having an amino acid sequence with at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO:12 and comprising a single mutation Q311K in the catalytic site; a Cre recombinase comprising a first recombinase enzyme having a single mutation K201R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:2, and a second recombinase enzyme having a single mutation Q311R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:5; a Cre recombinase comprising a first recombinase enzyme having a single mutation K201R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:2, and a second recombinase enzyme having a single mutation Q311K in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:4; a Vika recombinase comprising a first recombinase enzyme having a single mutation K219R in its catalytic site and having an amino acid sequence which is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 15, and a second recombinase enzyme having a single mutation Q330R in its catalytic site and having an amino acid sequence which is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 16; a Panto recombinase comprising a first recombinase enzyme having a single mutation K202R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 18, and a second recombinase enzyme having a single mutation Q312R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 19; a Dre recombinase comprising a first recombinase enzyme having a single mutation K202R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:21, and a second recombinase enzyme having a single mutation Q312R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:22; a Vcre recombinase comprising a first recombinase enzyme having a single mutation K221R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:24, and a second recombinase enzyme having a single mutation Q336R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:25, is selected from the group consisting of:

[0038] In another embodiment, the engineered DNA recombinase has the nucleic acid sequence: [ka] or a reverse complement thereof, and specifically recognizes a recombinase target sequence upstream of a loxF8 target site having the nucleic acid sequence: [ka] or a reverse complement thereof, which catalyzes the recombination of a gene sequence between the upstream recombinase target site sequence of SEQ ID NO: 65 of the loxF8 recombinase target site and the downstream recombinase target site sequence of SEQ ID NO: 66; and the ability to catalyze the recombination of a gene sequence between the upstream recombinase target sequence of SEQ ID NO: 65 of the loxF8 recombinase target site and the downstream recombinase target sequence of SEQ ID NO: 66 of the loxF8 recombinase target site is a) expressing an engineered DNA recombinase comprising a first recombinase enzyme and a second recombinase enzyme in a cell; and b) analyzing whether the engineered DNA recombinase expressed in step a) is capable of recombining DNA sequences on human chromosomes in said cells; The test is performed by a method including:

[0039] According to a further aspect, the present invention provides a nucleic acid molecule or a plurality of nucleic acid molecules each comprising or consisting of a nucleic acid sequence encoding an engineered DNA recombinase enzyme or a subunit thereof according to the invention.

[0040] According to yet another aspect, the present invention provides an expression vector comprising a nucleic acid molecule or a plurality of nucleic acid molecules according to the invention and expression control elements operably linked to said nucleic acid for promoting its expression.

[0041] According to a further aspect, the present invention provides a mammalian, insect, plant or bacterial host cell comprising the nucleic acid molecule or nucleic acid molecules according to the invention or an expression vector comprising a nucleic acid molecule according to the invention.

[0042] According to yet another aspect, the present invention provides a pharmaceutical composition comprising the engineered DNA recombinase according to the invention or the nucleic acid molecule or molecules of the invention, or the expression vector of the invention, or the cell of the invention, optionally further comprising one or more therapeutically acceptable diluents or carriers.

[0043] According to a further aspect, the present invention provides an engineered DNA recombinase according to the invention, or a nucleic acid molecule or nucleic acid molecules according to the invention, or an expression vector according to the invention, or a pharmaceutical composition according to the invention, for use in medicine.

[0044] According to yet another aspect, the present invention provides an engineered DNA recombinase according to the invention, or a nucleic acid molecule or molecules according to the invention, or an expression vector according to the invention, or a pharmaceutical composition according to the invention, for use in the treatment of haemophilia A. According to a preferred embodiment, the treatment is the treatment of severe haemophilia A.

[0045] According to a further aspect, the present invention relates to a method for inverting a DNA sequence at the genomic level in vitro in a cell comprising an engineered DNA recombinase according to the present invention, comprising the steps of: i. providing a nucleic acid molecule encoding a first recombinase enzyme of the present invention, wherein said first recombinase enzyme comprises at least one mutation that inactivates the catalytic activity of said first recombinase enzyme as a DNA recombinase, and said first recombinase monomer specifically recognizes a first half-site of a recombinase target site; ii. providing a nucleic acid molecule encoding a second recombinase enzyme of the invention, wherein said second recombinase enzyme comprises at least one mutation that inactivates the catalytic activity of said second recombinase enzyme as a DNA recombinase, and wherein said second recombinase monomer specifically recognizes a second half-site of a recombinase target site; iii. generating an expression vector by cloning a nucleic acid molecule encoding a first recombinase enzyme and a nucleic acid molecule encoding a second recombinase enzyme into the expression vector; iv. delivering the DNA sequence to be inverted, the expression vector of step iii) or an RNA molecule encoding the engineered DNA recombinase of the invention to a cell containing said expression vector; v. expressing the engineered DNA recombinase of the invention; vi. Inverting a DNA sequence to be inverted on a human chromosome in said cell using said engineered DNA recombinase of the present invention expressed in said cell. The method further comprises:

[0046] In a preferred embodiment, the cells are not human germ cells.

[0047] According to a further aspect, the present invention provides a method for treating or preventing a disease, said method comprising administering to a subject in need thereof an engineered DNA recombinase according to the invention, or a nucleic acid molecule or nucleic acid molecules according to the invention, or an expression vector according to the invention, or a host cell according to the invention, or a pharmaceutical composition according to the invention.

[0048] According to a further aspect, the present invention provides a method for treating or preventing hemophilia A comprising administering to a subject in need thereof an engineered DNA recombinase according to the invention, or a nucleic acid molecule or molecules according to the invention, or an expression vector according to the invention, or a host cell according to the invention, or a pharmaceutical composition according to the invention, wherein optionally hemophilia A is severe hemophilia A.

[0049] According to a further aspect, the present invention provides a method for recombination of a target DNA sequence in a cell, comprising the steps of: (a) a nucleic acid molecule encoding a first recombinase enzyme and a nucleic acid molecule encoding a second recombinase enzyme according to the invention; and / or (b) a first recombinase enzyme and a second recombinase enzyme according to the present invention wherein the target DNA sequence is recombined in the cell by introducing into the cell a gene encoding the target DNA sequence.

[0050] According to a preferred embodiment, the nucleic acid molecule encoding the first recombinase enzyme and / or the nucleic acid molecule encoding the second recombinase enzyme is mRNA.

[0051] According to an embodiment, the nucleic acid molecule encoding the first recombinase enzyme and / or the nucleic acid molecule encoding the second recombinase enzyme is an expression vector.

[0052] Further aspects and embodiments of the present invention will become apparent from the accompanying claims and the following detailed description. [Brief description of the drawings]

[0053] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]FIG. 1 shows the modification of the protein-protein interface of heterospecific D7 recombinase. (A) Nucleotide sequence of the relevant SSR target site. loxF8, loxF8L and loxF8R represent the target sequences of D7L and D7R recombinase, respectively. Highlighting indicates some features (bold - differences relative to loxP sites, underline - asymmetric residues of loxF8 site). (B) Bacterial assay showing the activity of co-expression of D7L and D7R (recombinase enzymes) on target sites loxF8, loxF8L and loxF8R (substrates). Recombination is indicated by bands aligned with one triangle and non-recombination events by bands aligned with two triangles. The concentration of L-arabinose (μg / mL) used to induce recombinase expression is indicated at the bottom. (C) Schematic of the desired obligate D7 recombinase activity. D7LA3 and D7RB2 have mutations that are required to allow heterodimerization only at the loxF8 target site. The mutations are required to prevent homodimerization of D7LA3 and D7RB2 at symmetric loxF8R and loxF8L sites. (D) Bacterial assay for recombination activity of co-expressed D7LA3 and D7RB2 for the indicated target sites. The concentration of L-arabinose (μg / mL) used to induce recombinase expression is shown at the bottom. [Diagram 2]FIG. 2 shows the selection method, where the activity of the initial library is compared to that of the final library, followed by analysis of acquired mutations. (A) Selection scheme where the library is subjected to positive selection pressure to select for active recombinases at the loxF8 site, and negative selection pressure to select for inactive mutants (indicated with an X) at the symmetric loxF8L or loxF8R site. (B) Bacterial assay showing the activity of the initial library (targeted library generated by applying the ISOR method to the D7 recombinase sequence) and the final library (library after 26 rounds of evolution and selection pressure) at the target sites (substrates) loxF8, loxF8L and loxF8R. The concentration of L-arabinose (μg / mL) used to induce recombinase expression is indicated at the bottom. (C) Mutation frequency calculated by the amount of times an amino acid position is mutated compared to the D7 sequence divided by the total amount of sequence. The amino acid positions of D7L and D7R are plotted along the x-axis, and the mutation frequency as a percentage of all sequences is plotted along the y-axis. Positions with mutation frequencies above 20% are displayed. Due to high background of sequencing reads, the plot position of D7R starts at the 6th amino acid position. (D) Activity of isolated D7 monomers on asymmetric site loxF8 and symmetric sites loxF8L and loxF8R. (E) Activity of D7 compared to heterodimer D7LK201R+D7RQ311R on asymmetric site loxF8 and symmetric sites loxF8L and loxF8R. Induction levels of L-arabinose (μg / ml) are listed along the bottom. [Diagram 3]Figure 3 shows obligate D7 activity in mammalian cells. (A) Schematic of mRNA expression constructs and reporter HEK293T cell line. Co-transfection of HEK293T cells with mRNA expressing NLS together with recombinase or BFP. Schematic of loxF8 integration reporter construct in HEK293T cells. LoxF8 sites are flanked by puromycin selection gene. After successful recombination (excision), mCherry is expressed from the SFFV promoter. (B) Representative bright field and mCherry fluorescence images of reporter HEK293T cells transfected with the indicated recombinases. 100 μm scale bar is indicated. (C) Recombination efficiency analyzed by FACS and shown by the percentage of mCherry expressing cells over total cells containing BFP. *Normalized to BFP. (D) Scheme of genomic inversion detection PCR. Two PCR programs were performed in parallel on genomic DNA extracted from HEK293T cells 48 hours after transfection of recombinase mRNA. The WT orientation is indicated by the amplification product generated by the primers. The inverted orientation is also indicated by the primers. (E) PCR results show untreated cells at the top and cells transfected with mRNA at the bottom row. Controls consist of HEK293T cells transfected with BFP but not recombinase mRNA (untreated), patient iPSCs carrying an inversion of exon 1 (inverted-Ctrl), non-inverted amplification product (WT-Ctrl) and water. (F) Sequences of predicted off-target sites in the human genome with high sequence similarity to loxF8L (HG1L and HG2L) and to loxF8R (HG1R and HG2R). (G) Recombination activity of D7 compared to the activity of D7K201R+D7RQ311R at predicted human off-target sites. [Figure 4]Figure 4 shows the broad application of obligate mutations to additional recombinase systems. (A) Recombination activity in bacteria of Cre compared to Cre with obligate mutations expressed on loxP as a heterodimer composed of both mutated subunits CreK201R and CreQ311R, or expressed alone on loxP as single mutated subunits of CreK201R or CreQ311R. (B) Recombination activity of Vika in bacteria compared to Vika with obligate associated mutations of K291R in one subunit and Q330R in the second mutated subunit. First, both Vika mutants are expressed together on vox or alone on vox as mutated subunits of VikaK219R or VikaQ330R. (C) mRNA expression constructs and HEK293T loxP reporter cell line. (D) Bright field and fluorescent mCherry cell images of HEK293T. (E) Recombination efficiency in HEK293T cells of combinations of Cre / loxP and obligate Cre subunits: CreK201R and CreQ311R expressed together, followed by the other mutant subunits CreK201R and subunit Q311R expressed alone. Percentage of recombination normalized to BFP. [Diagram 5]Figure 5 shows the structural details of Cre and the mutants investigated. (A) MD refined structure of the Cre complex (PDBID 3C29). One DNA loxP molecule is shown in the complex with two Cre monomers (active (A) and inactive (I) respectively). Residues K201 and Q311 are depicted as spheres. (B) The loxP sequence with the top strand (TS, SEQ ID NO:51) and bottom strand (BS, SEQ ID NO:139) is displayed. Arrows indicate the points of attack by the catalytic tyrosine (Y324) in each DNA strand, with the initial cleavage indicated by the top arrow. Bases in the spacer region are labelled. (C)-(F) Superposition of MD refined structures (taken from the last 50 ns of the simulation) of Cre and mutants CreK201R(A)-K201R(I) / loxP (C), CreQ311R(A)-Q311R(I) / loxP (D), CreK201R(A)-Q311R(I) / loxP (E) and CreQ311R(A)-K201R(I) / loxP (F). In the mutant structures, active (A) and inactive (I) monomers are shown on the left and right of the figure, respectively, and DNA is indicated. Side chains of relevant residues are shown as circles and sticks and numbered. Intermolecular H-bonds are indicated by dashed lines. The lack of an H-bond between H289 in the active monomer of CreQ311R(A)-Q311R(I) and the phosphate group of A4'TS in loxP is highlighted with two arrows pointing to those atoms (panel D). For clarity, the panel is made transparent and depicted in cartoon form. Check and cross symbols indicate active versus inactive mutants. (G) Results of a recombination test of a dimer containing monomers with mutations K201 and Q311. [Figure 6]Figure 6 shows a plasmid-based assay for visualizing recombination activity. A vector carrying a recombinase or recombinase library of interest and a target site. Upon recombination, a 750 bp fragment flanked by lox sites is excised. Protein-coding genes are indicated by arrows, the origin of replication (oriP15A) and pBAD promoter by rectangles, and the target site by triangles. Protein-coding genes include the chloramphenicol resistance gene (cmR), the arabinose regulatory protein (araC), and the gene encoding the recombinase(s) or library of interest. Restriction enzyme sites are indicated by dotted lines. NdeI and AvrII are used to select for recombined vectors. SacI and SbfI are used to visualize whether the plasmid is unrecombined or recombined. These enzymes excise a 2.2 KB fragment containing the recombinase and a 5 KB or 4.2 KB fragment if the plasmid is unrecombined or recombined, respectively. [Figure 7] Figure 7 shows the selection strategy. (A) Positive selection scheme modified for substrate binding directed evolution. Mutants with no activity at the loxF8 target site are removed from the library by digesting the purified plasmid with NdeI / AvrII and linearizing the non-recombined plasmid. Active mutants are then amplified with primers F and R1 and error-prone Taq polymerase to add mutations and pass the mutants through the next evolutionary cycle. (B) Negative selection scheme modified to remove mutants that can recombine at loxF8L or loxF8R symmetric sites. Only the loxF8L site is shown here because the selection method is the same for both sites. Mutants are amplified with primers F and R2 that bind between the symmetric sites and only those that cannot recombine are amplified. Taq polymerase is used to add mutations and pass the mutants through the next evolutionary cycle. [Figure 8]Figure 8 shows blue-white screening of the library and activity testing of selected colony samples. (A) The selection plasmid contains a transcription terminator adjacent to the symmetry site upstream of LacZa. Upon recombination at the symmetry site, the transcription terminator is excised and transcription of LacZa is driven by the constitutive cat promoter. Inactive variants are removed by NdeI and AvrII digestion. (B) Blue colonies contain mutants that are active at the symmetry site, therefore white colonies are selected that contain mutants that do not recombine at the symmetry site but rather recombine at the loxF8 site. (C) A sample of 75 white colonies, colony PCR showed colonies with the desired activity profile with a 1.7 KB band. [Figure 9] Figure 9 shows the gating strategy to evaluate the recombination efficiency of SSR for HEK293T reporter cell line. (A) FACS gating strategy. Single cells were gated cells from live cells. Dead cells were gated out. BFP+ cells were gated from single cells and finally mCherry+ cells were gated. (B) Schematic of mRNA expression construct and reporter HEK293T cell line. Shown are the adopted mRNA and tag BFP mRNA expressing a nuclear localization signal (NLS) fused to the recombinase and with the indicated features (5' cap and polyA tail). The stable reporter cell line harbors two loxP sites (triangles) flanking a puromycin selection gene (puro). Upon successful excision by recombination, mCherry is expressed from the SFFV promoter (arrow). (C) Representative microscopy images as overlays showing the distribution of bright field, BFP, mCherry and recombination. (D) FACS plot of a representative mRNA-transfected HEK293T reporter cell line where BFP fluorescence indicates transfected cells and mCherry fluorescence indicates gated BFP+ cells where recombination has occurred. [Figure 10]FIG. 10 shows MD-based analysis of hydrogen bonds and van der Waals contacts in Cre and the CreK201R and CreQ311R mutants examined. (A) Hydrogen bonds and van der Waals occupancies in the last 100 ns of the MD simulation. The top panel shows the loxP sequence with the top strand (TS) (SEQ ID NO:51) and bottom strand (BS) (SEQ ID NO:139) labeled. (B-F) Hydrogen bonds and van der Waals contact profiles in the last 100 ns of the MD simulation for (B) Cre, (C) CreK201R(A)-K201R(I), (D) CreQ311R(A)-Q311R(I), (E) CreK201R(A)-Q311R(I) and (F) CreQ311R(A)-K201R(I). The black loxP bases correspond to the spacer. The loxP bases in light and dark grey correspond to those that interact with the active (A) and inactive (I) protein monomers, respectively. The interacting protein residues of the active (A) and inactive (I) monomers are listed in light and dark grey, respectively. Residues involved in H-bond formation are highlighted in bold, residues involved in van der Waals contacts are highlighted in bold and underlined, and residues involved in H-bonds and van der Waals contacts are simultaneously highlighted in asterisks and bold. The phosphate group involved in Y324 recognition is highlighted with a circle. Active versus inactive mutants are highlighted with check and cross symbols, respectively. [Figure 11] Figure 11 shows a scheme of a Cre molecule binding to its target loxP site. The loxP site consists of two inverted repeats (13 bp) flanked by a non-palindromic spacer (8 bp). Two Cre molecules bind to the loxP site, each binding to one of the half-sites. [Figure 12]Figure 12 depicts a scheme of a typical tyrosine SSR recombination reaction. (A) Schematic of the stepwise recombination mechanism. Four recombinase molecules bind to two DNA substrates, forming a tetrameric complex (1). The recombinase in a "cleavable" conformation is shown in light grey, and the recombinase in a "non-cleavable" conformation is shown in dark grey. The nucleophilic tyrosine is shown as Y in a light grey circle, and is shown only for the active monomer. The activated nucleophilic tyrosine attacks the dissociable phosphate (shown as P), forming a 3'-phosphotyrosine bond, resulting in the release of a free 5'OH (2). The released 5'OH attacks the adjacent phosphotyrosine, forming a Holliday junction intermediate, resulting in strand exchange (3). When the complex isomerizes, the active monomer changes conformation to inactive and vice versa (4). The cleavage and strand exchange steps are repeated (5, 6). (B) Possible outcomes of the recombination reaction. Recombination reactions can result in excision / integration or inversion of DNA fragments depending on the orientation of the target site (spacer sequences). Target sites are shown as black triangles, and their directionality indicates the orientation of the target site. (Meinke et al., 2016). [Figure 13] Figure 13 shows the mutation combinations that result in obligately catalytically active D7 heterotetramers by bacterial recombination assays with substrates loxF8, loxF8L and loxF8R. The recombination activities of recombinases D7L+-D7R, D7LK201R+D7RQ311R and D7LK201R+D7RQ311K are shown. [Figure 14] Figure 14 shows obligate Dre activity in bacteria visualized by a PCR-based detection method (plasmid on the left). Recombination activity of recombinase Dre, DreK202R+DreQ312R, DreK202R and DreQ312R at rox sites is shown. [Figure 15]Figure 15 shows the evolution and selection scheme. A library is created by targeting diversification of amino acid positions 25, 29, 32 and 33 in the D7L recombinase and positions 69, 72 and 76 in the D7R recombinase. The two libraries are cloned together into an expression vector carrying the target sites of interest, then transformed into E. coli and the recombinase is expressed overnight. The resulting recombinant and non-recombined plasmids are purified to begin selection. (A) Selection for recombination at the symmetric site is performed by selecting for inactive mutants. Primer p4 binds between the target sites, and only inactive mutants are amplified at that site. (B) Selection for active mutants at that site occurs in two steps. First, the purified plasmid is digested with a restriction enzyme that cuts between the two target sites. Amplification will result only in products from active recombinase. The isolated mutants with the desired activity are then cloned back into a vector carrying the target sites of interest and carried forward to the next cycle. Each new cycle of selection rotated between selection of active mutants in loxF8 and inactive mutants in loxF8l and loxF8R (e.g., the first cycle selected for active mutants in loxF8, the second cycle selected for inactive mutants in loxF8L, and the third cycle selected for inactive mutants in loxF8R). [Figure 16] FIG. 16 shows an alignment of the amino acid sequences of wild-type DNA recombinases used to prepare mutated DNA recombinases according to the present invention. [Figure 17]FIG. 17 shows the 3D structure of a wild-type Cre recombinase-loxP synapse. The dimeric complex before cleavage is shown in A) with black highlighting indicating regions of high mutation frequency in obligate Cre-like recombinases. Regions include positions 129-136, 163-181, 289-301, 310-316 and 321-324. B) shows exemplary positions in the Cre dimeric complex where mutations result in obligate Cre-like recombinases (shown as spheres), including positions 129, 133, 173, 176, 201, 289, 292, 311, 315 and 324. Positions on the right monomeric subunit are shown in italics. [Figure 18] FIG. 18 is a summary showing single point mutations and their effect on recombinase activity of single recombinase monomers and recombinase obligate dimer complexes. [Figure 19] Figure 19 is a sequence alignment of the Cre, Vika and Dre recombinases showing the catalytic region for each recombinase, consisting of six catalytic sites (shown in boxes). Conserved amino acids between the different recombinases are highlighted in grey. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] Exemplary Recombinase Sequences Table 1 below provides an exemplary summary of sequences of recombinases used in the present invention. SEQ ID NOs indicating nucleic acid sequences are cited in parentheses, for example, SEQ ID NO: 1 indicates the amino acid sequence of the wt Cre recombinase monomer, and SEQ ID NO: 26 (quoted in parentheses) indicates the respective nucleic acid sequence encoding the Cre recombinase protein of SEQ ID NO: 1.

