Methods for site-specific introduction of genetic elements in engineered loci by dual mode recombinase mediated cassette exchange (BIRMCE)

Abstract

Systems and methods for mammalian cell genetic manipulation are described herein. Non-human animal models utilizing the systems, methods of generating the non-human animals, and methods of using the non-human animals are also described.

Description

Cross Reference to Related Applications

[0001] This application contains priority claims under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 523,550, filed June 27, 2023, the entire contents of which are hereby incorporated by reference. TECHNICAL FIELD

[0002] The present invention relates to genetic manipulation; for example, in cells, organoids, and non-human animal models. BACKGROUND

[0003] All publications herein are 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 description herein contains information useful to understand the present invention. It is not an admission that any information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Stable integration and / or editing of genetic elements to specific loci is a very difficult task. Despite different technologies trying to achieve this, it is still not done in a fast, clean, specific, cheap, and easy way. MADR technology is a technology that is able to do this with the mentioned characteristics. One of the “Achilles heels” of MADR and other similar technologies is that it relies on the elimination of exogenous genetic elements to turn off the system and express only one exogenous genetic element. Therefore, the aim of biRMCE is to accelerate the stable expression of one exogenous genetic element even in the presence of different genetic elements. This principle enhances the use of genetic cassette exchange in different applications where it is desired to stably integrate and / or edit genetic elements to specific loci.

[0005] Genetic cassette exchange by recombinases is a technology for integrating exogenous genetic elements into engineered loci of different types of cells. For example, one recombinase is used that targets its heterotypic recognition site, however, it has been found that cross-recombination between the heterotypic sites can occur, causing interference. Other similar works use integrase to target their respective recognition sites, but the main problem with using these systems is that they not only integrate the genetic cassette element, but also the complete genetic vector carrying undesired sequences. One existing solution is to use different types of recombinases, such as Flp & Cre, sequentially or simultaneously, to avoid cross-recombination between the recognition sites and to increase the efficiency of genetic cassette exchange. However, when using genetic cassettes carrying different types of genetic elements, the recombinases perform a reversible recombination reaction, making the system unstable and inefficient.

[0006] Accordingly, there remains a need in the art for robust and highly stable methods and systems for site-specific introduction of genetic elements in engineered loci. SUMMARY

[0007] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting.

[0008] Various embodiments provide a system comprising: (a) a donor vector comprising: (i) one or more polyadenylation signals or transcription termination elements upstream of a transgene or a nucleic acid encoding an RNA, (ii) the transgene or the nucleic acid encoding the RNA, and (iii) a recombinase recognition site comprising at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site; and (b) two recombinases specific for the recombinase recognition site.

[0009] In various embodiments, the donor vector can further comprise at least a third recombinase recognition site, and the system can further comprise at least a third recombinase specific for the at least a third recombinase recognition site.

[0010] In various embodiments, the system can further comprise a mammalian cell comprising a locus targeted by the donor vector and the two recombinases, and optionally the at least a third recombinase.

[0011] In various embodiments, the two recombinases can be provided by: (i) a single expression vector comprising two genes encoding recombinases specific for their recognition sites, or (ii) two expression vectors, a first expression vector comprising one gene encoding a first recombinase specific for the unidirectional recombinase recognition site, and a second expression vector comprising one gene encoding a second recombinase specific for the bidirectional recombinase recognition site, or (iii) a single mRNA encoding two recombinases specific for their recognition sites, or (iv) two mRNAs, a first mRNA encoding a first recombinase specific for the unidirectional recombinase recognition site, and a second mRNA encoding a second recombinase specific for the bidirectional recombinase recognition site, or (v) A single viral vector containing two genes, said two genes encoding recombinases specific to their recognition sites, or (vi) Two viral vectors, the first viral vector containing a gene encoding a first recombinase specific to the unidirectional recombinase recognition site, and the second viral vector containing a gene encoding a second recombinase specific to the bidirectional recombinase recognition site, or (vii) A single recombinant protein comprising the single-direction recombinase and the double-direction recombinase, or (viii) Two recombinant proteins, a first recombinase protein that is specific to the unidirectional recombinase recognition site, and a second recombinase protein that is specific to the bidirectional recombinase recognition site.

[0012] In various embodiments, in (i) the single expression containing two genes, wherein the two genes encode recombinases specific to their recognition sites, and the encoded recombinases may be fused together. In various embodiments, in (iii) the single mRNA encoding two recombinases specific to their recognition sites, the encoded two recombinases may be fused together. In various embodiments, in (v) the single viral vector containing two genes, wherein the two genes encode recombinases specific to their recognition sites, and the encoded recombinases may be fused together. In various embodiments, in (viii) the two recombinant proteins may be fused together.

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

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

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

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

[0017] In various embodiments, the unidirectional recombinase recognition site may be located upstream of the bidirectional recombinase recognition site. In various embodiments, 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, the unidirectional recombinase recognition site is embedded in the intron or a portion of 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 mutant thereof.

[0020] In various embodiments, the bidirectional recombinase may be Flp. In various embodiments, the unidirectional recombinase may be Bxb1 and the bidirectional recombinase is selected from FLp, Cre, VCre, SCre, Nigri, Panto, Vika, or mutants 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 mutants 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 flippaser 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-terminal 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). In various embodiments, the viral vector may be an adeno-associated virus (AAV) vector.

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

[0027] In various implementations, the expression vector containing the recombinase can be controlled by a tissue-specific promoter.

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

[0029] In various embodiments, the mammalian cell may be a human cell, and the locus may be the AAVS1 locus, H11 locus, HPRT1 locus, Rogi1 locus, Rogi2 locus, GAPDH locus, TATA box-binding protein (TBP) locus, kinin family member (KIF11) locus, TRAC locus, ZAP-70 locus, T cell activation adaptor protein (LAT) locus, or lymphocyte cytoplasmic protein 2 (LCP2) locus, and the method may be an in vitro, ex vivo, or in vivo method.

[0030] In various embodiments, the locus may include a first polynucleotide encoding a first protein, 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 of 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 located downstream of the gene at the locus.

[0031] In various embodiments, the mammalian cell may be a mouse cell, 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 activating adaptor protein (LAT) locus, or lymphocyte cytoplasmic protein 2 (LCP2) locus, and the method may be an in vitro, ex vivo, or in vivo method.

[0032] In various embodiments, the locus may include a first polynucleotide encoding a first protein, 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 of a second protein.

[0033] Various embodiments provide methods for genetic manipulation of mammalian cells, the methods including: transfecting or transducing the mammalian cells using any of the systems of the present invention.

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

[0035] In various implementations, unidirectional recombination may be located upstream of bidirectional recombination at the 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, kinin family member (KIF11) locus, TRAC locus, ZAP-70 locus, T cell activation adaptor protein (LAT) locus, or lymphocyte cytoplasmic protein 2 (LCP2) locus, and the method is an in vitro, ex vivo, or in vivo method.

[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, kinin family member (KIF11) locus, TRAC locus, Zap-70 locus, T cell activating adaptor protein (LAT) locus, or lymphocyte cytoplasmic protein 2 (LCP2) locus, and the method is an in vitro, ex vivo, or in vivo method.

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

[0039] In various embodiments, the one or more recombinases may include Bxb1 recombinase, Cre recombinase, flippase recombinase, Nigri recombinase, Panto recombinase, Vika recombinase, VCre recombinase, or SCre recombinase.

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

[0041] Various implementations provide non-human animal models, including: non-human animals incorporating the system of the present invention.

[0042] In various embodiments, the non-human animal model may be a personalized non-human animal model for a human subject's cancer, and the transgene or RNA is based on the human subject's cancer. In various embodiments, the non-human animal model may be a personalized non-human animal model for a human subject's disease or condition, and the transgene or RNA is based on the human subject's 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 of tumor suppressor genes, gain-of-function (GOF) mutations of proto-oncogenes, pseudogenes, siRNA, shRNA, sgRNA, pegRNA, crRNA, lncRNA, miRNA, epigenetic modifications, non-coding genetic or epigenetic abnormalities associated with human diseases, and combinations thereof.

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

[0045] In various embodiments, the system can target loci in the non-human animal model, and the loci include a recombinase recognition site containing at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site. In various embodiments, unidirectional recombination is located upstream of bidirectional recombination on the genome.

[0046] Various implementations provide methods for generating non-human animal models, the methods including: transfecting or transducing non-human animal models using the system of the present invention.

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

[0048] Various embodiments provide methods for evaluating the effects of drug candidates, the methods comprising: providing a non-human animal model of the present invention; administering the drug candidate to the non-human animal model; and evaluating the effect of the drug candidate on the non-human animal model.

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

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

[0051] Other features and advantages of the invention will become apparent from the following detailed description, which, in conjunction with the accompanying drawings, illustrates various features of embodiments of the invention by way of example. Attached Figure Description

[0052] Exemplary embodiments are illustrated in the accompanying drawings. It is intended that the embodiments and drawings disclosed herein be considered illustrative rather than restrictive.

[0053] Figures 1A-1B illustrate strategies comparing the efficiency of integrating transgenic elements into the Rosa 26 locus using Bxb1 alone or with FlpO in the presence of different Bxb1 recognition sites. Figure 1A shows the Rosa26 locus carrying the PuroR gene, flanked by the "open" attP and FRT sites. A promoter-less donor vector carries the mScarlet transgene, flanked by the attB and FRT sites. Recombination is expected between the genomic locus and the donor vector in the presence of Bxb1 or Bxb1+FlpO. Figure 1B shows the Rosa26 locus carrying the PuroR gene, flanked by the "locked" attR and FRT sites. A promoter-less donor vector carries the mScarlet transgene, flanked by the attB and FRT sites. Recombination is expected between the genomic locus and the donor vector in the presence of Bxb1 or Bxb1+FlpO. PuroR represents the puromycin resistance gene. Bxb1+FlpO is expressed in a separate plasmid.

