A method for site-specific introduction of gene elements at an engineered gene locus by bimodal recombinase-mediated cassette exchange (BIRMCE).

Abstract

This specification describes systems and methods for genetically manipulating mammalian cells. It also describes non-human animal models utilizing these systems, methods for producing such non-human animals, and methods for using such non-human animals. TIFF2026524180000002.tif96170

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 523,550, filed on June 27, 2023, which is hereby incorporated by reference in its entirety.

[0002] Field of the Invention The present invention relates to genetic engineering, for example, related to genetic engineering in cells, in organ models, and in non - human animal models.

Background Art

[0003] Background All publications herein are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. No information provided herein is admitted to be prior art to the claimed invention, nor is it admitted to be related to the claimed invention, nor is any publication specifically or implicitly referenced admitted to be prior art.

[0004] The stable integration and / or editing of gene elements at a specific locus is an extremely difficult task. While various techniques exist to achieve this, it remains impossible to accomplish it in a way that is rapid, error-free, specific, inexpensive, and easy. MADR technology is a technique that has the potential to accomplish this with the aforementioned characteristics. One of the "Achilles' heels" of MADR and other similar technologies is that in order to terminate the system and express only one foreign gene element, another foreign gene element must be removed. Therefore, the aim of bimodal recombinase-mediated cassette exchange (biRMCE) is to enable the stable expression of a single foreign gene element even in the presence of another gene element. This principle increases the use of gene cassette exchange for a variety of applications where the stable integration and / or editing of gene elements at a specific locus is desired.

[0005] Recombinase-mediated gene cassette exchange is a technique used in various cell types to incorporate foreign gene elements into manipulated loci. However, it has been found that cross-recombination can occur between heterotype sites when using, for example, a single recombinase targeting a heterotype recognition site, which poses a problem. Other similar studies use multiple integrases targeting different recognition sites, but the main problem with these systems is that these integrases incorporate not only the gene cassette element but also the entire gene vector carrying unwanted sequences. One existing solution is to use different types of recombinases, such as Flp and Cre, sequentially or simultaneously, to prevent cross-recombination between recognition sites and to increase the efficiency of gene cassette exchange. However, when gene cassettes carrying different types of gene elements are used, the reversible recombination reaction by the recombinases makes the system unstable and inefficient.

[0006] Therefore, in this field, there is still a need for robust and highly stable methods and systems for site-specifically introducing gene elements at manipulated gene loci. [Overview of the project]

[0007] Summary of the present invention The following embodiments and aspects are described and illustrated in relation to compositions and methods, but they are intended to be illustrative and not to limit the scope.

[0008] Various embodiments provide a system that includes the following: (a) (i) One or more polyadenylation signals or transcription stop elements located upstream of the nucleic acid encoding the transgene or RNA, (ii) nucleic acids encoding a transgene or RNA, and (iii) Recombinase recognition site comprising at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site donor vectors, including; and (b) Two types of recombinases specific to the recombinase recognition site.

[0009] In various embodiments, the donor vector may further include at least a third recombinase recognition site, and the system may further include at least a third recombinase specific to at least the third recombinase recognition site.

[0010] In various ways, the system The donor vector and the locus targeted by two types of recombinases, and optionally at least a third recombinase. It may further contain mammalian cells that include [the specified substance].

[0011] In various embodiments, two types of recombinases may be provided as follows: (i) One expression vector containing two genes encoding recombinases specific to the recognition site, or (ii) Two expression vectors, namely, a first expression vector containing one gene encoding a first recombinase specific to the unidirectional recombinase recognition site, and a second expression vector containing one gene encoding a second recombinase specific to the bidirectional recombinase recognition site, or (iii) One mRNA that encodes two types of recombinases specific to the recognition site, or (iv) Two types of mRNA, namely, a first mRNA encoding a first recombinase specific to the unidirectional recombinase recognition site, and a second mRNA encoding a second recombinase specific to the bidirectional recombinase recognition site, or (v) A viral vector containing two genes that encode a recombinase specific to that recognition site, or (vi) Two types of viral vectors, namely, a first viral vector containing one gene encoding a first recombinase specific to a unidirectional recombinase recognition site, and a second viral vector containing one gene encoding a second recombinase specific to a bidirectional recombinase recognition site, or (vii) One recombinant protein comprising a unidirectional recombinase and a bidirectional recombinase, or (viii) Two recombinant proteins, namely, a first recombinase protein specific to a unidirectional recombinase recognition site, and a second recombinase protein specific to a bidirectional recombinase recognition site.

[0012] In various embodiments, (i) in an expression vector containing two genes encoding recombinases specific to the recognition site, the encoded recombinases may be fused together. In various embodiments, (iii) in an mRNA containing two recombinases specific to the recognition site, the encoded recombinases may be fused together. In various embodiments, (v) in a viral vector containing two genes encoding recombinases specific to the recognition site, the encoded recombinases may be fused together. In various embodiments, in (viii), the two recombinant proteins may be fused together.

[0013] In various embodiments, one of the recombinases may be fused with one or more proteins other than recombinases. In various embodiments, one of the two fused recombinases may be further fused with one or more proteins other than recombinases.

[0014] In various embodiments, at least a third recombinase may be provided by: (iv) One expression vector containing a gene encoding at least a third recombinase specific to the third recombinase recognition site, or (x) One mRNA that encodes at least a third recombinase specific to at least a third recognition site, or (xi) A viral vector comprising a gene encoding at least a third recombinase specific to at least a third recombinase recognition site, or (xii) A recombinant protein comprising at least a third recombinase specific to at least a third recombinase recognition site.

[0015] In various embodiments, (iv) an expression vector comprising a gene encoding at least a third recombinase specific to a third recombinase recognition site, wherein the expression vector further comprises a gene encoding one or more proteins other than the third recombinase, and the encoded third recombinase is fused with the encoded one or more proteins.

[0016] In various embodiments, (x) a single mRNA encoding at least a third recombinase specific to at least a third recognition site, wherein the mRNA further encodes one or more proteins other than the third recombinase, and the encoded recombinase is further fused with the encoded one or more proteins. In various embodiments, (xi) a single viral vector comprising a gene encoding at least a third recombinase specific to at least a third recombinase recognition site, wherein the single viral vector further encodes one or more proteins other than the third recombinase, and the encoded recombinase is further fused with the encoded one or more proteins. In various embodiments, (xii) a single recombinant protein comprising at least a third recombinase specific to at least a third recombinase recognition site, which is fused with one or more proteins other than the third recombinase.

[0017] In various aspects, the unidirectional recombinase recognition site may be located upstream of the bidirectional recombinase recognition site. In various aspects, the unidirectional recombinase recognition site may be located downstream of the promoter.

[0018] In various embodiments, the donor vector may further include an intron, a portion of an intron, or at least one splice acceptor site, and optionally, a unidirectional recombinase recognition site is embedded in or in the intron.

[0019] In various embodiments, the unidirectional recombinase may be Bxb1. In various embodiments, the unidirectional recombinase may be selected from Bxb1, Phic31, PhiBT1, PhiC1, MR11, R4, TP901-1, A118, FC1, PhiRV, TG1, Phi370.1, Wβ, BL3, SPBc, K38, and any variants thereof.

[0020] In various embodiments, the bidirectional recombinase may be Flp. In various embodiments, the unidirectional recombinase may be Bxb1, and the bidirectional recombinase may be selected from FLp, Cre, VCre, SCre, Nigri, Panto, Vika, or variants thereof.

[0021] In various embodiments, the third recombinase may be selected from Bxb1, Phic31, PhiBT1, PhiC1, MR11, R4, TP901-1, A118, FC1, PhiRV, TG1, Phi370.1, Wβ, BL3, SPBc, K38, FLp, Cre, VCre, SCre, Nigri, Panto, Vika, or variants thereof.

[0022] In various embodiments, the unidirectional recombinase recognition site may be attB. In various embodiments, the unidirectional recombinase recognition site may be attP.

[0023] In various embodiments, the bidirectional recombinase recognition site may be a flippase recognition target (FRT), loxP, VloxP, SloxP, nox, or pox.

[0024] In various embodiments, one or both of the recombinase recognition sites may contain mutations.

[0025] In various embodiments, the donor vector may be selected from the group consisting of plasmids, linear PCR, linear single-stranded DNA, closed-end double-stranded DNA, circular single-stranded DNA, circular double-stranded DNA, RNA, minicircles, viral vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and human artificial chromosomes (HACs). In various embodiments, the viral vector may be an adeno-associated virus (AAV) vector.

[0026] In various embodiments, the donor vector may include at least four polyadenylation signals upstream of the nucleic acid encoding the transgene or RNA. In various embodiments, the donor vector may include an intron or a portion of an intron upstream and / or downstream of the nucleic acid encoding the transgene or RNA. In various embodiments, the donor vector may further include a post-transcriptional regulatory element. In various embodiments, the donor vector may further include a polyadenylation signal downstream of the nucleic acid encoding the transgene or RNA. In various embodiments, the donor vector may further include an open reading frame (ORF) beginning with a splice acceptor. In various embodiments, the donor vector may further include a fluorescent reporter.

[0027] In various embodiments, the expression vector containing the recombinase may be located under a tissue-specific promoter.

[0028] In various embodiments, RNA may be siRNA, shRNA, sgRNA, crRNA, pegRNA, lncRNA, or miRNA. In various embodiments, the transgene or RNA may contain disease-related mutations. In various embodiments, the transgene or RNA may contain gain-of-function (GOF) gene mutations, loss-of-function (LOF) gene mutations, or both.

[0029] In various embodiments, the mammalian cells may be human cells, and the gene locus may be the AAVS1 locus, H11 locus, HPRT1 locus, Rogi1 locus, Rogi2 locus, GAPDH locus, TATA box-binding protein (TBP) locus, kinesin family member (KIF11) locus, TRAC locus, ZAP-70 locus, Linker of Activation of T cells (LAT) locus, or Lymphocyte Cytosolic Protein 2 (LCP2) locus, and the method may be in vitro, ex vivo, or in vivo.

[0030] In various embodiments, the locus may include a secondary cistron containing a first polynucleotide encoding a first protein, a promoter, a recombinase recognition site recognized by a recombinase in the system, and a second polynucleotide encoding an open reading frame for a second protein. In various embodiments, the first protein, the second protein, or both may be fluorescent proteins. In various embodiments, the first polynucleotide encoding the protein may be downstream of the gene in the locus.

[0031] In various embodiments, the mammalian cells may be mouse cells, and the locus may be the ROSA26 locus, Hipp11 locus, Tigre locus, ColA1 locus, HPrt locus, GAPDH locus, TATA box-binding protein (TBP) locus, kinesin family member (KIF11) locus, TRAC locus, Zap-70 locus, T cell activation linker (LAT) locus, or lymphocyte cytoplasmic protein 2 (LCP2) locus, and the method may be in vitro, ex vivo, or in vivo.

[0032] In various embodiments, the locus may include a secondary cistron comprising a first polynucleotide encoding a first protein, a promoter, a recombinase recognition site recognized by a recombinase in the system, and a second polynucleotide encoding an open reading frame for a second protein.

[0033] Various embodiments provide methods for genetically modifying mammalian cells, each method comprising the step of transfecting or transfecting mammalian cells using any one of the systems of the present invention.

[0034] In various embodiments, the system may target a locus, which includes a recombinase recognition site comprising at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site.

[0035] In various embodiments, unidirectional recombination may be upstream of bidirectional recombination at a gene locus.

[0036] In various embodiments, the mammalian cells may be human cells, the system targets the AAVS1 locus, H11 locus, HPRT1 locus, Rogi1 locus, Rogi2 locus, GAPDH locus, TATA box-binding protein (TBP) locus, kinesin family member (KIF11) locus, TRAC locus, ZAP-70 locus, T cell activation linker (LAT) locus, or lymphocyte cytoplasmic protein 2 (LCP2) locus, and the method is in vitro, ex vivo, or in vivo.

[0037] In various embodiments, the mammalian cells may be mouse cells, and the system targets the ROSA26 locus, Hipp11 locus, Tigre locus, ColA1 locus, HPrt locus, GAPDH locus, TATA box-binding protein (TBP) locus, kinesin family member (KIF11) locus, TRAC locus, Zap-70 locus, T cell activation linker (LAT) locus, or lymphocyte cytoplasmic protein 2 (LCP2) locus, and the method is in vitro, ex vivo, or in vivo.

[0038] In various embodiments, the method may further include the step of administering one or more recombinase enzymes to cells.

[0039] In various aspects, one or more recombinase enzymes may include Bxb1 recombinase, Cre recombinase, flipperze recombinase, Nigri recombinase, Panto recombinase, Vika recombinase, VCre recombinase, or SCre recombinase.

