Method for site-specific introduction of genetic elements in engineered loci by bimodal recombinase-mediated cassette exchange (birmce)

Abstract

Described herein are systems and methods for genetic manipulation of mammalian cells. Also described are non-human animal models utilizing these systems, methods of generating these non-human animals, and methods of using these non-human animals.

Description

METHOD FOR SITE-SPECIFIC INTRODUCTION OF GENETIC ELEMENTS IN ENGINEERED LOCI BY BIMODAL RECOMBINASE-MEDIATED CASSETTE EXCHANGE(BIRMCE)CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application includes a claim of priority under 35 U.S.C. § 119(e) to U.S. provisional patent application No. 63 / 523,550, filed June 27, 2023, the entirety of which is hereby incorporated by reference.FIELD OF INVENTION

[0002] This invention relates to genetic manipulation: for example, in cells, organ models, and non-human animal models.BACKGROUND

[0003] All publications herein are incorporated by reference to tire same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Tire following description includes information that may be useful in understanding the present invention. It is not an admission that any of the 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 edition of genetic elements into specific loci is a very difficult task. Although there are different technologies trying to achieve this, it is still not possible to do it in a quick, clean, specific, cheap, and effortless way. MADR technology is a technology that can do it with the aforementioned features. One of the “Achilles heels” of MADR, and other similar technologies, is that it depends on the elimination of the exogenous genetic elements in order to close the system and to have the expression of only one exogenous genetic element. Tirus, the purpose of Bimodal Recombinase-Mediated Cassette Exchange (biRMCE) is that it can speed the stable expression of one exogenous genetic element even in the presence of different genetic elements. This principle enhances the use of genetic cassette exchanges for different applications where stable integration and / or edition of genetic elements into specific loci is desired.

[0005] Genetic cassette exchanges through recombinases arc techniques used to integrate exogenous genetic elements into engineered loci in different types of cells. For example, the use of one recombinase targeting its heterotypic recognition sites, nevertheless, it has been found that there can becross recombination between the heterotypic sites causing confounds. Other similar works use integrases targeting their respective recognition sites but the main issue with these systems is that they integrate not only the genetic cassettes elements but also the complete genetic vector which carries non-desired sequences. One existing solution is the sequential or simultaneous use of different types of recombinases, e.g., Flp & Cre, to avoid cross recombination between the recognition sites and to increase the efficiency of genetic cassette exchanges. However, recombinases perform reversible recombination reactions that make the system non-stable and not efficient when genetic cassettes earn ing different types of genetic elements are used.

[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 OF THE INVENTION

[0007] Tire 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 in scope.

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

[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 to the at least 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 third recombinase.

[0011] In various embodiments, the two recombinases can be provided by(i) one expression vector, comprising two genes encoding recombinases specific to their recognition sites, or(ii) two expression vectors, a first expression vector comprising one gene encoding a first recombinase that is specific to the unidirectional recombinase recognition site, and a second expression vector comprising one gene encoding a second recombinase that is specific to the bidirectional recombinase recognition site, or(iii) one mRNA encoding the two recombinases specific to their recognition sites, or(iv) two mRNA. a first mRNA encoding a first recombinase that is specific to the unidirectional recombinase recognition site, and the second mRNA encoding a second recombinase that is specific to the bidirectional recombinase recognition site, or(v) one viral vector comprising two genes encoding recombinases specific to their recognition sites, or(vi) two viral vectors, a first viral vector comprising one gene encoding a first recombinase that is specific to the unidirectional recombinase recognition site, and a second viral vector comprising one gene encoding a second recombinase that is specific to the bidirectional recombinase recognition site, or(vii) one recombinant protein comprising the unidirectional recombinase and the bidirectional 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 one expression vector comprising two genes encoding recombinases specific to their recognition sites, the encoded recombinases can be fused together. In various embodiments, in (iii) the one mRNA encoding the two recombinases specific to their recognition sites, the encoded two recombinases can be fused together. In various embodiments, in (v) the one viral vector comprising two genes encoding recombinases specific to their recognition sites, the encoded recombinases can be fused together. In various embodiments, in (viii) the two recombinant proteins can be fused together.