[0054] Figures 2A-2B The insertion of transgenic elements via Bxb1 integration or Bxb1+FlpO is shown to depend on the attP recognition site embedded in the Rosa26 locus. 2A shows that mScarlet integration and expression can be achieved in the presence of an "open" attP site in the Rosa26 locus, an attB site on the donor vector, and in the presence of Bxb1 or Bxb1+FlpO (Rx 1.1 and Rx 1.2). 2B shows that mScarlet integration and expression are significantly reduced in the presence of a "locked" attR site in the Rosa26 locus, while still having an attB site on the donor vector, and in the presence of Bxb1 or Bxb1+FlpO (Rx 2.1 and Rx 2.2). Recombinase-free samples were used as negative controls (Rx 1.3 and Rx 2.3). Epifluorescence microscopy images were taken 48 h post-nuclear transfection. mScarlet expression was measured in 10,000 cells by flow cytometry 48 h post-nuclear transfection. Hybrid mouse neural stem cells carrying the corresponding “2in1” landing region at the Rosa26 locus were used (see Figure 1).

[0055] Figures 3A-3BThe figures show the number of cells expressing mScarlet derived from Bxb1 or Bxb1+FlpO recombination. Figure 3A shows a graph comparing the quantification of cells expressing mScarlet after recombination with Bxb1 into the Rosa26 locus with either an "open" attP site or a "locked" attR site. Figure 3B shows a graph comparing the quantification of cells expressing mScarlet after recombination with Bxb1+FlpO into the Rosa26 locus with either an "open" attP site or a "locked" attR site. These data indicate that integration of the transgenic element depends on recombination at the attP / attB recognition site via Bxb1.

[0056] Figures 4A-4B illustrate strategies comparing the efficiency of integrating transgenic elements into the Rosa 26 locus, either alone or with Cre, in the presence of different Bxb1 recognition sites. Figure 4A shows the Rosa26 locus carrying the PuroR gene, flanked by a loxP site and an "open" attP site. A promoterless donor vector carries the mScarlet transgene, flanked by a loxP site and an attB site. Recombination is expected between the genomic locus and the donor vector in the presence of Bxb1 or Bxb1+Cre. Figure 4B shows the Rosa26 locus carrying the PuroR gene, flanked by a loxP site and a "locked" attR site. A promoterless donor vector carries the mScarlet transgene, flanked by a loxP site and an attB site. Recombination is expected between the genomic locus and the donor vector in the presence of Bxb1 or Bxb1+Cre. PuroR represents the puromycin resistance gene. Bxb1+Cre is expressed in a separate plasmid.

[0057] Figures 5A-5BThe insertion of transgenic elements via Bxb1 integration or Bxb1+Cre is shown to depend on the loxP recognition site embedded in the Rosa26 locus. 5A shows that mScarlet integration and expression can be achieved in the presence of an "open" attP site in the Rosa26 locus, the presence of an attB site on the donor vector, and the presence of Bxb1+Cre (Rx 3.2). 5B shows reduced mScarlet integration and expression in the presence of a "locked" attL site in the Rosa26 locus, the continued presence of an attB site on the donor vector, and the presence of Bxb1+Cre (Rx 4.2). Recombinases not expressed were used as negative controls (Rx 3.3 and Rx 4.3). Epifluorescence microscopy images were taken 48 h post-transfection. mScarlet expression was measured by flow cytometry in 10,000 cells 48 h post-transfection. Heterozygous mouse neural stem cells carrying the corresponding “2in1” landing region at the Rosa26 locus were used, see Figure 4.

[0058] Figures 6A-6B The figures show the number of cells expressing mScarlet derived from Bxb1 or Bxb1+Cre recombination. Figure 6A shows a graph comparing the quantification of cells expressing mScarlet after recombination into the Rosa26 locus via Bxb1 with either an "open" attP site or a "locked" attL site. Figure 6B shows a graph comparing the quantification of cells expressing mScarlet after recombination into the Rosa26 locus via Bxb1+Cre with either an "open" attP site or a "locked" attL site. These data indicate that the integration of the transgenic element depends on Cre / loxP recombination.

[0059] Figures 7A-7B The expression of mScarlet+ cells at different time points is shown by Bxb1+Cre recombination into the Rosa26 locus with different Bxb1 recognition sites. 7A shows the expression and quantification of mScarlet in cells with an "open" attP site downstream of the PuroR gene and expressing Bxb1+Cre (Rx 3.2). 7B shows the expression and quantification of mScarlet in cells with a "locked" attL site downstream of the PuroR gene and expressing Bxb1+Cre (Rx 4.2). Epifluorescence microscopy images were taken at 2, 4, and 7 days post-nuclear transfection. mScarlet expression was measured by flow cytometry in 10,000 cells at 2, 4, and 7 days post-nuclear transfection.

[0060] Figure 8A graph comparing the percentage of mScarlet expression in the recombination reactions shown in Figure 7 at 2, 4, and 7 days post-nuclear transfection is presented. The dashed line indicates that integration of transgenic elements mediated by Cre / loxP recombination is unstable, with less than 1% of cells expressing mScarlet at 7 days post-nuclear transfection. The solid line indicates that integration of transgenic elements mediated by both Cre / loxP and Bxb1 / attP / attB recombination reactions is more stable. The Y-axis represents the percentage of mScarlet+ cells. The X-axis represents the number of days post-recombination reaction induction. This data suggests that transgenic elements can be integrated through reversible / bidirectional and irreversible / unidirectional recombination reactions, namely: Cre / loxP reversible / bidirectional recombination and Bxb1 / attP / attB irreversible / unidirectional recombination.

[0061] Figure 9 A simplified version is shown from Figure 1 to... Figure 8 The data are presented in a table. Reactions 1.1 through 2.3 demonstrate that attP / attB recombination is necessary for irreversible / unidirectional integration via Bxb1. Reactions 3.1 through 4.3 indicate the possible existence of a dual-mode recombinase-mediated cassette exchange (biRMCE), namely: two recombination modes: reversible / bidirectional recombination of Cre / loxP and irreversible / unidirectional recombination of Bxb1 / attP / attB.

[0062] Figures 10A-10D illustrate schematic diagrams comparing strategies for full plasmid integration and recombinase-mediated cassette exchange. Figure 10A shows a schematic diagram of the "landing region" 2in1-loxP-attP-TagBFP2-nls-FRT in the Rosa26 locus. Figure 10B shows pDonor-2in1-loxP-attB-mScarlet-FRT carrying different recombination sites. Figure 10C shows the Rosa26 locus after recombination with pDonor-2in1-loxP-attB-mScarlet-FRT mediated by different recombinase expressions. Figure 10D shows the Rosa26 locus after Cre / loxP recombination.

[0063] Figures 11A-11F illustrate the validation of DNA cassette exchange via biRMCE. Subfigures 11A-11D show the expression of cytoplasmic red fluorescent protein (mScarlet) and / or nuclear blue fluorescent protein (TagBFP2). Nuclear TagBFP2 expression reveals cells that previously integrated the transgenic mScarlet via whole plasmid integration rather than DNA cassette exchange. TagBFP2 expression depends on Cre / loxP excision of the mScarlet cassette. Subfigure 11A shows several TagBFP2+ cells, revealing previous whole plasmid integration via Bxb1. Subfigures 11B-11C show almost no TagBFP+ cells, demonstrating previous DNA cassette exchanges via biRMCE using Bxb1-FlpO or FlpO-Bxb1, respectively. Subfigure 11D shows a small number of TagBFP+ cells, indicating previous DNA cassette exchanges via dRMCE using FlpO-Cre. Epifluorescence microscopy images were acquired 48 h post-Cre expression. Subfigure 11E shows a graph with the percentage of cells expressing TagBFP2 from subfigures A to D, confirming that Bxb1 expression alone induces full plasmid integration rather than cassette exchange. Subfigure 11F shows a graph with the percentage of cells expressing mScarlet from subfigures B to D. This indicates that both biRMCE and dRMCE efficiently (>95%) integrate transgenic elements via DNA cassette exchange. Flow cytometry was performed 48 h post-nuclear transfection to quantify cells expressing the fluorescent reporter gene. These data demonstrate that biRMCE and dRMCE integrate transgenic elements into the identified loci via DNA cassette exchange.

[0064] Figure 12 The results show that biRMCE is more stable and efficient than dRMCE in integrating transgenic elements into the recipient genomic DNA. The graphs show the efficiency of transgenic element expression at the Rosa26 locus at different time points. The Y-axis describes the percentage of mScarlet+ cells. The X-axis describes the number of days after recombination induction. The dashed line shows the kinetics of dRMCE. The solid line shows the kinetics of biRMCE. These results demonstrate that biRMCE is a stable response for integrating transgenic elements into a defined locus.

[0065] Figure 13A schematic diagram of the strategy for validating the locking of DNA cassette exchange via biRMCE is shown. A colorless cell line with a landing zone was used to compare dRMCE and biRMCE in parallel. This cell line has a CAG promoter at the Rosa26 locus, followed by a loxP site, an attP site, a puromycin resistance gene (PuroR), and an FRT site. This cell line was named Rosa26-2in1-loxP-attP-PuroR-FRT. For dRMCE, three plasmids were used (1: promoterless donor vector-loxP-EGFP-nls-FRT; 2: promoterless donor vector-loxP-Scarlet-nls-FRT; 3: pCag-FlpO-Cre). For biRMCE, three plasmids were used (1: promoterless donor vector-attB-EGFP-nls-FRT; 2: promoterless donor vector-attB-Scarlet-nls-FRT; 3: pCag-Bxb1-FlpO).

[0066] Figure 14 illustrates how biRMCE locks DNA cassette exchange in the Rosa26 locus landing region early on. The top subfigure shows cells expressing nuclear mScarlet and / or nuclear EGFP via dRMCE (A) or biRMCE (B). Arrows indicate cells expressing both mScarlet and EGFP. The bottom subfigure shows flow cytometry quantification of cells expressing nuclear mScarlet and / or nuclear EGFP via dRMCE (C) or biRMCE (D); yellow cells are double-positive for mScarlet and EGFP. Images were taken using epifluorescence microscopy two days after nuclear transfection. Flow cytometry was performed two days after nuclear transfection. These data demonstrate that biRMCE locks DNA cassette exchange from an early stage.

[0067] Figures 15A-15D demonstrate that biRMCE maintained the lock-in of DNA cassette exchange in the Rosa26 locus landing region at late time. The upper subfigure shows cells expressing nuclear mScarlet and / or nuclear EGFP from dRMCE (A) or biRMCE (B). Arrows indicate cells expressing both mScarlet and EGFP. The lower subfigure shows flow cytometry quantification of cells expressing nuclear mScarlet and / or nuclear EGFP from dRMCE (C) or biRMCE (D). Yellow cells are double-positive for mScarlet and EGFP. Note the fewer colored cells under dRMCE conditions, indicating less stable integration than biRMCE. Images were taken using epifluorescence microscopy eight days post-nuclear transfection. Flow cytometry was performed eight days post-nuclear transfection. These data demonstrate that biRMCE maintained the lock-in of DNA cassette exchange.