[0040] In various embodiments, mammalian cells may include blood cells, tumor cells, non-tumor cells, embryonic stem cells, adult stem cells, induced pluripotent stem cells, or tissue progenitor cells.

[0041] Various embodiments provide non-human animal models, which include non-human animals incorporating the system of the present invention.

[0042] In various embodiments, the non-human animal model may be a non-human animal model personalized for a human target cancer, and the transgene or RNA is based on the human target cancer. In various embodiments, the non-human animal model may be a non-human animal model personalized for a human target disease or condition, and the transgene or RNA is based on the human target disease or condition.

[0043] In various embodiments, the transgene or RNA may be selected from the group consisting of oncogenes, loss-of-function (LOF) mutations in tumor suppressor genes, gain-of-function (GOF) mutations in proto-oncogenes, pseudogenes, siRNA, shRNA, sgRNA, pegRNA, crRNA, lncRNA, miRNA, epigenetic modifications, non-coding or epigenetic abnormalities associated with human diseases, and combinations thereof.

[0044] In various embodiments, the transgene or RNA may be selected from a group consisting of gain-of-function (GOF) mutations, loss-of-function (LOF) mutations, or both.

[0045] In various embodiments, the system may target a locus in a non-human animal model, the locus comprising a recombinase recognition site including at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site. In various embodiments, unidirectional recombination is upstream of bidirectional recombination at the locus.

[0046] Various embodiments provide methods for producing non-human animal models, the methods comprising the step of transfecting or transfecting a non-human animal model using the system of the present invention.

[0047] Various embodiments provide non-human animal models prepared by the method of the present invention.

[0048] Various embodiments provide a method for evaluating the effects of a candidate drug, the method comprising the following steps: A step of providing a non-human animal model of the present invention; The step of administering candidate drugs to non-human animal models; and This stage involves evaluating the effects of candidate drugs on non-human animal models.

[0049] Various embodiments provide mammalian cells comprising the system of the present invention.

[0050] Various embodiments provide non-mammalian cells comprising the system of the present invention.

[0051] Other features and advantages of the present invention will be revealed in the following detailed description, in conjunction with the accompanying drawings which illustrate various features of the embodiments of the invention, for example. [Brief explanation of the drawing]