[0013] In various embodiments, any one of tire recombinase can be fused to one or more proteins other than the recombinase. In various embodiments, any one of the two fused recombinases can be further fused to one or more proteins other than the recombinase.

[0014] In various embodiments, the at least a third recombinases can be provided by(iv) one expression vector, comprising a gene encoding the at least third recombinase specific to the third recombinase recognition site, or(x) one mRNA encoding the at least third recombinase specific to the at least third recognition site, or(xi) one viral vector comprising a gene encoding the at least third recombinase specific to the at least third recombinase recognition site, or(xii) one recombinant protein comprising the at least third recombinase that is specific to the at least third recombinase recognition site.

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

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

[0017] In various embodiments, the unidirectional recombinase recognition site can be upstream from the bidirectional recombinase recognition site. In various embodiments, the unidirectional recombinase recognition site can be downstream to a promoter.

[0018] In various embodiments, the donor vector can further comprise an intron, part of an intron, or at least one splice acceptor site, and optionally, the unidirectional recombinase recognition site is embedded into an intron or part of the intron.

[0019] In various embodiments, the unidirectional recombinase can be Bxbl. In various embodiments, the unidirectional recombinase can be selected from Bxbl, Phic31. PhiBTl, PhiCl, MR11, R4, TP901-1, Al 18. FC1, PhiRV, TGI, Phi370.1. Wp, BL3, SPBc, K38, and any mutants thereof.

[0020] In various embodiments, the bidirectional recombinase can be Flp. In various embodiments, the unidirectional recombinase can be Bxb 1 and the bidirectional recombinase is selected from FLp, Cre, VCre, SCre, Nigri, Panto, Vika, or a mutant thereof.

[0021] In various embodiments, the third recombinase can be selected from Bxbl, Phic31, PhiBTl. PhiCl, MR11, R4, TP901-1, Al 18. FC1, PhiRV, TGI, Phi370.1, W , BL3. SPBc, K38, FLp. Cre, VCre, SCre, Nigri, Panto, Vika, or a mutant thereof.

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

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

[0024] In various embodiments, one or both of the recombinase recognition sites can comprise a mutation.

[0025] In various embodiments, the donor vector can be selected from tire group consisting of plasmid, linear PCR, linear single -stranded DNA, close-ended double-stranded DNA, circular singlestranded DNA, circular double-stranded DNA, RNA, minicircle, viral vector, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), and human artificial chromosome (HAC). In various embodiments, the viral vector can be an adeno-associated viral (AAV) vector.

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

[0027] In various embodiments, the expression vector comprising recombinases can be under tissue-specific promoters.

[0028] In various embodiments, the RNA can be siRNA, shRNA, sgRNA, crRNA, pegRNA, IncRNA or miRNA. In various embodiments, the transgene or the RNA can comprise disease associated mutations. In various embodiments, the transgene or the RNA can comprise a gain-of-function (GOF) gene mutation, loss-of-fiinction (LOF) gene mutation, or both.

[0029] In various embodiments, the mammalian cell can be a human cell, and the locus is an AAVS1 locus, Hl l locus, HPRT1 locus, Rogil locus, Rogi2 locus, GAPDH locus, TATA-Box Binding Protein (TBP) locus, Kincsin Family Member (KIF11) Locus, TRAC locus, ZAP-70 locus, Linker of Activation of T cells (LAT) locus, or Lymphocyte Cytosolic Protein 2 (LCP2 ) locus, and the method is an in vitro, ex vivo, or in vivo method.

[0030] In various embodiments, the locus can comprise a first polynucleotide encoding a first protein, a secondary cistron comprising a promoter, recombinase recognition sites recognized by arecombinases in the system, a second polynucleotide encoding open reading frame for a second protein. In various embodiments, the first protein or the second protein or both can be fluorescent proteins. In various embodiments, the first polynucleotide encoding the protein can be downstream of a gene of the locus.

[0031] In various embodiments, the mammalian cell can be a mouse cell, and the locus is ROSA26 locus, Hipp 11 locus, Tigre locus, ColAl locus. Hprt locus, GAPDH locus, TATA-Box Binding Protein (TBP) locus, Kinesin Family Member (KIF11) Locus, TRAC locus, Zap-70 locus, Linker of Activation of T cells (LAT) locus, or Lymphocyte Cytosolic Protein 2 (LCP2 ) locus, and the method is an in vitro, ex vivo, or in vivo method.