[0068] Figures 16A-16H illustrate the strategy and validation of genetically element-containing landing regions compatible with dRMCE / MADR and intron biRMCE. Figure 16A shows a schematic diagram of the mTmG landing region of the Rosa26 locus and a promoter-free donor vector ready for insertion of its side-attached DNA cassette via dRMCE / MADR. Figure 16B shows a novel landing region of the Rosa26 locus containing introns between the first ATG and the remaining open reading frames (ATG-free TagBFP-nls), with attP sites on the introns. This novel landing region possesses all elements compatible with dRMCE / MADR and intron biRMCE. Sub-figures C-E illustrate the expression of nuclear Tag-BFP-WPRE in heterozygous mTmG neural stem cells following a dRMCE / MADR response, as demonstrated by fluorescence microscopy and flow cytometry. Subfigures F-H illustrate the expression of nuclear Tag-BFP without the WPRE sequence in heterozygous mTmG neural stem cells after a dRMCE / MADR reaction, as demonstrated by fluorescence microscopy and flow cytometry. Arrows indicate cells expressing nuclear Tag-BFP. Images were taken using epifluorescence microscopy two days post-transfection. Flow cytometry was performed two days post-transfection.

[0069] Figures 17A-17H illustrate the strategies and validation of genetic landing regions compatible with dRMCE / MADR and intron biRMCE. Figure 17A shows a schematic diagram of the “2in1-loxP-ATG-in-attP-TRON-(ATG-less-TagBFP-nls)-FRT” landing region of the Rosa26 locus and several promoterless donor vectors ready to be inserted into their side-attached DNA cassettes via dRMCE / MADR or intron biRMCE. Figure 17B shows the new landing region of the Rosa26 locus after dRMCE / MADR or intron biRMCE. Figures 17C-17E show the expression of nuclear miRFP-670 in heterozygous TagBFP neural stem cells after a dRMCE / MADR reaction, as demonstrated by fluorescence microscopy and flow cytometry. Figures 17F-17H illustrate the expression of nuclear miRFP-670 in heterozygous TagBFP neural stem cells after intronic biRMCE reaction, as demonstrated by fluorescence microscopy and flow cytometry. Arrows indicate cells expressing nuclear miRFP-670. Images were taken using epifluorescence microscopy two days after nuclear transfection. Flow cytometry was performed two days after nuclear transfection.

[0070] Figure 18A schematic diagram illustrates the strategy for verifying the locking of DNA cassette exchanges via intron biRMCE. Locking of the intron biRMCE was verified using intronization of the landing region of the Rosa26 locus and the donor plasmid. The landing region contains a CAG promoter, followed by a loxP site, an ATG start codon, an intron embedded with attP, any ATG-less genetic element, and an FRT site. This landing region is named Rosa26-2in1-loxP-in-attP-TRON-(ATG-less-Gene)-FRT. The promoterless donor plasmid contains a genetic element without the ATG start codon, flanked by an attB site with a portion of an intron and an FRT site. Bxb1 and Flp recombinases are essential for intron biRMCE.

[0071] Figure 19 (sub-figures A-F) illustrates a strategy for validating the minimum recognition site of biRMCE. A) shows a schematic diagram of the minimum recognition site of biRMCE, where the "irreversible" recognition site is immediately upstream of the "reversible" recognition site. B) shows a schematic diagram of the "landing region" 3in1-loxP-(attP / VloxP)-TagBFP2-nls-FRT in the Rosa26 locus. C) shows a hybrid neural stem cell line expressing nuclear TagBFP2 carrying the landing region described in Figure B. D) shows pDonor-2in1-loxP-attB-mScarlet-VloxP carrying different specific recombination sites. E) shows the Rosa26 locus after recombination of pDonor-2in1-loxP-attB-mScarlet-VloxP with Bxb1. F) shows the Rosa26 locus after recombination of pDonor-2in1-loxP-attB-mScarlet-VloxP by Bxb1 and VCre.

[0072] Figure 20 (sub-figures A-D) illustrates DNA cassette exchange validation using the minimal recognition site of biRMCE. A) shows a heterozygous mScarlet cell line carrying a landing region obtained after recombination with Bxb1. B) shows a heterozygous mScarlet cell line carrying a landing region obtained after recombination with Bxb1 and VCre. C) shows the mScarlet cell line described in Figure A expressing nuclear TagBFP2 after Cre recombination. Flow cytometry quantification showed that 98% of the cells were TagBFP2 positive. The expected landing region is shown at the bottom of the sub-figure. D) shows the mScarlet cell line described in Figure B expressing nuclear TagBFP2 after Cre recombination. Flow cytometry quantification showed that 3.5% of the cells were TagBFP2 positive. The expected landing region is shown at the bottom of the sub-figure. Images in C and D were taken using epifluorescence microscopy two days after induction. Flow cytometry was performed two days after induction.

[0073] Figure 21 (sub-figures A-D) illustrates the knock-in of the MADR and biRMCE compatible element into the human GAPDH locus. A) shows a schematic diagram spanning exons six through nine of the endogenous human GAPDH locus. B) shows a schematic diagram of the human GAPDH locus after knock-in. The edited locus carries TagBFP2 below the GAPDH promoter, and a secondary cistron containing the CAG promoter, upstream of which is miRFP670 flanked by MADR and biRMCE-specific recombination sites. C) shows the HEK [GAPDH-TagBFP2nls-Cag-2in1(loxP-attP)-miRFP67nls-FRT] cell line after appropriate knock-in and several rounds of purification. D) shows the genotyping of HEK cells before and after knock-in. PCR fragments indicate the left homologous arm, right homologous arm, Cag-miRFP670 cistron, and the unedited GAPDH locus. WT = wild-type unedited HEK cells, KI = knock-in edited HEK cells. The colored chains indicate the size of the corresponding highlighted PCR fragment.

[0074] Figure 22 (sub-figures A-D) illustrates the validation and efficiency of MADR and biRMCE at the human GAPDH locus. A) Schematic diagram of the strategy for validating and comparing MADR and biRMCE at the human GAPDH locus using the mScarlet donor vector. B) Schematic diagram of the expected human GAPDH locus after mScarlet cassette exchange via MADR or biRMCE. C) Schematic diagram of mScarlet expression in the HEK [GAPDH-TagBFP2nls-Cag-2in1(loxP-attP)-miRFP67nls-FRT] cell line after MADR or biRMCE. D) Flow cytometry quantification of cells expressing mScarlet after MADR or biRMCE. Images were taken using epifluorescence microscopy ten days post-induction. Flow cytometry was performed ten days post-induction.

[0075] Figure 23 ,and Figure 13 Similarly, a schematic diagram of the strategy for verifying the locking of DNA cassette exchanges via biRMCE is shown. A colorless cell line with a landing zone was used for side-by-side comparison of dRMCE / MADR and biRMCE. This cell line contains the CAG promoter, loxP site, attP site, puromycin resistance gene (PuroR), and FRT site at the Rosa26 locus. This cell line was named Rosa26-2in1-loxP-attP-PuroR-FRT. For dRMCE / MADR, three plasmids were used (1: promoterless donor vector-loxP-miRFP670-nls-FRT; 2: promoterless donor vector-loxP-BFP-nls-FRT; 3: pCag-FlpO-Cre). For biRMCE, three plasmids were used (4: promoterless donor vector-attB-miRFP670-nls-FRT; 5: promoterless donor vector-attB-BFP-nls-FRT; 6: pCag-Bxb1-FlpO).

[0076] Figure 24 (subplots A-D), similar to Figure 14, illustrates how biRMCE locks DNA cassette exchanges in the Rosa26 locus landing region at an early stage. The upper subplot shows cells expressing nuclear miRFP670 and / or nuclear BFP, obtained two days post-induction via dRMCE / MADR (A) or biRMCE (B). Arrows indicate cells expressing both miRFP670 and BFP. The lower subplot shows flow cytometry quantification of cells expressing nuclear miRFP670 and / or nuclear BFP, obtained two days post-induction via dRMCE / MADR (C) or biRMCE (D); purple dots represent miRFP670-positive cells, blue dots represent BFP-positive cells, and red dots represent double-positive cells of miRFP670 and BFP. Images were taken using epifluorescence microscopy two days post-nuclear transfection. Flow cytometry was performed two days post-induction. These data demonstrate that biRMCE locks DNA cassette exchanges from an early time point.

[0077] Figure 25 (subplots A-D), similar to Figure 15, shows that biRMCE maintains the lock-in of DNA cassette exchange in the Rosa26 locus landing region at later time points. The upper subplot shows flow cytometry quantification of cells expressing nuclear miRFP670 and / or nuclear BFP at four days post-induction via dRMCE / MADR (A) or biRMCE (B). The lower subplot shows flow cytometry quantification of cells expressing nuclear miRFP670 or nuclear BFP at eight days post-induction via dRMCE / MADR (C) or biRMCE (D). Purple dots represent miRFP670-positive cells, and blue dots represent BFP-positive cells. Note that under dRMCE / MADR conditions, fewer colored cells were observed at eight days post-induction, indicating less stable integration than with biRMCE. Flow cytometry was performed at four and eight days post-induction. These data demonstrate that biRMCE maintains the lock-in of DNA cassette exchange at later time points. Detailed Implementation

[0078] All references cited herein are incorporated herein by reference in their entirety as if fully described. Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology, 3rd Edition, Revised Edition, J. Wiley & Sons (NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 7th Edition, J. Wiley & Sons (NY 2013); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012) provide general guidance to those skilled in the art regarding many of the terms used in this application.

[0079] Those skilled in the art will recognize that many methods and materials similar to or equivalent to those described and used herein can be applied in the practice of this invention. In fact, this invention is by no means limited to the methods and materials described herein. For the purposes of this invention, the following terms are defined below.

[0080] As used herein, the term "about," when associated with a referred numerical indication, means up to 5% of the referred numerical indication plus or minus the referred numerical indication, unless otherwise specifically specified herein. For example, the phrase "about 50%" covers a range of 45% to 55%. In various embodiments, if specifically specified in the claims, the term "about," when associated with a referred numerical indication, may mean up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the referred numerical indication plus or minus the referred numerical indication.