[0055] Table 1: Amino acid and nucleic acid sequences of recombinases [Table 1]

[0056] (General definition) Preferably, the terms used herein are defined as set forth in A multilingual glossary of biotechnological terms: (IUPAC Recommendations), Leuenberger, HGW, Nagel, B. and Klbl, H. (eds.) (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0057] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not to exclude any other integers or steps or group of integers or steps.

[0058] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of introductions. It is understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where a different designation is intended, this will be clear from the context.

[0059] As used herein, the terms "polypeptide," "peptide," and "protein" are interchangeable and are defined to mean a biomolecule made up of amino acids joined by peptide bonds.

[0060] When peptides or amino acid sequences are referred to herein, each amino acid residue is represented by a one-letter or three-letter name that corresponds to the common name of the amino acid according to the following conventional list: [Table 2]

[0061] As used herein, the terms "a," "an," and "the" are defined to mean "one or more" and include the plural forms unless the context is inappropriate.

[0062] The term "about" as used in connection with numerical values ​​is meant to encompass numerical values ​​in a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value.

[0063] As used herein, the term "and / or" is meant to refer to any / all of the options cited in the context of the term.

[0064] The term "at least one" as used herein refers to one or more of the respective items, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. According to a preferred embodiment, the term "at least one" refers to exactly one. In a particularly preferred embodiment with respect to the number of mutations in the catalytic region of the recombinase, the term "at least one mutation" preferably means a single mutation (particularly preferably a single amino acid substitution) and no further mutations in the catalytic region.

[0065] The term "subject" as used herein refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.

[0066] As used herein, the term "therapeutically effective amount" means an amount of an active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is desired by a researcher, veterinarian, physician or other clinician, including alleviation of the symptoms of the disease or disorder being treated.

[0067] The term "pharmaceutical composition" as used herein refers to a substance and / or combination of substances used for the identification, prevention or treatment of a disease or tissue condition. A pharmaceutical composition is formulated to be suitable for administration to a patient to prevent and / or treat a disease. Moreover, a pharmaceutical composition refers to a combination of an active agent with an inert or active carrier, making the composition suitable for therapeutic use. Such carriers are also referred to as pharma- ceutical acceptable. Pharmaceutical compositions can be formulated for oral, parenteral, topical, inhalation, rectal, sublingual, transdermal, subcutaneous or vaginal routes of application according to their chemical and physical properties. Pharmaceutical compositions include solids, semi-solids, liquids, transdermal therapeutic systems (TTS). Solid compositions are selected from the group consisting of tablets, coated tablets, powders, granules, pellets, capsules, effervescent tablets or transdermal therapeutic systems. Liquid compositions are also included, selected from the group consisting of solutions, syrups, infusions, extracts, solutions for intravenous application, solutions for infusion of the carrier system of the invention or solutions of the carrier system of the invention. Semisolid compositions which can be used in the context of the present invention include emulsions, suspensions, creams, lotions, gels, globules, buccal tablets and suppositories.

[0068] As used herein, the term "pharmaceutical acceptable" encompasses use in both human and veterinary medicine: for example, the term "pharmaceutical acceptable" encompasses a compound that is acceptable as a veterinary drug or a compound that is acceptable in human medicine and health care.

[0069] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where a portion of the sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. This percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matching positions, dividing the number of matched positions by the total number of positions within the comparison window, and multiplying the resulting value by 100 to generate the percentage of sequence identity.

[0070] The term "identical" is used herein in the context of two or more nucleic acid or polypeptide sequences to refer to two or more sequences or subsequences that are the same, i.e., contain the same sequence of nucleotides or amino acids. Sequences are "identical" to each other if they have a certain percentage of nucleotides or amino acid residues that are the same. According to the present invention, at least 70% identical includes at least 75%, at least 80, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a particular sequence when compared and aligned for maximum matching over a comparison window, or to a designated region as measured using one of the following sequence comparison algorithms, or by manual alignment and visual inspection. These definitions also refer to the complement of a test sequence. Thus, the term "at least 70% sequence identity" is used throughout the present specification in relation to comparison of polypeptide and polynucleotide sequences. This expression preferably refers to a sequence identity of at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective reference polypeptide or respective reference polynucleotide.

[0071] The sequence identities disclosed herein preferably refer to amino acid sequences or amino acids or their positions outside the catalytic region of the recombinase, and do not include amino acids or their positions within the catalytic region specified herein.

[0072] The term "sequence comparison" is used herein to refer to a process in which one sequence serves as a reference sequence to be compared with a test sequence. When using sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. Default program parameters are generally used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters. When two sequences are compared and no reference sequence is specified relative to the one for which the sequence identity percentage is to be calculated, unless otherwise specifically indicated, sequence identity should be calculated with reference to the longer of the two sequences to be compared. When a reference sequence is indicated, sequence identity is determined based on the full length of the reference sequence, which is indicated by the sequence number, unless otherwise specifically indicated.

[0073] In sequence alignment, the term "comparison window" refers to a stretch of contiguous positions of a sequence that is compared to a reference stretch of contiguous positions of the sequence having the same number of positions. Typically, the number of contiguous positions ranges from about 20 to 100 contiguous positions, about 25 to 90 contiguous positions, about 30 to 80 contiguous positions, about 40 to about 70 contiguous positions, about 50 to about 60 contiguous positions. According to the present invention, when comparing a sequence with a sequence of the present invention, such as SEQ ID NO: 1, for identity percentage, if the reference sequence has the same length or is longer than the SEQ ID NO of the present invention, preferably the full length of the SEQ ID NO: 1 will be compared to the reference sequence. If the reference sequence is shorter than the SEQ ID NO of the present invention, the full length of the reference sequence must be compared to the full length of the SEQ ID NO of the present invention.

[0074] Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be carried out, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)). Suitable algorithms for determining sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al., Nuc. Acids Res. 25:3389-402, 1977, and Altschul et al., J. Mol. Biol. 215:403-10, 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in the database sequences.T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. The cumulative score is calculated using M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of word hits in each direction is stopped when the cumulative alignment score falls an amount X below the maximum achieved: when the cumulative score becomes zero or less due to the accumulation of one or more negative scoring residue alignments; or when either end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) with an alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-87, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance.For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability in a comparison of a test nucleic acid to the reference nucleic acid is less than about 0.2, typically less than about 0.01, and more typically less than about 0.001.

[0075] A nucleic acid in the context of the present invention may be comprised in one nucleic acid molecule or may be divided into two or more nucleic acid molecules, each nucleic acid molecule comprising at least one of one or more sequences encoding a polypeptide or protein of the present invention. In some embodiments, one nucleic acid molecule encodes one part or monomer of a DNA recombinase of the present invention, and another nucleic acid molecule encodes another part or monomer of a DNA recombinase of the present invention. In some embodiments, a nucleic acid encodes two or more DNA recombinase polypeptides. A nucleic acid encoding multiple DNA recombinases of the present invention may comprise a nucleic acid cleavage site between two sequences encoding DNA recombinase polypeptides, may comprise a transcription initiation site or a translation initiation site, such as an internal ribosome entry site (IRES), between two sequences encoding DNA recombinase polypeptides, and / or may encode a proteolytic target site between two or more DNA recombinase polypeptides. When two or more DNA recombinase polypeptides are encoded on one nucleic acid molecule, the two or more DNA recombinase polypeptides may be under the control of the same promoter or under the control of separate promoters.

[0076] The term "nucleic acid", in the context of the present invention, refers to a single- or double-stranded oligomer or polymer of deoxyribonucleotide or ribonucleotide bases or both. A nucleotide monomer is composed of a nucleobase, a five-carbon sugar (such as, but not limited to, ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, nucleic acids are formed through phosphodiester bonds between individual nucleotide monomers, and in the context of the present invention, the term nucleic acid includes, but is not limited to, ribonucleic acid (RNA) molecules and deoxyribonucleic acid (DNA) molecules, as well as synthetic forms of nucleic acids containing other bonds (e.g., peptide nucleic acids as described in Nielsen et al., Science 254:1497-1500, 1991). Typically, nucleic acids are single- or double-stranded molecules and are composed of naturally occurring nucleotides. Representation of a single strand of a nucleic acid also defines (at least in part) the sequence of the complementary strand. Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Exemplary double-stranded nucleic acid molecules may have 3' or 5' overhangs, and thus are not required or expected to be completely double-stranded over their entire length. The term nucleic acid includes chromosomes or chromosome segments, vectors (e.g., expression vectors), expression cassettes, naked DNA or RNA polymers, primers, probes, cDNA, genomic DNA, recombinant DNA, cRNA, mRNA, tRNA, microRNA (miRNA), or small interfering RNA (siRNA). Nucleic acids may be, for example, single-stranded, double-stranded, or triple-stranded, and are not limited to any particular length. Unless otherwise indicated, a particular nucleic acid sequence includes or encodes complementary sequences in addition to any sequence explicitly indicated. Nucleic acids may be isolated or recombinant nucleic acids.

[0077] The nucleic acid may be present in whole cells, in a cell lysate, or may be in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially purified" when it has been purified away from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques.

[0078] The terms "vector", "cloning vector" and "expression vector" refer to a vehicle capable of introducing DNA or RNA sequences (e.g., foreign genes) into a host cell to transform the host and promote expression (e.g., transcription and translation) of the introduced sequences. A variety of expression vectors can be employed to express the polynucleotides encoding the DNA recombinase and the DNA recombinase of the present invention. Both viral-based and non-viral expression vectors can be used to produce the DNA recombinase and DNA recombinase described herein, for example, in mammalian host cells. Non-viral vectors and systems include plasmid(s), plasmids, cosmids, episomes, artificial chromosomes, phages or viral vectors. Such vectors may include regulatory elements, such as promoters, enhancers, terminators, etc., to express or direct the expression of the polypeptide upon administration to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include SV40 early promoter and enhancer (Mizukami T. et al., 1987), Moloney murine leukemia virus LTR promoter and enhancer (Kuwana Y et al., 1987), antibody heavy chain promoter (Mason JO et al., 1985) and enhancer (Gillies SD et al., 1983), etc. For example, non-viral vectors useful for expressing the polynucleotides and polypeptides described herein in mammalian (e.g., human or non-human) cells include all suitable vectors known in the art for expressing proteins. Other examples of plasmids include replicating plasmids containing an origin of replication, or integrating plasmids, such as pUC, pcDNA, pBR, etc.

[0079] The term "viral vector" refers to a nucleic acid vector construct that contains at least one element from a virus, has the ability to be packaged into a viral vector particle, and encodes at least an exogenous nucleic acid. The vectors and / or particles can be utilized to introduce a nucleic acid of interest into cells either in vitro or in vivo. Many forms of viral vectors are known in the art. Useful viral vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, SV40-based vectors, papilloma viruses, Epstein-Barr viruses, vaccinia virus vectors, and Semliki Forest virus (SFV)-based vectors. Recombinant viruses can be generated by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, and the like. Detailed protocols for generating such replication-defective recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, U.S. Patent Nos. 5,882,877, 6,013,516, 4,861,719, 5,278,056 and WO 94 / 19478.

[0080] The terms "recombinase", "DNA recombinase", "DNA recombinase enzyme" and "recombinase enzyme" are used interchangeably herein and each refer to what is understood in the art as a monomer of a protein complex that allows genetic recombination. The term includes monomeric subunits from site-specific recombinases (SSRs), particularly the tyrosine recombinase family. A functional complex that includes at least two DNA recombinases or monomers is also called a "DNA recombinase". Naturally occurring DNA recombinases, particularly site-specific recombinase (SSR) systems (such as tyrosine-type SSRs), generally consist of four identical monomeric subunits or monomers. Such complexes are called homotetramers.

[0081] As used herein, the term "complex" or "recombinase complex" of DNA recombinase refers to a combination of at least two monomeric recombinase subunits, also called recombinase enzymes. A complex of two subunits is called a dimer and a complex of four subunits is called a tetramer. Naturally occurring recombinase complexes consist of identical recombinase monomers or subunits; in the case of two identical subunits, the complex is called a homodimer and in the case of four identical subunits, the complex is called a homotetramer). According to a preferred embodiment of the present invention, the recombinase complex comprises at least two different recombinase subunits and thus exists as a heterodimer or heterotetramer.

[0082] In general, such recombinase complexes modify DNA between two specific target sequences. These sequences typically range from 30-200 base pairs in length and consist of two inverted repeat recombinase binding regions flanking a central spacer sequence where DNA cleavage and replication occur (Meinke et al., 2016). An example of such a target sequence, also referred to herein as a target site, is shown in FIG. 11, which shows an SSR Cre / loxP binding complex, in which Cre recombinase binds to a 34 base pair loxP target sequence. The loxP site consists of two 13 base pair inverted repeat Cre binding elements flanked by an 8 base pair spacer region. The binding elements are distinguished by their position with reference to the spacer sequence. The left half site is the 13 base pair binding element to the left of the spacer, and the right half site is the 13 base pair binding element to the right of the spacer. Depending on the number and relative orientation of target sites and their spacers, DNA recombinases either excise, integrate, invert or replace gene content (Figure 12; reviewed in Meinke et al., 2016). To initiate a recombination event, the recombinase complex recognizes a first target site and a second target site on the DNA duplex. Target sites are also referred to as upstream and downstream recognition sites, depending on their location on the DNA duplex. Thus, the term "half-site" referred to herein refers to the left and right sections of a DNA sequence, respectively, separated by a spacer sequence, to which the recombinase enzyme binds. Half-sites are also referred to as binding regions or binding elements. The term "target site" or "target sequence" as used herein refers to a sequence element that includes the left half-site, the spacer sequence, and the right half-site.

[0083] The term "type" as used in the context of a recombinase refers to a specific recombinase subunit, preferably selected from the group consisting of Cre-, Dre-, VCre-, SCre-, Vika-, lambda-Int-, Flp-, R-, Kw-, Kd-, B2-, B3-, Nigri- and Panto-recombinases.

[0084] The term "single mutation in the catalytic region" as used herein excludes any additional mutations in the catalytic region, but does not exclude any mutations (substitutions, deletions, insertions) in the remainder of the amino acid sequence of the respective recombinase enzyme. Thus, for a recombinase with a certain percentage identity to a given SEQ ID NO and at the same time a single amino acid substitution in the catalytic region, the percentage identity refers to the region outside the catalytic region and does not allow for additional mutations within the catalytic region.

[0085] As used herein, "upstream" refers to a 5' target site of a recombinase in DNA, including a first half site, such as the left half site, and a second half site, such as the right half site, wherein the first half site and the second half site are separated by a spacer sequence.

[0086] As used herein, "downstream" refers to a 3' target site of a recombinase in DNA, a first half site, such as a left half site, and a second half site, such as a right half site, wherein the first half site and the second half site are separated by a spacer sequence.

[0087] In a symmetric target site, the first half site, such as the left half site, and the second half site, such as the right half site, are either identical or palindromic (reverse complement). In an asymmetric target site, the first half site, such as the left half site, and the second half site, such as the right half site, are neither identical nor palindromic, i.e., they differ from each other in at least one nucleotide or nucleic acid.

[0088] "Obligate" proteins are complexes composed of multiple subunits. These subunits cannot function alone and are catalytically inactive in their isolated form. When the subunits come together, they form a functional complex. Thus, the presence of all obligate protein subunits is essential for the functionality of the protein, i.e., the recombinase activity of the recombinase complex of the present invention.

[0089] As used herein, the term "does not exhibit catalytic activity of DNA recombinase" or similar terms such as "does not have catalytic activity" used in the context of a DNA recombinase enzyme with a single point mutation in its catalytic region means that the mutant DNA recombinase enzyme exhibits catalytic activity that is less than about 90% of the activity of the same DNA recombinase enzyme without the mutation. The activity of the recombinase enzyme is preferably determined using a plasmid-based assay in E. coli. The plasmid DNA used in this assay contains two target sites for a given DNA recombinase enzyme (e.g., loxP target sites for Cre recombinase). If the DNA recombinase enzyme is active upon expression, the DNA substrate (plasmid DNA) is recombined by the DNA recombinase enzyme. Since the size and sequence of the recombination and non-recombination substrates are different, the recombination activity is calculated based on the ratio of the recombination substrate to the non-recombination substrate using the following formula: recombination activity (%) = 100 x (recombination substrate / (recombination substrate + non-recombination substrate)). These non-recombinant and recombinant DNA fragments can be distinguished either by gel electrophoresis or by sequencing.

[0090] The practice of the present invention will employ, unless otherwise indicated, conventional biochemistry, cell biology, and recombinant DNA techniques as described in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd ed., J. Sambrook et al. (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0091] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "etc.") are intended merely to better illustrate the invention and do not otherwise limit the scope of the invention as claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0092] Detailed Description of the Invention Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein, which may vary. It should also be understood that the terms used herein are merely for the purpose of describing specific embodiments, and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0093] In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects, unless expressly indicated to the contrary. In particular, any feature indicated as optionally preferred or advantageous may be combined with any other feature or features indicated as optionally preferred or advantageous.

[0094] Several documents are cited throughout the text of this specification. Whether supra or infra, each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, descriptions, etc.) is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by prior invention. Some documents cited herein are characterized as "incorporated by reference." In the event of a conflict between a definition or teaching of such an incorporated reference and a definition or teaching cited herein, the text of this specification shall control.

[0095] Each element of the present invention is described below. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be construed as limiting the present invention to only the embodiments explicitly described. The description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all described elements in this application should be considered to be disclosed by the description of this application unless the context indicates otherwise.

[0096] The present invention provides an engineered DNA recombinase for efficient and specific genome editing, comprising an obligate complex of recombinase, said complex comprising at least a first recombinase enzyme and at least a second recombinase enzyme, said first recombinase enzyme and said second recombinase enzyme specifically recognizing a first half-site and a second half-site of an upstream target site and / or a downstream target site of a DNA recombinase, said first recombinase enzyme and said second recombinase enzyme each comprising at least one mutation in a catalytic site, said first recombinase enzyme and said second recombinase enzyme carrying said at least one mutation in a catalytic site do not exhibit catalytic activity of a DNA recombinase when expressed alone, and said catalytic activity as a DNA recombinase is complemented in the obligate complex of recombinase.

[0097] Preferably, said first half site and said second half site of the upstream target site and / or the downstream target site of a DNA recombinase are non-identical and non-palindromic.

[0098] The present invention further provides an engineered DNA recombinase comprising a complex of at least a first DNA recombinase enzyme and at least a second DNA recombinase enzyme, wherein the first DNA recombinase enzyme and the second DNA recombinase enzyme specifically recognize a first half-site and a second half-site of an upstream target site and / or a downstream target site of the DNA recombinase, and wherein the first DNA recombinase enzyme and the second DNA recombinase enzyme each comprise a single amino acid substitution in their catalytic domain, wherein the first DNA recombinase enzyme and the second DNA recombinase enzyme carrying the single amino acid substitution do not exhibit DNA recombinase catalytic activity when expressed alone, and the first DNA recombinase enzyme and the second DNA recombinase enzyme carrying the single amino acid substitution form a complex and exhibit DNA recombinase catalytic activity when co-expressed.

[0099] According to one embodiment, the at least one first recombinase and the at least one second recombinase are of the same type. In this context, the same type means that the first and the second recombinase are derived from the same recombinase, which is preferably selected from the group consisting of Cre-, Dre-, VCre-, SCre-, Vika-, lambda-Int-, Flp-, R-, Kw-, Kd-, B2-, B3-, Nigri- and Panto-recombinases.

[0100] In a particularly preferred embodiment, the DNA recombinase is a complex of recombinase subunits. Such a complex may have a conformation as described herein. A preferred configuration for such a complex is a heterotetramer.

[0101] Surprisingly, it was found that introducing exactly one mutation into the catalytic site of each monomer of a DNA recombinase is sufficient to inactivate the catalytic activity of said monomers, but that pairing two mutated monomers with each other complements (restores) the catalytic activity of the DNA recombinase in an obligate complex of the recombinase. In other words, when a first recombinase having at least one mutation in its catalytic site is co-expressed with a second recombinase having at least one mutation in its catalytic site, and both form a heterocomplex, this heterocomplex is catalytically active and exhibits the activity of a DNA recombinase that does not have a mutation in the catalytic region of the recombinase monomer subunit. Therefore, in a more preferred embodiment, the present invention provides an engineered DNA recombinase for efficient and specific genome editing, comprising an obligate complex of recombinase, said complex comprising at least a first recombinase enzyme and at least a second recombinase enzyme, said first recombinase enzyme and said second recombinase enzyme specifically recognizing a first half-site and a second half-site of an upstream target site and / or a downstream target site of a DNA recombinase, said first recombinase enzyme and said second recombinase enzyme each comprising exactly one mutation in a catalytic site, said first recombinase enzyme and said second recombinase enzyme carrying exactly one mutation in a catalytic site do not exhibit the same catalytic activity of a DNA recombinase when expressed alone, and in the obligate complex of recombinase the catalytic activity as a DNA recombinase is complemented. Of course, the first and second recombinase enzymes may comprise further mutations outside the catalytic site and / or that do not impair their catalytic activity. Preferably, said first and second half-sites of the upstream and / or downstream target sites of the DNA recombinase are non-identical and non-palindromic.