[0032] In various embodiments, the locus can comprise a first polynucleotide encoding a first protein, a secondary cistron comprising a promoter, a recombinase recognition sites recognized by a recombinases in the system, a second polynucleotide encoding open reading frame for a second protein.

[0033] Various embodiments provide for a method of genetic manipulation of a mammalian cell, comprising: transfecting or transducing the mammalian cell with any one of the systems of the present invention.

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

[0035] In various embodiments, a unidirectional recombination can be upstream from a bidirectional recombination on tire locus.

[0036] In various embodiments, the mammalian cell can be a human cell, the system targets an AAVS1 locus, Hl l locus, HPRT1 locus, Rogil locus, Rogi2 locus, GAPDH locus, TATA-Box Binding Protein (TBP) locus, Kinesin Family Member (KIF11) Locus, TRAC locus, ZAP-70 locus, Linker of Activation of T cells (LAT) locus, or Lymphocyte Cytosolic Protein 2 (LCP2 ) locus, and the method is an in vitro, ex vivo, or in vivo method.

[0037] In various embodiments, the mammalian cell can be a mouse cell, and the system targets a ROSA26 locus. Hippl 1 locus, Tigre locus, ColAl locus, Hprt locus, GAPDH locus, TATA-Box Binding Protein (TBP) locus, Kinesin Family^ Member (KIF11) Locus, TRAC locus, Zap-70 locus, Linker of Activation of T cells (LAT) locus, or Lymphocyte Cytosolic Protein 2 (LCP2 ) locus, and the method is an in vitro, ex vivo, or in vivo method.

[0038] In various embodiments, the method can further comprise administering to the cell one or more recombinase enzymes.

[0039] In various embodiments, the one or more recombinase enzymes can comprise a Bxbl recombinase, a Cre recombinase, a flippase recombinase, a Nigri recombinase, a Panto recombinase, a Vika recombinase, VCrc recombinase, or SCrc recombinase.

[0040] In various embodiments, the mammalian cell can comprise a blood cell, a tumor cell, a non-tumor cell, an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a tissue precursor cell.

[0041] Various embodiments provide for a non-human animal model, comprising: a non-human animal comprising a system of the present invention.

[0042] In various embodiments, the non-human animal model can 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 can be a personalized non-human animal model 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 can be selected from the group consisting of an oncogene, loss-of-function (LOF) mutation of a tumor suppressor gene, gain-of-function (GOF) mutation of a proto-oncogene, pseudogene, siRNA, shRNA, sgRNA, pegRNA, crRNA, IncRNA, miRNA, epigenetic modification, non-coding genetic or epigenetic abnormality associated with human disease, and combinations thereof.

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

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

[0046] Various embodiments provide for a method of generating the non-human animal model, comprising: transfecting or transducing the non-human animal model with a system the present invention.

[0047] Various embodiments provide for a non-human animal model generated by the method of the present invention.

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

[0049] Various embodiments provide for a mammalian cell comprising the system of the present invention.

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

[0051] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.BRIEF DESCRIPTION OF THE FIGURES

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

[0053] Figures 1A-1B show a diagram of the strategy to compare the efficiency of Bxbl separately or together with FlpO to integrate transgenic elements in the presence of different Bxbl recognition sites into the Rosa 26 locus. 1A shows the Rosa26 locus bearing a PuroR gene flanked by an “open” attP site and by an FRT site. Promoter-less donor vectors carry an mScarlet transgene flanked by an attB site and an FRT site. Recombination between the genomic locus and donor vectors is expected in the presence of Bxbl or Bxbl+FlpO. IB shows the Rosa26 locus bearing a PuroR gene flanked by a “locked” attR site and by an FRT site. Promoter-less donor vectors cany' an mScarlet transgene flanked by an attB site and an FRT site. Recombination between the genomic locus and donor vectors is expected in the presence of Bxbl or Bxbl+FlpO. PuroR stands for a puromycin resistance gene. Bxbl+FlpO are expressed in separate plasmids.