[0081] "Mammal," as used herein, means any member of the class Mammalia, including but not limited to humans and non-human primates such as chimpanzees, and other ape and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domesticated mammals such as dogs and cats; and laboratory animals, including rodents such as mice, rats, and guinea pigs. This term does not indicate a specific age or sex. Therefore, adult and newborn subjects, whether male or female, are intended to be included within the scope of this term. In some embodiments, the subject is a human.

[0082] This invention describes a bimodal recombinase-mediated cassette exchange (biRMCE). BiRMCE utilizes both unidirectional and bidirectional recombination reactions present simultaneously in the system, solving many problems associated with integrating genetic elements using only bidirectional or unidirectional recombination reactions. First, there is no cross-recombination between the bidirectional and unidirectional recombinase recognition sites, ensuring system specificity. Second, it ensures the insertion of genetic cassettes flanking the unidirectional and bidirectional recombinase recognition sites, avoiding integration of the entire genetic vector. Because there is one irreversible reaction and one reversible reaction, this ensures system lock-in by preventing additional integration of different genetic cassettes. To illustrate, BiRMCE can be described as a competition between sperm and egg; only one sperm can enter the egg, therefore, the genetic cassette is equivalent to the "sperm," and the targeting locus is equivalent to the "egg." Furthermore, BiRMCE and dRMCE can be performed at the same locus, providing greater flexibility than previous MADR techniques.

[0083] The advantages of Bi-RMCE include, but are not limited to: (1) It is a one-step reaction to insert the side-attached DNA cassette. (2) The system is locked, allowing for high-throughput insertion of the DNA payload and immediate downstream analysis and / or applications, such as screening cells that have integrated the DNA cassette much faster, mainly because the invader DNA cassette does not carry a promoter. Downstream applications can be performed within hours of biRMCE. (3) There is no risk of cross-recombination between recombinase recognition sites, as the process relies on different recombination reactions. (4) It ensures that only one DNA cassette embedded in the plasmid is inserted into the host genome, as it relies on both irreversible and reversible reactions. Insertion of the DNA cassette via two unidirectional / irreversible reactions (such as the heterologous site of the integrase) may result in the integration of two 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.

[0084] Furthermore, it is more robust, stable, and multiplexed than dRMCE (as described in Osterwalder et al., Dual RMCE for efficient re-engineering of mouse mutant alleles, Nat Methods 7, 893-895 (2010)). Compared to 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, Vol. 104, 2019; Blanch-Asensio et al., STRAIGHT-IN enables high-throughput targeting of large DNA payloads in human pluripotent stem cells, Cell Reports Methods, Vol. 2, No. 10, 2022), it is faster and easier. Moreover, it is more reliable than using two heterologous integrase recognition sites to exchange cassettes. (See, for example, Inniss et al., A novel Bxb1 integrase RMCE system for high fidelity site-specific integration of mAb expression cassettein CHO Cells, Biotechnology and Bioengineering, Vol. 114, No. 8, August 2017, pp. 1837-1846; Low et al., Efficient targeted transgenesis of large donor DNA into multiplemouse genetic backgrounds using bacteriophage Bxb1 integrase, Sci Rep 12, 5424 (2022)).

[0085] As discussed in this paper, the locking properties of biRMCE are superior to dRMCE / MADR at 2 and 4 days post-induction. (See also...) Figure 13 -Figure 15 and Figure 23 -Figure 25.) Figure 23- In the data shown in Figure 25, the "color" of the genetic reporter genes was altered in the experiment to make them more distant in the color spectrum. This facilitates analysis and provides better resolution and output. (In generating...) Figure 13 - In the experiment with the data in Figure 15, red and green reporter genes, which are closer in spectral density, were used.

[0086] This paper also shows (e.g., Figures 19 and 20) that only ~80 bp of DNA is required to achieve biRMCE. This small amount of nucleotides is sufficient because we have demonstrated that biRMCE can be performed if the specific recombination sites are closely linked to each other and there are no other DNA sequences between them. This is crucial because integrating only ~80 bp of DNA (e.g., using prime editing to insert this ~80 bp of DNA) becomes easier and safer. Therefore, this method inserts ~80 bp of DNA, followed by the addition of a large amount of DNA, including a complete synthetic chromosome, via biRMCE. This is also helpful when adding very small DNA sequences flanking the biRMCE recombination sites, such as small sequences encoding sgRNAs.

[0087] As further illustrated (e.g., Figures 21 and 22), biRMCE is functional in the human genome. To demonstrate this, we inserted the biRMCE recombination site into the GAPDH locus of the human HEK-293T cell line.

[0088] Therefore, many embodiments of the present invention are based in part on these designs and findings. Systems for biRMCE

[0089] Various embodiments provide a system for biRMCE. The system may comprise a variety of compositions; therefore, the system can be considered as a combination of compositions.

[0090] Various embodiments provide a system comprising: (a) a donor vector comprising (i) one or more polyadenylation signaling or transcription termination elements located upstream of a transgene or nucleic acid encoding RNA; (ii) the transgene or nucleic acid encoding the RNA; and (iii) a recombinase recognition site containing at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site; and (b) two recombinases specific to the recombinase recognition site. In various embodiments, the system further comprises an intron, a portion of an intron, or at least one splice acceptor site.

[0091] In various embodiments, the donor vector of the system further comprises at least a third recombinase recognition site, and the system further comprises at least a third recombinase that is specific to the at least third recombinase recognition site.

[0092] 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 4th, 5th, 6th, 7th, 8th, 9th, or 10th recombinase recognition site, and the system further comprises a 4th, 5th, 6th, 7th, 8th, 9th, or 10th recombinase recognition site, respectively.

[0093] In various embodiments, the system further comprises mammalian cells and optionally at least a third recombinase, the mammalian cells containing loci targeted by the donor vector and the two recombinases. In various embodiments, the mammalian cells are located within a mammal.

[0094] In various embodiments, the two recombinases are provided by: (i) A single expression vector containing two genes, said two genes encoding a recombinase that is specific to the recombinase recognition site, or (ii) Two expression vectors, the first expression vector containing a gene encoding a first recombinase specific to the unidirectional recombinase recognition site, and the second expression vector containing a gene encoding a second recombinase specific to the bidirectional recombinase recognition site, or (iii) A single mRNA encoding the two recombinases, or (iv) Two mRNAs, the first mRNA encoding a first recombinase specific to the unidirectional recombinase recognition site, and the second mRNA encoding a second recombinase specific to the bidirectional recombinase recognition site, or (v) A single viral vector containing two genes, said two genes encoding a recombinase that is specific to the recombinase recognition site, or (vi) Two viral vectors, the first viral vector containing a gene encoding a first recombinase specific to the single-recombinase recognition site, and the second viral vector containing a gene encoding a second recombinase specific to the double-recombinase recognition site, or (vii) A single recombinant protein containing both a one-way recombinase and a two-way recombinase, or (viii) Two recombinant proteins, a first recombinase protein being specific for the unidirectional recombinase recognition site and a second recombinase protein being specific for the bidirectional recombinase recognition site.

[0095] In various embodiments, in (i) an expression vector containing two genes encoding recombinases specific to their recognition sites, the encoded recombinases are fused together. In various embodiments, in (iii) a single mRNA encoding two recombinases specific to their recognition sites, the encoded two recombinases are fused together. In various embodiments, in (v) a single viral vector containing two genes encoding recombinases specific to their recognition sites, the encoded recombinases are fused together. In various embodiments, in (viii) the two recombinant proteins are fused together.

[0096] In various embodiments, any one of the recombinases is fused with one or more proteins other than the recombinase. In various embodiments, any one of the two fused recombinases is further fused with one or more proteins other than the recombinase.

[0097] 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. In various embodiments, 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.

[0098] In other embodiments, the one or more proteins may be a combination of any two or three of nucleases, reverse transcriptases, polymerases, and transposases. Non-limiting examples include nucleases fused to reverse transcriptases, fused to unidirectional recombinases, and fused to bidirectional recombinases (e.g., Cas9, nicking enzymes such as dCas9).

[0099] In various embodiments, one or more proteins other than the recombinase 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. Additional examples include, but are not limited to, etanercept, bevacizumab, rituximab, adalimumab, infliximab, trastuzumab, insulin glargine, epoetin alfa, pegylated filgrastim, ranibizumab, daboetin alpha, interferon β-1a (Avonex), interferon β-1a (Rebif), insulin aspart, recombinant human insulin, octanediol alpha, lispro insulin, cetuximab, pegylated interferon alpha-2a, interferon β-1b, epoetin alpha, insulin aspart, botulinum toxin type A, epoetin β, recombinant antihemophilic factor, filgrastim, insulin detemir, natezumab, insulin (humulin), and palizumab.

[0100] In various embodiments, the at least third recombinase is provided by: (iv) A single expression vector comprising a gene encoding at least a third recombinase that is specific to the third recombinase recognition site, or (x) A single mRNA encoding at least a third recombinase that is specific to at least the third recognition site, or (xi) A single viral vector, said single viral vector comprising a gene encoding at least a third recombinase that is specific to the recognition site of said at least a third recombinase, or (xii) A single recombinant protein comprising at least a third recombinase that is specific to the recognition site of the at least third recombinase.

[0101] In various embodiments, in (iv) a single expression vector comprising a gene encoding at least a third recombinase specific to the third recombinase recognition site, the expression vector further comprises a gene encoding one or more proteins other than the third recombinase, and the encoded third recombinase is fused to the encoded one or more proteins. In various embodiments, in (x) a single mRNA encoding at least a 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 to the encoded one or more proteins. In various embodiments, in (xi) a single viral vector comprising a gene encoding at least a third recombinase specific to the at least third recombinase recognition site, the single viral vector further encodes one or more proteins other than the third recombinase, and the encoded recombinase is further fused to the encoded one or more proteins. In various embodiments, in (xii) a single recombinant protein comprising at least a third recombinase specific to the at least third recombinase recognition site, fused to one or more proteins other than the third recombinase.

[0102] 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. In various embodiments, 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.

[0103] In other embodiments, the one or more proteins may be a combination of any two or three of nucleases, reverse transcriptases, polymerases, and transposases. Non-limiting examples include nucleases fused with reverse transcriptases, fused with unidirectional recombinases, and fused with bidirectional recombinases (e.g., Cas9, nicking enzymes such as dCas9).