[0052] Exemplary embodiments are shown in the referenced figures. The embodiments and figures disclosed herein are intended to be illustrative and not limiting. [Figure 1] Figures 1A and 1B show schematic diagrams of strategies for comparing the efficiency of Bxb1 alone or Bxb1 and FlpO in combination when incorporating a transgene element in the presence of different Bxb1 recognition sites within the Rosa26 locus. 1A shows the Rosa26 locus carrying the PuroR gene flanked by "open" attP and FRT sites. The promoter-less donor vector carries the mScarlet transgene flanked by the attB and FRT sites. Recombination between this locus on the genome and the donor vector is predicted in the presence of Bxb1 or Bxb1+FlpO. 1B shows the Rosa26 locus carrying the PuroR gene flanked by "locked" attR and FRT sites. The promoter-less donor vector carries the mScarlet transgene flanked by the attB and FRT sites. Recombination between the gene locus on the genome and the donor vector is predicted in the presence of Bxb1 or Bxb1+FlpO. PuroR represents the puromycin resistance gene. Bxb1+FlpO is expressed in separate plasmids. [Figure 2]Figures 2A and 2B show that insertion of the transgene element via integration in Bxb1 or Bxb1+FlpO requires the recognition site attP embedded in the Rosa26 locus. 2A shows that integration and expression of mScarlet are achievable in the presence of an "open" attP site in the Rosa26 locus, an attB site in the donor vector, and Bxb1 or Bxb1+FlpO (Rx 1.1 and 1.2). 2B shows that integration and expression of mScarlet were significantly reduced in the presence of a "locked" attR site in the Rosa26 locus, an attB site in the donor vector, and Bxb1 or Bxb1+FlpO (Rx 2.1 and 2.2). No recombinase expression was used as a negative control (Rx 1.3 and 2.3). Epifluorescence microscopy images were acquired 48 hours after nucleofection. mScarlet expression was measured by flow cytometry in 10,000 cells 48 hours after nucleofection. Heterozygous mouse neural stem cells carrying each of the "2-in-1" landing pads at the Rosa26 locus were used; see Figure 1 for details. [Figure 3] Figures 3A and 3B show the number of cells expressing mScarlet at the Rosa26 locus via recombination with Bxb1 or recombination with Bxb1+FlpO. 3A is a graph comparing the quantification of mScarlet-expressing cells after recombination with Bxb1 to a Rosa26 locus with an "open" attP site or a Rosa26 locus with a "locked" attR site. 3B is a graph comparing the quantification of mScarlet-expressing cells after recombination with Bxb1+FlpO to a Rosa26 locus with an "open" attP site or a Rosa26 locus with a "locked" attR site. This data indicates that integration of the transgene element requires recombination by Bxb1 at the recognition sites attP / attB. [Figure 4]Figures 4A-4B show schematic diagrams of strategies for comparing the efficiency of Bxb1 alone versus Bxb1 and Cre combined when incorporating a transgene element in the presence of different Bxb1 recognition sites within the Rosa26 locus. 4A shows the Rosa26 locus carrying the PuroR gene, flanked by a loxP site and an "open" attP site. A promoter-less donor vector carries the mScarlet transgene, flanked by a loxP site and an attB site. Recombination between this genomic locus and the donor vector is predicted in the presence of Bxb1 or Bxb1+Cre. 4B shows the Rosa26 locus carrying the PuroR gene, flanked by a loxP site and a "locked" attR site. A promoter-less donor vector carries the mScarlet transgene, flanked by a loxP site and an attB site. Recombination between the gene locus on the genome and the donor vector is predicted in the presence of Bxb1 or Bxb1+Cre. PuroR represents the puromycin resistance gene. Bxb1+Cre is expressed in separate plasmids. [Figure 5]Figures 5A–5B show that insertion of the transgene element via integration with Bxb1 or Bxb1+Cre requires the recognition site loxP embedded in the Rosa26 locus. 5A shows that integration and expression of mScarlet are achievable in the presence of an "open" attP site at the Rosa26 locus, an attB site in the donor vector, and Bxb1+Cre (Rx 3.2). 5B shows that integration and expression of mScarlet were reduced in the presence of a "locked" attL site at the Rosa26 locus, an attB site in the donor vector, and Bxb1+Cre (Rx 4.2). No recombinase expression was used as a negative control (Rx 3.3 and 4.3). Epifluorescence microscopy images were acquired 48 hours after nucleofection. mScarlet expression was measured by flow cytometry in 10,000 cells 48 hours after nucleofection. Heterozygous mouse neural stem cells carrying each of the "2-in-1" landing pads at the Rosa26 locus were used; see Figure 4 for details. [Figure 6] Figures 6A and 6B show the number of cells expressing mScarlet at the Rosa26 locus via recombination with Bxb1 or with Bxb1+Cre. 6A is a graph comparing the quantification of mScarlet-expressing cells after Bxb1 recombination to a Rosa26 locus with an "open" attP site or a Rosa26 locus with a "locked" attL site. 6B is a graph comparing the quantification of mScarlet-expressing cells after Bxb1+Cre recombination to a Rosa26 locus with an "open" attP site or a Rosa26 locus with a "locked" attL site. This data indicates that the incorporation of the transgene element requires Cre / loxP recombination. [Figure 7]Figures 7A and 7B show the expression of mScarlet+ cells via Bxb1+Cre recombination to the Rosa26 locus with different Bxb1 recognition sites at different time points. 7A shows mScarlet expression and quantification in cells with an "open" attP site downstream of the PuroR gene when Bxb1+Cre is expressed (Rx 3.2). 7B shows mScarlet expression and quantification in cells with a "locked" attL site downstream of the PuroR gene when Bxb1+Cre is expressed (Rx 4.2). Epifluorescence microscopy images were acquired 2, 4, and 7 days after nucleofection. mScarlet expression was measured by flow cytometry in 10,000 cells 2, 4, and 7 days after nucleofection. [Figure 8] Figure 8 shows a graph comparing the percentage expression of mScarlet from the recombination reactions described in Figure 7 at 2, 4, and 7 days after nucleofection. The dashed line indicates that the integration of the transgene element mediated by Cre / loxP recombination is unstable, and that less than 1% of cells express mScarlet at 7 days after nucleofection. The solid line indicates that the integration of the transgene element mediated by both Cre / loxP and Bxb1 / attP / attB recombination reactions is more stable. The Y-axis shows the percentage of mScarlet+ cells. The X-axis shows the number of days after induction of recombination. This data suggests that the transgene element can be integrated by both reversible / bidirectional recombination and irreversible / unidirectional recombination, i.e., by the reversible / bidirectional recombination of Cre / loxP and the irreversible / unidirectional recombination of Bxb1 / attP / attB. [Figure 9]Figure 9 shows a simplified table of the data from Figures 1 to 8. Reactions 1.1 to 2.3 demonstrate that attP / attB recombination is necessary for irreversible / unidirectional recombination by Bxb1. Reactions 3.1 to 4.3 suggest that bimodal recombinase-mediated cassette exchange (biRMCE) is possible, i.e., two types of recombination: reversible / bidirectional recombination of Cre / loxP and irreversible / unidirectional recombination of Bxb1 / attP / attB. [Figure 10] Figures 10A–10D show schematic diagrams of strategies for comparing whole plasmid integration with recombinase-mediated cassette exchange. 10A illustrates 2in1-loxP-attP-TagBFP2-nls-FRT, the "landing pad" at the Rosa26 locus. 10B shows pDonor-2in1-loxP-attB-mScarlet-FRT carrying a different recombination site. 10C shows the Rosa26 locus after recombination with pDonor-2in1-loxP-attB-mScarlet-FRT mediated by the expression of a different recombinase. 10D shows the Rosa26 locus after Cre / loxP recombination. [Figure 11-1]Figures 11A–11F show verification of DNA cassette exchange via biRMCE. Panels 11A–11D show the expression of red fluorescent protein (mScarlet) in the cytoplasm and / or blue fluorescent protein (TagBFP2) in the nucleus. TagBFP2 expression in the nucleus reveals cells in which the transgene mScarlet was previously incorporated by whole plasmid integration rather than DNA cassette exchange. TagBFP2 expression is dependent on the excision of the mScarlet cassette by Cre / loxP. Panel 11A shows several TagBFP2+ cells, indicating that whole plasmid integration via Bxb1 occurred previously. Panels 11B–11C show very few TagBFP+ cells, demonstrating that DNA cassette exchange via biRMCE occurred previously using Bxb1-FlpO or FlpO-Bxb1, respectively. Panel 11D shows a small number of TagBFP+ cells, indicating that DNA cassette exchange via dRMCE occurred previously using FlpO-Cre. Epifluorescence microscopy images were acquired 48 hours after Cre expression. Panel 11E shows a graph of the percentage of cells expressing TagBFP2 in panels A–D, demonstrating that Bxb1 expression alone induces whole plasmid integration rather than cassette exchange. Panel 11F shows a graph of the percentage of cells expressing mScarlet in panels B–D, demonstrating that both biRMCE and dRMCE efficiently (>95%) integrate transgene elements via DNA cassette exchange. Flow cytometry was performed to quantify cells expressing the fluorescent reporter 48 hours after nucleofection. These data demonstrate that both biRMCE and dRMCE integrate transgene elements into designated loci via DNA cassette exchange. [Figure 11-2] Please refer to the explanation in Figure 11-1. [Figure 12]Figure 12 shows that biRMCE is more stable and efficient than dRMCE in the integration of transgene elements into recipient genomic DNA. The graph shows the efficiency of transgene element expression at the Rosa26 locus at different time points. The Y-axis shows the percentage of mScarlet+ cells. The X-axis shows the number of days after recombination induction. The dashed line shows the kinetics of dRMCE. The solid line shows the kinetics of biRMCE. This result demonstrates that biRMCE is a stable response in the integration of transgene elements into a given locus. [Figure 13] Figure 13 shows a schematic diagram of the strategy for verifying lock-in of DNA cassette exchange by biRMCE. A colorless cell line with one landing pad was used to compare dRMCE and biRMCE side by side. The cell line has a CAG promoter, followed by a loxP site, an attP site, a puromycin resistance gene (PuroR), and an FRT site at the Rosa26 locus. The cell line is named Rosa26-2in1-loxP-attP-PuroR-FRT. Three plasmids were used for dRMCE (1: promoter-less donor vector-loxP-EGFP-nls-FRT; 2: promoter-less donor vector-loxP-Scarlet-nls-FRT; 3: pCag-FlpO-Cre). Three plasmids were used for biRMCE (1: promoter-less donor vector-attB-EGFP-nls-FRT; 2: promoter-less donor vector-attB-Scarlet-nls-FRT; 3: pCag-Bxb1-FlpO). [Figure 14]Figure 14 shows that biRMCE locks DNA cassette exchange at the Rosa26 locus landing pad in the early stages. The upper panel shows cells expressing mScarlet and / or EGFP in the nucleus via dRMCE (A) or biRMCE (B). Arrows indicate cells expressing both mScarlet and EGFP. The lower panel shows the quantification by flow cytometry of cells expressing mScarlet and / or EGFP in the nucleus via dRMCE (C) or biRMCE (D), with yellow cells being double-positive for mScarlet and EGFP. Images were acquired using epifluorescence microscopy 2 days after nucleofection. Flow cytometry was performed 2 days after nucleofection. These data demonstrate that biRMCE locks DNA cassette exchange from the early time point. [Figure 15] Figures 15A–15D show that biRMCE maintains lock-in of DNA cassette exchange at the Rosa26 locus landing pad in later stages. The upper panel shows cells expressing mScarlet and / or EGFP in the nucleus via dRMCE (A) or biRMCE (B). Arrows indicate cells expressing both mScarlet and EGFP. The lower panel shows the quantification by flow cytometry of cells expressing mScarlet and / or EGFP in the nucleus via dRMCE (C) or biRMCE (D), with yellow cells being double-positive for mScarlet and EGFP. Note that there are fewer colored cells under dRMCE conditions, indicating that integration is not as stable as with biRMCE. Images were acquired using epifluorescence microscopy 8 days after nucleofection. Flow cytometry was performed 8 days after nucleofection. These data demonstrate that biRMCE maintains the lock-in of DNA cassette exchange. [Figure 16-1]Figures 16A–16H illustrate the strategies and validations for genetic landings containing gene elements compatible with both dRMCE / MADR and intronic biRMCE. Figure 16A shows a schematic diagram of the mTmG landing pad at the Rosa26 locus and a promoter-less donor vector ready for insertion of its surrounding DNA cassette via dRMCE / MADR. Figure 16B shows a novel landing pad at the Rosa26 locus containing an intron with an attP site, located between the initial ATG and the rest of the open reading frame (ATG-less TagBFP-nls). This novel landing pad contains elements that are fully compatible with both dRMCE / MADR and intronic biRMCE. Panels C–E show Tag-BFP-WPRE expression in the nucleus after dRMCE / MADR response in heterozygous mTmG neural stem cells, as observed by fluorescence microscopy and flow cytometry. Panels F–H show WPRE sequence-free Tag-BFP expression in the nucleus of heterozygous mTmG neural stem cells after dRMCE / MADR reaction, as observed by fluorescence microscopy and flow cytometry. Arrows indicate several cells expressing Tag-BFP in the nucleus. Images were acquired using epifluorescence microscopy two days after nucleofection. Flow cytometry was performed two days after nucleofection. [Figure 16-2] Please refer to the explanation in Figure 16-1. [Figure 16-3] Please refer to the explanation in Figure 16-1. [Figure 17-1]Figures 17A–17H show strategies and validations for a genetic landing pad compatible with both dRMCE / MADR and intronic biRMCE. Figure 17A shows a schematic diagram of the Rosa26 locus landing pad "2in1-loxP-ATG-in-attP-TRON-(ATG-less-TagBFP-nls)-FRT" and a pair of promoter-less donor vectors that can insert the DNA cassette they enclose via dRMCE / MADR or intronic biRMCE. Figure 17B shows the new landing pad at the Rosa26 locus after dRMCE / MADR or intronic biRMCE. Figures 17C–17E show the expression of miRFP-670 in the nucleus after dRMCE / MADR response in heterozygous TagBFP neural stem cells, as observed by fluorescence microscopy and flow cytometry. Figures 17F–17H show the expression of miRFP-670 in the nuclei of heterozygous TagBFP neural stem cells after intronic biRMCE reaction, as observed by fluorescence microscopy and flow cytometry. Arrows indicate cells expressing miRFP-670 in the nucleus. Images were acquired using epifluorescence microscopy two days after nucleofection. Flow cytometry was performed two days after nucleofection. [Figure 17-2] Please refer to the explanation in Figure 17-1. [Figure 18]Figure 18 shows a schematic diagram of a strategy for verifying lock-in of DNA cassette exchange by intronized biRMCE. Intronization of both the Rosa26 locus landing pad and donor plasmid is used to verify lock-in of intronized biRMCE. The landing pad has a CAG promoter, followed by a loxP site, an ATG start codon, an intron in which attP is embedded, an arbitrary ATG-less gene element, and an FRT site. The landing pad is named Rosa26-2in1-loxP-in-attP-TRON-(ATG-less-Gene)-FRT. The promoter-less donor plasmid contains a gene element without the ATG start codon, which is flanked by an attB site with part of an intron and an FRT site. The recombinases Bxb1 and Flp are required to perform intronized biRMCE. [Figure 19] Figure 19 (Panels A-F) shows schematic diagrams of strategies for verifying the minimum recognition site for biRMCE. A) illustrates the minimum recognition site for biRMCE, where the "irreversible" recognition site is immediately upstream of the "reversible" recognition site. B) illustrates 3in1-loxP-(attP / VloxP)-TagBFP2-nls-FRT, the "landing pad" in the Rosa26 locus. C) shows a heterozygous neural stem cell line expressing TagBFP2 in the nucleus, which carries the landing pad described in Figure B. D) shows pDonor-2in1-loxP-attB-mScarlet-VloxP carrying different specific recombination sites. E) shows the Rosa26 locus after recombination with pDonor-2in1-loxP-attB-mScarlet-VloxP via Bxb1. F) shows the Rosa26 locus after recombination via Bxb1 and VCre in pDonor-2in1-loxP-attB-mScarlet-VloxP. [Figure 20]Figure 20 (Panels A-D) shows the validation of DNA cassette exchange using the smallest recognition site for biRMCE. A) shows a heterozygous mScarlet cell line carrying a landing pad obtained after recombination with Bxb1. B) shows a heterozygous mScarlet cell line carrying landing pads obtained after recombination with Bxb1 and VCre. C) shows the mScarlet cell line described in Figure A, expressing TagBFP2 in the nucleus after recombination with Cre. Quantification by flow cytometry shows that 98% of cells are TagBFP2 positive. The predicted landing pad is shown at the bottom of the panel. D) shows the mScarlet cell line described in Figure B, expressing TagBFP2 in the nucleus after recombination with Cre. Quantification by flow cytometry shows that 3.5% of cells are TagBFP2 positive. The predicted landing pad is shown at the bottom of the panel. Images C and D were acquired using epifluorescence microscopy two days after induction. Flow cytometry was performed two days after induction. [Figure 21]Figure 21 (Panels A-D) shows the knock-in of elements compatible with MADR and biRMCE into the human GAPDH locus. A) shows a schematic diagram of exons 6 through 9 of the endogenous human GAPDH locus. B) shows a schematic diagram after knock-in into the human GAPDH locus. The edited locus carries a secondary cistron containing TagBFP2 below the GAPDH promoter and a CAG promoter upstream of miRFP670, flanked by MADR and biRMCE-specific recombination sites. C) shows the cell line HEK [GAPDH-TagBFP2nls-Cag-2in1(loxP-attP)-miRFP67nls-FRT] after appropriate knock-in and several rounds of purification. D) shows genotyping of HEK cells before and after knock-in. The PCR fragments show the left homology arm, right homology arm, Cag-miRFP670 cistron, and unedited GAPDH locus. WT = wild-type unedited HEK cells, KI = knock-in edited HEK cells. The colored lines indicate the size of each highlighted PCR fragment. [Figure 22] Figure 22 (Panels A-D) shows the validation and efficiency of MADR and biRMCE at the human GAPDH locus. A) shows a schematic diagram of strategies for validation and comparison of MADR and biRMCE at the human GAPDH locus using the mScarlet donor vector. B) shows a schematic diagram of the predicted human GAPDH locus after mScarlet cassette exchange via MADR or biRMCE. C) shows mScarlet expression in the cell line HEK [GAPDH-TagBFP2nls-Cag-2in1(loxP-attP)-miRFP67nls-FRT] after MADR or biRMCE. D) shows the quantification of cells expressing mScarlet after MADR or biRMCE by flow cytometry. Images were acquired using epifluorescence microscopy 10 days after induction. Flow cytometry was performed 10 days after induction. [Figure 23]Figure 23, like Figure 13, shows a schematic diagram of the strategy for verifying lock-in of DNA cassette exchange by biRMCE. A colorless cell line with one landing pad was used to compare dRMCE / MADR and biRMCE side by side. The cell line has a CAG promoter, loxP site, attP site, puromycin resistance gene (PuroR), and FRT site in the Rosa26 locus. The cell line is named Rosa26-2in1-loxP-attP-PuroR-FRT. Three plasmids were used for dRMCE / MADR (1: promoter-less donor vector-loxP-miRFP670-nls-FRT; 2: promoter-less donor vector-loxP-BFP-nls-FRT; 3: pCag-FlpO-Cre). Three plasmids were used for biRMCE (4: promoter-less donor vector -attB-miRFP670-nls-FRT; 5: promoter-less donor vector -attB-BFP-nls-FRT; 6: pCag-Bxb1-FlpO). [Figure 24] Figure 24 (Panels A-D), like Figure 14, shows that biRMCE locks DNA cassette exchange at the Rosa26 locus landing pad in the early stages. The upper panel shows cells expressing miRFP670 and / or BFP in the nucleus via dRMCE / MADR (A) or biRMCE (B) two days after induction. Arrows indicate cells expressing both miRFP670 and BFP. The lower panel shows flow cytometry quantification of cells expressing miRFP670 and / or BFP in the nucleus via dRMCE / MADR (C) or biRMCE (D) two days after induction; purple dots indicate miRFP670-positive cells, blue dots indicate BFP-positive cells, and red dots indicate double-positive cells for both miRFP670 and BFP. Images were acquired using epifluorescence microscopy two days after nucleofection. Flow cytometry was performed two days after induction. These data demonstrate that biRMCE locks DNA cassette exchange from the initial time point. [Figure 25] Figure 25 (Panels A-D), like Figure 15, shows that biRMCE maintains lock-in of DNA cassette exchange at the Rosa26 locus landing pad at later time points. The upper panel shows the quantification by flow cytometry of cells expressing miRFP670 and / or BFP in the nucleus via dRMCE / MADR (A) or biRMCE (B) 4 days after induction. The lower panel shows the quantification by flow cytometry of cells expressing miRFP670 or BFP in the nucleus via dRMCE / MADR (C) or biRMCE (D) 8 days after induction. Purple dots indicate miRFP670-positive cells, and blue dots indicate BFP-positive cells. Note that under the dRMCE / MADR condition, there are fewer colored cells at 8 days after induction, indicating that its integration is not as stable as with biRMCE. Flow cytometry was performed at 4 and 8 days after induction. These data demonstrate that biRMCE maintains DNA cassette exchange lock-in at later time points. [Modes for carrying out the invention]

[0053] Description of the present invention All references cited herein are incorporated by reference in the same way they are cited in their entirety. Unless otherwise defined, technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention pertains. The following provides a general guide to many of the terms used herein: Singleton et al., "Dictionary of Microbiology and Molecular Biology" 3 rded., Revised, J. Wiley & Sons (New York, NY 2006);March, "Advanced Organic Chemistry Reactions, Mechanisms and Structure" 7 th ed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, "Molecular Cloning: A Laboratory Manual" 4 th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012).

[0054] Those skilled in the art will understand that there are many methods and materials similar to or equivalent to those described herein that can be used in carrying out the present invention. In fact, the present invention is by no means limited to the methods and materials described herein. The following terms are defined below in relation to the present invention:

[0055] As used herein, the term “about” means, when used in relation to a referenced numerical expression, a referenced numerical expression that is up to 5% greater or less than the referenced numerical expression, unless otherwise specifically provided herein. For example, the expression “about 50%” encompasses a range of 45% to 55%. In various embodiments, when used in relation to a referenced numerical expression, and when the term is specifically used in the claims, the term “about” may mean a referenced numerical expression that is up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% greater or less than the referenced numerical expression.

[0056] As used herein, “mammal” means any member of the class Mammalia, including humans, as well as non-human primates such as chimpanzees and other apes and monkey species; domestic animals such as cattle, sheep, pigs, goats, and horses; domesticated animals such as dogs and cats; and laboratory animals such as rodents such as mice, rats, and guinea pigs. The term does not imply any particular age or sex. Therefore, both adult and newborn subjects, whether male or female, are intended to be included within the scope of the term. In some embodiments, the subject is human.