[0102] It was most surprising that position 201 was mutated to arginine instead of lysine in the first recombinase enzyme. What makes this change interesting is that lysine 201 is highly conserved across the tyrosine SSR family and has previously been described as essential for the catalytic activity of Cre by promoting DNA cleavage during recombination. Thus, a recombinase in which position 201 is altered would not be expected to be functional. Mutation of the catalytic K201 residue inactivates the SSR when expressed as a monomer. Based on these observations, it was even more surprising that the recombinase activity could be rescued by the presence of the paired Q311R mutation on the second recombinase enzyme. Only when the paired mutations are applied as single mutations in the catalytic region of each recombinase enzyme (subunit), the engineered SSR is able to efficiently recombine the intended target sequence, whereas the recombinase enzyme (subunit) carrying the point mutation is inactive when expressed alone.

[0103] As mentioned above, by altering the DNA specificity of Cre through engineering and directed evolution, it is possible to generate separate SSR variants that recombine together as heterotetramers with asymmetric target sequences (6, 8-10). The generation of such heterotetrameric SSR systems substantially expands the possible sequences that can be targeted in the genome. However, possible combinations of different subunits may lead to active SSR by-products that can catalyze off-target recombination. Previously, prevention of homotetramer formation was achieved through structure-guided redesign of several residues involved in the protein-protein interaction interface between different recombinase monomers (16). Thus, this approach to generate obligate SSR systems is limited to enzymes with available crystal structures and is therefore not easily adaptable to engineered or distantly related recombinases.

[0104] The present invention further relates to engineered DNA recombinases that specifically recognize upstream and downstream target sequences of a loxF8 recombinase target site and catalyze the inversion of a genetic sequence between these upstream and downstream target sequences of the loxF8 recombinase target site.

[0105] The present invention further relates to nucleic acid molecules encoding the engineered DNA recombinase according to the invention.

[0106] In a further embodiment, the present invention provides a mammalian, insect, plant or bacterial host cell comprising said nucleic acid molecule or molecules encoding an engineered DNA recombinase according to the invention.

[0107] The engineered DNA recombinase or nucleic acid molecule according to the invention may be used as a pharmaceutical and therefore may be included in a pharmaceutical composition, optionally in combination with one or more therapeutically acceptable diluents or carriers.

[0108] The engineered DNA recombinase or pharmaceutical composition according to the invention is suitable for the treatment of diseases that can be cured by genome editing, in particular for the treatment of hemophilia A.

[0109] In a further embodiment, a method is provided for determining recombination at the genome level in a host cell culture or in a patient comprising an engineered DNA recombinase according to the invention.

[0110] Employing directed molecular evolution, we surprisingly discovered that obligate SSR systems could also be generated by mutating residues involved in recombinant catalysis. Importantly, this novel method of generating obligate SSRs required only the modification of one conserved residue in each separate SSR monomer. This simplified approach could potentially be applied to many engineered or natural SSRs without prior structural knowledge of the enzyme.

[0111] The finding that identified mutations can convert naturally occurring SSRs into obligate enzymes, including Cre recombinase, can be usefully explored for sophisticated genetic and synthetic biology studies. A large number of conditional knockout mouse models have been generated based on the Cre / loxP system (28, 29). Typically, animals carrying a floxed allele are crossed with mice expressing Cre from a tissue-specific promoter to inactivate the gene in a specific organ or tissue. This approach can be further refined by expressing CreK201R, CreQ311K, and CreQ311R from two different promoters. Here, deletion of the gene will only occur in cells in which both promoters are active. Similarly, further improvement in the accuracy of gene lineage tracing studies (30) has been achieved by employing the obligate CreK201R / CreR282E and CreQ311K / CreQ311R systems. Similarly, obligate SSRs may enable the generation of more sophisticated circuits for synthetic biology, where SSRs are frequently used to construct biosensors and biological mechanisms (31, 32).

[0112] For example, it is shown herein that the mutations K201R, Q311K and Q311R identified in the Cre system to provide an obligate recombinase complex result in obligate complexes of other recombinases, in particular obligate complexes comprising at least two recombinase enzymes, wherein a first recombinase enzyme comprises said at least one mutation K201R, wherein the numbering of the amino acid positions refers to the amino acid sequence of the wild-type Cre recombinase monomer of SEQ ID NO:1, and a second recombinase enzyme comprises said at least one mutation selected from the group consisting of Q311K and Q311R, wherein the numbering of the amino acid positions refers to the amino acid sequence of the wild-type Cre recombinase monomer of SEQ ID NO:1.

[0113] Preferred recombinase sequences with mutations are listed below: The mutated first recombinase enzyme containing the K201R mutation has SEQ ID NO:2. The mutated second recombinase enzyme containing the Q311K mutation has SEQ ID NO:4. The mutated second recombinase enzyme containing the Q311R mutation has SEQ ID NO:5.

[0114] The recombinase enzyme contained in the obligate complex is preferably a DNA recombinase, which may be a naturally occurring recombinase (i.e., a recombinase isolated from any type of biological sample) or a designer recombinase, such as a recombinase evolved by directed molecular evolution or rational design, or any combination thereof. Methods for generating designer recombinases are known in the art. For example, WO 2018 / 229226 A1 teaches vectors and methods for making designer DNA recombinases by directed molecular evolution. WO 2008 / 083931 A1 discloses the directed molecular evolution of a tailored recombinase (Tre 1.0) using a sequence in the long terminal repeat (LTR) of HIV as a recognition site (loxLTR Tre 1.0). Further development of this approach using asymmetric target sites was described in WO 2011 / 147590 A2 (Tre 3.0) and WO 2016 / 034553 A1 (Tre 3.1 and Tre / Brec1) and in the publication Karpinski J et al., 2016 (Brec1). Methods for engineering naturally occurring or designer recombinases by rational design are also known in the art (e.g., Abi-Ghanem et al., 2013; Karimova et al., 2016).

[0115] According to a preferred embodiment of the present invention, the engineered DNA recombinase is a mutant of naturally occurring site-specific recombinase or a mutant of designer DNA recombinase.Naturally occurring site-specific recombinases include, but are not limited to, Cre-, Dre-, VCre-, SCre-, Vika-, lambda-Int-, Flp-, R-, Kw-, Kd-, B2-, B3-, Nigri- or Panto-recombinase.Designer DNA recombinases are disclosed, for example, in WO 2014 / 016248 and WO 2018 / 229226.

[0116] According to the present invention, at least one, preferably a single amino acid substitution in a DNA recombinase is located at a position within the catalytic region of said DNA recombinase. The catalytic region of a recombinase, specifically a tyrosine-type SSR recombinase, consists of six catalytic sites. These catalytic sites are shown in the alignment in FIG. 19 as boxes with numbered positions referring to Cre recombinase. Table 2 below identifies the amino acids, their positions within the complete sequence of each DNA recombinase shown, and the corresponding SEQ ID NOs belonging to the catalytic region. Table 2. Catalytic domains in exemplary DNA recombinases [Table 3]

[0117] Based on the alignment of different recombinases (see, for example, FIG. 19), catalytic regions in other recombinases can be identified. The skilled artisan will easily identify suitable alignment tools, such as Clustal Omega or EMBOSS Needle, well-known tools from the European Bioinformatics Institute of the European Molecular Biology Laboratory. For the sequence alignment shown in FIG. 19, the default settings of Clustal Omega v1.2.4 have been used. Based on such an alignment, a consensus sequence of the catalytic region can also be determined. Thus, according to the invention, the catalytic region can also be defined by the following amino acid sequence: [ka] Here, the amino acids in parentheses indicate alternative amino acids at the respective positions.

[0118] According to a preferred embodiment, at least one mutation, specifically a single amino acid substitution, in the DNA recombinase is at a conserved amino acid position in the catalytic domain of the DNA recombinase. Conserved amino acids within the catalytic domain of a recombinase can be derived from an alignment of recombinases, such as the alignment shown in Figure 19. Conserved amino acids within the catalytic region of the recombinase preferably include positions E129, R130, Q133, A134, A167, Y168, T170, L171, L172, R173, E176, K201, T202, H289, S290, A291, R292, V293, G294, A295, A296, R297, D298, M310, Q311, G313, W315, V321, M322, N323 and Y324 of SEQ ID NO:1, and corresponding positions in another recombinase, preferably one or more of SEQ ID NO:14, SEQ ID NO:17 and SEQ ID NO:20.

[0119] The term "amino acid position corresponding to position" as used in the context of the present invention refers to an amino acid position aligned in an amino acid sequence alignment with the amino acid sequence of the recombinase described herein, preferably the full-length amino acid sequence of SEQ ID NO: 1. For example, the skilled artisan can easily align further recombinases to the alignment shown in Figure 19, allowing the identification of amino acids corresponding to the amino acid positions identified for SEQ ID NO: 1 and also for SEQ ID NOs: 14, 17 and 20. For example, as can be derived from the alignment, the position corresponding to E129 in SEQ ID NO: 1 is E146 in SEQ ID NO: 14, E130 in SEQ ID NO: 17 and E131 in SEQ ID NO: 20. Thus, further corresponding amino acid positions of other recombinase enzymes can be identified by the skilled artisan without undue burden.

[0120] According to a particularly preferred embodiment, at least one mutation in the DNA recombinase, in particular a single amino acid substitution, is present at a position selected from the group consisting of positions E129, Q133, R173, E176, K201, H289, R292, Q311, W315 and Y324 of SEQ ID NO: 1, or at a corresponding position of another recombinase, wherein said amino acid position in the other recombinase corresponds to positions E129, Q133, R173, E176, K201, H289, R292, Q311, W315 or Y324 of SEQ ID NO: 1. Preferably, said other recombinase comprises a sequence according to one of SEQ ID NO: 14, SEQ ID NO: 17 and SEQ ID NO: 20. The corresponding positions are shown in the alignment in FIG. 19. In particular, the corresponding positions in SEQ ID NO: 14 are located at positions E146, Q151, R191, N194, K219, H308, R311, Q330, W334, and Y343, in SEQ ID NO: 17 they are located at positions E130, Q134, R174, E177, K202, H290, R293, Q312, W316, and Y325, and in SEQ ID NO: 20 they are located at positions E131, Q135, R175, E178, K202, H290, R293, Q312, W316, and Y325. From the alignment shown in Figure 19, the skilled artisan can align further DNA recombinases and identify their catalytic regions and the specific positions shown herein.

[0121] According to a particularly preferred embodiment, the single substitution in the DNA recombinase is selected from the group consisting of E129R, Q133H, R173A, R173C, R173D, R173E, R173F, R173G, R173I, R173K, R173L, R173M, R173N, R173P, R173Q, R173S, R173T, R173V, R173W, R173Y, E176H, E176I, E176L of SEQ ID NO:1. , E176M, E176V, E176W, E176Y, K201A, K201C, K201C, K201D, K201F, K201G, K201H, K201I, K201L, K201M, K20 1N, K201P, K201Q, K201R, K201S, K201T, K201V, K201W, K201Y, H289D, H289E, H289I, H289K, H289R, H289W, R2 92A, R292C, R292E, R292F, R292G, R292H, R292I, R292L, R292M, R292N, R292P, R292Q, R292S, R292T, R292V, R292W, R292Y, Q311R, W315C, W315E, W315G, W315I, W315K, W315L, W315M, W315N, W315Q, W315R, W315S, W315T , W315V, Y324A, Y324C, Y324E, Y324F, Y324H, Y324I, Y324K, Y324L, Y324M, Y324N, Y324Q, Y324R, Y324S, Y324T, Y324V and Y324W, or is present at the corresponding position in another recombinase, preferably at the corresponding position in SEQ ID NO:14, SEQ ID NO:17 or SEQ ID NO:20.

[0122] Particularly preferred combinations of single substitutions in the catalytic domains of the first and second DNA recombinases are highlighted in Table 6 and FIG.

[0123] Highly preferred combinations of single amino acid substitutions in the first and second DNA recombinases are single substitutions occurring at positions R173 and Q311, K201 and Q311, Q311 and R292, W315 and Y324, and positions Q311 and Y324 of SEQ ID NO:1, respectively, or at the corresponding positions of another recombinase, preferably at the corresponding positions of SEQ ID NO:14, SEQ ID NO:17 or SEQ ID NO:20.

[0124] In a preferred embodiment, the first recombinase enzyme comprises the at least one mutation selected from K201R, and the numbering of the amino acid positions refers to the amino acid sequence of the wild-type Cre recombinase monomer of SEQ ID NO: 1, and the second recombinase enzyme comprises the at least one mutation at a position selected from Q311K and Q311R, and the numbering of the amino acid positions refers to the amino acid sequence of the wild-type Cre recombinase monomer of SEQ ID NO: 1. The engineered obligate DNA recombinase of the present invention catalyzes DNA recombination events such as excision, replacement or inversion of a target sequence. The present invention specifically discloses an obligate complex of recombinase that catalyzes the inversion of a DNA sequence present in the int1h region on the human X chromosome.

[0125] In a most preferred embodiment, said first recombinase enzyme comprises said at least one or exactly one mutation at position K201R, wherein the numbering of the amino acid positions refers to the amino acid sequence of the wild-type Cre recombinase monomer of SEQ ID NO:1, and said second recombinase enzyme comprises said at least one or exactly one mutation Q311K and Q311R, wherein the numbering of the amino acid positions refers to the amino acid sequence of the wild-type Cre recombinase monomer of SEQ ID NO:1.

[0126] In a most preferred embodiment, the recombinase contained in the complex was generated using the methods described herein, and therefore the features and embodiments relating to the recombinase target sites and recombinases described later in this specification also apply to the described methods of the present invention.

[0127] A recombinase complex, such as a DNA recombinase of the present invention, also typically comprises four recombinase enzymes, i.e., four recombinase monomers. Compositions of the recombinase complex within the scope of the present invention include, for example: · All four recombinase monomers are different; ·Three recombinase monomers are identical and one monomer is different; · Two recombinase monomers are identical and the other two monomers are different; the complex comprises two different homodimers; the complex comprises two different heterodimers; the complex comprises two identical heterodimers; the complex comprises two different monomers; or The complex contains two identical monomers.

[0128] "Different" in this context means that the monomers are not identical in their primary structure, i.e. show differences in their amino acid sequence, in that they carry at least one, preferably a single amino acid substitution in their catalytic region; and / or show high specificity for one of the four half-sites of the upstream and downstream target sites of the recombinase, advantageously resulting in a surprisingly increased specificity of the engineered DNA recombinase of the invention. When the DNA recombinase of the invention consists of four monomers, preferably at least two monomers carry a first amino acid substitution in their catalytic region, for example a K201R mutation or a corresponding mutation thereto, and the other two monomers carry a second amino acid substitution in their catalytic region, for example a Q311K or Q311R mutation or a corresponding mutation thereto.

[0129] In a preferred embodiment, the recombinase complex is an obligate dimer, more preferably an obligate heterodimer, preferably capable of recognizing a first target sequence and a second target sequence of an upstream or downstream recombinase target site in DNA.

[0130] In a further preferred embodiment, the recombinase complex of the invention is a tetramer, more preferably a heterotetramer, for recognition of upstream and downstream target sites of a recombinase in DNA, said tetramer consisting of two obligate heterodimers as described herein, the monomers of said heterodimer being preferably linked to each other, e.g. via peptide bonds or protein-protein interactions, and said obligate heterodimer exhibiting DNA recombinase activity.

[0131] According to a further preferred embodiment of the invention, a monomer of said heterodimer has been further evolved by directed evolution or rational design to specifically recognize the first or second half-site of a recombinase target site, such that the first heterodimer in the complex of two obligate heterodimers specifically recognizes the first and second half-sites of an upstream recombinase target site in DNA, and the second obligate heterodimer specifically recognizes the first and second half-sites of a downstream recombinase target site.

[0132] More preferably, the monomer of said heterodimer is a tyrosine site-specific recombinase.Thus, a preferred embodiment of the present invention provides an engineered DNA recombinase comprising a recombinase subunit which is a site-specific recombinase, most preferably a tyrosine site-specific recombinase.

[0133] Preferably, the tyrosine site-specific recombinase is selected from the group consisting of Cre, Dre-, VCre-, SCre-, Vika-, lambda-Int-, Flp-, R-, Kw-, Kd-, B2-, B3-, Nigri- and Panto-recombinases. The recognition target sites of these bacterial and yeast T-SSR systems are discussed in Meinke et al., 2016 and Karimova et al., 2016, and are shown in Table 3 below. Table 3. Recognition target sites of bacterial and yeast T-SSR systems [Table 4] TIFF2024544843000009.tif105170Underlined solid line: Left half area Dashed underline: right half area Bold: spacer sequence

[0134] In a further preferred embodiment, an engineered DNA recombinase of the invention is provided, wherein said recombinase target site is a target site of a tyrosine site-specific recombinase evolved by directed evolution, such as Cre-, Dre-, VCre-, SCre-, Vika-, lambda-Int-, Flp-, R-, Kw-, Kd-, B2-, B3-, Nigri- and Panto-recombinase. In a more preferred embodiment, an engineered DNA recombinase of the invention is provided, wherein said recombinase target site is a target site of a tyrosine site-specific recombinase evolved by directed evolution, such as Cre-, Dre-, VCre-, Vika- and Panto-recombinase. Preferably, the corresponding single amino acid substitutions are at the following positions in the catalytic domains of the first and second recombinase enzymes in Dre-, VCre-, Vika- and Panto-recombinases: · Dre-recombinase: mutation K202R in the first recombinase enzyme and mutation Q312R in the second recombinase enzyme; · VCre-recombinase: mutation K221R in the first recombinase enzyme and mutation Q336R in the second recombinase enzyme; Vika recombinase: the mutation K219R in the first recombinase enzyme and the mutation Q330R in the second recombinase enzyme; and Panto-recombinase: Mutation K202R in the first recombinase enzyme and mutation Q312R in the second recombinase enzyme It will be introduced in.

[0135] Most preferably, said engineered DNA recombinase is selected from the group comprising: A genetically engineered DNA recombinase enzyme comprising a first recombinase enzyme and a second recombinase enzyme, wherein the first recombinase enzyme is a polypeptide having an amino acid sequence with at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO: 7 and comprising a mutation corresponding to the mutation at position K201R, and the second recombinase enzyme is a polypeptide having an amino acid sequence with at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO: 11 and comprising a mutation corresponding to the mutation at position Q311R. In this embodiment, the mutation at position K201R was introduced into the polypeptide of SEQ ID NO: 6 and the mutation at position Q311R was introduced into the polypeptide of SEQ ID NO: 10. Sequence identity in this context refers to the amino acid sequence outside the catalytic region of the recombinase, i.e. the catalytic region preferably comprises only a single amino acid substitution and no further mutations. A genetically engineered DNA recombinase enzyme comprising a first recombinase enzyme and a second recombinase enzyme, wherein the first recombinase enzyme is a polypeptide having an amino acid sequence with at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO: 7 and comprising a mutation corresponding to the mutation at position K201R, and the second recombinase enzyme is a polypeptide having an amino acid sequence with at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO: 12 and comprising a mutation corresponding to the mutation at position Q311K. In this embodiment, the mutation at position K201R was introduced into the polypeptide of SEQ ID NO: 6 and the mutation at position Q311K was introduced into the polypeptide of SEQ ID NO: 10. Sequence identity in this context refers to the amino acid sequence outside the catalytic region of the recombinase, i.e. the catalytic region preferably comprises only a single amino acid substitution and no further mutations.

[0136] According to a preferred embodiment, the mutation K201R is the only mutation in the catalytic domain of the first recombinase and the mutation Q311K or Q311R is the only mutation in the catalytic domain of the second recombinase.

[0137] In a further preferred embodiment, there is provided an engineered DNA recombinase of the invention, which is a variant of a naturally occurring site-specific recombinase selected from: a Cre recombinase comprising a K201R mutation in a first recombinase enzyme, said Cre recombinase having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 2; and a Q311R mutation in a second recombinase enzyme, said second recombinase having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence of SEQ ID NO: 5. The wild-type Cre recombinase from which the mutated SEQ ID NOs: 2 and 5 are derived has the sequence of SEQ ID NO: 1, the sequence identity referring to the amino acid sequence outside the catalytic region of the recombinase, i.e. the catalytic region preferably comprises only a single amino acid substitution and no further mutations; a Cre recombinase comprising a K201R mutation in a first recombinase enzyme, said Cre recombinase having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 2; and a Q311K mutation in a second recombinase enzyme, said second recombinase having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence of SEQ ID NO: 4. The wild-type Cre recombinase from which the mutated SEQ ID NOs: 2 and 4 are derived has the sequence of SEQ ID NO: 1, the sequence identity referring to the amino acid sequence outside the catalytic region of the recombinase, i.e. the catalytic region preferably comprises only a single amino acid substitution and no further mutations; a Vika recombinase comprising a K219R mutation in a first recombinase enzyme, said Vika recombinase having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 15; and a Q330R mutation in a second recombinase enzyme, said second recombinase enzyme having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence of SEQ ID NO: 16. The wild type Vika recombinase from which the mutated SEQ ID NOs: 15 and 16 are derived has the sequence of SEQ ID NO: 14, the sequence identity referring to the amino acid sequence outside the catalytic region of the recombinase, i.e. the catalytic region preferably comprises only single amino acid substitutions and no further mutations; a Panto recombinase comprising a K202R mutation in a first recombinase enzyme and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 18; and a Q312R mutation in a second recombinase enzyme having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence of SEQ ID NO: 19. The wild-type Panto recombinase from which the mutated SEQ ID NOs: 18 and 19 are derived has the sequence of SEQ ID NO: 17, the sequence identity referring to the amino acid sequence outside the catalytic region of the recombinase, i.e. the catalytic region preferably comprises only a single amino acid substitution and no further mutations; a Dre recombinase comprising a K202R mutation in a first recombinase enzyme and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 21; and a Q312R mutation in a second recombinase enzyme having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 22. The wild type Dre recombinase from which the mutated SEQ ID NOs: 21 and 22 are derived has SEQ ID NO: 20, the sequence identity referring to the amino acid sequence outside the catalytic region of the recombinase, i.e. the catalytic region preferably contains only a single amino acid substitution and no further mutations; and a Vcre recombinase comprising a K221R mutation in a first recombinase enzyme, said Vcre recombinase having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 24; and a Q336R mutation in a second recombinase enzyme, said second recombinase having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence of SEQ ID NO: 25. The wild type Vcre recombinase from which the mutated SEQ ID NOs: 24 and 25 are derived has SEQ ID NO: 23, the sequence identity referring to the amino acid sequence outside the catalytic region of the recombinase, i.e. the catalytic region preferably comprises only a single amino acid substitution and no further mutations.