[0104] In various embodiments, one or more proteins other than the third recombinase are therapeutic proteins. Non-limiting examples of therapeutic proteins are provided herein.

[0105] In various embodiments, the unidirectional recombinase recognition site is located upstream of the bidirectional recombinase recognition site. In various embodiments, the unidirectional recombinase recognition site is located downstream of the promoter.

[0106] In various embodiments, the unidirectional recombinase is Bxb1 or any mutant 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 mutant thereof.

[0107] In various embodiments, the unidirectional recombinase is PhiC31, PhiBT1, PhiC1, MR11, R4, TP901-1, A118, FC1, PhiRV, TG1, Phi370.1, Wβ, BL3, SPBc, K38, or any mutant thereof.

[0108] 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 mutants thereof.

[0109] In various embodiments, the first recombinase recognition site is attB or any mutant thereof. Alternatively, the first recombinase recognition site may be attP or any mutant thereof.

[0110] In various embodiments, the second recombinase recognition site of the recombinase recognition site is a flippase target recognition (FRT), loxP, VloxP, SloxP, nox, or pox. In other embodiments, the second recombinase recognition site of the recombinase recognition site is a modified loxP, a flippase target recognition (FRT), VloxP, SloxP, nox, or pox.

[0111] In various embodiments, the one-way recombinase is PhiC31 and the recombinase recognition sites are attB and attP.

[0112] In various implementations, one or both of the recombinase recognition sites contain mutations.

[0113] In various embodiments, the third recombinase recognition site is attB or any mutant thereof, attP or any mutant thereof. In various embodiments, the third recombinase recognition site is a flippase target recognition (FRT), loxP, VloxP, SloxP, nox, or pox. In various embodiments, the third recombinase recognition site is a modified loxP, flippase target recognition (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 contains a mutation.

[0114] In various embodiments, at least one additional recombinase recognition site is attB or any mutant thereof, attP or any mutant thereof. In various embodiments, at least one additional recombinase recognition site is a flippase target recognition (FRT), loxP, VloxP, SloxP, nox, or pox. In various embodiments, at least one additional recombinase recognition site is a modified loxP, flippase target recognition (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 contains a mutation. At least one additional recombinase recognition site is, for example, the 4th, 5th, 6th, 7th, 8th, 9th, or 10th recombinase recognition site.

[0115] In various embodiments, the donor vector is selected from the group consisting of plasmids, linear DNA (e.g., PCR fragments, synthetic linear DNA), microcircles, viral vectors, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and human artificial chromosomes (HACs). A non-limiting example of a viral vector is an adeno-associated virus (AAV) vector. In various embodiments, the donor vector is selected from the group consisting of linear single-stranded DNA, closed-terminal double-stranded DNA, circular single-stranded DNA, circular double-stranded DNA, and RNA.

[0116] In various embodiments, the donor vector contains at least four polyadenylation signals located upstream of the transgene or the nucleic acid encoding RNA. In other embodiments, the donor vector contains at least one, at least two, or at least three polyadenylation signals located upstream of the transgene or the nucleic acid encoding RNA.

[0117] In other embodiments, the donor vector contains an intron or a portion of an intron located upstream of the transgene or the nucleic acid encoding RNA.

[0118] In various embodiments, the donor vector further includes a post-transcriptional regulatory element. In various embodiments, the donor vector further includes a polyadenylation signal downstream of the transgene or nucleic acid encoding RNA. In various embodiments, the donor vector further includes an open reading frame (ORF) beginning with a splice acceptor. In various embodiments, the donor vector further includes a fluorescent reporter gene.

[0119] In various implementations, the expression vector containing the recombinase is controlled by a tissue-specific promoter.

[0120] In various implementations, the RNA is siRNA, shRNA, sgRNA, crRNA, pegRNA, lncRNA, or miRNA.

[0121] In many embodiments, the transgene or RNA contains a disease-related mutation. In many embodiments, the transgene or RNA contains a gain-of-function (GOF) gene mutation, a loss-of-function (LOF) gene mutation, or both.

[0122] 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, kinin family member (KIF11) locus, TRAC locus, ZAP-70 locus, T cell activating adaptor protein (LAT) locus, or lymphocyte cytoplasmic protein 2 (LCP2) locus, and the method is an in vitro, ex vivo, or in vivo method. Although examples are listed, any human genomic locus can be used according to various embodiments of the present invention.

[0123] In various embodiments, the locus includes a first polynucleotide encoding a first protein, 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 of the first protein. In various embodiments, the first polynucleotide encoding the second protein is located downstream of the gene at the locus.

[0124] Examples of promoters include, but are not limited to, CAG, CMV, EF1a, PGK, TRE, U6, and UAS.

[0125] 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, KusabiraOrange, and Kusabira. Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express (T1), DsRed-Monomer, mTang erine, mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum and AQ143.

[0126] In various embodiments, the first fluorescent protein is TagBFP2, the promoter is the CAG promoter, and the second fluorescent protein is miRFP670.

[0127] In various embodiments, the mammalian cells are mouse cells, and the loci are ROSA26, Hipp11, Tigre, ColA1, Hprt, GAPDH, TATA box-binding protein (TBP), kinesin family member (KIF11), TRAC, Zap-70, T-cell activating adaptor protein (LAT), or lymphocyte cytoplasmic protein 2 (LCP2), and the method is in vitro, ex vivo, or in vivo. Although examples are listed, any mouse genomic locus can be used according to various embodiments of the invention.

[0128] In various embodiments, the locus in the mouse cell contains a first polynucleotide encoding a first protein, 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 of 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 described herein. Methods of genetic manipulation

[0129] Various embodiments of the present invention provide methods for genetic manipulation of mammalian cells, comprising: transfecting or transducing mammalian cells using any of the systems of the present invention described herein.

[0130] In various embodiments, the system targets a locus containing a recombinase recognition site having at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site. In various embodiments, unidirectional recombination is located upstream of bidirectional recombination at the locus.

[0131] In various embodiments, the locus includes a third recombinase recognition site. In various embodiments, the locus includes additional recombinase recognition sites; for example, a fourth, fifth, sixth, seventh, eighth, ninth, or tenth recombinase recognition site.

[0132] 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 adaptor protein) locus, or LCP2 (lymphocyte cytoplasmic protein 2, also known as SLP-76) locus, and the method is an in vitro, ex vivo, or in vivo method. 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 adaptor protein) locus, or Lcp2 (lymphocyte cytoplasmic protein 2) locus, and the method is an in vitro, ex vivo, or in vivo method.

[0133] In various embodiments, the method further includes administering one or more recombinant enzymes to the cells.

[0134] In various embodiments, one or more recombinases include Bxb1 recombinase, Cre recombinase, flippase recombinase, Nigri recombinase, Panto recombinase, Vika recombinase, VCre recombinase, or SCre recombinase.

[0135] In various embodiments, the mammalian cells include blood cells, tumor cells, non-tumor cells, embryonic stem cells, adult stem cells, induced pluripotent stem cells, or tissue precursor cells. Non-human animal models & methods for generating non-human animal models

[0136] Various implementations provide non-human animal models, which include: non-human animals containing any of the systems of the present invention described herein.

[0137] In various embodiments, the non-human animal model is a personalized non-human animal model for cancer in a human subject, and the transgene or RNA is based on the cancer of that human subject.

[0138] In various embodiments, the non-human animal model is a personalized non-human animal model for a disease or condition of a human subject, and the transgene or RNA is based on the disease or condition of that human subject.

[0139] In various embodiments, the non-human animal model includes gain-of-function (GOF) mutations, loss-of-function (LOF) mutations, or both.

[0140] In various embodiments, the transgene or RNA is selected from the group consisting of: oncogenes, loss-of-function (LOF) mutations of tumor suppressor genes, gain-of-function (GOF) mutations of proto-oncogenes, pseudogenes, siRNA, shRNA, sgRNA, pegRNA, crRNA, lncRNA, miRNA, epigenetic modifications, non-coding genetic or epigenetic abnormalities associated with human diseases, and combinations thereof.

[0141] In various embodiments, the system targets loci in non-human animal models, and the loci contain a recombinase recognition site having at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site.

[0142] In various implementations, unidirectional recombination is located upstream of bidirectional recombination at the locus.

[0143] 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 apes).

[0144] Various embodiments of the present invention provide methods for generating non-human animal models of the present invention, said methods being performed by transfecting or transducing a non-human animal model using any of the systems of the present invention.

[0145] Various embodiments of the present invention provide non-human animal models generated by any of the methods of the present invention. Drug screening

[0146] Various embodiments provide methods for evaluating the effects of drug candidates, the methods comprising: providing a non-human animal model of the present invention; administering a drug candidate to the non-human animal model; and evaluating the effects of the drug candidate on the non-human animal model.

[0147] Various embodiments provide mammalian cells incorporating the system of the present invention.