[0057] Aspects of the present invention describe bimodal recombinase-mediated cassette exchange (biRMCE). BiRMCE takes advantage of the benefit of performing unidirectional and bidirectional recombination reactions simultaneously in the system and solves many problems associated with using either bidirectional or unidirectional recombination reactions alone to incorporate gene elements. First, no cross-recombination occurs between the bidirectional recombinase recognition site and the unidirectional recombinase recognition site, thus ensuring the specificity of the system. Second, BiRMCE ensures the insertion of the gene cassette sandwiched between the unidirectional and bidirectional recombinase recognition sites, thereby preventing the integration of the entire gene vector. Because one irreversible reaction and one reversible reaction occur, it becomes impossible to incorporate another gene cassette, thereby ensuring the system is locked. To use an analogy, BiRMCE can be described as a competition among sperm vying for an egg, where only one sperm can penetrate the egg. In this analogy, the gene cassette is the "sperm," and the target gene locus is the "egg." Furthermore, it is possible to perform both BiRMCE and dRMCE at the same gene locus, which offers greater flexibility than previous MADR technologies.

[0058] The strengths of Bi-RMCE include, but are not limited to, the following: (1) It is a one-step reaction for inserting the sandwiched DNA cassette. (2) The system is locked, thereby enabling high-throughput insertion of the DNA payload and rapid downstream analysis and / or application. For example, cells into which the DNA cassette has been incorporated can be selected much more quickly, mainly because the invading DNA cassette does not carry a promoter. Downstream applications can be performed within hours of biRMCE. (3) Because the process requires separate recombination reactions, there is no risk of cross-recombination between recombinase recognition sites. (4) Because it requires irreversible and reversible reactions, it ensures that only one DNA cassette embedded in the plasmid is inserted into the host genome. Insertion of a DNA cassette via two types of unidirectional / irreversible reactions, such as a heterotype site for integrase, can result in the incorporation of two types of plasmids if the "trans" reaction outweighs the "cis" reaction. (5) It is more robust and stable than reactions that rely solely on reversible recombination reactions.

[0059] In addition, it is more robust, stable, and multiplexed than dRMCE, for example, as described below: Osterwalder et al., "Dual RMCE for efficient re-engineering of mouse mutant alleles", Nat Methods 7, 893-895 (2010). It is also faster and easier than DROID and STRAIT-IN technologies (see, for example, Neil et al., "Assembly of large mobilizable genetic cargo by double recombinase operated insertion of DNA (DROID)", Plasmid, Volume 104, 2019; Blanch-Asensio et al., "STRAIGHT-IN enables high-throughput targeting of large DNA payloads in human pluripotent stem cells", Cell Reports Methods, Volume 2, Issue 10, 2022). Furthermore, it is more reliable than using two different heterotype recognition sites for integrase to change cassettes. (For example, see: Inniss et al., "A novel Bxb1 integrase RMCE system for high fidelity site-specific integration of mAb expression cassette in CHO Cells", Biotechnology and Bioengineering, Vol. 114, Issue 8, August 2017, pages 1837-1846; Low et al., "Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase", Sci Rep 12, 5424 (2022).

[0060] As discussed herein, the lock-in properties of biRMCE are superior to those of dRMCE / MADR at 2 and 4 days post-induction (see Figures 13–15 and 23–25). In the data shown in Figures 23–25, the experiments were conducted using the "color" changes of gene reporters that are far apart from each other in the color spectrum. This facilitated analysis and resulted in better resolution and output. In the experiments that yielded the data in Figures 13–15, red and green gene reporters that are closer in the spectrum were used.

[0061] Furthermore, as shown herein (for example, Figures 19 and 20), only about 80 bp of DNA is required to induce biRMCE. This short nucleotide is sufficient because we have demonstrated that biRMCE can be performed even when specific recombination sites are adjacent to each other without other DNA sequences in between. This is important because inserting only about 80 bp of DNA, for example by using prime editing to insert about 80 bp of DNA, is very simple and safe. Thus, this method inserts about 80 bp of DNA and then adds a longer DNA, including the entire synthetic chromosome, via biRMCE. This method is also useful for adding very short DNA sequences that are sandwiched between biRMCE recombination sites, such as short sequences encoding sgRNA.

[0062] As further shown (for example, Figures 21 and 22), the biRMCE is functional in the human genome. To demonstrate this, we inserted a biRMCE recombination site into the GAPDH gene locus of the human HEK-293T cell line.

[0063] Thus, various aspects of the present invention are, in part, based on the designs and findings described above.

[0064] System for biRMCE Various embodiments provide a system for biRMCE. Since the system may include various compositions, it can also be considered a combination of compositions.

[0065] Various embodiments provide a system that includes the following: (a) (i) one or more polyadenylation signal or transcription stop elements located upstream of the nucleic acid encoding the transgene or RNA, (ii) the nucleic acid encoding the transgene or RNA, and (iii) a recombinase recognition site comprising at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site. donor vectors, including; and (b) Two types of recombinases specific to the recombinase recognition site. In various embodiments, the system further includes an intron, or a portion of an intron, or at least one splice acceptor region.

[0066] In various embodiments, the system's donor vector further comprises at least a third recombinase recognition site, and the system further comprises at least a third recombinase specific to at least the third recombinase recognition site.

[0067] In various embodiments, the donor vector of the system further comprises one or more additional recombinase recognition sites, and the system further comprises one or more additional recombinases specific to the one or more additional recombinase recognition sites. For example, the donor vector comprises a fourth, fifth, sixth, seventh, eighth, ninth, or tenth recombinase recognition site, and the system further comprises a fourth, fifth, sixth, seventh, eighth, ninth, or tenth recombinase, each specific to the fourth, fifth, sixth, seventh, eighth, ninth, or tenth recombinase recognition site.

[0068] In various ways, the system The donor vector and the locus targeted by two types of recombinases, and optionally at least a third recombinase. Further includes mammalian cells containing [the specified element]. In various embodiments, mammalian cells are found within mammals.

[0069] In various embodiments, two types of recombinases are provided as follows: (i) One expression vector containing two genes encoding recombinases specific to the recombinase recognition site, or (ii) Two expression vectors, namely, a first expression vector containing one gene encoding a first recombinase specific to the unidirectional recombinase recognition site, and a second expression vector containing one gene encoding a second recombinase specific to the bidirectional recombinase recognition site, or (iii) One mRNA encoding two types of recombinase, or (iv) Two types of mRNA, namely, a first mRNA encoding a first recombinase specific to the unidirectional recombinase recognition site, and a second mRNA encoding a second recombinase specific to the bidirectional recombinase recognition site, or (v) A viral vector containing two genes encoding recombinases specific to the recombinase recognition site, or (vi) Two types of viral vectors, namely, a first viral vector containing one gene encoding a first recombinase specific to a unidirectional recombinase recognition site, and a second viral vector containing one gene encoding a second recombinase specific to a bidirectional recombinase recognition site, or (vii) One recombinant protein comprising a unidirectional recombinase and a bidirectional recombinase, or (viii) Two recombinant proteins, namely, a first recombinase protein specific to a unidirectional recombinase recognition site, and a second recombinase protein specific to a bidirectional recombinase recognition site.

[0070] In various embodiments, (i) in an expression vector containing two genes encoding recombinases specific to their recognition site, the encoded recombinases are fused together. In various embodiments, (iii) in an mRNA containing two recombinases specific to their recognition site, the encoded recombinases are fused together. In various embodiments, (v) in a viral vector containing two genes encoding recombinases specific to their recognition site, the encoded recombinases are fused together. In various embodiments, (viii) in which two recombinant proteins are fused together.

[0071] In various embodiments, one of the recombinases is fused with one or more other proteins. In various embodiments, one of the two fused recombinases is further fused with one or more other proteins.

[0072] In various embodiments, one or more proteins other than the recombinase may be nucleases. In various embodiments, one or more proteins other than the recombinase may be reverse transcriptases. One or more proteins other than the recombinase may be polymerases. In various embodiments, one or more proteins other than the recombinase may be transposases.

[0073] In yet another embodiment, the one or more proteins may be any combination of two or three of the nucleases, reverse transcriptases, polymerases, and transposases. In yet another embodiment, the one or more proteins may be a combination of the nucleases, reverse transcriptases, polymerases, and transposases. Non-limiting examples include nucleases (e.g., Cas9, nickase-like dCas9) fused to reverse transcriptases, fused to unidirectional recombinases, fused to bidirectional recombinases.

[0074] In various embodiments, one or more proteins other than recombinases may be therapeutic proteins. Examples of therapeutic proteins include, but are not limited to, antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. Further examples include, but are not limited to, etanercept, bevacizumab, rituximab, adalimumab, infliximab, trastuzumab, insulin glargine, epoetin α, pegfilgrastim, ranibizumab, darbepoetin α, interferon β-1a (Avonex), interferon β-1a (Levif), insulin aspart, Rhu insulin, octocog α, insulin lispro, cetuximab, peginterferon α-2a, interferon β-1b, eptacog α, insulin aspart, onabotulinum toxin A, epoetin β, Rec antihemophilia factor, filgrastim, insulin detemir, natalizumab, insulin (Humulin), and palivizumab.

[0075] In various embodiments, at least a third recombinase is provided by: (iv) One expression vector containing a gene encoding at least a third recombinase specific to the third recombinase recognition site, or (x) One mRNA that encodes at least a third recombinase specific to at least a third recognition site, or (xi) A viral vector comprising a gene encoding at least a third recombinase specific to at least a third recombinase recognition site, or (xii) A recombinant protein comprising at least a third recombinase specific to at least a third recombinase recognition site.

[0076] In various embodiments, (iv) an expression vector comprising a gene encoding at least a third recombinase specific to a third recombinase recognition site, wherein the expression vector further comprises a gene encoding one or more proteins other than the third recombinase, and the encoded third recombinase is fused with the encoded one or more proteins. In various embodiments, (x) an mRNA comprising a gene encoding at least a third recombinase specific to at least a third recognition site, wherein the mRNA further encodes one or more proteins other than the third recombinase, and the encoded recombinase is further fused with the encoded one or more proteins. In various embodiments, (xi) a viral vector comprising a gene encoding at least a third recombinase specific to at least a third recombinase recognition site, wherein the viral vector further encodes one or more proteins other than the third recombinase, and the encoded recombinase is further fused with the encoded one or more proteins. In various embodiments, (xii) a recombinant protein comprising at least a third recombinase specific to at least a third recombinase recognition site is fused with one or more proteins other than the third recombinase.

[0077] In various embodiments, one or more proteins other than the recombinase may be nucleases. In various embodiments, one or more proteins other than the recombinase may be reverse transcriptases. One or more proteins other than the recombinase may be polymerases. In various embodiments, one or more proteins other than the recombinase may be transposases.

[0078] In yet another embodiment, the one or more proteins may be any combination of two or three of the nucleases, reverse transcriptases, polymerases, and transposases. In yet another embodiment, the one or more proteins may be a combination of the nucleases, reverse transcriptases, polymerases, and transposases. Non-limiting examples include nucleases (e.g., Cas9, nickase-like dCas9) fused to reverse transcriptases, fused to unidirectional recombinases, fused to bidirectional recombinases.

[0079] In various embodiments, one or more proteins other than the third recombinase are therapeutic proteins. Non-limiting examples of therapeutic proteins are those provided herein.

[0080] In various aspects, the unidirectional recombinase recognition site is located upstream of the bidirectional recombinase recognition site. In various aspects, the unidirectional recombinase recognition site is located downstream of the promoter.

[0081] In various embodiments, the unidirectional recombinase is Bxb1 or any variant thereof. In various embodiments, the unidirectional recombinase is Bxb1 and the bidirectional recombinase is Flp. In various embodiments, the unidirectional recombinase is Bxb1 and the bidirectional recombinase is Cre, VCre, SCre, Nigri, Panto, Vika, or a variant thereof.

[0082] In various embodiments, the unidirectional recombinases are PhiC31, PhiBT1, PhiC1, MR11, R4, TP901-1, A118, FC1, PhiRV, TG1, Phi370.1, Wβ, BL3, SPBc, K38, or any of their variants.

[0083] In various embodiments, the third recombinase is selected from Bxb1, Phic31, PhiBT1, PhiC1, MR11, R4, TP901-1, A118, FC1, PhiRV, TG1, Phi370.1, Wβ, BL3, SPBc, K38, FLp, Cre, VCre, SCre, Nigri, Panto, Vika, or their variants.

[0084] In various embodiments, the first recombinase recognition site among the recombinase recognition sites is attB or any variant thereof. Alternatively, the first recombinase recognition site among the recombinase recognition sites may be attP or any variant thereof.

[0085] In various embodiments, the second recombinase recognition site among the recombinase recognition sites is a flippase recognition target (FRT), loxP, VloxP, SloxP, nox, or pox. In other embodiments, the second recombinase recognition site among the recombinase recognition sites is a modified loxP, a flippase recognition target (FRT), VloxP, SloxP, nox, or pox.