[0138] According to a further preferred embodiment, the first recombinase enzyme comprises a mutation selected from the group consisting of the mutation K201R of SEQ ID NO: 1, 2 or 7, the mutation K202R of SEQ ID NO: 18 or 21, the mutation K219R of SEQ ID NO: 15 and the mutation K221R of SEQ ID NO: 24; and the second recombinase enzyme comprises a mutation selected from the group consisting of the mutation Q311K of SEQ ID NO: 4 or 11, the mutation Q311R of SEQ ID NO: 5 or 12, the mutation Q312R of SEQ ID NO: 19 or 22, the mutation Q330R of SEQ ID NO: 16 and the mutation Q336R of SEQ ID NO: 25.

[0139] Generally, the engineered DNA recombinase according to the invention recognises a recombinase target site, where the upstream recombinase target site and the downstream recombinase target site are asymmetric.

[0140] Most preferably, the engineered DNA recombinase according to the present invention comprises the nucleic acid sequence: [ka] or a reverse complement thereof, and specifically recognizes a recombinase target sequence upstream of a loxF8 target site having the nucleic acid sequence: [ka] or specifically recognizes a recombinase target sequence downstream of the loxF8 target site having a reverse complementary sequence thereof.

[0141] Even most preferably, the engineered DNA recombinase enzyme according to the present invention catalyzes the inversion of the genetic sequence between the upstream recombinase target sequence of SEQ ID NO:65 and the downstream recombinase target sequence of SEQ ID NO:66 of the loxF8 recombinase target site.

[0142] The ability of the loxF8 recombinase target site to catalyze the inversion of a gene sequence between the upstream recombinase target sequence of SEQ ID NO:65 and the downstream recombinase target sequence of SEQ ID NO:66 is a) expressing an engineered DNA recombinase comprising a first recombinase enzyme and a second recombinase enzyme in a cell; and b) analyzing whether the engineered DNA recombinase expressed in step a) is capable of inverting a DNA sequence on a human chromosome in said cell; The test can be performed by a method including:

[0143] A preferred engineered DNA recombinase or recombinase complex according to the present invention is a DNA recombinase capable of catalysing the inversion of a genetic sequence between an upstream recombinase target sequence of SEQ ID NO: 65 and a downstream recombinase target sequence of SEQ ID NO: 66 of a loxF8 recombinase target site, wherein a first recombinase enzyme has an amino acid sequence having at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8, and / or said second recombinase enzyme has an amino acid sequence having at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12.

[0144] The most preferred engineered DNA recombinase or recombinase complex according to the present invention is capable of catalysing the inversion of a genetic sequence between an upstream recombinase target sequence of SEQ ID NO: 65 and a downstream recombinase target sequence of SEQ ID NO: 66 of a loxF8 recombinase target site, and comprises a first recombinase enzyme having an amino acid sequence having at least 70%, preferably 80%, more preferably 90% sequence identity to the sequence set forth in SEQ ID NO: 7, and a second recombinase enzyme having an amino acid sequence having at least 70%, preferably 80%, more preferably 90% sequence identity to the sequence set forth in SEQ ID NO: 11, wherein the sequence identity preferably refers to the amino acid sequence outside the catalytic region of the recombinase.

[0145] Substrate-binding directed evolution techniques can be employed to generate DNA recombinases that recombine target sequences upstream and downstream of loxF8 target sites (Buchholz and Stewart 2001). Introducing mutations in such evolved DNA recombinases at positions corresponding to the mutations K201R, G282E or R282E in the first Cre recombinase monomer of SEQ ID NO: 1 and at positions corresponding to the mutations Q311K or Q311R in the second Cre recombinase monomer of SEQ ID NO: 1, respectively, results in the engineered recombinase monomers of SEQ ID NO: 2, 3, 4 and 5 of the present invention.

[0146] Said engineered DNA recombinase according to the invention recombines nucleic acid sequences, in particular DNA sequences, by recognizing two target sites and causing deletion, insertion, inversion or substitution of DNA sequences. Advantageously, the engineered DNA recombinase according to the invention recognizes asymmetric recognition sites of the loxF8 sequence according to SEQ ID NO: 65 (upstream) and SEQ ID NO: 66 (downstream). These recognition sites are not present anywhere in the human genome and can therefore be used for specific DNA recombination. Advantageously, the engineered DNA recombinase according to the invention does not require target sites to be artificially introduced into the genome. Even more advantageously and most preferably, the engineered DNA recombinase according to the invention causes an inversion of a DNA sequence. A further advantage is that the engineered DNA recombinase according to the invention allows precise genome editing without triggering endogenous DNA repair pathways. The invention further relates to a nucleic acid molecule, such as a polynucleotide or a nucleic acid or a plurality of nucleic acid molecules, each of which comprises or consists of a nucleic acid sequence encoding a DNA recombinase, such as a first and a second DNA recombinase according to the invention, or a genetically modified DNA recombinase or a subunit thereof.

[0147] The coding sequence encoding the polypeptide may be identical to the coding sequence of the polypeptide shown in SEQ ID NOs: 27 to 30, 32, 33, 36, 37, 40, 41, 43, 44, 46, 47, 49 and 50, preferably SEQ ID NOs: 27 to 30, 32, 33, 36 and 37, or may be a different coding sequence that encodes the same polypeptide as a result of redundancy or degeneracy of the genetic code or single nucleotide polymorphisms.

[0148] For example, it may be an RNA transcript of SEQ ID NO: 27-30, 32, 33, 36, 37, 40, 41, 43, 44, 46, 47, 49, or 50, which comprises the full length of the coding sequence of the polypeptide of the present invention. In a preferred embodiment, the "polynucleotide" according to the present invention is one of SEQ ID NO: 27-30, 32, 33, 36, or 37.

[0149] The wild type or original polypeptide used for genetic engineering according to the present invention is encoded by the following polynucleotide: the polynucleotide of SEQ ID NO:26 encodes the wild-type Cre DNA recombinase monomer of SEQ ID NO:1; the polynucleotide of SEQ ID NO:31 encodes the evolved D7L DNA recombinase monomer of SEQ ID NO:6; the polynucleotide of SEQ ID NO:35 encodes the evolved D7R DNA recombinase monomer of SEQ ID NO:10; the polynucleotide of SEQ ID NO: 39 encodes the wild-type Vika DNA recombinase monomer of SEQ ID NO: 14; the polynucleotide of SEQ ID NO: 42 encodes the wild-type Panto DNA recombinase monomer of SEQ ID NO: 17; the polynucleotide of SEQ ID NO: 45 encodes the wild-type Dre DNA recombinase monomer of SEQ ID NO: 20; and The polynucleotide of SEQ ID NO: 48 encodes the wild-type Cre DNA recombinase monomer of SEQ ID NO: 23.

[0150] Nucleic acids encoding the polypeptides of SEQ ID NO: 2-5, 7, 8, 11, 12, 15, 16, 18, 19, 21, 22, 24 or 25, preferably SEQ ID NO: 2-5, 7, 8, 11 or 12, may include, but are not limited to, a coding sequence for the polypeptide alone; a coding sequence for the polypeptide and additional coding sequences such as a leader sequence or secretory sequence or a proprotein sequence; and a coding sequence for the polypeptide (and optionally additional coding sequences) and non-coding sequences such as introns or non-coding sequences 5' and / or 3' of the coding sequence for the polypeptide. Nucleic acids encoding the polypeptides of SEQ ID NO: 2-5, 7, 8, 11, 12, 15, 16, 18, 19, 21, 22, 24 or 25, preferably SEQ ID NO: 2-5, 7, 8, 11 or 12, include nucleic acids that are codon-optimized for expression in human cells. They may further include a nuclear localization sequence.

[0151] Therefore, the term "polynucleotide encoding a polypeptide" or "nucleic acid encoding a polypeptide" should be understood to encompass polynucleotides or nucleic acids that comprise only the coding sequence of the DNA recombinase enzyme of the invention, e.g., the polypeptide of SEQ ID NO: 2-5, 7, 8, 11, 12, 15, 16, 18, 19, 21, 22, 24 or 25, preferably SEQ ID NO: 2-5, 7, 8, 11 or 12, as well as those that comprise additional coding and / or non-coding sequences. The terms polynucleotide and nucleic acid are used interchangeably.

[0152] The invention also includes polynucleotides in which the coding sequence of a polypeptide may be fused in the same reading frame to a polynucleotide sequence that aids in the expression and secretion of the polypeptide from a host cell; for example, a leader sequence may be so fused, which functions as a secretory sequence to control the transport of the polypeptide from the cell. Polypeptides with such leader sequences are called preproteins or preproproteins, and the leader sequence may be cleaved by the host cell to form the mature form of the protein. These polynucleotides may have a 5' extension region to code for a proprotein, which is a mature protein with additional amino acid residues added to the N-terminus. An expression product with such a prosequence is called a proprotein, which is an inactive form of the mature protein; however, when the prosequence is cleaved, an active mature protein remains. Additional sequences may also be attached to the protein and become part of the mature protein. Thus, for example, the polynucleotides of the invention may code for a polypeptide, or a protein with a prosequence, or a protein with both a prosequence and a presequence (such as a leader sequence).

[0153] The polynucleotides of the invention may also have the coding sequence fused in frame to a marker sequence that allows for purification of the polypeptide of the invention. The marker sequence can be an affinity tag or an epitope tag, such as a polyhistidine tag, a streptavidin tag, an Xpress tag, a FLAG tag, a cellulose or chitin binding tag, a glutathione-S-transferase tag (GST), a hemagglutinin (HA) tag, a c-myc tag or a V5 tag.

[0154] The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984), and the c-myc tag may be an epitope from the human Myc protein (Evans et al., 1985).

[0155] When the nucleic acid of the invention is an mRNA, particularly for use as a pharmaceutical, delivery of mRNA therapeutics has been facilitated by significant advances in maximizing the translation and stability of the mRNA, preventing its immunostimulatory activity, and developing in vivo delivery techniques. The 5' cap and 3' poly(A) tail are the primary contributors to efficient translation and extended half-life of mature eukaryotic mRNA. Incorporation of cap analogs such as ARCA (anti-reverse cap analog) and 120-150 bp poly(A) tails into in vitro transcribed (IVT) mRNA has significantly improved the expression of the encoded protein and the stability of the mRNA. New types of cap analogs such as 1,2-dithiodiphosphate modified caps are resistant to RNA decapping complexes and can further improve the efficiency of RNA translation. Replacing rare codons in the mRNA protein coding sequence with frequently occurring synonymous codons, so-called codon optimization, promotes more efficient protein synthesis and limits mRNA destabilization by rare codons, thus preventing accelerated degradation of the transcript. Similarly, engineering the 3' and 5' untranslated regions (UTRs), which contain sequences involved in the recruitment of RNA-binding proteins (RBPs) and miRNAs, can increase the levels of protein products. Interestingly, UTRs can be purposefully modified to encode regulatory elements (e.g., K-turn motifs and miRNA binding sites), providing a means to control RNA expression in a cell-specific manner. Some RNA base modifications, such as N1-methyl-pseudouridine, have been shown to not only serve to mask mRNA immunostimulatory activity, but also to increase mRNA translation by enhancing translation initiation. In addition to the observed effects on protein translation, base modifications and codon optimization affect the secondary structure of mRNA, which in turn affects translation. Respective modifications of the nucleic acid molecules of the present invention are also contemplated by the present invention.

[0156] The RNA or RNAs preferably code for either the DNA recombinase or its subunits. Specific methods for delivering and expressing nucleic acids and specifically RNA are disclosed, for example, in EP2590676 and EP3115064. The RNA may be present in a particle, and is preferably self-replicating. After the particle is administered in vivo, the RNA is released from the particle and translated in the cell to provide either the DNA recombinase or its monomeric subunits.

[0157] A self-replicating RNA molecule (replicon), when delivered to a vertebrate cell without any proteins, can result in the production of multiple daughter RNAs by transcription from itself (through antisense copies it produces). These daughter RNAs, and colinear subgenomic transcripts, can themselves be translated to provide in situ expression of the encoded polypeptide, or transcribed to provide additional transcripts in the same sense as the delivered RNA that is translated to provide in situ expression of the polypeptide. The overall result of this transcriptional sequence is a large amplification of the number of replicon RNAs introduced, and thus the encoded polypeptide becomes the major polypeptide product of the cell.

[0158] A preferred self-replicating RNA molecule encodes (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule and (ii) a polypeptide of the invention. The polymerase may be, for example, an alphavirus replicase, including one or more of the alphavirus proteins nsP1, nsP2, nsP3 and nsP4. A self-replicating RNA molecule of the invention preferably does not encode an alphavirus structural protein. Thus, a preferred self-replicating RNA is capable of generating its own genomic RNA copy in a cell, but is unable to generate RNA-containing virions. A self-replicating RNA molecule useful in the context of the present invention may have two open reading frames. The first (5') open reading frame encodes the replicase and the second (3') open reading frame encodes the polypeptide of the invention. In some embodiments, the RNA may have additional (e.g., downstream) open reading frames, for example, to further encode accessory polypeptides.

[0159] Such RNA is particularly suitable for use in gene therapy generally, and in the treatment of genetic disorders or diseases in particular.

[0160] The present invention further contemplates providing polynucleotides that hybridize to the sequences described hereinabove, where there is at least 70%, preferably at least 90%, more preferably at least 95% identity or similarity between the sequences, and thus encode proteins with similar biological activity. Furthermore, as is known in the art, "similarity" exists between two polypeptides when the amino acid sequences contain identical or conserved amino acid substitutions for individual residues in the sequence. Identity and similarity can be measured using sequence analysis software (e.g., ClustalW from PBIL (Pole Bioinformatique Lyonnais) http: / / npsa-pbil.ibcp.fr). The present invention particularly provides such polynucleotides that hybridize under stringent conditions to the polynucleotides described hereinabove.

[0161] Appropriate stringent conditions can be defined, for example, by the concentrations of salt or formamide in the prehybridization and hybridization solutions, or the hybridization temperature, and are well known in the art. In particular, stringency can be increased by lowering the concentration of salt, by increasing the concentration of formamide, and / or by raising the hybridization temperature.

[0162] For example, hybridization under high stringency conditions may employ about 50% formamide at about 37°C to 42°C, while hybridization under reduced stringency conditions may employ about 35% to 25% formamide at about 30°C to 35°C. One particular set of conditions for hybridization under high stringency conditions employs 50% formamide, 5x SSPE, 0.3% SDS, and 200 μg / ml sheared and denatured salmon sperm DNA at 42°C. For hybridization under reduced stringency, similar conditions may be used with 35% formamide at a reduced temperature of 35°C. The temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly. Variations of the above ranges and conditions are well known in the art. Preferably, hybridization should occur only where there is at least 95%, more preferably at least 97% identity between the sequences. In a preferred embodiment, a polynucleotide that hybridizes to the polynucleotides described herein above encodes a polypeptide that exhibits substantially the same biological function or activity as the mature protein of SEQ ID NO: 2-5, 7, 8, 11, 12, 15, 16, 18, 19, 21, 22, 24 or 25, preferably SEQ ID NO: 2-5, 7, 8, 11 or 12.

[0163] As mentioned above, suitable polynucleotide probes may have at least 14 bases, preferably 30 bases, more preferably at least 50 bases, and hybridize to the polynucleotides of the invention having identity thereto as described herein above. For example, such polynucleotides may be employed as probes for hybridizing to polynucleotides encoding the polypeptides of SEQ ID NOs: 2-5, 7, 8, 11, 12, 15, 16, 18, 19, 21, 22, 24 or 25, preferably SEQ ID NOs: 2-5, 7, 8, 11 or 12, respectively, for example, for the recovery of such polynucleotides, or as diagnostic probes, or as PCR primers. Therefore, the present invention includes polynucleotides having at least 70% identity, preferably at least 90% identity, more preferably at least 95% identity to the polynucleotides of SEQ ID NOs: 2 to 5, 7, 8, 11, 12, 15, 16, 18, 19, 21, 22, 24 or 25, preferably SEQ ID NOs: 2 to 5, 7, 8, 11 or 12, and fragments thereof preferably having at least 30 bases, more preferably at least 50 bases.

[0164] The terms "homology" or "identity," used interchangeably herein, refer to the sequence similarity between two polynucleotide sequences or two polypeptide sequences, with identity being the more strict comparison. The phrases "percent identity or homology" and "identity or homology" refer to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences. "Sequence similarity" refers to the percent similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0 to 100% similar, or any integer value therebetween. Identity or similarity can be determined by comparing positions in each sequence that can be aligned for purposes of comparison. If a position in the compared sequences is occupied by the same nucleotide base or amino acid, the molecules are identical at that position. The degree of similarity or identity between polynucleotide sequences is a function of the number of identical or matching nucleotides at positions shared by the polynucleotide sequences.

[0165] A degree of identity of polypeptide sequences is a function of the number of identical amino acids at positions shared by the polypeptide sequences. A degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at positions shared by the polypeptide sequences. As used herein, the term "substantially identical" refers to an identity or homology of at least 70%, 75%, at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0166] The degree of sequence identity is determined by selecting one sequence as a query sequence and aligning it with homologous sequences retrieved from GenBank using the blastp algorithm (NCBI) using the internet-based tool ClustalW.

[0167] As is well known in the art, the genetic code is redundant in that a particular amino acid is coded for by more than one nucleotide triplet (codon), and the present invention includes polynucleotide sequences that code for the same amino acid using different codons than those specifically exemplified in the sequences herein. Such polynucleotide sequences are referred to herein as "equivalent" polynucleotide sequences. The present invention further includes variants of the polynucleotides described herein above that code for fragments, such as part or all of the proteins, analogs, and derivatives of the polypeptides of SEQ ID NOs: 2-5, 7, 8, 11, or 12. Variant forms of polynucleotides can be allelic variants of naturally occurring polynucleotides or variants of non-naturally occurring polynucleotides. For example, variants of nucleic acids can simply differ in the codon sequence for an amino acid due to the degeneracy of the genetic code, or there can be deletion variants, substitution variants, and addition or insertion variants. As is known in the art, allelic variants are alternative forms of a polynucleotide sequence and can have substitutions, deletions, or additions of one or more nucleotides that do not substantially alter the biological function of the encoded polypeptide.

[0168] In a further embodiment, the polynucleotide of the invention encodes an obligate heterodimer, said heterodimer comprising a first recombinase enzyme having an amino acid sequence having at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO: 2, 3, 7, 8, 15, 18, 21 or 24 for recognition of upstream and downstream target sequences of a recombinase target site, and a second recombinase enzyme having an amino acid sequence having at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO: 4, 5, 11, 12, 16, 19, 22 or 25.

[0169] In one embodiment, a polynucleotide of the invention comprises the nucleic acid of SEQ ID NO:27 and the nucleic acid of SEQ ID NO:29.

[0170] In a further embodiment, the polynucleotide of the invention comprises the nucleic acid of SEQ ID NO:27 and the nucleic acid of SEQ ID NO:30.

[0171] In a further embodiment, the polynucleotide of the invention comprises the nucleic acid of SEQ ID NO:28 and the nucleic acid of SEQ ID NO:30.

[0172] In a further embodiment, the polynucleotide of the invention comprises the nucleic acid of SEQ ID NO:32 and the nucleic acid of SEQ ID NO:36.

[0173] In a further embodiment, the polynucleotide of the invention comprises the nucleic acid of SEQ ID NO:32 and the nucleic acid of SEQ ID NO:37.

[0174] In a further embodiment, the polynucleotide of the invention comprises the nucleic acid of SEQ ID NO:33 and the nucleic acid of SEQ ID NO:36.

[0175] In a further embodiment, the polynucleotide of the invention comprises the nucleic acid of SEQ ID NO:40 and the nucleic acid of SEQ ID NO:41.

[0176] In a further embodiment, the polynucleotide of the invention comprises the nucleic acid of SEQ ID NO:43 and the nucleic acid of SEQ ID NO:44.

[0177] In a further embodiment, the polynucleotide of the invention comprises the nucleic acid of SEQ ID NO:46 and the nucleic acid of SEQ ID NO:47.

[0178] In a further embodiment, the polynucleotide of the invention comprises the nucleic acid of SEQ ID NO:49 and the nucleic acid of SEQ ID NO:50.