[0148] Various embodiments provide methods for evaluating the effects of drug candidates, the methods comprising: providing mammalian cells comprising the system of the present invention; contacting the drug candidate with the mammalian cells; and evaluating the effects of the drug candidate on the mammalian cells. Example

[0149] The following embodiments are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. References to specific materials are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can develop equivalent means or reactants without inventiveness and without departing from the scope of the invention. Example 1 Experimental process mice

[0150] All mice used were maintained and euthanized in accordance with the guidelines of the Cedars-Sinai Animal Care and Use Committee. mT / mG (Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo / J) mice (Muzumdar, Tasic, Miyamichi, Li, & Luo, 2007) were mated with C57BL / 6J mice to produce heterozygous mice. Downstream experiments were conducted using male and female pups from day 0 to day 2 after birth (P). plasmid cloning

[0151] The pDonor plasmid was derived from MADR-pDonors and used with NEBuilder HiFi DNA Assembly Master Mix (NEB) combined with standard restriction digestion techniques (Kim et al., 2019). In short, a specific recombination site was created via oligonucleotide synthesis and inserted into the MADR-pDonor. The vector expressing the recombinase was derived from the previously validated pCag-FlpO-2A-Cre EV (Addgene 129419) (Kim et al., 2019). The pCag-NLS-HA-Bxb1 (Addgene 51271) plasmid was used as a template for Bxb1 PCR. Downstream plasmid generation was accomplished by removing the existing ORF and adding a new cassette using HiFi DNA Assembly. PCR was performed using standard protocols and KAPA HiFi PCR reagents. Cell line generation

[0152] The brains of heterozygous P0-P2 mT / mG juvenile mice were dissociated to establish the polyclonal mouse neural stem cell line (mNSC) used in this study. Sex was not recorded due to a lack of reliable visual methods. Cell line generation and maintenance were 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), an antibiotic-antifungal agent (Life Technologies 15240), human epidermal growth factor (hEGF) (Sigma E9644), heparin (Sigma H3393), and basic fibroblast growth factor (bFGF) (Millipore GF003). Recipient cell lines were de novo generated by nuclear transfection of the pCag-FlpO-2A-Cre EV plasmid and the corresponding MADR-pDonors (Kim et al., 2019) via a dual recombinase-mediated cassette exchange (Osterwalder et al., 2010) targeting the ROSA26 locus in mTmG cells. Cell line selection and purification were performed by flow cytometry. nuclear transfection

[0153] mNSC nuclear transfection was performed using a Nucleofector 2b device and a Mouse Neural Stem Cell Kit, as recommended by the manufacturer (Lonza AG). The nuclear transfection mixture contained either 10 µg or 1 µg of plasmid or mRNA. Epifluorescence microscopy

[0154] Images were captured using a fluorescence microscope (ECHO Revolve) with the appropriate fluorescence channels and filters. Flow cytometry

[0155] Cells were collected as previously described (Kim et al., 2019). Cells were dissociated using Accutase (Millipore), precipitated at 800g for 3 min, and resuspended in EDTA. FACS was performed on a Beckman Coulter MoFlo on the Cedars-Sinai flow cytometry core platform. Example 2 result Indirect biRMCE reaction verification

[0156] To indirectly test biRMCE, we first attempted to confirm that Bxb1 / attB / attP recombination is irreversible / unidirectional (Ghosh, Wasil, & Hatfull, 2006; Merrick, Zhao, & Rosser, 2018). To confirm this, we created two heterozygous new recipient cell lines with either an attP or attR site at the Rosa26 locus, between the Cag promoter and the puromycin resistance gene (PuroR), followed by an FRT site (Fig. 1A-Fig. B). To prepare these cell lines, we performed dRMCE / MADR using the corresponding pDonors, exchanging the mTmG cassette for a new one. Next, we developed a promoterless donor vector containing the reporter gene mScarlet, flanked by both the attB and FRT sites (pDonor-attB-mScarlet-FRT). Then, with or without plasmids expressing recombinases Bxb1 or FlpO, we performed nuclear transfection with pDonor-attB-mScarlet-FRT (Fig. 1A-Fig. 1B, reactions 1.1-2.3). Cells carrying the “open” attP site expressed mScarlet in the presence of Bxb1 or both Bxb1 and FlpO. Figure 2A (Reactions 1.1 and 1.2). In cells with a "locked" attR site, mScarlet expression is almost nonexistent in the presence of Bxb1 or Bxb1 and FlpO. Figure 2B(Reactions 2.1 and 2.2). In the absence of recombinase, no mScarlet was expressed ( Figures 2A-2B (Reactions 1.1 and 2.3). Quantitative comparisons of Scarlet expression in reactions 1.1 and 1.2, and reactions 2.1 and 2.2, clearly showed that the increase or decrease in recombination depended on the attP or attR site ( Figures 3A-3B These results confirm that the attR site (i.e., the product of recombination between attP and attB mediated by Bxb1) almost does not allow for recombination to recur.

[0157] We employed a similar strategy to visualize the possibility of recombinase-mediated cassette exchange rather than whole plasmid integration. To confirm this, we created two heterozygous novel recipient cell lines with either an attP or attR site at the Rosa26 locus, between the polyadenylation signal and the FRT site of the PuroR gene (Fig. 4A–Fig. 4B). To prepare these cell lines, we performed dRMCE / MADR using the corresponding pDonors, exchanging the mTmG cassette for the new one. Next, we developed a promoter-free donor vector containing the reporter gene mScarlet, flanked by a loxP site and an attB site (pDonor-loxP-mScarlet-attB). Then, with or without plasmids expressing recombinases Bxb1 or Cre, we performed nuclear transfection with pDonor-loxP-mScarlet-attB (Fig. 4A–Fig. B, Reactions 3.1–4.3). In cells that express only Bxb1, there is almost no expression of mScarlet, regardless of whether the attP or attR site is "open" (or "locked") Figures 5A-5B (Reactions 3.1 and 4.1). This is possible because Bxb1 itself allows full plasmid integration, but mScarlet is not expressed because the pDonor vector carries multiple polyadenylation signals upstream of the open reading frame. Interestingly, mScarlet is expressed in both cell lines when both Bxb1 and Cre are expressed. Figures 5A-5B (Reactions 3.2 and 4.2), indicating that pDonors integration is mediated by Cre / loxP recombination. In the absence of recombinase, no mScarlet is expressed ( Figures 5A-5B (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, there is almost no mScarlet expression (…). Figure 6ASurprisingly, comparing mScarlet expression between responses 3.2 and 4.2 showed that more mScarlet-positive cells were observed in cell lines containing “open” attP sites. Figure 6B Therefore, we compared reactions 3.2 and 4.2 at different time points. Two days and seven days after nuclear transfection, we observed a decrease in the mScarlet signal over time in both reactions. Figures 7A-7B However, when comparing the changes in mScarlet quantification over time in the two reactions, it was clear that cells carrying the "locked" attR site no longer expressed mScarlet on day 7 post-nuclear transfection, demonstrating that all initial mScarlet expression was due to Cre / loxP recombination-mediated full plasmid integration and excision. Figure 8 (dashed line). On the other hand, in cell lines carrying the "open" attP site, some cells still expressed mScarlet on day seven after nuclear transfection. Figure 8 (Solid line). These data indicate that the integration of the mScarlet transgene is mediated by two different modes of recombination: reversible / bidirectional recombination via Cre / loxP and irreversible / unidirectional recombination via Bxb1 / attP / attB, termed bimodal recombinase-mediated cassette exchange (biRMCE). All previous strategies, data, and results validating indirect biRMCE reactions are available in […]. Figure 9 The table has been simplified. Note that all 12 responses strongly indicate that biRMCE occurred at the Rosa26 locus. biRMCE confirmed

[0158] We validated biRMCE by making some adjustments to our previous strategy. First, we prepared a novel heterozygous recipient cell line with an attP site at the Rosa26 locus, between the Cag promoter and the nuclear TagBFP2 gene, followed by an FRT site (Fig. 10A). To prepare this cell line, we performed dRMCE / MADR using the corresponding pDonor-2in1-loxP-attP-TagBFP2-nls-FRT to exchange the mTmG cassette for the new cassette. Next, we created a promoterless donor vector containing the reporter gene mScarlet, flanked upstream by the loxP and attB sites and downstream by the FRT site, named pDonor-2in1-loxP-attB-mScarlet-FRT (Fig. 10B). Then, we developed two novel plasmids, each expressing two recombinases under the same promoter within the same plasmid. These plasmids were pCag-Bxb1-FlpO and pCag-FlpO-Bxb1. As controls, we used pCag-FlpO-Cre and pCag-Bxb1 (Fig. 10C). Subsequently, we induced Cre expression to observe the excision of the mScarlet cassette in the presence of two loxP sites at the Rosa26 locus (Fig. 10D). We then proceeded to transfect the pDonor-2in1-loxP-attB-mScarlet-FRT nucleus into the heterozygous cell line 2in1-loxP-attP-TagBFP2-nls-FRT, using four different plasmids encoding the corresponding recombinases. Because the loxP and attP sites are upstream of nuclear TagBFP2 in the recipient cell line, while in pDonor the loxP and attB sites are upstream of mScarlet, we ensured mScarlet expression and loss of nuclear TagBFP2 in these target cells regardless of the type of recombinase plasmid used. We observed cells expressing only mScarlet under different conditions and purified the cells to generate four distinct cell lines. We theorized that when using the pCag-Bxb1 plasmid, the nuclear TagBFP2 cassette should still be present at the Rosa26 locus, as it promotes full plasmid integration; however, when using a plasmid expressing both recombinases in the same cistron, if dRMCE or biRMCE is present, the nuclear TagBFP2 cassette should not be present at the Rosa26 locus. To confirm this, we transfected CremRNA nuclei into different cell lines to observe Cre / loxP excision of the mScarlet cassette while simultaneously re-expressing nuclear TagBFP2.We observed numerous cells expressing nuclear TagBFP2 in cell lines previously induced with pCag-Bxb1 (Fig. 11A), while almost none expressed nuclear TagBFP2 in cell lines previously induced with pCag-FlpO-Cre via dRMCE (Fig. 11D). We also observed a minority of nuclear TagBFP2 cells in cell lines previously induced with pCag-Bxb1-FlpO or pCag-FlpO-Bxb1, indicating that previous mScarlet integration was mediated by biRMCE (Fig. 11B–11C). Quantitative analysis of nuclear TagBFP2 after Cre / loxP recombination confirmed that pCag-Bxb1 integrated the entire plasmid (Fig. 11E). Quantitative analysis of mScarlet after Cre / loxP recombination confirmed that both biRMCE and dRMCE integrated DNA flanked by their respective specific recombination sites (Fig. 11F). Stability and efficiency of biRMCE and dRMCE

[0159] After validating biRMCE, we compared its stability and efficiency with the well-characterized dRMCE / MADR (Anderson, Voziyanova, & Voziyanov, 2012; Kim et al., 2019; Osterwalder et al., 2010). For this purpose, we transfected the Rosa26-2in1-loxP-attP-TagBFP2-nls-FRT cell line with pDonor-2in1-loxP-attB-mScarlet-FRT and pCag-FlpO-Cre or pCag-Bxb1-FlpO, and tracked mScarlet expression at different time points. At the early time point, two days after nuclear transfection, dRMCE was more efficient than biRMCE. However, at the mid- and late time points, seven and eleven days after nuclear transfection, biRMCE maintained its efficiency compared to the significantly decreased dRMCE. Figure 12 This data demonstrates that biRMCE is more stable and therefore more effective than dRMCE in integrating transgenic elements. DNA integration locking via biRMCE