[0086] In various aspects, the unidirectional recombinase is PhiC31, and the recombinase recognition sites are attB and attP.

[0087] In various embodiments, one or both of the recombinase recognition sites include mutations.

[0088] In various embodiments, the third recombinase recognition site is attB or any variant thereof, attP or any variant thereof. In various embodiments, the third recombinase recognition site is a flippase recognition target (FRT), loxP, VloxP, SloxP, nox, or pox. In various embodiments, the third recombinase recognition site is a modified loxP, a flippase recognition target (FRT), VloxP, SloxP, nox, or pox. In various embodiments, the third recombinase recognition site is attB and attP. In various embodiments, the third recombinase recognition site includes mutations.

[0089] In various embodiments, at least one additional recombinase recognition site is attB or any variant thereof, attP or any variant thereof. In various embodiments, at least one additional recombinase recognition site is a flippase recognition target (FRT), loxP, VloxP, SloxP, nox, or pox. In various embodiments, at least one additional recombinase recognition site is a modified loxP, a flippase recognition target (FRT), VloxP, SloxP, nox, or pox. In various embodiments, at least one additional recombinase recognition site is attB and attP. In various embodiments, at least one additional recombinase recognition site includes a mutation. At least one additional recombinase recognition site is, for example, a fourth, fifth, sixth, seventh, eighth, ninth, or tenth recombinase recognition site.

[0090] In various embodiments, donor vectors are selected from the group consisting of plasmids, linear DNA (e.g., PCR fragments, synthetic linear DNA), minicircles, viral vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and human artificial chromosomes (HACs). A non-limiting example of a viral vector is the adeno-associated virus (AAV) vector. In various embodiments, donor vectors are selected from the group consisting of linear single-stranded DNA, closed-end double-stranded DNA, circular single-stranded DNA, circular double-stranded DNA, and RNA.

[0091] In various embodiments, the donor vector includes at least four polyadenylation signals upstream of the nucleic acid encoding the transgene or RNA. In other embodiments, the donor vector includes at least one, at least two, or at least three polyadenylation signals upstream of the nucleic acid encoding the transgene or RNA.

[0092] In another embodiment, the donor vector contains an intron or a portion of an intron upstream of the nucleic acid encoding the transgene or RNA.

[0093] In various embodiments, the donor vector further comprises a post-transcriptional regulatory element. In various embodiments, the donor vector further comprises a polyadenylation signal downstream of the nucleic acid encoding the transgene or RNA. In various embodiments, the donor vector further comprises an open reading frame (ORF) beginning with a splice acceptor. In various embodiments, the donor vector further comprises a fluorescent reporter.

[0094] In various embodiments, expression vectors containing recombinases are located under a tissue-specific promoter.

[0095] In various forms, RNA is siRNA, shRNA, sgRNA, crRNA, pegRNA, lncRNA, or miRNA.

[0096] In various embodiments, the transgene or RNA contains disease-related mutations. In various embodiments, the transgene or RNA contains gain-of-function (GOF) gene mutations, loss-of-function (LOF) gene mutations, or both.

[0097] In various embodiments, the mammalian cell is a human cell, and the locus is the AAVS1 locus, H11 locus, HPRT1 locus, Rogi1 locus, Rogi2 locus, GAPDH locus, TATA box-binding protein (TBP) locus, kinesin family member (KIF11) locus, TRAC locus, ZAP-70 locus, T cell activation linker (LAT) locus, or lymphocyte cytoplasmic protein 2 (LCP2) locus, and the method is in vitro, ex vivo, or in vivo. While examples are listed, any locus on the human genome can be used in various embodiments of the present invention.

[0098] In various embodiments, the locus comprises a first polynucleotide encoding a first protein, and a secondary cistron containing a promoter, a recombinase recognition site recognized by a recombinase in the system, and a second polynucleotide encoding an open reading frame for the first protein. In various embodiments, the first polynucleotide encoding the second protein is downstream of the gene in the locus.

[0099] Examples of promoters include, but are not limited to, CAG, CMV, EF1a, PGK, TRE, U6, and UAS.

[0100] In various embodiments, the first protein, the second protein, or both are fluorescent proteins. Examples of fluorescent proteins include, but are not limited to, miRFP670, EGFP, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TagBFP2, TurboGFP, AcGFP, ZsGreen, T-Sapphire, EBFP, EBFP2, Azurite, mTagBFP, ECFP, mECFP, Cerulean, mTurquoise, CyPet, AmCyan1, Midori-Ishi Cyan, TagCFP, mTFP1(Teal), EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, mBanana, Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express(T1), DsRed-Monomer, mTangerine, mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, and AQ143.

[0101] In various embodiments, the first fluorescent protein is TagBFP2, the promoter is the CAG promoter, and the second fluorescent protein is miRFP670.

[0102] In various embodiments, the mammalian cells are mouse cells, and the loci are the ROSA26 locus, Hipp11 locus, Tigre locus, ColA1 locus, HPrt locus, GAPDH locus, TATA box-binding protein (TBP) locus, kinesin family member (KIF11) locus, TRAC locus, Zap-70 locus, T cell activation linker (LAT) locus, or lymphocyte cytoplasmic protein 2 (LCP2) locus, and the method is in vitro, ex vivo, or in vivo. While examples are listed, any locus on the mouse genome can be used in various embodiments of the present invention.

[0103] In various embodiments, a gene locus in mouse cells comprises a first polynucleotide encoding a first protein, a promoter, a recombinase recognition site recognized by a recombinase in the system, and a secondary cistron containing a second polynucleotide encoding an open reading frame for a second protein. In various embodiments, the first protein, the second protein, or both are fluorescent proteins. Examples of fluorescent proteins, promoters, and recombinase recognition sites are as described herein.

[0104] Genetic manipulation methods Various aspects of the present invention provide methods for genetically modifying mammalian cells, the methods comprising the step of transfecting or transducing mammalian cells using any one of the systems of the present invention described herein.

[0105] In various embodiments, the system targets a locus, which includes a recombinase recognition site comprising at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site. In various embodiments, unidirectional recombination is upstream of bidirectional recombination at the locus.

[0106] In various embodiments, the locus includes a third recombinase recognition site. In various embodiments, the locus includes an additional recombinase recognition site, which is, for example, a fourth, fifth, sixth, seventh, eighth, ninth, or tenth recombinase recognition site.

[0107] In various embodiments, the mammalian cells are human cells, the system targets the AAVS1 locus, H11 locus, or HPRT1 locus, Rogi1 locus, Rogi2 locus, GAPDH locus, TATA box-binding protein (TBP) locus, kinesin family member (KIF11) locus, TRAC locus, ZAP-70 locus, LAT (T cell activation linker) locus, or LCP2 (lymphocyte cytoplasmic protein 2, also known as SLP-76) locus, and the method is in vitro, ex vivo, or in vivo. In various embodiments, the mammalian cells are mouse cells, and the system targets the ROSA26 locus, Hipp11 locus, Tigre locus, ColA1 locus, HPrt locus, GAPDH locus, TATA box-binding protein (TBP) locus, kinesin family member (KIF11) locus, Trac locus, Zap-70 locus, Lat (T cell activation linker) locus, or Lcp2 (lymphocyte cytoplasmic protein 2) locus, and the method is in vitro, ex vivo, or in vivo.

[0108] In various embodiments, the method further includes the step of administering one or more recombinase enzymes to cells.

[0109] In various aspects, one or more recombinase enzymes include Bxb1 recombinase, Cre recombinase, flipperze recombinase, Nigri recombinase, Panto recombinase, Vika recombinase, VCre recombinase, or SCre recombinase.

[0110] In various aspects, mammalian cells include blood cells, tumor cells, non-tumor cells, embryonic stem cells, adult stem cells, induced pluripotent stem cells, or tissue progenitor cells.

[0111] Non-human animal models and methods for creating non-human animal models Various embodiments provide non-human animal models, which include non-human animals comprising any one of the systems of the present invention described herein.

[0112] In various embodiments, the non-human animal model is a personalized non-human animal model for a human target cancer, and the transgene or RNA is based on the human target cancer.

[0113] In various embodiments, the non-human animal model is a personalized non-human animal model for a human disease or condition, and the transgene or RNA is based on the human disease or condition.

[0114] In various aspects, non-human animal models include gain-of-function (GOF) mutations, loss-of-function (LOF) mutations, or both.

[0115] In various embodiments, the transgene or RNA is selected from the group consisting of oncogenes, loss-of-function (LOF) mutations in tumor suppressor genes, gain-of-function (GOF) mutations in proto-oncogenes, pseudogenes, siRNA, shRNA, sgRNA, pegRNA, crRNA, lncRNA, miRNA, epigenetic modifications, non-coding or epigenetic abnormalities associated with human diseases, and combinations thereof.

[0116] In various embodiments, the system targets a locus in a non-human animal model, the locus comprising a recombinase recognition site including at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site.

[0117] In various embodiments, unidirectional recombination lies upstream of bidirectional recombination at a gene locus.

[0118] Examples of non-human animals include mice, rats, dogs, guinea pigs, rabbits, hamsters, pigs, sheep, and non-human primates (e.g., monkeys (e.g., macaques, rhesus monkeys), and great apes).

[0119] Various aspects of the present invention provide a method for producing a non-human animal model of the present invention, which involves transfecting or transfecting a non-human animal model using any one of the systems of the present invention.

[0120] Various aspects of the present invention provide non-human animal models prepared by any one of the methods of the present invention.

[0121] Drug screening Various embodiments provide a method for evaluating the effects of a candidate drug, the method comprising the following steps: A step of providing a non-human animal model of the present invention; The step of administering candidate drugs to non-human animal models; and This stage involves evaluating the effects of candidate drugs on non-human animal models.

[0122] Various embodiments provide mammalian cells comprising the system of the present invention.

[0123] Various embodiments provide a method for evaluating the effects of a candidate drug, the method comprising the following steps: A step of providing mammalian cells comprising the system of the present invention; The step of bringing the candidate drug into contact with mammalian cells; and This stage involves evaluating the effects of candidate drugs on mammalian cells. [Examples]

[0124] The following examples are provided to illustrate the claimed invention in more detail and should not be construed as limiting the scope of the invention. Where specific materials are mentioned, they are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can obtain equivalent means or reactants without demonstrating any particular originality and without departing from the scope of the invention.

[0125] Example 1 Experimental Procedure mouse All mice used were maintained and euthanized in accordance with the Cedars-Sinai Institutional Animal Care and Use Committee. Heterozygous mice were generated by crossing mT / mG (Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo / J) mice (Muzumdar, Tasic, Miyamichi, Li, & Luo, 2007) with C57BL / 6J mice. Male and female offspring between postnatal days (P)0 and P2 were used in subsequent experiments.

[0126] Plasmid cloning The pDonor plasmid was created from MADR-pDonor using NEBuilder HiFi DNA Assembly Master Mix (NEB) in combination with standard restriction digestion techniques (Kim et al., 2019). Briefly, a specific recombination site was created by oligo synthesis and inserted into MADR-pDonor. The recombinase expression vector was created from the previously validated (Kim et al., 2019) pCag-FlpO-2A-Cre EV (Addgene 129419). Plasmid pCag-NLS-HA-Bxb1 (Addgene 51271) was used as a template for PCR of Bxb1. Subsequent steps in plasmid construction were performed by removing existing ORFs and adding new cassettes using HiFi DNA Assembly. PCR was performed using KAPA HiFi PCR reagents and a standard protocol.

[0127] cell line creation The brains of heterozygous mT / mG offspring (P0-P2) were dissected to establish the polyclonal mouse neural stem cell line (mNSC) used in this study. Sex was not recorded due to the lack of a reliable visual method. The cell line was constructed and maintained as previously described (Breunig et al., 2015). Cells were cultured in flasks treated with CELLstart CTS (Thermo Fisher Scientific, Waltham, MA). Cells were grown in a medium containing Neurobasal-A medium (Life Technologies 10888-022) supplemented with vitamin A-free B-27 (Life Technologies 12587-010), GlutaMAX (Life Technologies 35050), antibiotics-antifungal agents (Life Technologies 15240), human epidermal growth factor (hEGF) (Sigma E9644), heparin (Sigma H3393), and basic fibroblast growth factor (bFGF) (Millipore GF003). De novo production of recipient cell lines was achieved by targeting the ROSA26 locus of mTmG cells via dual recombinase-mediated cassette exchange (Osterwalder et al., 2010) and by nucleofection of the plasmid pCag-FlpO-2A-Cre EV with their respective MADR-pDonors (Kim et al., 2019). Cell line selection and purification were performed by flow cytometry.