[0179] The invention also provides vectors, preferably expression vectors, comprising such polynucleotides, host cells genetically engineered with such vectors or comprising a nucleic acid molecule or nucleic acid molecules according to the invention, and the production of a polypeptide of the invention, such as SEQ ID NO: 2-5, 7, 8, 11, 12, 15, 16, 18, 19, 21, 22, 24 or 25, preferably SEQ ID NO: 2-5, 7, 8, 11 or 12, by recombinant techniques. The host cells are genetically engineered (transduced or transformed or transconjugated or transfected) with such vectors, which may for example be cloning vectors or expression vectors. The vectors may be in the form of, for example, plasmids, conjugated plasmids, viral particles, phages, etc. The vectors or genes may be integrated into the chromosome at specific or non-specific sites. Methods for genomic integration of recombinant DNA, such as homologous recombination or transposase-mediated integration, are well known in the art. The engineered host cells may be cultured in conventional nutrient media, modified as appropriate to activate promoters, select for transformants, or amplify the genes of the invention. Culture conditions such as temperature, pH, etc. are those commonly used with the host cell selected for expression, which are well known to those of skill in the art. The host cell may be a mammalian cell, an insect cell, a plant cell, or a bacterial host cell that contains a nucleic acid or recombinant polynucleotide molecule or an expression vector described herein.

[0180] The polynucleotide sequence in the expression vector is operably linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. Non-limiting representative examples of such promoters include LTR or SV40 promoter, E. coli lac, ara, rha or trp, the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.

[0181] One of skill in the art can select a vector based on desired properties for production in a particular cell, such as, for example, a mammalian cell or a bacterial cell.

[0182] Any of a variety of inducible promoters or enhancers can be included in vectors for expression of the antibodies or regulatable nucleic acids of the present invention. Such inducible systems include, for example, the tetracycline inducible system; the metallothionein promoter induced by heavy metals; insect steroid hormones responsive to ecdysone or related steroids such as muristerone; mouse mammary tumor virus (MMTV) induced by steroids such as glucocorticoids and estrogens; and heat shock promoters inducible by temperature changes; the rat neuron-specific enolase gene promoter; the human β-actin gene promoter; the human platelet-derived growth factor B (PDGF-B) chain gene promoter; the rat sodium channel gene promoter; the human copper-zinc superoxide dismutase gene promoter; and promoters for members of the mammalian POU domain regulatory gene family.

[0183] Regulatory elements, including promoters or enhancers, can be constitutive or regulated depending on the nature of the regulation. Regulatory sequences or regulatory elements are operably linked to one of the polynucleotide sequences of the present invention such that the physical and functional relationship between the polynucleotide sequence and the regulatory sequence allows transcription of the polynucleotide sequence. Vectors useful for expression in eukaryotic cells can include regulatory elements, including, for example, CAG promoter, SV40 early promoter, cytomegalovirus (CMV) promoter, mouse mammary tumor virus (MMTV) steroid-inducible promoter, Pgtf, Moloney murine leukemia virus (MMLV) promoter, thy-1 promoter, etc.

[0184] Optionally, the vector may include a selection marker. As used herein, "selection marker" refers to a genetic element that provides a selectable phenotype to a cell into which the selection marker is introduced. A selection marker is generally a gene whose gene product inhibits cell growth or provides resistance to a drug that kills the cell. A variety of selection markers can be used in the DNA construct of the present invention, including, for example, Neo, Hyg, hisD, Gpt and Ble genes, as described, for example, in Ausubel et al., 1999 and U.S. Pat. No. 5,981,830. Drugs useful for selecting the presence of the selection marker include, for example, G418 for Neo, hygromycin for Hyg, histidinol for hisD, xanthine for Gpt, and bleomycin for Ble. The DNA construct of the present invention may incorporate a positive selection marker, a negative selection marker, or both.

[0185] Various mammalian cell culture systems can also be employed to express recombinant proteins. An example of a mammalian expression system includes the COS-7 line of monkey kidney fibroblasts. Other cell lines that can express compatible vectors include, for example, C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors generally also include an origin of replication, a suitable promoter and enhancer, and any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from SV40 splice and polyadenylation sites can be used to provide the necessary nontranscribed genetic elements.

[0186] Polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphorylated cellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Recovery can be facilitated if the polypeptide is expressed on the surface of the cell, but this is not a prerequisite. It may also be desirable to recover cleavage products that are cleaved after expression of longer polypeptide forms. Protein refolding steps, as known in the art, can be used to complete configuration of the mature protein, if necessary. High performance liquid chromatography (HPLC) can be employed for final purification steps.

[0187] According to a further embodiment of the present invention, there is provided a gene therapy vector for use in, for example, increasing the expression of an engineered protein of the present invention systemically or locally in a subject. The gene therapy vector is used to prevent, alleviate, ameliorate, reduce, inhibit, and / or treat a disease treatable by genome editing, particularly hemophilia A. The gene therapy vector typically comprises an expression cassette comprising an engineered polynucleotide encoding a DNA recombinase of the present invention. In one embodiment, the vector is a viral vector. In a preferred embodiment, the viral vector is derived from a virus selected from the group consisting of adenovirus, retrovirus, lentivirus, herpesvirus, and adeno-associated virus (AAV). In a more preferred embodiment, the vector is derived from one or more of adeno-associated virus (AAV) serotypes 1-11, or any subgroup or any engineered form thereof. In another embodiment, the viral vector is encapsulated in an anionic liposome.

[0188] In another embodiment, the vector is a non-viral vector. In a preferred embodiment, the non-viral vector is selected from the group consisting of naked DNA, cationic liposome complexes, cationic polymer complexes, cationic liposome-polymer complexes, and exosomes.

[0189] When the vector is a viral vector, the expression cassette suitably comprises a first inverted terminal repeat operably linked in a 5' to 3' direction (in terms of the transcribed mRNA), an enhancer, a promoter, a polynucleotide encoding an engineered DNA recombinase of the invention, a 3' untranslated region, a polyadenylation (polyA) signal, and a second inverted terminal repeat. The promoter is, for example, selected from the group consisting of a cytomegalovirus (CMV) promoter and a chicken beta actin (CAG) promoter. The polynucleotide preferably comprises DNA or cDNA or RNA or mRNA. In a preferred embodiment, the polynucleotide encoding an engineered DNA recombinase of the invention comprises one or more of the polypeptides of SEQ ID NOs: 27-30, 32, 33, 36, 37, 40, 41, 43, 44, 46, 47, 49 and 50. In a most preferred embodiment, a polynucleotide encoding an engineered DNA recombinase of the present invention has at least about 75%, 80%, 85% or 90% sequence identity to one or more of SEQ ID NOs: 27-30, 32, 33, 36 and 37, for example, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.

[0190] The present invention further relates to an engineered DNA recombinase of the invention or a nucleic acid molecule or molecules of the invention, or a recombinant polynucleotide, or an expression vector of the invention for use as a medicament. In a more preferred embodiment, the present invention further relates to an engineered DNA recombinase of the invention or a nucleic acid molecule or molecules of the invention, or a recombinant polynucleotide, or an expression vector of the invention for use in the prevention or treatment of diseases that can be treated by genome editing, in particular hemophilia A.

[0191] In a further embodiment, the present invention relates to the use of an engineered DNA recombinase of the present invention or a nucleic acid molecule or molecules of the present invention, or a recombinant polynucleotide, or an expression vector, for the preparation of a medicament for the prevention or treatment of a disease that can be treated by genome editing. According to a preferred embodiment, said disease is a genetic disease or disorder. According to a particularly preferred embodiment, said disease or disorder is hemophilia A.

[0192] In a further embodiment, the present invention relates to a method for the prevention or treatment of a disease that can be treated by genome editing, in particular hemophilia A, comprising administering a therapeutically effective amount of an engineered DNA recombinase of the invention or a nucleic acid molecule or molecules of the invention, or a recombinant polynucleotide, or an expression vector to a patient in need thereof.

[0193] The engineered DNA recombinase of the invention or the nucleic acid molecule or molecules of the invention or the recombinant polynucleotide or the expression vector can be used for the treatment of a genetic disorder or a disease caused by a genetic disorder. A particularly preferred embodiment relates to the treatment of severe forms of hemophilia A.

[0194] The present invention also provides a method for producing an obligate DNA recombinase enzyme complex. According to a preferred embodiment, such a method comprises: (i) introducing a single amino acid substitution into a catalytic domain of a first DNA recombinase enzyme, wherein said single amino acid substitution renders the first DNA recombinase enzyme catalytically inactive; (ii) introducing a single amino acid substitution into the catalytic domain of a second DNA recombinase enzyme, wherein said single amino acid substitution renders the second DNA recombinase enzyme catalytically inactive; (iii) co-expressing both the mutated first DNA recombinase enzyme and the mutated second DNA recombinase enzyme in the host cell; (iv) isolating the mutated first DNA recombinase enzyme and the mutated second DNA recombinase enzyme from the host cell. Includes.

[0195] The present invention provides a method for producing an obligate DNA recombinase for genome editing, preferably for recombination of DNA sequences, comprising the steps of: i. providing a nucleic acid molecule encoding a first recombinase enzyme and a nucleic acid molecule encoding a second recombinase enzyme, wherein the first recombinase enzyme binds to a first half-site of an asymmetric recombinase target site and the second recombinase enzyme binds to a second half-site of the asymmetric recombinase target site, wherein the first recombinase enzyme and the second recombinase enzyme form a heterodimer capable of inducing site-specific DNA recombination of a sequence of interest at the asymmetric recombinase target site in a DNA sequence, wherein the asymmetric recombinase target site comprises a first half-site and a second half-site of a recombinase target site which are non-identical and non-palindromic; ii. performing mutagenesis to generate a library of nucleic acid molecules encoding a first mutant recombinase enzyme and nucleic acid molecules encoding a second mutant recombinase enzyme, where mutations are introduced into the first recombinase enzyme and the second recombinase enzyme; iii. generating an expression vector by cloning the library of nucleic acid molecules encoding a first mutant recombinase enzyme and the library of nucleic acid molecules encoding a second mutant recombinase enzyme into an expression vector, wherein said expression vector carries the DNA sequences of interest to be recombined; iv. transfecting a cell with the expression vector of step iii) and expressing the first mutant recombinase enzyme and the library of second mutant recombinase enzymes in the same cell, thereby forming a recombinase heterodimer comprising the first mutant recombinase enzyme and the second mutant recombinase enzyme; v. performing a positive selection screening for heterodimers obtained in step iv that are capable of inducing site-specific DNA recombination of the sequence of interest at the asymmetric recombinase target site in the DNA; vi. performing a negative selection screening for heterodimers obtained in step iv or v that are unable to induce site-specific DNA recombination of the sequence of interest at an off-target site, preferably a symmetric recombinase target site in the DNA; vii. selecting an obligate DNA recombinase that is capable of recombining a DNA sequence of interest at a recombinase target site in the DNA at an asymmetric recombinase target site and is incapable of recombining a DNA sequence of interest at an off-target recombinase target site in the DNA; The method further comprises:

[0196] Surprisingly, it was found that interfacial mutations between the monomers not only prevented homodimer formation but also dramatically reduced the recombination activity of the heterodimer at the asymmetric recombinase target site, whereas mutations in the catalytic domains of the first and second recombinase enzymes led to the formation of heterodimers with high activity at the asymmetric recombinase target site.

[0197] Thus, in a preferred embodiment of the method of the present invention, the first mutant recombinase enzyme and the second mutant recombinase enzyme of the obligate DNA recombinase obtained in step vii each comprise at least one mutation in a catalytic site, which renders the first recombinase enzyme and the second recombinase enzyme catalytically inactive when expressed alone.

[0198] More preferably, said first mutant recombinase enzyme and said second mutant recombinase enzyme of said obligate DNA recombinase obtained in step vii do not comprise inter-monomer interface mutations.

[0199] The first recombinase enzyme and the second recombinase enzyme according to steps ii to vi are preferably evolved by substrate-binding directed evolution (SLiDE) or directed evolution. Evolution of recombinases using substrate-binding protein evolution (SLiDE) is known in the art and described, for example, in Buchholz and Stewart, 2001; Sakara et al., 2007; Karpinski et al., 2016; and Lansing et al., 2020 and WO 2018 / 229226 A1, herein below.

[0200] In a preferred embodiment, the selection according to steps v and vi of the method of the invention is repeated between the selection of obligate heterodimers that are catalytically active at an asymmetric target site (positive selection) and between heterodimers that are catalytically inactive at an off-target site, preferably a symmetric target site.

[0201] The method according to the invention comprises the generation of positive selection pressure for activity at an asymmetric target site and the generation of negative selection pressure at a symmetric target site, where the generation of positive selection pressure and the generation of said negative selection pressure are achieved by diversifying two libraries of DNA recombinase enzymes via error-prone PCR (e.g., error-prone MyTaq DNA polymerase, Bioline) and selecting mutant pairs of a first recombinase enzyme and a second recombinase enzyme that have activity at the desired asymmetric target site, as described in more detail in Example 2 and Figures 2 and 15.

[0202] More preferably, at least 10, more preferably at least 15, and most preferably at least 20 SLiDE cycles of positive and negative selection are performed.

[0203] In a further preferred embodiment of the method of the invention, the positive and negative selection screen comprises purification of the expression vector after culturing the cells obtained in step iv and analysis of recombinant and non-recombinant vectors.

[0204] Most preferably, the positive selection screen is performed against the asymmetric target site loxF8 and the negative selection screen is performed against the symmetric target sites loxF8R and loxF8L.

[0205] The method according to the invention comprises the steps of: viii. identifying potential off-target sites for the desired obligate DNA recombinase; ix. Analyzing the recombinase activity of the desired obligate DNA recombinase at the identified off-target sites; and x. Selecting an obligate DNA recombinase that does not exhibit recombinase activity at at least one off-target site It may further include:

[0206] The present invention further relates to an obligate DNA recombinase identified or obtained using any of the aforementioned methods. The properties, characteristics and embodiments described above for the obligate DNA recombinase apply equally to the method of generating an obligate DNA recombinase for genome editing and to the obligate DNA recombinase obtained by said method.

[0207] The genetically engineered DNA recombinase of the invention or the nucleic acid molecule, recombinant polynucleotide or expression vector or host cell of the invention can further be comprised in a pharmaceutical composition, which may optionally further contain one or more therapeutically acceptable diluents or carriers.

[0208] According to a further aspect, the present invention provides a pharmaceutical composition for use in preventing or treating a disorder that can be treated by genome editing, such as, for example, hemophilia A. The pharmaceutical composition of the present invention comprises at least one first DNA recombinase enzyme and / or at least one second DNA recombinase enzyme of the invention, or a DNA recombinase enzyme of the invention, or a nucleic acid molecule or nucleic acid molecules of the invention, or a multiple recombination polynucleotide, or an expression vector, or a host cell, and one or more therapeutically acceptable diluents or carriers.

[0209] A pharmaceutical composition according to a preferred embodiment comprises a therapeutically effective amount of a vector comprising a nucleic acid sequence of a polynucleotide encoding one or more engineered proteins according to the invention, or comprising a nucleic acid encoding a therapeutically active amount of an engineered recombinant DNA recombinase of the invention, or comprising a therapeutically active amount of an engineered recombinant DNA recombinase of the invention, or comprising a therapeutically active amount of a host cell(s) of the invention (together termed a "therapeutically active agent").

[0210] The present invention also provides a method of treating a disease or disorder, particularly a genetic disease or disorder, by administering a therapeutically effective amount of an engineered DNA recombinase, or a nucleic acid molecule of the invention, or a recombinant polynucleotide, or an expression vector, or a host cell to a subject in need thereof.

[0211] It will be understood that the single or total daily dosage of the therapeutically active agents and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed, the age, weight, general health, sex and diet of the patient; the duration, route of administration and excretion rate of the specific compound employed; the duration of treatment; drugs used in combination or with the specific nucleic acid or polypeptide employed; as well as factors well known in the medical arts. For example, it is well within the skill of the art to begin administering the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the product may vary over a wide range per adult per day. The therapeutically effective amount of the therapeutically active agent, such as the vector according to the present invention, to be administered, as well as the dosage for treating a pathological condition with the numerous viral or non-viral particles and / or pharmaceutical compositions described herein, will depend on a number of factors, including the age and condition of the patient, the severity of the disturbance or disorder, the method and frequency of administration, and the specific peptide used.

[0212] Pharmaceutical compositions containing the therapeutically active agents according to the invention may be in any form suitable for the selected mode of administration.

[0213] In one embodiment, the pharmaceutical compositions of the invention are administered parenterally.

[0214] The phrases "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include intraepidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, intrathecal, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion.

[0215] The therapeutically active agents of the present invention may be administered to animals and humans as the sole active agent or in combination with other active agents, in unit dosage forms, in admixture with conventional pharmaceutical supports.

[0216] In a further embodiment, the pharmaceutical composition contains a pharma- ceutically acceptable vehicle for an injectable formulation. These may be, in particular, isotonic sterile saline (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc., or mixtures of such salts), or a dry, in particular lyophilized, composition, which, upon addition of sterile water or saline, as the case may be, allows the constitution of an injectable solution.

[0217] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.In all cases, the form must be sterile and liquid.It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0218] Solutions containing the therapeutically active agent as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0219] The therapeutically active agents can be formulated into the compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), for example, formed with inorganic acids such as hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0220] The carrier may also be present as a solvent or dispersion medium, including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0221] Sterile injectable solution is prepared by incorporating the active polypeptide in the required amount in a suitable solvent together with some of the other ingredients listed above as necessary, followed by filtration sterilization.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other necessary ingredients from those listed above.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces powder of active ingredient plus any additional desired ingredients from a previously sterile-filtered solution.

[0222] Upon formulation, solutions can be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described above, but also drug release capsules and the like can be employed. Multiple doses can also be administered. Where appropriate, the therapeutically active agents described herein can be formulated in any suitable vehicle for delivery. For example, they can be placed in a pharma-ceutically acceptable suspension, solution or emulsion. Suitable media include saline and liposomal preparations. More specifically, pharma-ceutically acceptable carriers include sterile aqueous carriers for non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include, but are not limited to, water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.

[0223] Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0224] Colloidal dispersion systems can also be used for targeted gene delivery. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0225] The appropriate treatment regimen can be determined by a physician and depends on the age, sex, weight, and stage of the disease of the subject. As an example, for delivery of the nucleic acid sequence encoding the engineered DNA recombinase of the present invention using a viral expression vector, each unit dose of the engineered DNA recombinase expression vector may be, for example, 10 per ml. 11 ~10 16 The composition may comprise between 2.5 μl and 100 μl of a composition comprising a viral expression vector in a medicamentously acceptable fluid at a viral genome concentration in the range of 100 μl to 200 μl.

[0226] Effective dosages and dosing regimens for administering the engineered DNA recombinase of the present invention in the form of a recombinant polypeptide will depend on the disease or condition to be treated and can be determined by one of skill in the art. Exemplary, non-limiting ranges for therapeutically effective amounts of the engineered DNA recombinase of the present invention are about 0.1-10 mg / kg / body weight, 0.1-5 mg / kg / body weight, etc., such as 0.1-2 mg / kg / body weight, 0.1-1 mg / kg / body weight, etc., such as about 0.15, about 0.2, about 0.5, about 1, about 1.5 or about 2 mg / kg / body weight.

[0227] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the effective amount of pharmaceutical composition required. For example, the physician or veterinarian can start with a dose of the therapeutically active agent of the present invention employed in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. In general, the appropriate daily dose of the composition of the present invention is the amount of the delivery system that is the lowest dose effective to produce a therapeutic effect. Such an effective dose generally depends on the factors described above. Administration may be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, for example, administered proximal to the target site. If desired, the effective daily dose of the pharmaceutical composition may be administered as two, three, four, five, six or more subdoses, optionally in unit dosage form, administered separately at appropriate intervals throughout the day. Although it is possible to administer the delivery system of the present invention alone, it is preferred to administer the delivery system as a pharmaceutical composition as described above.

[0228] Further provided is a kit comprising the therapeutically active agent described above and herein.In one embodiment, the kit provides the therapeutically active agent in one or more unit dosage forms, for example in a preloaded syringe or ampoule, ready for administration to a subject.In another embodiment, the therapeutically active agent is provided in lyophilized form.

[0229] Nucleic acid sequences that are potential target sites for DNA recombinases capable of inducing site-specific DNA recombination of a sequence of interest in a genome can be identified according to the methods described in WO 2018 / 229226 A1, which can be, for example, a) screening the genome or a part thereof comprising the sequence of interest for two sequences which are potential spacer sequences having a length of at least 5 and at most 12 bp, where one of the potential spacer sequences is upstream of the sequence of interest and the other potential spacer sequence is downstream of the sequence of interest, the two sequences having a maximum distance of no more than 2 megabases, preferably no more than 1.5 megabases or no more than 1 megabase, more preferably 900 kb, 800 kb, 700 kb, 600 kb or 500 kb, most preferably 400 kb or 300 kb, and a minimum distance of 150 bp; b) identifying potential target sites by determining, for each potential spacer sequence, the adjacent nucleotides on one side, preferably 10-20 nucleotides, more preferably 12-15 nucleotides, most preferably 13 nucleotides, that form a potential first half-site, and the adjacent nucleotides on the other side, preferably 10-20 nucleotides, more preferably 12-15 nucleotides, most preferably 13 nucleotides, that form a potential second half-site, both of which form potential target sites; c) the substep of further screening the potential target sites identified in step b) to select potential target sequences that do not occur (elsewhere) in the genome of the host to ensure sequence-specific recombination, preferably inversion; Includes.

[0230] Preferably, the sequences of the potential target sites identified for the DNA recombinase occur naturally in the genome.

[0231] The first and second recombinase enzymes can be evolved by directed evolution or rational design, preferably by substrate-binding directed evolution (SLiDE), e.g. as described in WO 2018 / 229226 A1, until at least one first designer DNA recombinase enzyme active at a first target site selected in a) is obtained and at least one second designer DNA recombinase enzyme active at a second target site is obtained. a) selecting a nucleotide sequence upstream of the nucleotide sequence to be modified as a first target site and selecting a nucleotide sequence downstream of the nucleotide sequence to be modified as a second target site, the sequences of the target sites preferably not being identical, but each target site comprising a first half-site and a second half-site each having 10-20 nucleotides separated by a spacer sequence of 5-12 nucleotides; b) applying molecular directed evolution to at least one library of DNA recombinases using vectors comprising the first target site selected in a) and the second target site as substrates. Includes.