[0160] biRMCE is mediated by two different modes of recombination: an irreversible / unidirectional recombination and a reversible / bidirectional recombination. Therefore, as long as attP / attB recombination occurs between the promoter and the open reading frame, biRMCE should prevent further integration of DNA boxes into the Rosa26 locus once the first recombination has occurred. To demonstrate this, we compared biRMCE with dRMCE, as the latter is based on two reversible / bidirectional recombination reactions. We sought a strategy that uses two different pDonors, each containing a different reporter gene, namely nuclear mScarlet and nuclear EGFP. Each pDonor is flanked by a specific recombination site recognized only by the corresponding recombinase. Figure 13 At an early time point, two days after induction with both dRMCE / MADR and biRMCE, there were clearly more double-positive cells expressing mScarlet and EGFP under dRMCE conditions (Fig. 14A–14B). Flow cytometry analysis confirmed that there were significantly fewer double-positive cells under biRMCE conditions (Fig. 14C–14D). At a later time point, eight days after induction, there were even fewer double-positive cells under biRMCE conditions (Fig. 15). This demonstrates that biRMCE allows for rapid and stable transgenesis while reliably locking the system and preventing additional trans-recombination between the genome landing site and the exogenous intruder pDonor. Intron biRMCE verification

[0161] We developed an intronic version of biRMCE by adding the attP site embedded in an intron of the Rosa26 genome and adding a portion of the same intron downstream of the attB site in pDonor. This strategy allows for the removal of the Kozak sequence and the first “ATG” of the open reading frame from pDonor. To validate the intronic biRMCE, we created a new cell line by targeting the endogenous Rosa26 locus in mT / mG cells via dRMCE / MADR and pDonor-2in1-loxP-ATG-in-attP-TRON-(ATG-less-TagBFP-nls)-FRT (Fig. 16A). After cassette insertion, the resulting genetic “landing zone” of the Rosa26 locus should have a loxP site, followed by the “ATG” sequence, the attP site (embedded in the intron), the TagBFP-nls sequence (without the first ATG), and the FRT site (Fig. 16B). We continued nuclear transfection of heterozygous mT / mG cells using plasmid pCag-FlpO-Cre and two versions of pDonor-2in1-loxP-ATG-in-attP-TRON-(ATG-less-TagBFP-nls)-FRT. The difference between the pDonor versions was that one had the WPRE sequence while the other did not. We observed several cells expressing nuclear Tag-BFP using different versions of pDonor (Fig. 16C–Fig. 16H). These data suggest that the intron located between the first “ATG” exon and the open reading frame without the “ATG” sequence (with the attP site embedded) allows for the correct expression of the open reading frame (nuclear Tag-BPF). Because the resulting genetic “landing zone” of the Rosa26 locus has loxP, attP, and FRT sites, it should be compatible with both dRMCE / MADR and intron biRMCE by using the corresponding pDonor and recombinase (Fig. 17A). Two distinct genetic “landing regions” can be developed following either dRMCE / MADR or intron biRMCE (Fig. 17B). To confirm this, we isolated cells expressing nuclear Tag-BFP to prepare new cell lines. We then transfected the cells with the corresponding plasmids to confirm either dRMCE / MADR or intron biRMCE. We observed several cells expressing nuclear miRFP-670 using the corresponding pDonor and recombinase (Figs. 17C–17H). This data confirms that the “2-in-1” genetic landing region is compatible with both dRMCE / MADR and intron biRMCE. It also confirms that pDonor can be used to carry transgenes that are correctly expressed via intron biRMCE, even without the Kozak sequence and the first “ATG” of the open reading frame. Example 3 Experimental process Commercial cell lines

[0162] HEK293T cells derived from human embryonic kidneys (purchased from ATCC) were used to validate MADR and biRMCE in vitro. This cell line was maintained in DMEM high-glucose medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS, GlutaMAX (Life Technologies 35050), and penicillin-streptomycin-amphotericidal (Thermo Fisher Scientific, Waltham, MA). GAPDH-MADR / biRMCE human cell line generation

[0163] A GAPDH targeting vector was prepared via DNA synthesis. P2A-TagBFP2nls-Cag-loxP-attP-miRFP670nls-FRT was inserted into the GAPDH vector and used for transfection of human cells. The GAPDH targeting vector was designed to knock into the gene locus via homology-directed repair (HDR) after double-strand breaks (DSBs) induced by the Cas9 / sgRNA complex. Cell selection was performed by selecting TagbBFP2 and miRFP670 double-positive cells using flow cytometry. Selected stable cell lines were co-transfected with the 2in1-MADR / biRMCE-mScarlet donor vector and pCag-FlpO-Cre or pCag-Bxb1-FlpO to induce MADR or biRMCE, respectively. Cas9 protein, sgRNA and ribonucleoprotein (RNP) complex

[0164] Cas9 and sgRNA were purchased from IDT. The Cas9 protein and sgRNA were compounded in a 1:1 ratio to prepare the RNP complex. Cell lipid transfection

[0165] HEK cells were plated using coated plates containing 0.01% poly-L-lysine. When confluence was approximately 60-70%, cells were transfected using lipofectamine 3000 (Thermo Fisher Scientific). The culture medium was changed 24 hours after transfection and every 48 hours thereafter. Genotyping

[0166] PCR was used to verify the correct targeted integration at the GAPDH locus. PCR primers were designed to amplify the left and right sides of the knock-in site, the endogenous unedited GAPDH locus, and the second cistron across the CAG promoter and open reading frame. The PCR fragments were loaded onto a 1% agarose gel for visualization. The PCR fragments were extracted from the agarose gel and sent for Sanger sequencing for further validation. Example 4 result DNA integration locking via biRMCE

[0167] biRMCE is mediated by two different modes of recombination: an irreversible / unidirectional recombination and a reversible / bidirectional recombination. Therefore, as long as attP / attB recombination occurs between the promoter and the open reading frame, biRMCE should prevent further integration of DNA boxes into the Rosa26 locus once the first recombination has occurred. To demonstrate this, we compared biRMCE with dRMCE, as the latter is based on two reversible / bidirectional recombination reactions. We sought a strategy that uses two different pDonors, each containing a different reporter gene, namely nuclear miRFP670 and nuclear BFP. Each pDonor is flanked by a specific recombination site recognized only by the corresponding recombinase. Figure 23 At early time points, namely two days after dRMCE / MADR and biRMCE induction, there were clearly more double-positive cells expressing miRFP670 and BFP under dRMCE conditions (Fig. 24A-24B). Flow cytometry analysis confirmed that there were significantly fewer double-positive cells under biRMCE conditions (Fig. 24C-24D). At later time points, namely four and eight days after induction, no double-positive cells were observed under biRMCE conditions (Fig. 25). This demonstrates that biRMCE allows for rapid and stable transgenesis while reliably locking the system and preventing additional trans-recombination between the genome landing site and the exogenous intruder pDonor. Minimum recognition site of biRMCE

[0168] Previously, we validated and confirmed biRMCE, which utilizes specific recombination sites flanking open reading frames spanning hundreds to thousands of base pairs of DNA. Therefore, we sought to elucidate the minimal recognition site for biRMCE, namely, two specific recombination sites (irreversible and reversible sites) tightly linked together without any other DNA sequence in between (Fig. 19A). To demonstrate this, we prepared a heterozygous recipient cell line carrying four specific recombination sites at the Rosa26 locus. The attP recognition site is immediately upstream of the VloxP recognition site, followed by the TagBFP2 open reading frame and the FRT site. A loxP site is also located near the CAG promoter for downstream analysis (Fig. 19B). After several rounds of purification, we obtained a cell line with the novel Rosa26 locus (Fig. 19C). Next, we created a promoterless donor vector containing the reporter gene mScarlet, flanked upstream by loxP and attB sites and downstream by VloxP site, named pDonor-2in1-loxP-attB-mScarlet-VloxP (Fig. 19D). Then, the entire mScarlet vector was integrated using the pCag-Bxb1 plasmid, while only the mScarlet cassette flanked by attB and VloxP was inserted using the pCag-Bxb1-VCre plasmid (Figs. 19E-19F). After appropriate vector integration via Bxb1 or appropriate cassette exchange via Bxb1 and VCre, we selected mScarlet-positive cells to prepare pure cell lines carrying the new recombinant Rosa26 locus. If full vector integration occurs, two loxP sites are expected flanking the mScarlet open reading frame, while if cassette exchange via biRMCE is present, only one loxP site should be upstream of the mScarlet open reading frame (Fig. 20A-20B). Finally, Cre recombinase was induced in both cell lines to verify full plasmid integration or biRMCE (Fig. 20C-20D). In the mScarlet cell line prepared using Bxb1, several TagBFP2-positive cells (98%) were observed, indicating full integration of the pDonor vector. This signifies that the mScarlet open reading frame was excised, and the TagBFP2 open reading frame was returned to the vicinity of the CAG promoter for appropriate expression (Fig. 20C). On the other hand, in cell lines prepared using Bxb1 and VCr, TagBFP2-positive cells were very rare (3.5%), meaning that most mScarlet cells carry only one loxP site at the Rosa26 locus because cassette exchange avoids the incorporation of a second loxP site (Fig. 20D). All these data demonstrate that biRMCE can be performed when specific recombination sites are closely linked to each other and there are no other DNA sequences in between. biRMCE is compatible with the human genome.

[0169] We have previously validated biRMCE in the mouse genome, which, despite being a mammalian genome, differs significantly from the human genome. Therefore, it is important to incorporate transgenic elements into the human genome for multiple purposes, including therapeutics. Thus, we decided to incorporate the biRMCE element into the human genome to validate its functionality. To test biRMCE in the human genome, we engineered the human HEK293T cell line targeting the GAPDH locus (Figure 21A). The knock-in locus contains several genetic elements to facilitate cell selection and biRMCE testing. We added the TagBFP2 sequence downstream of exon nine of the GAPDH gene, along with a secondary cistron carrying the CAG promoter, loxP site, attP site, miRFP670 open reading frame, and FRT site (Figure 21B). After selecting cells that were double-positive for both nuclear TagBFP2 and nuclear miRFP670, we obtained the HEK [GAPDH-TagBFP2nls-Cag-2in1(loxP-attP)-miRFP67nls-FRT] cell line (Fig. 21C). PCR genotyping confirmed the correct knock-in at the human GAPDH locus (Fig. 21D). Because the second cistron carries the genetic element for testing MADR and biRMCE, we decided to use pDonor-2in1-loxP-attB-mScarlet-FRT, which is compatible with both MADR and biRMCE, to compare them side-by-side using a specific recombinase (Fig. 22A). Following MADR or biRMCE, the expected recombinant GAPDH site should express mScarlet in the second cistron (Fig. 22B). Transfection with pDonor-2in1-loxP-attB-mScarlet-FRT and recombinase plasmids PCag-FlpO-Cre or pCag-Bxb1-FlpO showed mScarlet-positive cells under both conditions (Fig. 22C). Quantitative analysis by flow cytometry showed that biRMCE was an order of magnitude more efficient than MADR (Fig. 22D). These data demonstrate that biRMCE can be performed in the human genome and is more efficient than MADR.