[0128] Cellular nucleofection Nucleofection of mNSCs was achieved using the Nucleofector 2b instrument and mouse neural stem cell kit, as recommended by the manufacturer (Lonza AG). Nucleofection mixtures contained either 10 μg or 1 μg of plasmid or mRNA in total weight, respectively.

[0129] Observation with reflected fluorescence microscope Images were acquired using a fluorescence microscope (ECHO Revolve) with each fluorescence channel and filter.

[0130] Flow cytometry Cells were collected as previously described (Kim et al., 2019). The cells were dissociated using Accutase (Millipore), pelletized at 800 g for 3 minutes, and resuspended in EDTA. FACS was performed using Beckman Coulter's MoFlo in a Cedars-Sinai Flow Cytometry Core.

[0131] Example 2 result Indirect verification of the biRMCE reaction To indirectly test biRMCE, the inventors first attempted to demonstrate that Bxb1 / attB / attP recombination is irreversible / unidirectional (Ghosh, Wasil, & Hatfull, 2006; Merrick, Zhao, & Rosser, 2018). To demonstrate this, the inventors created two new heterozygous recipient cell lines in which either the attP or attR site was supported between the Cag promoter and the puromycin resistance gene (PuroR) at the Rosa26 locus, followed by an FRT site (Figure 1A-B). To create these cell lines, the inventors performed dRMCE / MADR using each pDonor and replaced the mTmG cassette with a new cassette. Next, the inventors developed a promoter-free donor vector (pDonor-attB-mScarlet-FRT) containing the reporter gene mScarlet, which is sandwiched between the attB and FRT sites. Subsequently, the inventors performed nucleofection of pDonor-attB-mScarlet-FRT with or without plasmids expressing the recombinases Bxb1 or FlpO (Figures 1A-B, reactions 1.1-2.3). Cells carrying an "open" attP site expressed mScarlet in the presence of Bxb1, or in the presence of both Bxb1 and FlpO (Figure 2A, reactions 1.1 and 1.2). Cells with a "locked" attR site expressed little to no mScarlet, both in the presence of Bxb1 and in the presence of both Bxb1 and FlpO (Figure 2B, reactions 2.1 and 2.2). In the absence of recombinase, mScarlet was not expressed (Figure 2A-B, reactions 1.3 and 2.3). Quantitative comparisons of Scarlet expression between reactions 1.1 and 1.2, and between reactions 2.1 and 2.2, clearly show an increase or decrease in recombination at the attP or attR site (Figure 3A-B). These results demonstrate that the attR site, the product of Bxb1-mediated recombination between attP and attB, has little tendency to tolerate de novo recombination.

[0132] The inventors pursued a similar strategy to visualize whether recombinase-mediated cassette exchange is possible rather than whole plasmid integration. To demonstrate this, the inventors created two novel heterozygous recipient cell lines, each carrying either an attP or attR site between the polyadenylation signal and the FRT site of the PuroR gene at the Rosa26 locus (Figure 4A-B). To create these cell lines, the inventors performed dRMCE / MADR using each pDonor to exchange the mTmG cassette with a new cassette. Next, the inventors developed a promoter-free donor vector (pDonor-loxP-mScarlet-attB) containing the reporter gene mScarlet, flanked by the loxP and attB sites. Subsequently, the inventors performed nucleofection of pDonor-loxP-mScarlet-attB with or without plasmids expressing the recombinase Bxb1 or Cre (Figures 4A-B, reactions 3.1-4.3). In cells expressing Bxb1 alone, mScarlet was hardly expressed, whether carrying the "open" attP site or the "locked" attR site (Figures 5A-B, reactions 3.1 and 4.1). This is a possible result because Bxb1 alone enables the integration of the entire plasmid, while the pDonor vector does not express mScarlet because it carries several polyadenylation signals upstream of the open reading frame. Interestingly, when Bxb1 and Cre were expressed, mScarlet was expressed in both cell lines (Figures 5A-B, reactions 3.2 and 4.2), suggesting that pDonor integration is mediated by Cre / loxP recombination. In the absence of the recombinase, mScarlet was not expressed (Figures 5A-B, reactions 3.3 and 4.3). A comparison of mScarlet expression in reactions 3.1 and 4.1 clearly shows that when attP or attR is downstream of the polyadenylation signal and only Bxb1 is expressed, mScarlet is hardly expressed (Figure 6A).Interestingly, a comparison of mScarlet expression in reaction 3.2 and reaction 4.2 shows that more mScarlet-positive cells are present in cell lines containing "open" attP sites (Figure 6B). Therefore, we compared reaction 3.2 and reaction 4.2 at different time points. Two and seven days after nucleofection, we observed a time-dependent decrease in mScarlet signaling in both reactions (Figures 7A-B). However, when comparing the quantification of mScarlet over time in both reactions, it is clear that cells carrying "locked" attR sites no longer express mScarlet seven days after nucleofection, demonstrating that all initial mScarlet expression is attributable to whole plasmid integration and excision mediated by Cre / loxP recombination (Figure 8, dashed line). On the other hand, in cell lines carrying the "open" attP site, some cells still expressing mScarlet remained 7 days after nucleofection (Figure 8, solid line). These data suggest that the integration of the transgene mScarlet is mediated by two different modes of recombination: reversible / bidirectional recombination by Cre / loxP and irreversible / unidirectional recombination by Bxb1 / attP / attB, which we call bimodal recombinase-mediated cassette exchange (biRMCE). All of the strategies, data, and results described above for indirect verification of the biRMCE reaction are summarized in the table in Figure 9. Note that all 12 reactions strongly suggest the presence of biRMCE at the Rosa26 locus.

[0133] Proof of biRMCE The inventors demonstrated biRMCE by making several adjustments to the above-described strategy. First, the inventors created a novel heterozygous recipient cell line in which the attP site was located between the Cag promoter and the nuclear-expressed TagBFP2 gene at the Rosa26 locus, followed by an FRT site (Figure 10A). To create this cell line, the inventors performed dRMCE / MADR using each pDonor-2in1-loxP-attP-TagBFP2-nls-FRT and replaced the mTmG cassette with a new cassette. Next, the inventors created a promoter-less donor vector containing the reporter gene mScarlet, sandwiched between the upstream loxP and attB sites and the downstream FRT site, and named it pDonor-2in1-loxP-attB-mScarlet-FRT (Figure 10B). Next, the inventors developed two novel plasmids, each expressing two different recombinases under the same promoter within the same plasmid. The plasmids are pCag-Bxb1-FlpO and pCag-FlpO-Bxb1. As controls, the inventors used pCag-FlpO-Cre and pCag-Bxb1 (Figure 10C). Subsequently, the inventors induced Cre expression and observed that the mScarlet cassette was excised when two loxP sites were present at the Rosa26 locus (Figure 10D). The inventors then proceeded with nucleofection of the heterozygous cell line 2in1-loxP-attP-TagBFP2-nls-FRT with pDonor-2in1-loxP-attP-mScarlet-FRT using four different plasmids encoding their respective recombinases. In recipient cell lines, the loxP and attP sites are upstream of nuclear-expressed TagBFP2, and in pDonor, the loxP and attB sites are upstream of mScarlet. Therefore, in these target cells, regardless of the type of recombinase plasmid used, we ensure that mScarlet is expressed while nuclear-expressed TagBFP2 is lost.The inventors observed cells expressing only mScarlet under different conditions and then purified these cells to create four different cell lines. The inventors hypothesized that if plasmid pCag-Bxb1 was used, the plasmid would promote the integration of the entire plasmid, and therefore the nuclear-expressed TagBFP2 cassette should still be present at the Rosa26 locus. On the other hand, if plasmids expressing two types of recombinases in the same cistron were used, the inventors hypothesized that if dRMCE or biRMCE occurred, the nuclear-expressed TagBFP2 cassette should not be present at the Rosa26 locus. To verify this point, the inventors nucleofected different cell lines with Cre mRNA and observed whether the mScarlet cassette was excised by Cre / loxP while TagBFP2 was reexpressed in the nucleus. The inventors observed many nuclear-expressing TagBFP2 cells in cell lines previously induced with pCag-Bxb1 (Figure 11A), and observed only a few cells expressing TagBFP2 in the nucleus in cell lines previously induced via dRMCE using pCag-FlpO-Cre (Figure 11D). The inventors also observed only a small amount of nuclear-expressing TagBFP2 in cell lines previously induced with pCag-Bxb1-FlpO or pCag-FlpO-Bxb1, indicating that mScarlet integration was previously mediated by biRMCE (Figures 11B-11C). Quantification of nuclear-expressing TagBFP2 after Cre / loxP recombination demonstrates that pCag-Bxb1 integrated the entire plasmid (Figure 11E). Quantifying mScarlet after Cre / loxP recombination demonstrates that both biRMCE and dRMCE incorporate DNA flanked by recombination sites specific to each of them (Figure 11F).

[0134] Stability and efficiency of biRMCE and dRMCE After validating biRMCE, the inventors compared its stability and efficiency with the well-characterized dRMCE / MADR (Anderson, Voziyanova, & Voziyanov, 2012; Kim et al., 2019; Osterwalder et al., 2010). To do this, the inventors nucleofected the cell line Rosa26-2in1-loxP-attP-TagBFP2-nls-FRT with pDonor-2in1-loxP-attB-mScarlet-FRT along with pCag-FlpO-Cre or pCag-Bxb1-FlpO, and examined mScarlet expression at different time points. At the initial time point, 2 days after nucleofection, dRMCE was more efficient than biRMCE. Nevertheless, at the mid- and late-term time points, 7 and 11 days after nucleofection, biRMCE maintained its efficiency compared to dRMCE, while dRMCE's efficiency significantly decreased (Figure 12). This data demonstrates that biRMCE is more stable and therefore more efficient than dRMCE in the integration of transgene elements.

[0135] Lock-in of DNA integration via biRMCE biRMCE is mediated by two different modes of recombination: irreversible / unidirectional recombination and reversible / bidirectional recombination. Therefore, as long as attP / attB recombination occurs between the promoter and the open reading frame, once the first recombination occurs, biRMCE should prevent further incorporation of DNA cassettes into the Rosa26 locus. To demonstrate this point, we compared biRMCE with dRMCE, as the latter is based on two types of reversible / bidirectional recombination reactions. We pursued a strategy using two different pDonors, each containing nuclear-expressed mScarlet and nuclear-expressed EGFP, which are different reporters. Each pDonor is flanked by specific recognition sites that are recognized only by their respective recombinases (Figure 13). At the initial time points, two days after induction with dRMCE / MADR and two days after induction with biRMCE, it is clear that there are more double-positive cells expressing mScarlet and EGFP under the dRMCE condition (Figure 14A-B). Flow cytometry analysis confirmed that there are significantly fewer double-positive cells under the biRMCE condition (Figure 14C-14D). At the later time point, eight days after induction, there are even fewer double-positive cells under the biRMCE condition (Figure 15). This demonstrates that biRMCE enables rapid and stable genetic recombination while also demonstrating reliability in locking the system, preventing additional trans-recombination between the genomic landing pad and the foreign invader, pDonor.

[0136] Verification of intron-type biRMCE By adding the attP site and embedding it in the intron of the Rosa26 locus, and by adding a portion of the same intron downstream of the attB site of the pDonor, the inventors developed an intronic version of biRMCE. This strategy makes it possible to remove the Kozak sequence and the first "ATG" of the open reading frame from the pDonor. To validate the intronic biRMCE, the inventors created a new cell line by targeting the endogenous Rosa26 locus of mT / mG cells with dRMCE / MADR and pDonor-2in1-loxP-ATG-in-attP-TRON-(ATG-less-TagBFP-nls)-FRT (Figure 16A). The genetic "landing pad" of the Rosa26 locus obtained after cassette insertion should contain a loxP site, followed by an "ATG" sequence, an attP site (embedded in an intron), a TagBFP-nls sequence (without the initial ATG), and an FRT site (Figure 16B). The inventors proceeded with nucleofection of heterozygous mT / mG cells using the plasmid pCag-FlpO-Cre and two versions of pDonor-2in1-loxP-ATG-in-attP-TRON-(ATG-less-TagBFP-nls)-FRT. The different versions of pDonor are based on the fact that one has a WPRE sequence and the other does not. The inventors observed several cells expressing Tag-BFP in the nucleus using these different versions of pDonor (Figures 16C-16H). These data indicate that an intron with an embedded attP site, located between the first "ATG" exon and an open reading frame without an "ATG" sequence, enables the correct expression of the open reading frame (nuclear-expressed Tag-BPF). The resulting genetic "landing pad" at the Rosa26 locus, possessing loxP, attP, and FRT sites, should be compatible with both dRMCE / MADR and intronic biRMCE, performed using their respective pDonor and recombinase (Figure 17A).Two different genetic "landing pads" can emerge after dRMCE / MADR or intronic biRMCE (Figure 17B). To demonstrate this, the inventors isolated cells expressing Tag-BFP in the nucleus to create new cell lines. Subsequently, the inventors performed nucleofection on these cells using their respective plasmids to validate dRMCE / MADR or intronic biRMCE. The inventors observed several cells expressing miRFP-670 in the nucleus using their respective pDonors and recombinases (Figures 17C-17H). This data demonstrates that the "2-in-1" genetic landing pad is compatible with both dRMCE / MADR and intronic biRMCE. This data also demonstrates that a pDonor lacking both the Kozak sequence and the first "ATG" of the open reading frame can be used to deliver a transgene that can be correctly expressed via intronic biRMCE.