[0232] The choice of asymmetric target sites provides an opportunity to compare two different evolution strategies: a single recombinase can be evolved to recognize both 10-20 bp, more preferably 12-15 bp, most preferably 13 bp half-sites, or two recombinases can be evolved in parallel for each half-site. By combining the two recombinases, a functional heterodimer capable of recombining the asymmetric sites can be formed.

[0233] In one embodiment of the invention, it is preferred that a single recombinase is evolved to recognise both 10-20 bp, more preferably 12-15 bp, most preferably 13 bp half-sites.

[0234] In a further embodiment of the present invention, it is preferred to evolve two recombinases in parallel for each half-site resulting in a heterodimer. The heterodimer is composed of two recombinases that can form either a heterodimer or two different homodimers, thus increasing the amount of potential recognition sequences. This approach can be disadvantageous as it increases the chance of unintended recombination at off-target sites. It was the goal of the present invention to restrict the monomers from homodimerizing in order to reduce the chance of recombination at off-target sites. To achieve this goal, the recombinase monomers were physically fused / linked to each other to force the desired heterodimer assembly enabled by the mutations as disclosed herein.

[0235] In order to select engineered DNA recombinases that are highly specific for a desired recombinase target site, i.e., that exhibit reduced off-target recombination, the method of the invention may in a further embodiment comprise: i. identifying potential off-target sites for a desired engineered DNA recombinase; ii. analyzing the recombinase activity of the desired engineered DNA recombinase at the off-target sites identified in step i; and iii. Selecting engineered DNA recombinases that do not exhibit recombinase activity at at least one off-target site. may include.

[0236] The off-target site of recombinase can be identified, for example, by using bioinformatics methods known to those skilled in the art. Other methods include ChIP-Seq-based assays to identify putative off-targets in humans, followed by validation and DNA enrichment by qPCR. These methods are also known to those skilled in the art.

[0237] The recombinase activity of the engineered DNA recombinase at these potential off-target sites can be experimentally tested, for example, by cloning the genomic sequence as an excision substrate into a bacterial reporter vector, as described herein below. Recombination at the off-target sites can then be detected by monitoring the expression of the reporter gene, for example, using a PCR-based assay. Such assays can also be performed in human tissue culture to determine whether the off-target sites are altered by the engineered DNA recombinase in vivo.

[0238] Most preferably, the engineered DNA recombinase of the present invention exhibits high specificity for loxF8 target sites having target sequences of SEQ ID NOs: 65 and 66 and exhibits no activity at off-target sites at high induction levels. Preferably, the off-target sites not recognized by the engineered DNA recombinase of the present invention are selected from the group consisting of SEQ ID NOs: 67 to 83, as shown in Table 4. Table 4: Nucleic acid sequences of off-target sites [Table 5] Bold: sequence not homologous to the target loxF8 sequence (SEQ ID NO:65) Underline: spacer sequence

[0239] A particular advantage of the present invention is that any recombinase target site can be used to evolve a recombinase enzyme of engineered DNA recombinase that exhibits specific activity for this recombinase target site. The method of the present invention includes the provision of a target site-specific obligate recombinase complex, where the monomers of the recombinase heterodimer are specifically adapted by introducing a single mutation into an evolved or naturally occurring recombinase. The method of the present invention further has the advantage that the undesired off-target activity of the recombinase complex, i.e., the engineered DNA recombinase, can be dramatically reduced, preferably completely eliminated. This makes the obligate recombinase complex, i.e., the engineered DNA recombinase, particularly suitable for use in gene therapy.

[0240] In a further embodiment, the present invention provides a method for determining recombination at the genome level in a host cell culture, comprising an engineered DNA recombinase for efficient and specific genome editing according to the present invention, i. providing a nucleic acid molecule encoding a first recombinase enzyme, wherein said first recombinase enzyme has been evolved by directed evolution or rational design to specifically recognize a first half-site of a recombinase target site, said first recombinase enzyme comprising at least one mutation that inactivates said catalytic activity of said first recombinase enzyme as a DNA recombinase; ii. providing a nucleic acid molecule encoding a second recombinase enzyme, wherein said second recombinase enzyme has been evolved by directed evolution or rational design to specifically recognize a second half-site of a recombinase target site, said second recombinase enzyme comprising at least one mutation that inactivates said catalytic activity of said second recombinase enzyme as a DNA recombinase; iii. cloning the nucleic acid molecule encoding the first recombinase enzyme of step i and the nucleic acid molecule encoding the second recombinase enzyme of step ii into an expression vector further comprising a first reporter gene for expressing a first reporter protein to prepare an expression vector; iv. transfecting a host cell with the expression vector of step iii, and transfecting the host cell with a reporter plasmid comprising a second reporter gene for expressing a second reporter protein; v. expressing an engineered DNA recombinase comprising the first recombinase enzyme and the second recombinase enzyme fused to the first reporter gene; and expressing a second reporter protein; vi. identifying cells that exhibit dual expression of the first reporter protein and the second reporter protein, indicating successful recombination and thereby complementation of the DNA recombinase function by forming an engineered obligate DNA recombinase enzyme that includes the first recombinase enzyme and the second recombinase enzyme. The method further comprises:

[0241] A suitable first reporter gene according to step iii is the gene encoding EGFP, so that a suitable first reporter protein is EGFP.

[0242] A suitable second reporter gene according to step iv is the gene encoding mCherry, so that a suitable second reporter protein is mCherry.

[0243] This system has the advantage that the transfection efficiency of cells transfected with both expression and reporter plasmids can be measured based on GFP fluorescence. GFP and mCherry double-positive cells reflect the recombination of the reporter in human cells. To calculate the recombination efficiency of the reporter plasmid in human cells, the double-positive cells can be normalized to the transfection efficiency.

[0244] The genetically engineered first and second recombinase enzymes suitable for use in the method for determining recombination at the genome level in a host cell culture and the single mutations that inactivate their DNA recombinase activity are described hereinabove.Similarly, suitable pairs of first and second recombinase enzymes that form obligate DNA recombinase complexes with complementary DNA recombinase activity are also described hereinabove.

[0245] The engineered DNA recombinase described herein was developed, for example, to correct a large gene inversion of exon 1 in the F8 gene causing hemophilia A. To investigate the inversion potency of the heterodimer on the genome level, an in vitro recombinase assay was developed as described in Example 9. The inversion potency of the obligate heterodimer on the genome level in recombinase-expressing cells was found to be comparable to that of the non-obligate heterodimer. The same was demonstrated for the deletion efficiency of the obligate heterodimer according to the invention (see Example 6 and Figure 3, especially Figure 3C). The deletion efficiency of the obligate heterodimer according to the invention was slightly increased. This is surprising, since conventional obligate heterodimers generated by altering the protein-protein interface have significantly reduced recombination activity.

[0246] Thus, the present invention relates in a further embodiment to a method for inversion of a DNA sequence at the genomic level in a cell, comprising the steps of: i. providing a nucleic acid molecule encoding a first recombinase enzyme, wherein said first recombinase enzyme comprises at least one mutation in its catalytic domain that inactivates the catalytic activity of said first recombinase enzyme as a DNA recombinase, and wherein said first recombinase monomer specifically recognizes a first half-site of a recombinase target site; ii. providing a nucleic acid molecule encoding a second recombinase enzyme, wherein said second recombinase enzyme comprises at least one mutation in its catalytic domain that inactivates the catalytic activity of said second recombinase enzyme as a DNA recombinase, and wherein said second recombinase monomer specifically recognizes a second half-site of a recombinase target site; iii. generating an expression vector by cloning a nucleic acid molecule encoding a first recombinase enzyme and a nucleic acid molecule encoding a second recombinase enzyme into the expression vector; iv. delivering the expression vector of step iii) to a cell containing the DNA sequence to be inverted; v. expressing a first and a second recombinase enzyme in said cell; vi. Inverting the DNA sequence to be inverted on the chromosome in the cell in which the first and second recombinase enzymes are expressed. The method further comprises:

[0247] In a preferred embodiment, the cell is a human cell and the inversion occurs in a human chromosome in said cell. In a preferred embodiment, the cell is not a human germ cell.

[0248] Preferably, the DNA recombinase of step v is an engineered DNA recombinase of the invention as described herein, more preferably recognising a first half site and a second half site of an upstream and downstream target site of a recombinase, most preferably the upstream target site of SEQ ID NO: 65 and the downstream target site of SEQ ID NO: 66 of the loxF8 recombinase or their reverse complements.

[0249] In one embodiment, the expression vector of step iii) is delivered to said cells, for example by transfection.

[0250] In an alternative embodiment, the method does not include the step of generating an expression vector, but rather involves delivering to said cell an RNA molecule encoding an engineered DNA recombinase according to the invention.

[0251] In one embodiment, the method of inverting a DNA sequence at the genome level is carried out in a genetically engineered host cell.

[0252] In a preferred embodiment, the method of inversion of a DNA sequence at the genome level is carried out in vitro in human cells derived from a patient, more preferably a patient suffering from hemophilia A.

[0253] In a further preferred embodiment, the method of inversion of a DNA sequence at the genome level is carried out in vivo in a patient, in particular a patient suffering from hemophilia A.

[0254] The engineered first and second recombinase enzymes suitable for use in the method of inverting DNA sequences at the genome level in a host cell culture and the single mutations that inactivate their DNA recombinase activity are described hereinabove.Similarly, suitable pairs of first and second recombinase enzymes that form obligate DNA recombinase complexes with complementary DNA recombinase activity are also described hereinabove.

[0255] According to a further aspect, the present invention provides a method for treating or preventing a disease, said method comprising administering to a subject in need thereof an engineered DNA recombinase, or a nucleic acid molecule or nucleic acid molecules, or an expression vector, or a host cell, or a pharmaceutical composition of the present invention.

[0256] The present invention further provides a method of treating or preventing hemophilia A, comprising administering to a subject in need thereof an engineered DNA recombinase, or a nucleic acid molecule or molecules, or an expression vector, or a host cell, or a pharmaceutical composition of the present invention. Optionally, the hemophilia A is severe hemophilia A.

[0257] The present invention further provides a method for recombining a target DNA sequence in a cell, comprising the steps of: (a) a nucleic acid molecule encoding a first recombinase enzyme and a nucleic acid molecule encoding a second recombinase enzyme of the invention; and / or (b) the first recombinase enzyme and the second recombinase enzyme of the present invention into a cell, thereby recombining a target DNA sequence within the cell.

[0258] In one embodiment, the nucleic acid molecule encoding the first recombinase enzyme and / or the nucleic acid molecule encoding the second recombinase enzyme is mRNA.

[0259] According to a further embodiment, the nucleic acid molecule encoding the first recombinase enzyme and / or the nucleic acid molecule encoding the second recombinase enzyme is an expression vector, preferably an expression vector as described herein.

[0260] The present invention also relates to the following: Item 1. An engineered DNA recombinase enzyme comprising a complex of at least a first DNA recombinase enzyme and at least a second DNA recombinase enzyme, wherein the first DNA recombinase enzyme and the second DNA recombinase enzyme specifically recognize a first half-site and a second half-site of an upstream target site and / or a downstream target site of the DNA recombinase, the first DNA recombinase enzyme and the second DNA recombinase enzyme each comprise a single amino acid substitution in their catalytic domain, the first DNA recombinase enzyme and the second DNA recombinase enzyme carrying the single amino acid substitution do not exhibit DNA recombinase catalytic activity when expressed alone, and the first DNA recombinase enzyme and the second DNA recombinase enzyme carrying the single amino acid substitution form a complex and exhibit DNA recombinase catalytic activity when co-expressed. Preferably, the single amino acid substitution in the catalytic region of the first recombinase enzyme is different from the single amino acid substitution in the catalytic region of the second recombinase enzyme, more preferably, the single amino acid substitution in the catalytic region of the first recombinase enzyme is at a different position within the catalytic region than the single amino acid substitution in the catalytic region of the second recombinase. Item 2. The DNA recombinase according to Item 1, wherein the at least one first recombinase and the at least one second recombinase are of the same type. Item 3. The DNA recombination enzyme according to Item 2, wherein both the at least one first recombinase and the at least one second recombinase are Cre-, Dre-, VCre-, SCre-, Vika-, lambda-Int-, Flp-, R-, Kw-, Kd-, B2-, B3-, Nigri-, or Panto-recombinase. Item 4. The DNA recombinase according to any one of Items 1 to 3, wherein the DNA recombinase is a recombinase complex in the form of a heterotetramer. Item 5. The DNA recombination enzyme according to any one of Items 1 to 4, wherein the single amino acid substitutions in the at least one first recombinase enzyme and the at least one second recombinase enzyme are at conserved amino acid positions within the catalytic domain. Item 6. The single amino acid substitution in at least one first recombinase enzyme and at least one second recombinase enzyme is selected from the group consisting of E129R, Q133H, R173A, R173C, R173D, R173E, R173F, R173G, R173I, R173K, R173L, R173M, R173N, R173P, R173Q, R173S, R173T, R173V, R173W, R173Y, E176H, E177H, E178H, E179H, E180H, E181H, E182H, E183H, E184H, E185H, E186H, E187H, E188H, E189H, E190H, E191H, E192H, E193H, E194H, E195H, E196H, E197H, E198H, E199H, E200H, E201H, E202H, E203H, E204H, E205H, E206H, E207H, E208H, E210H, E211H, E212H, E213H, E214H, E215H, E221H, E222H, E223H, E224H, E225H, E230H, E231H, E232H, E233H, E234H, E235H, E236H, E237H, E238H, E240H, E241H, E242H, E243H, E244H, E245H, 176I, E176L, E176M, E176V, E176W, E176Y, K201A, K201C, K201C, K201D, K201F, K201G, K201H, K201I, K201L, K201M , K201N, K201P, K201Q, K201R, K201S, K201T, K201V, K201W, K201Y, H289D, H289E, H289I, H289K, H289R, H289W, R29 2A, R292C, R292E, R292F, R292G, R292H, R292I, R292L, R292M, R292N, R292P, R292Q, R292S, R292T, R292V, R292W, R292Y, Q311R, W315C, W315E, W315G, W315I, W315K, W315L, W315M, W315N, W315Q, W315R, W315S, W315T, W315V, Y324 A DNA recombinase according to any one of items 1 to 5, wherein the amino acid sequence of the recombinase is selected from the group consisting of A, Y324C, Y324E, Y324F, Y324H, Y324I, Y324K, Y324L, Y324M, Y324N, Y324Q, Y324R, Y324S, Y324T, Y324V, and Y324W, or is at a corresponding position in another recombinase, preferably at a corresponding position in SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 20. Item 7. A single amino acid substitution in at least one first recombinase enzyme and at least one second recombinase enzyme, (i) E129, Q133, R173, E176, K201, H289, R292, Q311, W315, and Y324 of SEQ ID NO: 1; (ii) E146, Q151, R191, N194, K219, H308, R311, Q330, W334, and Y343 of SEQ ID NO: 14; (iii) E130, Q134, R174, E177, K202, H290, R293, Q312, W316, and Y325 of SEQ ID NO: 17; (iv) E131, Q135, R175, E178, K202, H290, R293, Q312, W316, and Y325 of SEQ ID NO: 20; or (v) Amino acid position in another recombinase 7. The DNA recombinase according to any one of Items 1 to 6, wherein the amino acid positions in the other recombinase correspond to positions E129, Q133, R173, E176, K201, H289, R292, Q311, W315, or Y324 in SEQ ID NO: 1. Item 8. A genetically engineered DNA recombinase comprising a complex of at least a first DNA recombinase enzyme and at least a second DNA recombinase enzyme, (i) the first recombinase enzyme is a polypeptide having an amino acid sequence that has at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO:7 and that contains a single mutation K201R in the catalytic domain, and the second recombinase enzyme is a polypeptide having an amino acid sequence that has at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO:11 and that contains a single mutation Q311R in the catalytic domain; or (ii) the first recombinase enzyme is a polypeptide having an amino acid sequence with at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO: 7 and containing a single mutation K201R in the catalytic domain; and the second recombinase enzyme is a polypeptide having an amino acid sequence with at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO: 12 and containing a single mutation Q311K in the catalytic domain; The genetically engineered DNA recombinase. Item 9. (i) a first Cre recombinase comprising a single mutation K201R in the catalytic domain and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:2, and a second Cre recombinase comprising a single mutation Q311R in the catalytic domain and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:5; (ii) a first Cre recombinase comprising a single mutation K201R in the catalytic domain and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:2, and a second Cre recombinase comprising a single mutation Q311K in the catalytic domain and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:4; (iii) a first Vika recombinase comprising a single mutation K219R in its catalytic domain and having an amino acid sequence which is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 15, and a second Vika recombinase comprising a single mutation Q330R in its catalytic domain and having an amino acid sequence which is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 16; (iv) a first Panto recombinase comprising a single mutation K202R in the catalytic domain and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 18, and a second Panto recombinase comprising a single mutation Q312R in the catalytic domain and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 19; (v) a first Dre recombinase comprising a single mutation K202R in the catalytic domain and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:21, and a second Dre recombinase comprising a single mutation Q312R in the catalytic domain and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:22; (vi) a first Vcre recombinase comprising a single mutation K221R in the catalytic domain and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 24, and a second Vcre recombinase comprising a single mutation Q336R in the catalytic domain and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 25. A DNA recombinase comprising: Item 10. A nucleic acid molecule encoding at least one first DNA recombinase enzyme and / or at least one second DNA recombinase enzyme according to any one of items 1 to 9. Item 11. An expression vector comprising the nucleic acid molecule of Item 10 and one or more expression control elements operably linked to said nucleic acid to facilitate its expression. Item 12. A host cell comprising the nucleic acid molecule according to Item 10 or the expression vector according to Item 11. Item 13. A pharmaceutical composition comprising at least one first DNA recombinase enzyme and / or at least one second DNA recombinase enzyme according to any one of items 1 to 9, or the nucleic acid molecule according to item 10, or the expression vector according to item 11, or the host cell according to item 12, and one or more therapeutically acceptable diluents or carriers. Item 14. The DNA recombinase complex according to any one of items 1 to 9, or the nucleic acid molecule according to item 10, or the expression vector according to item 11, or the host cell according to item 12, or the pharmaceutical composition according to item 13, for use as a pharmaceutical. Item 15. The DNA recombinase complex according to any one of Items 1 to 9, or the nucleic acid molecule according to Item 10, or the expression vector according to Item 11, or the host cell according to Item 12, or the pharmaceutical composition according to Item 13, for use in the treatment of hemophilia A, preferably for use in the treatment of severe hemophilia A. Item 16. A method for producing an obligate DNA recombinase for genome editing, comprising: (i) providing a nucleic acid molecule encoding a first recombinase enzyme and a nucleic acid molecule encoding a second recombinase enzyme, wherein the first recombinase enzyme binds to a first half-site of an asymmetric recombinase target site and the second recombinase enzyme binds to a second half-site of the asymmetric recombinase target site, wherein the first recombinase enzyme and the second recombinase enzyme form a heterodimer capable of inducing site-specific DNA recombination of a sequence of interest at the asymmetric recombinase target site in a DNA sequence, wherein the asymmetric recombinase target site comprises a first half-site and a second half-site of an upstream target site and / or a downstream target site of a DNA recombinase, and wherein the first half-site and the second half-site are non-identical and non-palindromic; (ii) performing mutagenesis to generate a library of nucleic acid molecules encoding a first mutant recombinase enzyme and nucleic acid molecules encoding a second mutant recombinase enzyme, where mutations are introduced into the first recombinase enzyme and the second recombinase enzyme; (iii) generating an expression vector by cloning the library of nucleic acid molecules encoding a first mutant recombinase enzyme and the library of nucleic acid molecules encoding a second mutant recombinase enzyme into an expression vector, wherein said expression vector harbors the DNA sequences of interest to be recombined; (iv) transfecting a cell with the expression vector of step iii) to express the first mutant recombinase enzyme and the library of second mutant recombinase enzymes in the same cell, thereby forming a recombinase heterodimer comprising the first mutant recombinase enzyme and the second mutant recombinase enzyme; (v) performing a positive selection screening for heterodimers obtained in step iv that are capable of inducing site-specific DNA recombination of the sequence of interest at the asymmetric recombinase target site in the DNA; (vi) performing a negative selection screen for heterodimers obtained in step iv or v that are unable to induce site-specific DNA recombination of the sequence of interest at an off-target site, preferably a symmetric recombinase target site in the DNA; (vii) selecting an obligate DNA recombinase that is capable of recombining a DNA sequence of interest at a recombinase target site in DNA comprising a first half-site and a second half-site of the upstream target site and / or downstream target site of the DNA recombinase, and that is incapable of recombining a DNA sequence of interest at an off-target site, preferably a symmetric recombinase target site in the DNA; wherein in the obligate DNA recombinase obtained in step (vii), the first mutant recombinase enzyme and the second mutant recombinase enzyme, respectively, comprise at least one mutation in a catalytic site that renders the first recombinase enzyme and the second recombinase enzyme catalytically inactive when expressed alone. Item 17. A method for producing an obligate DNA recombinase enzyme complex, comprising: (i) introducing a single amino acid substitution into a catalytic domain of a first DNA recombinase enzyme, wherein said single amino acid substitution renders the first DNA recombinase enzyme catalytically inactive; (ii) introducing a single amino acid substitution into the catalytic domain of a second DNA recombinase enzyme, wherein said single amino acid substitution renders the second DNA recombinase enzyme catalytically inactive; (iii) co-expressing both the mutated first DNA recombinase enzyme and the mutated second DNA recombinase enzyme in the host cell; (iv) isolating the mutated first DNA recombinase enzyme and the mutated second DNA recombinase enzyme from the host cell. The method comprising: Item 18. A DNA recombinase obtainable by the method according to item 16 or 17. Item 19. A method for inverting a DNA sequence in a cell at the genome level in vitro, comprising: (i) providing a nucleic acid molecule encoding a first recombinase enzyme according to any one of items 1 to 9, wherein said first recombinase enzyme specifically recognizes a first half-site of a recombinase target site; (ii) providing a nucleic acid molecule encoding a second recombinase enzyme according to any one of items 1 to 9, wherein said second recombinase enzyme specifically recognizes a second half-site of the recombinase target site; (iii) generating an expression vector by cloning a nucleic acid molecule encoding the first recombinase enzyme and a nucleic acid molecule encoding the second recombinase enzyme into the expression vector; (iv) delivering said expression vector to a cell containing the DNA sequence to be inverted; (v) expressing a first recombinase enzyme and a second recombinase enzyme in the cell; (vi) allowing formation of a DNA recombinase complex comprising a first recombinase enzyme and a second recombinase enzyme; (vii) enabling inversion of the DNA sequence to be inverted within said cell The method comprising: EXAMPLES

[0261] (Example of the present invention) The following examples are provided for the sole purpose of illustrating various embodiments of the invention and are not intended to limit the invention in any manner.