[0170] Various embodiments of the present invention have been described in the specific embodiments. While these descriptions directly depict the embodiments described above, it should be understood that modifications and / or variations of the specific embodiments shown and described herein can be conceived by those skilled in the art. Any such modifications or variations falling within the scope of this description are intended to be included therein. Unless specifically stated otherwise, it is the inventor's intention that the words and phrases in the specification and claims have the common and customary meaning to those skilled in the art.

[0171] The description of various embodiments of the invention known to the applicant at the time of filing this application has been presented and is intended for illustrative and descriptive purposes. This description is not intended to be exhaustive, nor is it intended to limit the invention to the precise forms disclosed, and many modifications and variations are possible in accordance with the foregoing teachings. The described embodiments are intended to explain the principles of the invention and its practical application, and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications suitable for the particular intended use. Therefore, it is intended that the invention be practiced not be limited to the specific embodiments disclosed.

[0172] While specific embodiments of the invention have been shown and described, changes and modifications will be readily apparent to those skilled in the art based on the teachings herein without departing from the invention and its broader aspects. Therefore, the appended claims are intended to cover all such changes and modifications falling within the true spirit and scope of the invention. As used herein, the term “comprising” is used when referring to compositions, methods, and their respective components that are useful to embodiments, but inclusion of unspecified elements (whether useful or not) is open-ended. Those skilled in the art will understand that, generally, the terms used herein are generally meant as “open-ended” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “at least having,” the term “includes” should be interpreted as “including but not limited to,” etc.). Although the open-ended term “comprising” is used herein as a synonym for terms such as including, containing, or having, alternative terms such as “consisting of” or “consisting essentially of” may also be used to describe the invention or any embodiment thereof.

[0173] Unless otherwise stated, the terms “a / an” and “the,” and similar references used in the context of describing a particular embodiment of the application (especially in the context of the claims), may be interpreted as encompassing both the singular and the plural. The description of ranges of values ​​herein is intended only as a convenient way to individually refer to each individual value falling within that range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if described separately herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or the context clearly contradicts this. The use of any and all instances or exemplary language (e.g., “as”) provided for certain embodiments is intended only to better clarify the application and does not constitute a limitation on the scope of the additionally claimed application. The abbreviation “eg” derives from the Latin word *exempligratia* and is used herein to denote a non-limiting example. Therefore, the abbreviation “eg” is synonymous with the term “for example.” No language in the specification should be construed as indicating that any unclaimed element is essential for implementing the application.

[0174] "Optional" or "optionally" means that the situation described below may or may not occur, so the description includes instances where the situation occurs and instances where it does not occur.

[0175] The grouping of alternative elements or embodiments disclosed herein should not be construed as limiting. Each member of a group may be cited and claimed individually or in any combination with other members of that group or other elements present herein. One or more members of a group may be included in or removed from a group for convenience and / or patentability reasons. When any such inclusion or removal occurs, the specification herein is deemed to include the modified group, thereby satisfying the written description of all Markush groups used in the appended claims.

Claims

1. A system comprising: (a) A donor carrier, the donor carrier comprising: (i) One or more polyadenylation signaling or transcription termination elements located upstream of the transgene or the nucleic acid encoding RNA. (ii) the genetic modification or the nucleic acid encoding the RNA, and (iii) A recombinase recognition site, wherein the recombinase recognition site includes at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site; (b) Two recombinases that are specific to the recognition site of the recombinase.

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

3. The system of claim 1, further comprising a mammalian cell containing a locus targeted by the donor vector and the two recombinases; and optionally the at least a third recombinase.

4. The system according to any one of claims 1-3, wherein, The two recombinases are provided by the following: (i) A single expression vector containing two genes, said two genes encoding recombinases specific to their recognition sites, or (ii) Two expression vectors, the first expression vector containing a gene encoding a first recombinase specific to the unidirectional recombinase recognition site, and the second expression vector containing a gene encoding a second recombinase specific to the bidirectional recombinase recognition site, or (iii) A single mRNA encoding two recombinases that are specific to their recognition sites, or (iv) Two mRNAs, the first mRNA encoding a first recombinase specific to the unidirectional recombinase recognition site, and the second mRNA encoding a second recombinase specific to the bidirectional recombinase recognition site, or (v) A single viral vector containing two genes, said two genes encoding recombinases specific to their recognition sites, or (vi) Two viral vectors, the first viral vector containing a gene encoding a first recombinase specific to the unidirectional recombinase recognition site, and the second viral vector containing a gene encoding a second recombinase specific to the bidirectional recombinase recognition site, or (vii) A single recombinant protein comprising the single-direction recombinase and the double-direction recombinase, or (viii) Two recombinant proteins, a first recombinase protein that is specific to the unidirectional recombinase recognition site, and a second recombinase protein that is specific to the bidirectional recombinase recognition site.

5. The system according to claim 4, wherein In the single expression of two genes described in (i), wherein, The two genes encode recombinases that are specific to their recognition sites, and the encoded recombinases are fused together; or In (iii) a single mRNA encoding two recombinases specific to their recognition sites, the two recombinases are fused together; or In the single viral vector containing two genes described in (v), wherein the two genes encode recombinases specific to their recognition sites, the encoded recombinases being fused together; or In (viii), the two recombinant proteins are fused together.

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

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

8. The system according to any one of claims 2-7, wherein, The at least third recombinase is provided by: (iv) A single expression vector comprising a gene encoding at least the third recombinase that is specific to the recognition site of the third recombinase, or (x) A single mRNA encoding the at least third recombinase that is specific to the at least third recognition site, or (xi) A single viral vector comprising a gene encoding at least a third recombinase that is specific to the recognition site of the at least third recombinase, or (xii) A single recombinant protein comprising the at least third recombinase having specificity for the recognition site of the at least third recombinase.

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

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

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

12. The system according to any one of claims 1-11, 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 the intron or a portion of an intron.

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

14. The system according to any one of claims 1-12, 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 mutant thereof.

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

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

17. The system according to any one of claims 2-12, 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 mutants thereof.

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

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

20. The system according to any one of claims 1-17, 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 1-17, wherein, One or both of the recombinase recognition sites contain mutations.

22. The system according to any one of claims 1-21, wherein, The donor vector is selected from the group consisting of: plasmids, linear PCR, linear single-stranded DNA, closed-terminal double-stranded DNA, circular single-stranded DNA, circular double-stranded DNA, RNA, microcircles, 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 1-23, wherein, The donor vector contains at least four polyadenylation signals located upstream of the transgene or the nucleic acid encoding the RNA.

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

26. The system according to any one of claims 1-25, wherein, The donor vector further includes post-transcriptional regulatory elements.

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

28. The system according to any one of claims 1-27, wherein, The donor carrier further includes an open reading frame (ORF) beginning with the cuticle acceptor.

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

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

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

32. The system according to any one of claims 1-30, wherein, The genetic modification or the RNA contains disease-related mutations.

33. The system according to any one of claims 1-30, wherein, The transgene or the RNA contains 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 1-33, wherein, 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, kinin family member (KIF11) locus, TRAC locus, ZAP-70 locus, T cell activation adaptor protein (LAT) locus, or lymphocyte cytoplasmic protein 2 (LCP2) locus, and the method is an in vitro, ex vivo, or in vivo method.

35. The system according to claim 34, wherein, The locus contains a first polynucleotide encoding a first protein, 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 of a second protein.

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

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 locus.

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

39. The system according to claim 38, wherein, The locus contains a first polynucleotide encoding a first protein, 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 of a second protein.

40. A method for genetic manipulation of mammalian cells, the method comprising: The mammalian cells are transfected or transduced using the system described in any one of claims 1-39.

41. The method according to claim 40, wherein, The system targets a locus containing a recombinase recognition site, which includes 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, One-way recombination is located upstream of two-way recombination at the locus.

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

44. The method according to any one of claims 40-42, 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, kinin family member (KIF11) locus, TRAC locus, Zap-70 locus, T cell activating adaptor protein (LAT) locus, or lymphocyte cytoplasmic protein 2 (LCP2) locus, and the method is an in vitro, ex vivo, or in vivo method.

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

46. ​​The method according to any one of claims 40-45, wherein, The one or more recombinases include Bxb1 recombinase, Cre recombinase, flippase recombinase, Nigri recombinase, Panto recombinase, Vika recombinase, VCre recombinase, or SCre recombinase.

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

48. A non-human animal model, said non-human animal model comprising: Non-human animals containing the system of any one of claims 1 to 39.

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

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

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 of tumor suppressor genes, gain-of-function (GOF) mutations of proto-oncogenes, pseudogenes, siRNA, shRNA, sgRNA, pegRNA, crRNA, lncRNA, miRNA, epigenetic modifications, non-coding genetic or epigenetic abnormalities associated with human diseases, and combinations thereof.

52. The non-human animal model according to any one of claims 48-51, wherein the non-human animal model comprises gain-of-function (GOF) mutations, loss-of-function (LOF) mutations, or both.

53. The non-human animal model according to any one of claims 48-52, wherein, The system targets a locus in the non-human animal model, and the locus contains a recombinase recognition site, which includes 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 48-53, wherein, One-way recombination is located upstream of two-way recombination at the locus.

55. A method for generating non-human animal models, the method comprising: The non-human animal model is transfected or transduced using the system described in any one of claims 1-29.

56. A non-human animal model generated by the method of claim 55.

57. A method for evaluating the efficacy of a drug candidate, the method comprising: Provide a non-human animal model according to any one of claims 48-54 or 56; The drug candidate was administered to the non-human animal model; as well as The effects of the drug candidate on the non-human animal model were evaluated.

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

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

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