[0137] Example 3 Experimental Procedure Commercially available cell lines HEK293T, derived from human fetal kidney and purchased from ATCC, was used for in vitro validation of MADR and biRMCE. The cell line was maintained in high-glucose DMEM (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS, GlutaMAX (Life Technologies 35050), and penicillin-streptomycin-amphotericin (Thermo Fisher Scientific, Waltham, MA).

[0138] Creation of the GAPDH-MADR / biRMCE human cell line The GAPDH-targeting vector was constructed by DNA synthesis. P2A-TagBFP2nls-Cag-loxP-attP-miRFP670nls-FRT was inserted into the GAPDH vector and used for transfection in human cells. The GAPDH-targeting vector was designed to be knocked into the locus by homologous recombination repair (HDR) after induction of double-strand breaks (DSBs) via the Cas9 / sgRNA complex. Cells were selected by selecting double-positive cells for TagbBFP2 and miRFP670 via flow cytometry. The selected stable cell lines were transfected with the donor vector 2in1-MADR / biRMCE-mScarlet along with pCag-FlpO-Cre or pCag-Bxb1-FlpO to induce MADR or biRMCE, respectively.

[0139] Cas9 protein, sgRNA, and ribonucleoprotein (RNP) complex Cas9 and sgRNA were obtained from IDT. The Cas9 protein and sgRNA were combined in a 1:1 ratio to form an RNP complex.

[0140] Cellular lipofection Plates coated with 0.01% poly-L-lysine were used for plating HEK cells. Cells were transfected with Lipofectamine 3000 (Thermo Fisher Scientific) when confluence was approximately 60-70%. The culture medium was changed 24 hours after transfection and then every 48 hours thereafter.

[0141] Genotyping PCR was used to verify appropriate target-directed integration at the GAPDH locus. PCR primers were designed to amplify secondary cistrons from the left and right sides of the knock-in site, the unedited endogenous GAPDH locus, and the open reading frame from the CAG promoter. For visualization, PCR fragments were loaded onto 1% agarose gels. The PCR fragments were extracted from the agarose gels and subjected to Sanger sequencing for subsequent validation.

[0142] Example 4 result Lock-in of DNA integration via biRMCE biRMCE is mediated by two different modes of recombination: irreversible / unidirectional recombination and reversible / bidirectional recombination. Therefore, as long as attP / attB recombination occurs between the promoter and the open reading frame, once the first recombination occurs, biRMCE should prevent further incorporation of DNA cassettes into the Rosa26 locus. To demonstrate this point, we compared biRMCE with dRMCE, as the latter is based on two types of reversible / bidirectional recombination reactions. We pursued a strategy using two different pDonors, each containing nuclear-expressed miRFP670 and nuclear-expressed BFP, which are different reporters. Each pDonor is flanked by specific recognition sites that are recognized only by their respective recombinases (Figure 23). At the initial time points, two days after induction with dRMCE / MADR and two days after induction with biRMCE, it was clear that there were more double-positive cells expressing miRFP670 and BFP under the dRMCEE condition (Figure 24A-B). Flow cytometry analysis confirmed that there were significantly fewer double-positive cells under the biRMCE condition (Figure 24C-24D). At the later time points, four and eight days after induction, there were no double-positive cells under the biRMCE condition (Figure 25). This demonstrates that biRMCE enables rapid and stable gene recombination while also demonstrating the reliability of the system's locking mechanism, which prevents additional trans-recombination between the genomic landing pad and the foreign invader, pDonor.

[0143] Minimal recognition area for biRMCE As described above, the inventors verified and validated biRMCE using specific recombination sites that flank open reading frames of DNA containing hundreds to thousands of base pairs. Based on this, the inventors attempted to identify the minimum recognition site required for biRMCE. This involves ligating both irreversible and reversible recombination sites immediately after each other, without any other DNA sequences between them (Figure 19A). To demonstrate this, the inventors created a heterozygous recipient cell line carrying four specific recombination sites at the Rosa26 locus. Of these, the attP recognition site is located immediately upstream of the VloxP recognition site, followed by the TagBFP2 open reading frame and the FRT site. The loxP site is also located near the CAG promoter, which is useful for subsequent analysis (Figure 19B). After several rounds of purification, the inventors obtained a cell line with a new Rosa26 locus (Figure 19C). Next, the inventors constructed a promoter-less donor vector containing the reporter gene mScarlet, flanked by the upstream loxP and attB sites and the downstream VloxP site, and named it pDonor-2in1-loxP-attB-mScarlet-VloxP (Figure 19D). Subsequently, plasmid pCag-Bxb1 was used to incorporate the entire mScarlet vector, while plasmid pCag-Bxb1-VCre was used to insert only the mScarlet cassette flanked by attB and VloxP (Figures 19E-19F). After appropriate vector integration via Bxb1, or after appropriate cassette exchange via Bxb1 and VCre, the inventors selected mScarlet-positive cells to create a pure cell line carrying the new recombinant Rosa26 locus. If the entire vector is embedded, it is predicted that two loxP regions will be flanking the open reading frame of mScarlet, whereas if cassette swapping by biRMCE is present, there should be only one loxP region upstream of the open reading frame of mScarlet (Figures 20A-B).Finally, Cre recombinase was induced in both cell lines to verify whole plasmid integration or biRMCE (Figures 20C-20D). In the mScarlet cell line created using Bxb1, which incorporates the entire pDonor vector, a significant number of TagBFP2-positive cells (98%) were present, indicating that the open reading frame of mScarlet was excised and the open reading frame of TagBFP2 was returned to a position near the CAG promoter for proper expression (Figure 20C). On the other hand, in the cell lines created using Bxb1 and VCre, only a very small number of TagBFP2-positive cells (3.5%) were present, indicating that the majority of mScarlet cells carried only one loxP site at the Rosa26 locus, as cassette exchange prevented the integration of a second loxP site (Figure 20D). All of these data demonstrate that biRMCE is possible when specific recombination sites are adjacent to each other without other DNA sequences present between them.

[0144] biRMCE is compatible with the human genome. As described above, the inventors validated biRMCE in the mouse genome, but the mouse genome is a mammalian genome that has several significant differences from the human genome. Therefore, for several purposes, including therapeutic purposes, it is important to integrate the transgene element into the human genome. For this reason, the inventors decided to integrate the biRMCE element into the human genome and validate its functionality. To test biRMCE in the human genome, the inventors manipulated a human HEK293T cell line targeting the GAPDH locus (Figure 21A). This knock-in locus has several gene elements that facilitate cell selection and allow for testing biRMCE. The inventors added the TagBFP2 sequence downstream of exon 9 of the GAPDH gene, and further added a secondary cistron carrying the CAG promoter, loxP site, attP site, open reading frame of miRFP670, and FRT site (Figure 21B). After selecting double-positive cells expressing nuclear TagBFP2 and nuclear miRFP670, the inventors obtained the cell line HEK [GAPDH-TagBFP2nls-Cag-2in1(loxP-attP)-miRFP67nls-FRT] (Figure 21C). PCR genotyping confirmed appropriate knock-in to the human GAPDH locus (Figure 21D). Since the secondary cistron carries gene elements for testing MADR and biRMCE, the inventors decided to compare MADR and biRMCE side-by-side using pDonor-2in1-loxP-attB-mScarlet-FRT, which is compatible with MADR and biRMCE by using a specific recombinase (Figure 22A). The predicted recombinant GAPDH locus should express mScarlet in the secondary cistron after MADR or biRMCE (Figure 22B). Transfection of pDonor-2in1-loxP-attB-mScarlet-FRT with the recombinase plasmids PCag-FlpO-Cre or pCag-Bxb1-FlpO demonstrated the presence of mScarlet-positive cells under both conditions (Figure 22C).Quantitative analysis by flow cytometry showed that biRMCE was an order of magnitude more efficient than MADR (Figure 22D). These data demonstrate that biRMCE is feasible in the human genome and is more efficient than MADR.

[0145] Various aspects of the present invention are described in the detailed description above. While these descriptions directly illustrate the aspects described above, it will be understood that those skilled in the art will come up with modifications and / or variations of the specific aspects shown and described herein. Any such modifications or variations within the scope of this description are also intended to be included herein. Unless otherwise specified, the inventors intend that the words and expressions in this specification and in the claims have meanings that are common and customary to those skilled in the art in the applicable field.

[0146] The above description provides for various aspects of the Invention as understood by the applicant at the time of filing this application, and this description is intended for illustrative and explanatory purposes only. This description is not intended to be exhaustive, nor is it intended to limit the Invention to the disclosed form, and many modifications and changes are possible based on the teachings above. The described aspects are useful for illustrating the principles of the Invention and their practical applications, and are useful for those skilled in the art to utilize the Invention in various forms and with various modifications suitable for specific intended uses. Accordingly, the Invention is not intended to be limited to the specific aspects disclosed for carrying out the Invention.

[0147] While specific aspects of the present invention are shown and described herein, it will be apparent to those skilled in the art that modifications and alterations can be made based on the teachings herein without departing from the present invention and its broader scope, and therefore the appended claims encompass all such modifications and alterations that fall within the true spirit and scope of the present invention. Where used herein, the terms “comprising” or “comprises” are used in reference to compositions, methods, and their individual components that are useful in a particular aspect, but the terms allow for the inclusion of unspecified elements that may or may not be useful. Those skilled in the art will generally understand that the terms used herein are usually intended to be “open” (for example, “including” should be interpreted as “including, but not limited to,” “having” should be interpreted as “having at least,” and “includes” should be interpreted as “includes, but not limited to,” etc.). The open-ended term "comprising" is used herein to describe and claim the invention as a synonym for, for example, "including," "containing," or "having," while the invention or aspects thereof may also be described using other terms, for example, "consisting of" or "essentially consisting of."

[0148] Unless otherwise specified, the terms “a,” “an,” and “the,” as well as similar references, used in the context of describing certain aspects of this application (particularly in the context of the claims), may be interpreted as encompassing both their singular and plural forms. The descriptions of ranges of values ​​herein are intended solely as a way to omit the individual descriptions of each distinct value contained within those ranges. Unless otherwise specified herein, each individual value is incorporated herein in the same way as if those values ​​were described individually. All methods described herein may be performed in any appropriate order unless otherwise specified herein or unless it is clearly inconsistent with the context. The use of any examples or any exemplary expressions (e.g., “for example”) provided herein in relation to certain aspects is intended solely to provide a more detailed explanation of this application and does not constitute a limitation on the scope of this application as otherwise claimed. The abbreviation "e.g." derives from the Latin "exempli gratia" (for example), which is used herein to indicate a non-limiting example. Therefore, the abbreviation "e.g." is synonymous with the term "for example." No expression herein should be construed as indicating any element not covered by the claims that is essential to the practice of this application.

[0149] "Optional" or "optional" means that the circumstances described therein may or may not occur, and therefore such descriptions include examples in which the circumstances occur and examples in which they do not occur.

[0150] The grouping of optional elements or aspects of the disclosure disclosed herein should not be construed as limiting. Each member of a group may be referred to and claimed individually, or each member of a group may be referred to and claimed as any combination with other members of the group, or as any combination with other elements found herein. One or more members of a group may be included in or removed from a group for convenience and / or patentability. In the event of any such inclusion or removal, the modified group is deemed to be included herein, and thus this specification satisfies the description for all Markush groups used in the appended claims.

Claims

1. The system includes: (a) (i) One or more polyadenylation signals or transcription stop elements located upstream of the nucleic acid encoding the transgene or RNA, (ii) the nucleic acid encoding the transgene or RNA, and (iii) Recombinase recognition site comprising at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site donor vectors, including; (b) Two types of recombinases specific to the recombinase recognition site.

2. The system according to claim 1, wherein the donor vector further comprises at least a third recombinase recognition site, and the system further comprises at least a third recombinase specific to the at least third recombinase recognition site.

3. The donor vector and the two types of recombinases, and optionally the locus that is a target of the at least third recombinase, The system according to claim 1, further comprising mammalian cells containing the above.