[0262] Example 1: Plasmid construction Previously described plasmids containing loxF8, loxF8L and loxF8R target sites were used for evolution (pEVO-loxF8, pEVO-loxF8L and pEVO-loxF8R, respectively) (10). The mutations published by Zhang et al. (16) were introduced into the sequences of both D7 subunits via DNA fragment synthesis (Twist Bioscience), with mutation A3 (K25R, D29R, R32E, D33L, Q35R, R337E, E123L) applied to D7L and mutation B2 (E69D, R72E, L76E, E308R) applied to D7R. The synthesized fragments were inserted into the pEVO vector in two cloning steps. First, D7L was inserted into the pEVO vector. A3 has SacI and XhoI, then D7R B2 has BsrGI and XbaI (NEB). Upon confirmation of the correct sequence by Sanger sequencing using primers 1 and 2 (Table 5), both molecules were subcloned into three different pEVO vectors containing target sites for loxF8, loxF8L and loxF8R using the restriction enzymes SacI and SbfI (NEB). Table 5: Cloning primers [Table 6]

[0263] The vector used for library analysis, pEVO-lacZ, was adapted from a previously described selection plasmid (8). [ka] and loxF8R [ka] A loxP spacer sequence was added to prevent recombination of the symmetric target site with the loxF8 site. The symmetric target site is flanked by two strong transcriptional terminators (17). Upon recombination, removal of the terminator sequences allows transcription of the lacZa fragment driven by the constitutive cat promoter.

[0264] Example 2: Substrate binding directed evolution Recombinases were evolved using Substrate-Linked Protein Evolution (SLiDE) as previously described (Buchholz and Stewart, 2001; Sakara et al., 2007; Karpinski et al., 2016; Lansing et al., 2019). A counterselection strategy was established by altering the selection of active and inactive recombinases at loxF8 and symmetric sites, respectively.

[0265] Positive selection pressure for activity at the asymmetric site (loxF8) and negative selection pressure at the symmetric sites (loxF8L and loxF8R) were achieved by a modified method of substrate-binding directed evolution (6). Each cycle of evolution included diversifying the library by error-prone PCR (MyTaq DNA Polymerase Bioline) and selecting variants for the desired activity at the target site. The diversified library was cloned into the target site-containing pEVO, and the vector was then transformed into electrocompetent XL1-Blue E. coli to express the recombinase variants overnight via an arabinose-inducible promoter. Selection was repeated between positive and negative selection strategies. To perform positive selection for loxF8 recombination, the purified plasmid was digested with the enzymes NdeI and AvrII to linearize all non-recombining variants and then amplified with primers 1 and 3 (Table 5). Negative selection was achieved by a primer (primer 4, Table 5) that binds between the symmetric target sites and can amplify only recombinases that have not undergone a recombination event. For each round of evolution, selection alternated between the three target sites. Recombination efficiency was monitored by a plasmid-based activity assay. A scheme of the applied evolution method is shown in Figure 15.

[0266] Example 3: Recombinase activity assay: Plasmid-based blue-white screening To visualize the recombination activity of a recombinase or a recombinase library on a target site of interest, we used a plasmid-based assay as previously described ( 6 , 9 , 10 ).

[0267] For activity analysis of the final library and selection of active variants, blue-white activity screening was used. The library was cloned into pEVO LacZ counter selection plasmid with restriction enzymes SacI and SbfI. After plating on Xgal indicator plates containing selection antibiotic and arabinose, the activity of the recombinase variants was read as blue or white. White colonies represent inactive recombinase pairs or pairs highly specific for loxF8. To remove inactive recombinases, the library was induced overnight at low (10 μg / ml L-arabinose, Sigma) arabinose levels before blue-white screening. Purified plasmids were digested with NdeI and AvrII to linearize unrecombined plasmids, then retransformed and induced with higher levels of arabinose (100 μg / ml L-arabinose, Sigma) to allow sensitive detection of low levels of symmetric site activity. Blue colonies contained variants that were active at the symmetric site. Therefore, white colonies containing mutants that had not recombined the symmetric site but had recombined the LoxF8 site were selected. Eighty white colonies were selected, and colony PCR showed that 75 of the 80 selected colonies had the desired activity profile, showing a 1.7 KB band.

[0268] Example 4: Library design A3 residue positions K25, D29, R32, and D33 and B2 positions E69, R72, and L76 were targeted by ISOR (incorporating synthetic oligonucleotides via gene reassembly). To target diversity to the A3 and B2 positions, an adapted method of incorporating synthetic oligonucleotides via gene assembly (ISOR) was applied (18). The incorporated oligonucleotides were designed with degenerate codons (VNS, GHW, and MDG). VNS contains 16 possible amino acids (D, E, H, I, K, M, N, Q, S, A, G, L, P, T, V, R), GHW contains codons corresponding to four possible amino acid variants (D, E, A, V), and MDG contains codons corresponding to five amino acid variants (K, L, M, Q, R). The integrated A3 and B2 oligonucleotides (primers 5-20, Table 3) were applied in parallel to the shuffled D7L and D7R recombinases, respectively.

[0269] Example 5: Sequence Analysis To determine which mutations occur most frequently among the mutated recombinases, the amino acid sequence of the D7L mutant was aligned with the original D7L recombinase sequence, and the D7R mutant was aligned with the original D7R recombinase sequence. From the alignment, the amount of mutations occurring at each position was divided by the total amount of sequenced samples to determine the mutation frequency at each position (Figure 2C). For D7L, the most commonly mutated residue positions are (highlighted in dark gray) K25, D29, K201, and S305, and for D7, the most commonly mutated residue is Q311. For the D7R mutant, residue positions 1-10 were removed because they provided poor background reads. The primer binding site between the two recombinase libraries is not long enough to extract adequate reads while still keeping the mutated pair together. Mutations need to be applied to each monomer to control for complex formation. Without both monomers carrying unique mutations, D7L or D7R could still form functional homotetramers, resulting in undesired recombination of their correlated evolved half-sites, thus suggesting that the combination of mutations is important.

[0270] Example 6: Expression in mammalian cells and recombination efficiency in mammalian cells A fluorescence-based reporter assay was used to determine the recombination properties of the obligate monomers as previously described (10). HEK293T cells were seeded at a density of 350,000 cells / ml 1 day prior to transfection. mRNAs encoding the obligate monomers and blue fluorescent protein (BFP) were transfected into a HEK293 reporter cell line containing integrated lox sites flanked by repeated SV poly(A) sequences. Recombination of these sites results in expression of the downstream monomeric red fluorescent protein (mCherry). Recombinase activity was quantified by FACS using a MACSQuant VYB flow cytometer (Miltenyi Biotec) 48 h after transfection. Recombination rates were determined by the percentage of cells exhibiting red fluorescence within the blue fluorescent population.

[0271] Example 7: HEK293T cell culture HEK293T cells were cultured in 12-well format with DMEM (Gibco) supplemented with 10% FBS (Capricon Scientific) and 1% penicillin-streptomycin (ThermoFisher). When they reached 90% confluence, the cells were split. Each well was washed once with PBS and 100 μl of trypsin (Gibco) was added. After 3 min incubation at 37°C, the detached cells were collected in a 15 ml tube. The cells were counted with a Countess 3 FL Automated Cell Counter (ThermoFisher) and plated at a density of 75,000 cells / well in 1 ml medium. For transfection, the cells were plated at a density of 350,000 cells / well in 1 ml medium.

[0272] Example 8: mRNA transfection HEK293T cells were transfected 24 hours after seeding. For each transfection reaction, a total of 300 ng of mRNA (100 ng tag BFP mRNA and 200 ng recombinase mRNA) was prepared in a 1.5 ml tube. 100 μl of Opti-MEM I reduced serum medium was mixed with 1.5 μl of Lipofectamine MessangerMax (ThermoFisher) and added to the mRNA sample. The mixture was briefly vortexed and incubated at RT for 15 min. Meanwhile, the medium of the cells was replaced with fresh medium. The transfection mixture was then added to the cells. The medium was changed the next day and the cells were analyzed 2 days after transfection.

[0273] Example 9: PCR-based genomic inversion detection Inversion of the loxF8 locus after treatment of HEK293T cells with D7 recombinase dimer was detected as previously described ( 10 ).

[0274] Example 10: In vitro transcription (IVT) mRNA was generated using the HiScribe™ T7 ARCA mRNA kit (NEB) and purified with the Monarch RNA Cleanup kit (NEB) according to the manufacturer's instructions. D7 recombinase dimer and tag-BFP templates for IVT were generated as previously described (10). Templates for the different Cre mutants were generated using primers 21 and 22 (Table 5). Aliquots of 4 μg of mRNA were stored at -80°C for up to 6 months.

[0275] Example 11: Evaluation of single point mutations in the catalytic domain of recombinant synapses to generate obligate phenotypes To generate additional obligate recombinases, additional single mutations in the catalytic domain of recombinant synapse of Cre recombinase (Guo et al. 1997; Lee et al. 2002; Gibb et al. 2010; Meinke et al. 2016) (i.e., at amino acid positions 129-136, 163-181, 199-211, 289-301, 310-316 and 321-324 of SEQ ID NO:1, see FIG. 17A, darkened areas) were introduced as outlined in Table 6 below. A more detailed overview of the mutation positions in Cre is provided in FIG. 17B. Experiments show that an inactivating mutation in the catalytic domain of the first recombinase monomer can be rescued by a different inactivating mutation in the catalytic domain of the second recombinase monomer. In this example, a Cre recombinase mutant of SEQ ID NO:1 with a single amino acid substitution in the catalytic region was generated and expressed at its loxP target site to evaluate whether the introduced mutation rendered the monomer inactive. In a second step, two different inactive monomers were co-expressed at their loxP target sites to evaluate whether a combination of two inactive monomers could rescue the recombinase activity. The recombination activity of the recombinase on loxP was measured by a previously described plasmid-based assay (6, 9, 10). In the experiment, the recombinant pEVO is smaller in size compared to the non-recombined pEVO. The larger fragment (approximately 5 kb) represents a non-recombined substrate, while the smaller fragment (approximately 4.3 kb) represents a recombined substrate. These two different fragments can be distinguished by gel electrophoresis or by sequencing. Recombination efficiency (shown as dimeric and monomeric activity, respectively) was calculated based on the ratio of recombinant to non-recombinant substrates using the following formula: recombination activity (%) = 100 × (recombinant substrate / (recombinant substrate + non-recombinant substrate)). Table 6 [Table 7] TIFF2024544843000017.tif242170TIFF2024544843000018.tif242170TIFF2024544843000019.tif243170TIFF2024544843000020.tif192170

[0276] Results achieved by embodiments of the present invention 1. Intermonomer interface mutations reduce recombination activity To form obligate heterospecific Cre-type SSR complexes for asymmetric substrates, previous studies have focused on redesigning the interface between interacting monomeric proteins. In particular, Zhang et al. generated obligate Cre and Cre mutant heterotetramers to recombine an artificial asymmetric loxM7 / loxP target sequence (16). Key positions for interface redesign were selected from mutations predicted to form alternative interaction interfaces between wild-type Cre molecules and Cre mutants. To examine whether the same interface mutations could be adopted for other heterospecific Cre-type SSRs, a D7 mutant was generated by mutating two subunits to potentially form an obligate D7 recombinase. Thus, a D7L mutant (D7L A3 ) was generated by mutating positions K25R, D29R, R32E, D33L, Q35R, E123L and R337E, whereas the D7R mutant (D7R B2 ) harbored the mutations E69D, R72K, L76E, and E308R.

[0277] To compare the recombination efficiency before and after application of the mutations, we first co-expressed non-mutated D7L and D7R from vectors carrying either loxF8, loxF8L or loxF8R target sites as excision substrates (Figure 1A and Figure 6). As expected, recombination was efficiently achieved at the asymmetric loxF8 target site when both D7L and D7R monomers were co-expressed (Figure 1B). Recombination was also observed for both the symmetric loxF8L and loxF8R target sites (Figure 1B), presumably due to functional homotetramer formation of their corresponding evolved recombinases. The interface mutations (D7L A3 and D7R B2) blocks recombination at symmetric loxF8L and loxF8R sites by preventing the assembly of homotetramers, but the different subunits are still required to form active heterotetramers on asymmetric loxF8 sequences (Figure 1C). A3 and D7R B2 Co-expression of D7L on the asymmetric loxF8 site did not result in detectable recombination from vectors carrying symmetric loxF8L or loxF8R sequences (Figure 1D), indicating that these mutations prevented the formation of active homotetramers. A3 and D7R B2 Co-expression of D7L did not result in any observable activity compared to that of the original D7L+D7R complex at the same induction concentration of L-arabinose (100 μg / mL). A3 and D7R B2 To determine whether the recombinases of D7 are able to form an active complex, the induction was increased to 1000 μg / mL L-arabinose, resulting in very low activity at the loxF8 sites, proving that the complex is functional, simply very inefficient (Figure 1D). Thus, although the applied mutations are functional in principle, they also affect the recombination activity of the D7 heterotetramer. It was therefore sought to restore activity by searching for combinations of amino acid changes that are more suitable for the modified D7 system.

[0278] 2. Substrate-binding directed evolution to evolve an obligate D7 recombinase with high activity To explore beneficial residue changes, we started generating two libraries of D7L and D7R recombinase variants around previously described (16) residue positions involved in the protein-protein interface (A3 - K25, D29R, R32E, D33L, Q35R, E123L, and R337E and B2 - E69D, R72K, L76E, and E308R). The libraries were subjected to a well-established substrate-binding directed evolution (SLiDE) procedure (6, 18). To keep the library to a practical screening size, we directed diversity to a subset of residue positions located along the interface between the largest monomers: A3 positions K25, D29, R32, and D33, and B2 positions E69, R72, and L76. At each of these positions, mutations were restricted to a subset of amino acids (D, E, H, I, K, M, N, Q, S, A, G, L, P, T, V, R) previously predicted for interface redesign (16). The two initial libraries, D7L and D7R, were cloned into the corresponding vectors to initiate repeated positive selections active at the asymmetric site (loxF8) and negative selections at the symmetric sites (loxF8L and loxF8R) with a modified version of SLiDE (Figures 2A and 7A). By evolving the two libraries together, instead of in parallel, we were able to select functional pairs with the desired activity profile. After 26 cycles of SLiDE, we detected a significant increase in recombination activity towards the asymmetric site and negligible recombination at the symmetric site (Figure 2B), indicating that a heterodimeric pair of recombinases with the desired characteristics had evolved.

[0279] To eliminate carryover of inactive recombinase variants that leaked through selection, single mutant pairs were evaluated by using blue-white colony screening. The selection plasmid (pEVO-LacZa) allowed the simultaneous identification of mutants that showed high activity at the asymmetric loxF8 site while not recombining the symmetric site (Figure 8A-C). 75 white colonies were selected and the encoded recombinase pairs were sequenced. Surprisingly, none of the highly mutated amino acid positions (69, 72, 76, and 308) were found to be enriched in the evolved D7R recombinase. In contrast, glutamine at position 311 was altered in 41% (31 of 75 mutants) of the sequenced D7R recombinases, with 87% of the mutants (27 of 31) exhibiting an arginine at this position. The results indicate that the original targeted residues did not produce the desired results, and instead a single substitution that arose randomly during evolution was preferred (i.e., Q311R), which likely prevented recombination at the symmetric loxF8R site while maintaining activity on the asymmetric loxF8 sequence when co-expressed with the D7L mutant.

[0280] Sequencing of D7L-derived clones revealed five positions that were mutated in more than 20% of the sequenced clones (positions 25, 29, 20, 282, and 305; Fig. 2C). Positions 25 and 29 most likely originated from the initial target residues involved in protein-protein interactions, whereas positions 201, 282, and 305 suggested that they arose through random mutation and selection during directed evolution.

[0281] The most surprising result was the frequently mutated position 201, which was altered in 48% (36 of 75) of the clones, and all clones were found to have an arginine rather than a lysine at this position (Figure 2C). What makes this change intriguing is that lysine 201 is highly conserved across the tyrosine SSR family (19) and the residue has been described as essential for the catalytic activity of Cre (20-22). K201 has been observed to function as an active residue within the Cre complex to facilitate DNA cleavage during recombination (20, 23). Thus, it is expected that a recombinase with an altered position 201 would not exhibit any recombinase activity. Based on this study, we aimed to further examine whether mutating the catalytic K201 residue in D7L would inactivate the SSR when expressed as a monomer, and if so, whether the presence of the paired Q311R mutation on the D7R monomer would rescue activity and allow recombination to occur.

[0282] To determine the effect on recombination of the mutations applied to each monomer, we first assessed their ability to recombine their original target when expressed alone as monomers. K201R Or D7R Q311R Expression of D7L alone did not result in detectable recombination events, demonstrating that these mutations inactivate the enzyme when expressed alone (Figure 2D). K201R and D7R Q311R was inactive at the asymmetric loxF8 site when expressed alone (Fig. 2D). In stark contrast, D7L K201R and D7R Q311RWhen co-expressed with loxF8, efficient recombination was observed on the asymmetric loxF8 (Figure 2E), whereas no recombination was detectable on the symmetric loxF8L and loxF8R sites (Figure 2E). Importantly, the loxF8 sites were recombined at a similar rate compared to the original wild-type D7 clone (Figure 2E), indicating that the two mutations did not impair the activity of the entire recombinase complex. Thus, application of only one mutation to each recombinase monomer resulted in an obligate SSR complex with activity comparable to that of the wild-type D7 SSR.

[0283] 3. D7L K201R +D7R Q311R supports obligate recombination in mammalian cells Since the D7 recombinase is intended for application within the human genome, the next step is to develop an obligate D7L K201R +D7R Q311R The aim of this study was to investigate the activity of the complex. To allow for easy quantification, we measured the recombination efficiency in a HEK293T reporter cell line (10). This reporter cell line was cotransfected with mRNA carrying the recombinase together with mRNA encoding the tag BFP to monitor the transfection efficiency (Figure 3A). K201R +D7R Q311R Transfection of the molecules revealed a recombination efficiency of 81% compared to 79% for wild-type D7 (Figure 3B,C), suggesting that the introduction of the obligate mutations does not impair recombinase activity in human cells. Importantly, transfection of each mutant monomeric subunit alone did not result in significant loxF8 target site recombination (Figure 3B,C).

[0284] D7 recombinase was originally generated to correct a genomic int1h inversion frequently found in hemophilia A patients (24). The enzyme recognizes two loxF8 sequences found on the human X chromosome at a distance of 140 kb from each other. The first site is present in intron 1 of the factor VIII gene, and the second site is located 130 kb upstream of the factor VIII transcription start site (10, 25, 26). D7 has been shown to efficiently invert displaced exon 1 sequences adjacent to the loxF8 target sites upon expression in human cells (10). D7L acting on these sites at the endogenous locus K201R +D7R Q311R To confirm the ability of the mutant, D7L K201R and D7R Q311R We extracted genomic DNA from HEK293T cells transfected with the mRNA and performed a PCR-based assay designed to detect the inversion of exon 1 (Figure 3D). K201R +D7R Q311R Expression of β-lactamase resulted in an inversion of the genomic fragment (Figure 3E), demonstrating that this degenerative mutation does not interfere with the activity of the recombinase that recombines the inversion that causes the disease.

[0285] D7L K201R +D7R Q311R To assess whether the heterodimer improved target site specificity, we analyzed its activity against four predicted human off-target sites (Figure 3F) using a plasmid-based activity assay (Supplementary Figure 1). Consistent with previous data (10), the wild-type D7 recombinase showed no detectable activity at three of the four sequences, but did show activity against the HG2L off-target site (Figure 3G). In comparison, the obligate D7L K201R +D7R Q311R The complex showed no detectable activity at all four of the predicted off-target sites (Figure 3G), demonstrating its improved applied properties. Collectively, these results suggest that D7L K201R +D7R Q311RWe show that heterodimers promote target site specificity while maintaining comparable recombination efficiency for loxF8 target sites in mammalian cells.

[0286] 4. The K201R and Q311R mutations render Cre, Vika, and Dre recombinases obligate To explore the more general applicability and to gain insight into the molecular mechanisms of the identified obligate SSR system, we investigated the phenotypes of corresponding mutations in two naturally occurring homotetrameric SSR complexes, namely Cre and Vika (27). K201R and Cre Q311R Two mutant monomers, namely Vika K219R and Vika Q330R Obligatory mutations were also engineered into Vika at positions 219 and 330, following conserved sequences found in sequence alignments (27), to form Vika. Both were analyzed for activity against excision substrates in E. coli. K201R or Cre Q311R When Vika was expressed alone, no recombination was observed at the loxP target (Figure 4A). To test whether both mutations introduced into the same monomer would result in recombination, CreK201R+Q311R was generated. Analysis of the Cre double mutant on the excision substrate in E. coli showed no observable recombination of the loxP target sequence, indicating that the mutations must be present on different monomers to allow the formation of an active SSR complex (Figure 4A). K219R and Vika Q330R A similar activity profile was seen when coexpressed on a vox target sequence (albeit with some loss of activity), whereas no recombination was observed when the mutated monomers were expressed alone (Fig. 4B).

[0287] We next assessed whether the obligate Cre system could function efficiently in mammalian cells and maintain the recombination profile seen in bacteria. We transfected the HEK293T red fluorescent reporter cell line with SSR mRNA and assessed recombination activity (Figure 4C). When the Cre mutants were expressed alone, negligible recombination activity was detected (Figure 4D, 4E), whereas Cre expressed alone was not. K201R and Cre Q311R In sharp contrast, Cre was shown to be inactive. K201R and Cre Q311R Co-expression of loxP resulted in recombination efficiencies comparable to wild-type Cre (Figures 4D, 4E). Collectively, these results demonstrate that targeted mutations are not only applicable to the heterotetrameric D7 / loxF8 complex, but can also be applied to obtain an obligate system of wild-type SSRs found in nature.

[0288] Further applicability of the obligate SSR system was tested in the naturally occurring Dre / rox complex. Degenerate mutations were incorporated into Dre at positions 202 and 312 according to the conserved sequence found in the alignment to Cre (Figure 16), forming the DreK202R+DreQ312R / rox complex. Due to the low activity of wild-type Dre, a sensitive PCR-based detection method was used to test the functionality of the mutant complex in E. coli (Figure 14) (primers 23 and 24 in Table 3). Amplification on substrate confirms rox recombination when both DreK202R and DreQ312R are present, and minimal or complete loss of recombination when the obligate Dre mutant is expressed alone. The results of the PCR-based assay are shown in Figure 14.

[0289] 5. Molecular modeling supports a mechanism for catalyzing the promotion of obligate heterotetramer formation Based on the highest resolution co-crystal structure of Cre, Cre bound to loxP target sites K201R and Cre Q311RWe constructed a molecular model of the gene (PDB: 3C29) and then performed extensive molecular dynamics simulation analyses. These analyses revealed that the single mutant Cre K201R and Cre Q311R The reason why Cre is inactive has become clear (Fig. 5). K201R In the mutant, arginine 201 shifts dramatically in the inactive subunit to interact with the DNA backbone at base A2 of the bottom strand instead of interacting with base T5 on the top strand, thereby reducing recombination ability. Furthermore, catalytic tyrosine 324 of the active subunit displaces base T3' on the top strand and forms a hydrogen bond with base A4', thereby reducing the recombination capacity of Cre. K201R This explains why it is inactive.

[0290] Cre Q311R For the mutants, we observed that catalytic tyrosine 324 was replaced in both the active and inactive subunits, whereas K201 lost a critical interaction with the DNA backbone. In addition, we altered another residue known to play a key role in recombinant catalysis (H289) (Figure S10D).

[0291] We then analyzed a model in which the K201R mutation was introduced into the active subunit and the Q311R mutation was placed into the inactive subunit (Figure 10E). Surprisingly, this configuration resulted in a model that was not compatible with active recombination. Tyrosine 324 was displaced in the inactive subunit and interacted with base C3 on the bottom strand, while K201 was dramatically shifted to also interact with base C3 on the bottom strand. Thus, this configuration does not constitute an active recombinase.

[0292] Finally, they analyzed a model in which the K201R mutation was introduced into the inactive subunit and the Q311R mutation was placed into the active subunit: this configuration was consistent with an active enzyme in which all catalytic residues were positioned to allow recombination.

[0293] 6. Single point mutations in the catalytic domain of recombinant synapse lead to obligate phenotypes We further show that an inactivating mutation in the catalytic region of a monomer can be rescued by an inactivating mutation in the catalytic region of another monomer. Specifically, a single Cre monomer mutant (see Table 4) was inactive at the loxP target site when expressed alone (see FIG. 18, grey bars). However, activity on loxP was rescued when two different inactive monomers were co-expressed on loxP (see FIG. 18, black bars). The principle that catalytically impaired monomers can form obligate heterocomplexes was demonstrated for 186 combinations (Table 6). Furthermore, since the catalytic region is conserved in the family of T-SSRs (see, for example, the alignment in FIG. 19), this principle can be applied to other DNA recombinases, specifically other DNA recombinases, such as T-SSRs such as Vika, Dre and Panto recombinases.

[0294] Discussion of the results of the examples By altering the DNA specificity of Cre through engineering and directed evolution, separate SSR variants can be generated that recombine together as heterotetramers with asymmetric target sequences (6, 8-10). The generation of such heterotetrameric SSR systems substantially expands the potential sequences that can be targeted within a genome. However, possible combinations of subunits can lead to active SSR by-products that can catalyze off-target recombination. Previously, prevention of homotetramer formation was achieved by structure-guided redesign of several residues involved in the protein-protein interaction interface between different recombinase monomers (16). Thus, this approach to generate obligate SSR systems is limited to enzymes with available crystal structures and therefore cannot be easily adapted to engineered or distantly related recombinases. We show that obligate SSR systems can also be generated by mutating amino acid residues within the catalytic region. Importantly, this novel method to generate obligate SSRs only requires the modification of one single conserved residue within each separate SSR monomer. This simplified approach is potentially applicable to many engineered or natural SSRs without prior structural knowledge of the enzyme.

[0295] In summary, the present invention provides a simplified approach to reduce off-target recombination and improve the specificity of engineered wild-type SSRs. In particular, the D7L at the off-target site K201R and D7R Q311R Enhanced specificity of the system was demonstrated. The data provided further support the general concept that catalytically inactive monomers can be rescued when co-expressed with another catalytically inactive monomer. Importantly, this novel method to generate obligate SSRs requires only the modification of one residue within the catalytic region of each separate SSR monomer. This simplified approach can be applied to many engineered or natural DNA recombinases without prior structural knowledge of the enzyme.

[0296] References [Table 8] TIFF2024544843000022.tif173170

Claims

1. 1. A method for producing an obligate DNA recombinase for genome editing, comprising: i. providing a nucleic acid molecule encoding a first recombinase enzyme and a nucleic acid molecule encoding a second recombinase enzyme, wherein the first recombinase enzyme binds to a first half-site of an asymmetric recombinase target site and the second recombinase enzyme binds to a second half-site of the asymmetric recombinase target site, wherein the first recombinase enzyme and the second recombinase enzyme form a heterodimer capable of inducing site-specific DNA recombination of a sequence of interest at the asymmetric recombinase target site in a DNA sequence, wherein the asymmetric recombinase target site comprises a first half-site and a second half-site of an upstream target site and / or a downstream target site of a DNA recombinase, and wherein the first half-site and the second half-site are non-identical and non-palindromic; ii. performing mutagenesis to generate a library of nucleic acid molecules encoding a first mutant recombinase enzyme and nucleic acid molecules encoding a second mutant recombinase enzyme, wherein mutations are introduced into the first recombinase enzyme and the second recombinase enzyme; iii. creating an expression vector by cloning the library of nucleic acid molecules encoding the first mutant recombinase enzyme and the library of nucleic acid molecules encoding the second mutant recombinase enzyme into an expression vector, wherein the expression vector carries the DNA sequence of interest to be recombined; iv. transfecting cells with the expression vector of step iii. and expressing the library of first mutant recombinase enzymes and the library of second mutant recombinase enzymes in the same cells, thereby forming recombinase heterodimers comprising the first mutant recombinase enzymes and the second mutant recombinase enzymes; v. performing a positive selection screening for heterodimers obtained in step iv. that are capable of inducing site-specific DNA recombination of the sequence of interest at the asymmetric recombinase target site in DNA; vi. performing a negative selection screening for heterodimers obtained in step iv. or v. that are unable to induce site-specific DNA recombination of the sequence of interest at an off-target site in DNA, preferably a symmetric recombinase target site; vii. Selecting an obligate DNA recombinase that is capable of recombining a DNA sequence of interest at a recombinase target site in DNA comprising a first half-site and a second half-site of the upstream target site and / or downstream target site of the DNA recombinase, wherein the first half-site and the second half-site are not identical or palindromic; and that is incapable of recombining the DNA sequence of interest at an off-target site in DNA, preferably a symmetric recombinase target site. wherein in the obligate DNA recombinase obtained in step vii., the first mutant recombinase enzyme and the second mutant recombinase enzyme each comprise at least one mutation in a catalytic site that renders the first recombinase enzyme and the second recombinase enzyme catalytically inactive when expressed alone.

2. 2. The method of claim 1, wherein the first recombinase enzyme and the second recombinase enzyme described in steps ii. to vi. have been evolved by substrate-linked directed evolution (SLiDE) or directed evolution.

3. The method of claim 1, wherein the selection described in steps v. and vi. is cycled between selecting obligate heterodimers that are catalytically active at the asymmetric target site (positive selection) and selecting heterodimers that are catalytically inactive at the off-target site, preferably the symmetric target site.

4. 1. A genetically engineered DNA recombinase for genome editing, comprising an obligate complex of recombinases, the complex comprising at least a first recombinase enzyme and at least a second recombinase enzyme, wherein the first recombinase enzyme and the second recombinase enzyme specifically recognize a first half-site and a second half-site of an upstream target site and / or a downstream target site of a DNA recombinase, and the first recombinase enzyme and the second recombinase enzyme each comprise at least one mutation in a catalytic site, wherein the first recombinase enzyme and the second recombinase enzyme carrying the at least one mutation in a catalytic site do not exhibit catalytic activity of a DNA recombinase when expressed alone, and wherein the catalytic activity of the DNA recombinase is complemented in the obligate recombinase complex.

5. 5. The genetically engineered DNA recombinase of claim 4, wherein the at least one first recombinase and the at least one second recombinase are of the same type.

6. 6. The genetically engineered DNA recombinase of claim 5, wherein the at least one first recombinase and the at least one second recombinase are both Cre-, Dre-, VCre-, SCre-, Vika-, lambda-Int-, Flp-, R-, Kw-, Kd-, B2-, B3-, Nigri-, or Panto-recombinase.

7. The genetically engineered DNA recombinase according to any one of claims 4 to 6, wherein the DNA recombinase is a complex of recombinases in the form of a heterotetramer.

8. 7. The engineered DNA recombinase enzyme of any one of claims 4 to 6, wherein the at least one mutation is a single amino acid substitution in the catalytic domain of the recombinase, preferably the single amino acid substitution is at a conserved amino acid position within the catalytic domain.

9. (i) the first recombinase enzyme comprises a Cre recombinase and the second recombinase enzyme comprises a Cre recombinase, wherein each Cre recombinase comprises a single amino acid substitution within an amino acid region selected from the group consisting of SEQ ID NO:109 to SEQ ID NO:114; (ii) the first recombinase enzyme comprises a Vika recombinase and the second recombinase enzyme comprises a Vika recombinase, wherein each Vika recombinase comprises a single amino acid substitution within an amino acid region selected from the group consisting of SEQ ID NO:115 to SEQ ID NO:120; (iii) the first recombinase enzyme comprises a Dre recombinase and the second recombinase enzyme comprises a Dre recombinase, wherein each Dre recombinase comprises a single amino acid substitution within an amino acid region selected from the group consisting of SEQ ID NO:121 to SEQ ID NO:126; or (iv) the first recombinase enzyme comprises a Panto recombinase and the second recombinase enzyme comprises a Panto recombinase, wherein each Panto recombinase comprises a single amino acid substitution within an amino acid region selected from the group consisting of SEQ ID NO:127 to SEQ ID NO:132; The genetically engineered DNA recombinase according to any one of claims 4 to 6.

10. and wherein the at least one mutation or single amino acid substitution in the at least one first recombinase enzyme and the at least one second recombinase enzyme is selected from the group consisting of E129R, Q133H, R173A, R173C, R173D, R173E, R173F, R173G, R173I, R173K, R173L, R173M, R173N, R173P, R173Q, R173S, R173T, R173V, R173W ... 3Y, E176H, E176I, E176L, E176M, E176V, E176W, E176Y, K201A, K201C, K201C, K201D, K201F, K201G, K201H, K201I, K201 L, K201M, K201N, K201P, K201Q, K201R, K201S, K201T, K201V, K201W, K201Y, H289D, H289E, H289I, H289K, H289R, H289W, R292A, R292C, R292E, R292F, R292G, R292H, R292I, R292L, R292M, R292N, R292P, R292Q, R292S, R292T, R292V, R292W, R 292Y, Q311R, W315C, W315E, W315G, W315I, W315K, W315L, W315M, W315N, W315Q, W315R, W315S, W315T, W315V, Y324A, Y32 7. The engineered DNA recombinase of any one of claims 4 to 6, wherein the amino acid residues are selected from the group consisting of Y324C, Y324E, Y324F, Y324H, Y324I, Y324K, Y324L, Y324M, Y324N, Y324Q, Y324R, Y324S, Y324T, Y324V and Y324W, or at the corresponding amino acid positions of another recombinase, preferably at the corresponding positions of SEQ ID NO: 14, SEQ ID NO: 17 or SEQ ID NO:

20.

11. at least one mutation or single amino acid substitution in the at least one first recombinase enzyme and the at least one second recombinase enzyme; (i) E129, Q133, R173, E176, K201, H289, R292, Q311, W315, and Y324 of SEQ ID NO: 1; (ii) E146, Q151, R191, N194, K219, H308, R311, Q330, W334, and Y343 of SEQ ID NO: 14; (iii) E130, Q134, R174, E177, K202, H290, R293, Q312, W316, and Y325 of SEQ ID NO: 17; (iv) E131, Q135, R175, E178, K202, H290, R293, Q312, W316, and Y325 of SEQ ID NO: 20; or a position selected from the group consisting of (v) an amino acid position in another recombinase, wherein the amino acid position in the other recombinase corresponds to position E129, Q133, R173, E176, K201, H289, R292, Q311, W315, or Y324 of SEQ ID NO:

1. The genetically engineered DNA recombinase according to any one of claims 4 to 6,

12. The method of any one of claims 1 to 3 or the engineered DNA recombinase of any one of claims 4 to 6, wherein the first recombinase enzyme and the second recombinase enzyme do not comprise an interfacial mutation between the monomers.

13. The method of any one of claims 1 to 3 or the engineered DNA recombinase of any one of claims 4 to 6, wherein the engineered DNA recombinase is a mutant of a naturally occurring site-specific recombinase or a mutant of a designer DNA recombinase.

14. the first recombinase enzyme comprises a mutation selected from the group consisting of the mutation K201R of SEQ ID NO: 1, 2 or 7, the mutation K202R of SEQ ID NO: 18 or 21, the mutation K219R of SEQ ID NO: 15, and the mutation K221R of SEQ ID NO: 24; and the second recombinase enzyme comprises a mutation selected from the group consisting of the mutation Q311K of SEQ ID NO: 4 or 11, the mutation Q311R of SEQ ID NO: 5 or 12, the mutation Q312R of SEQ ID NO: 19 or 22, the mutation Q330R of SEQ ID NO: 16 and the mutation Q336R of SEQ ID NO: 25, The method of any one of claims 1 to 3 or the engineered recombinant DNA enzyme of any one of claims 4 to 6.

15. the recombinase target site is a target site for a tyrosine site-specific recombinase (e.g., Cre-, Dre-, VCre-, SCre-, Vika-, lambda-Int-, Flp-, R-, Kw-, Kd-, B2-, B3-, Nigri-, and Panto-recombinase), and the engineered DNA recombinase is a genetically engineered DNA recombinase enzyme comprising a first recombinase enzyme and a second recombinase enzyme, wherein the first recombinase enzyme is a polypeptide having an amino acid sequence with at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO: 7 and comprising a single mutation K201R in the catalytic site; and the second recombinase enzyme is a polypeptide having an amino acid sequence with at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO: 11 and comprising a single mutation at position Q311R in the catalytic site; a genetically engineered DNA recombinase enzyme comprising a first recombinase enzyme and a second recombinase enzyme, wherein the first recombinase enzyme is a polypeptide having an amino acid sequence with at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO:7 and comprising a single mutation K201R in the catalytic site, and the second recombinase enzyme is a polypeptide having an amino acid sequence with at least 70%, preferably 80%, more preferably 90% sequence identity with the sequence set forth in SEQ ID NO:12 and comprising a single mutation Q311K in the catalytic site; a Cre recombinase comprising a first recombinase enzyme having a single mutation K201R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:2, and a second recombinase enzyme having a single mutation Q311R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:5; a Cre recombinase comprising a first recombinase enzyme having a single mutation K201R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:2, and a second recombinase enzyme having a single mutation Q311K in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:4; - Vika recombinase, comprising a first recombinase enzyme having a single mutation K219R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 15, and a second recombinase enzyme having a single mutation Q330R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 16; - Panto recombinase, comprising a first recombinase enzyme having a single mutation K202R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 18, and a second recombinase enzyme having a single mutation Q312R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 19; - Dre recombinase, comprising a first recombinase enzyme having a single mutation K202R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 21, and a second recombinase enzyme having a single mutation Q312R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 22; and a Vcre recombinase comprising a first recombinase enzyme having a single mutation K221R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO: 24, and a second recombinase enzyme having a single mutation Q336R in its catalytic site and having an amino acid sequence that is at least 70%, preferably 80%, more preferably 90% identical to a polypeptide having the sequence set forth in SEQ ID NO:

25.

7. The method of any one of claims 1 to 3 or the engineered DNA recombinase of any one of claims 4 to 6, selected from the group consisting of:

16. The engineered DNA recombinase comprises the nucleic acid sequence: 【Chemical 1】 or a reverse complement thereof, and specifically recognizes a recombinase target sequence upstream of the loxF8 target site, and 【Chemistry 2】 or a reverse complementary sequence thereof, and catalyzes the recombination of a gene sequence between the upstream recombinase target site sequence of SEQ ID NO: 65 and the downstream recombinase target site sequence of SEQ ID NO: 66 of the loxF8 recombinase target site; The ability of the loxF8 recombinase target site to catalyze the recombination of a gene sequence between an upstream recombinase target sequence of SEQ ID NO: 65 and a downstream recombinase target sequence of SEQ ID NO: 66 is determined by: a) expressing the engineered DNA recombinase enzyme comprising a first recombinase enzyme and a second recombinase enzyme in a cell; and b) analyzing whether the engineered DNA recombinase expressed in step a) is capable of recombining DNA sequences on human chromosomes in the cells; 7. The method of any one of claims 1 to 3 or the engineered DNA recombinase of any one of claims 4 to 6, which is tested by a method comprising:

17. 5. One or more nucleic acid molecules each comprising or consisting of a nucleic acid sequence encoding the engineered DNA recombinase of claim 4 or a subunit thereof.

18. 18. An expression vector comprising one or more nucleic acid molecules of claim 17 and expression control elements operably linked to said nucleic acids to facilitate their expression.

19. 18. A host cell comprising one or more nucleic acid molecules of claim 17.

20. 19. A host cell comprising the expression vector of claim 18.

21. 21. A pharmaceutical composition comprising the engineered DNA recombinase of any one of claims 4 to 6, one or more nucleic acid molecules of claim 17, an expression vector of claim 18, or a host cell of claim 19 or 20.

22. 22. The pharmaceutical composition of claim 21, further comprising one or more therapeutically acceptable diluents or carriers.

23. 21. The genetically engineered DNA recombinase of any one of claims 4 to 6, one or more nucleic acid molecules of claim 17, an expression vector of claim 18, or a host cell of claim 19 or 20, for use in medicine.

24. 21. The genetically engineered DNA recombinase of any one of claims 4 to 6, one or more nucleic acid molecules of claim 17, an expression vector of claim 18, or a host cell of claim 19 or 20, for use in the treatment of hemophilia A, preferably for use in the treatment of severe hemophilia A.

25. 1. A method for inverting a DNA sequence at the genome level in an in vitro cell, comprising: i. providing a nucleic acid molecule encoding a first recombinase enzyme according to any one of claims 4 to 6, wherein said first recombinase enzyme comprises at least one mutation that inactivates the catalytic activity of said first recombinase enzyme as a DNA recombinase, and wherein a monomer of said first recombinase specifically recognizes a first half-site of a recombinase target site; ii. providing a nucleic acid molecule encoding a second recombinase enzyme according to any one of claims 4 to 6, wherein said second recombinase enzyme comprises at least one mutation that inactivates the catalytic activity of said second recombinase enzyme as a DNA recombinase, and wherein a monomer of said second recombinase specifically recognizes a second half-site of a recombinase target site; iii. generating an expression vector by cloning the nucleic acid molecule encoding the first recombinase enzyme and the nucleic acid molecule encoding the second recombinase enzyme into an expression vector; iv. delivering the DNA sequence to be inverted, the expression vector of step iii., or the RNA molecule encoding the engineered DNA recombinase of any one of claims 4 to 6 to a cell containing said expression vector; v. expressing the engineered DNA recombinase of any one of claims 4 to 6; vi. A step of inverting a DNA sequence to be inverted on a human chromosome in said cell using the genetically engineered DNA recombinase according to any one of claims 4 to 6 that has been expressed in said cell. The method comprising:

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