4. The two types of recombinases mentioned above (i) One expression vector containing two genes encoding recombinases specific to the recognition site, or (ii) Two types of expression vectors, namely, a first expression vector containing one gene encoding a first recombinase specific to the unidirectional recombinase recognition site, and a second expression vector containing one gene encoding a second recombinase specific to the bidirectional recombinase recognition site, or (iii) One mRNA that encodes the two recombinases specific to the recognition site, or (iv) Two types of mRNA, namely, a first mRNA encoding a first recombinase specific to the unidirectional recombinase recognition site, and a second mRNA encoding a second recombinase specific to the bidirectional recombinase recognition site, or (v) A viral vector containing two genes that encode a recombinase specific to that recognition site, or (vi) Two types of viral vectors, namely, a first viral vector containing one gene encoding a first recombinase specific to the unidirectional recombinase recognition site, and a second viral vector containing one gene encoding a second recombinase specific to the bidirectional recombinase recognition site, or (vii) One recombinant protein comprising the unidirectional recombinase and the bidirectional recombinase, or (viii) Two types of recombinant proteins, namely, a first recombinase protein specific to the unidirectional recombinase recognition site and a second recombinase protein specific to the bidirectional recombinase recognition site. The system according to any one of claims 1 to 3, provided by [company name].

5. (i) In one expression vector containing two genes encoding a recombinase specific to the recognition site, the encoded recombinases are fused together, or (iii) In one mRNA that encodes two recombinases specific to the recognition site, the two encoded recombinases are fused together, or (v) In a viral vector containing two genes that encode a recombinase specific to the recognition site, the encoded recombinases are fused together or (viii) In which the two recombinant proteins are fused together, The system according to claim 4.

6. The system according to claim 4, wherein one of the aforementioned recombinases is fused with one or more proteins other than the recombinase.

7. The system according to claim 5, wherein one of the two fused recombinases is further fused with one or more proteins other than the recombinase.

8. The above-mentioned at least third recombinase, (iv) One expression vector comprising a gene encoding at least the third recombinase specific to the third recombinase recognition site, or (x) One mRNA that encodes the at least third recombinase specific to the at least third recognition site, or (xi) A viral vector comprising a gene encoding the at least third recombinase specific to the at least third recombinase recognition site, or (xii) A recombinant protein comprising the at least third recombinase specific to the at least third recombinase recognition site. The system according to any one of claims 2 to 7, provided by [company name].

9. (iv) One expression vector comprising a gene encoding at least the third recombinase specific to the third recombinase recognition site, wherein the expression vector further comprises a gene encoding one or more proteins other than the third recombinase, and the encoded third recombinase is fused with the one or more proteins, or (x) In one mRNA encoding the at least third recombinase specific to the at least third recognition site, the mRNA further encodes one or more proteins other than the third recombinase, and the encoded recombinase is further fused with the one or more proteins encoded, or (xi) In one viral vector comprising a gene encoding the at least third recombinase specific to the at least third recombinase recognition site, the one viral vector further encodes one or more proteins other than the third recombinase, and the encoded recombinase is further fused with the one or more encoded proteins, or In (xii), one recombinant protein comprising the at least third recombinase specific to the at least third recombinase recognition site is fused with one or more proteins other than the third recombinase. The system according to claim 8.

10. The system according to any one of claims 1 to 9, wherein the unidirectional recombinase recognition site is located upstream of the bidirectional recombinase recognition site.

11. The system according to any one of claims 1 to 9, wherein the unidirectional recombinase recognition site is located downstream of the promoter.

12. The system according to any one of claims 1 to 11, wherein the donor vector further comprises an intron, a portion of an intron, or at least one splice acceptor site, and optionally the unidirectional recombinase recognition site is embedded in or in the intron.

13. The system according to any one of claims 1 to 12, wherein the unidirectional recombinase is Bxb1.

14. The system according to any one of claims 1 to 12, wherein the unidirectional recombinase is selected from Bxb1, Phic31, PhiBT1, PhiC1, MR11, R4, TP901-1, A118, FC1, PhiRV, TG1, Phi370.1, Wβ, BL3, SPBc, K38, and any variant thereof.

15. The system according to any one of claims 1 to 12, wherein the bidirectional recombinase is Flp.

16. The system according to any one of claims 1 to 12, wherein the unidirectional recombinase is Bxb1, and the bidirectional recombinase is selected from FLp, Cre, VCre, SCre, Nigri, Panto, Vika, or their variants.

17. The system according to any one of claims 2 to 12, wherein the third recombinase is selected from Bxb1, Phic31, PhiBT1, PhiC1, MR11, R4, TP901-1, A118, FC1, PhiRV, TG1, Phi370.1, Wβ, BL3, SPBc, K38, FLp, Cre, VCre, SCre, Nigri, Panto, Vika, or their variants.

18. The system according to any one of claims 1 to 17, wherein the unidirectional recombinase recognition site is attB.

19. The system according to any one of claims 1 to 17, wherein the unidirectional recombinase recognition site is attP.

20. The system according to any one of claims 1 to 17, wherein the bidirectional recombinase recognition site is a flippase recognition target (FRT), loxP, VloxP, SloxP, nox, or pox.

21. The system according to any one of claims 1 to 17, wherein one or both of the recombinase recognition sites include a mutation.

22. The system according to any one of claims 1 to 21, wherein the donor vector is selected from the group consisting of plasmids, linear PCR, linear single-stranded DNA, closed-end double-stranded DNA, circular single-stranded DNA, circular double-stranded DNA, RNA, minicircles, viral vectors, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), and human artificial chromosomes (HAC).

23. The system according to claim 22, wherein the viral vector is an adeno-associated virus (AAV) vector.

24. The system according to any one of claims 1 to 23, wherein the donor vector includes at least four polyadenylation signals upstream of the nucleic acid encoding the transgene or the RNA.

25. The system according to any one of claims 1 to 24, wherein the donor vector includes an intron or a portion of an intron upstream and / or downstream of the nucleic acid encoding the transgene or the RNA.

26. The system according to any one of claims 1 to 25, wherein the donor vector further comprises a post-transcriptional regulatory element.

27. The system according to any one of claims 1 to 26, wherein the donor vector further comprises a polyadenylation signal downstream of the nucleic acid encoding the transgene or the RNA.

28. The system according to any one of claims 1 to 27, wherein the donor vector further comprises an open reading frame (ORF) beginning with a splice acceptor.

29. The system according to any one of claims 1 to 28, wherein the donor vector further comprises a fluorescent reporter.

30. The system according to any one of claims 4 to 29, wherein the expression vector containing the recombinase is located under a tissue-specific promoter.

31. The system according to any one of claims 1 to 30, wherein the RNA is siRNA, shRNA, sgRNA, crRNA, pegRNA, lncRNA, or miRNA.

32. The system according to any one of claims 1 to 30, wherein the introduced gene or RNA includes a disease-related mutation.

33. The system according to any one of claims 1 to 30, wherein the introduced gene or RNA includes a gain-of-function (GOF) gene mutation, a loss-of-function (LOF) gene mutation, or both.

34. The system according to any one of claims 1 to 33, wherein the mammalian cells are human cells, and the gene locus is the AAVS1 locus, H11 locus, HPRT1 locus, Rogi1 locus, Rogi2 locus, GAPDH locus, TATA box-binding protein (TBP) locus, kinesin family member (KIF11) locus, TRAC locus, ZAP-70 locus, Linker of Activation of T cells (LAT) locus, or Lymphocyte Cytosolic Protein 2 (LCP2) locus, and the method is an in vitro method, an ex vivo method, or an in vivo method.

35. The system according to claim 34, wherein the gene locus comprises a secondary cistron comprising a first polynucleotide encoding a first protein, a promoter, a recombinase recognition site recognized by the recombinase in the system, and a second polynucleotide encoding an open reading frame for a second protein.

36. The system according to claim 35, wherein the first protein, the second protein, or both are fluorescent proteins.

37. The system according to claim 35 or claim 36, wherein the first polynucleotide encoding the protein is located downstream of the gene at the gene locus.

38. The system according to any one of claims 1 to 37, wherein the mammalian cells are mouse cells, and the gene locus is the ROSA26 locus, Hipp11 locus, Tigre locus, ColA1 locus, HPrt locus, GAPDH locus, TATA box-binding protein (TBP) locus, kinesin family member (KIF11) locus, TRAC locus, Zap-70 locus, T cell activation linker (LAT) locus, or lymphocyte cytoplasmic protein 2 (LCP2) locus, and the method is an in vitro method, an ex vivo method, or an in vivo method.

39. The system according to claim 38, wherein the gene locus comprises a secondary cistron comprising a first polynucleotide encoding a first protein, a promoter, a recombinase recognition site recognized by the recombinase in the system, and a second polynucleotide encoding an open reading frame for a second protein.

40. A method for genetically manipulating mammalian cells, The step of transfecting or transducing mammalian cells using the system described in any one of claims 1 to 39. The method, including the method described above.

41. The method according to claim 40, wherein the system targets a gene locus, and the gene locus includes a recombinase recognition site comprising at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site.

42. The method according to claim 40 or claim 41, wherein the unidirectional recombination is upstream of the bidirectional recombination at the gene locus.

43. The method according to any one of claims 40 to 42, wherein the mammalian cells are human cells, and the system targets the AAVS1 locus, H11 locus, HPRT1 locus, Rogi1 locus, Rogi2 locus, GAPDH locus, TATA box-binding protein (TBP) locus, kinesin family member (KIF11) locus, TRAC locus, ZAP-70 locus, T cell activation linker (LAT) locus, or lymphocyte cytoplasmic protein 2 (LCP2) locus, and the method is an in vitro method, an ex vivo method, or an in vivo method.

44. The method according to any one of claims 40 to 42, wherein the mammalian cells are mouse cells, and the system targets the ROSA26 locus, Hipp11 locus, Tigre locus, ColA1 locus, HPrt locus, GAPDH locus, TATA box-binding protein (TBP) locus, kinesin family member (KIF11) locus, TRAC locus, Zap-70 locus, T cell activation linker (LAT) locus, or lymphocyte cytoplasmic protein 2 (LCP2) locus, and the method is an in vitro method, an ex vivo method, or an in vivo method.

45. The method according to any one of claims 40 to 44, further comprising the step of administering one or more types of recombinase enzymes to the cells.

46. The method according to any one of claims 40 to 45, wherein the one or more recombinase enzymes include Bxb1 recombinase, Cre recombinase, flipperjelly recombinase, Nigri recombinase, Panto recombinase, Vika recombinase, VCre recombinase, or SCre recombinase.

47. The method according to any one of claims 40 to 46, wherein the mammalian cells include blood cells, tumor cells, non-tumor cells, embryonic stem cells, adult stem cells, induced pluripotent stem cells, or tissue progenitor cells.

48. Non-human animals comprising the system described in any one of claims 1 to 39 Non-human animal models, including those mentioned above.

49. The non-human animal model according to claim 48, wherein the non-human animal model is a non-human animal model personalized for a human target cancer, and the transgene or RNA is based on the human target cancer.

50. The non-human animal model according to claim 48, wherein the non-human animal model is a non-human animal model personalized for a human disease or condition, and the introduced gene or RNA is based on the human disease or condition.

51. The non-human animal model according to claim 49 or 50, wherein the transgene or RNA is selected from the group consisting of oncogenes, loss-of-function (LOF) mutations in tumor suppressor genes, gain-of-function (GOF) mutations in proto-oncogenes, pseudogenes, siRNA, shRNA, sgRNA, pegRNA, crRNA, lncRNA, miRNA, epigenetic modifications, non-coding or epigenetic abnormalities associated with human diseases, and combinations thereof.

52. A non-human animal model according to any one of claims 48 to 51, comprising a gain-of-function (GOF) mutation, a loss-of-function (LOF) mutation, or both.

53. The non-human animal model according to any one of claims 48 to 52, wherein the system targets a locus in a non-human animal model, and the locus includes a recombinase recognition site comprising at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site.

54. The non-human animal model according to any one of claims 48 to 53, wherein unidirectional recombination is upstream of bidirectional recombination at the gene locus.

55. A method for creating non-human animal models, The step of transfecting or transfecting a non-human animal model using the system described in any one of claims 1 to 29. The method, including the method described above.

56. A non-human animal model prepared by the method described in claim 55.

57. A method for evaluating the effects of candidate drugs, A step of providing a non-human animal model according to any one of claims 48 to 54 or claim 56; The step of administering candidate drugs to non-human animal models; and Steps to evaluate the effects of the candidate drug on non-human animal models. The method, including the method described above.

58. A mammalian cell comprising the system described in any one of claims 1 to 39.

59. A non-mammalian cell comprising the system described in any one of claims 1 to 39.

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