[0054] Figures 2A-2B show that the insertion of transgenic elements via Bxb 1 integration or Bxb 1+FlpO relies on the attP recognition site embedded in the Rosa26 locus. 2A shows that the integration and expression of mScarlet can be achieved in the presence of an “open” attP site in the Rosa26 locus, in the presence of an attB site on tire donor vectors, and in the presence of Bxbl or Bxbl+FlpO (Rx 1.1 and 1.2). 2B shows a greatly reduced integration and expression of mScarlet in the presence of a “locked” attR site in the Rosa26 locus, while still having the presence of an attB site on the donor vectors, and in the presence of Bxbl or Bxbl+FlpO (Rx 2.1 and 2.2). The non-expression of the recombinases was used as negative control(Rx 1.3 and 2.3). Epifluorescence microscopy images were taken 48h post nucleofection. mScarlet expression was measured in 10,000 cells by flow cytometry 48h post nucleofection. Heterozygous mouse neural stem cells carrying the respective ‘2inl’ landing pad in the Rosa26 locus, see Figure 1, were used.

[0055] Figures 3A-3B show the number of cells expressing mScarlet into the Rosa26 locus via Bxbl or Bxbl+FlpO recombination. 3A shows a graph comparing the quantification of cells expressing mScarlet after Bxbl recombination into the Rosa26 locus with an “open” attP site or a “locked” attR site. 3B shows a graph comparing the quantification of cells expressing mScarlet after Bxbl+FlpO recombination into tire Rosa26 locus with an “open” attP site or a “locked” attR site. This data shows that integration of the transgenic elements relies on the recombination of attP / attB recognition sites through Bxbl.

[0056] Figures 4A-4B show a diagram of the strategy to compare the efficiency of Bxbl separately or together with Cre to integrate transgenic elements in tire presence of different Bxbl recognition sites into the Rosa 26 locus. 4A shows the Rosa26 locus bearing a PuroR gene flanked by a loxP site and by an “open” attP site. Promoter-less donor vectors carry an mScarlet transgene flanked by a loxP site and by an attB site. Recombination between the genomic locus and donor vectors is expected in the presence of Bxbl or Bxbl+Cre. 4B shows the Rosa26 locus bearing a PuroR gene flanked by a loxP site and by a “locked” attR site. Promoter-less donor vectors carry an mScarlet transgene flanked by a loxP site and by an attB site. Recombination between the genomic locus and donor vectors is expected in the presence of Bxbl or Bxbl+Cre. PuroR stands for a puromycin resistance gene. Bxbl+Cre are expressed in separate plasmids.

[0057] Figure 5A-5B shows that the insertion of transgenic elements via Bxbl integration or Bxbl+Cre relies on the loxP recognition site embedded in the Rosa26 locus. 5 A shows that the integration and expression of mScarlet can be achieved in the presence of an “open” attP site in the Rosa26 locus, in the presence of an attB site on the donor vectors, and in the presence of Bxbl+Cre (Rx 3.2). 5B shows a reduced integration and expression of mScarlet in the presence of a “locked” attL site in the Rosa26 locus, while still having the presence of an attB site on the donor vectors, and in the presence of Bxbl+Cre (Rx 4.2). The non-expression of the recombinases was used as negative control (Rx 3.3 and 4.3). Epifluorescence microscopy images were taken 48h post nucleofection. mScarlet expression was measured in 10,000 cells by flow cytometry 48h post nucleofection. Heterozygous mouse neural stem cells carrying the respective “2inl” landing pad in the Rosa26 locus, see Figure 4, were used.

[0058] Figures 6A-6B show the number of cells expressing mScarlet into the Rosa26 locus via Bxbl or Bxbl+Cre recombination. 6A shows a graph comparing the quantification of cells expressing mScarlet after Bxbl recombination into the Rosa26 locus with an “open” attP site or a “locked” attL site. 6B shows a graph comparing the quantification of cells expressing mScarlet after Bxb 1+Cre recombination into the Rosa26 locus with an “open” attP site or a “locked” attL site. This data shows that integration of the transgenic elements relies on tire Cre / loxP recombination.

[0059] Figures 7A-7B show the expression of mScarlet+ cells at different time points via Bxbl+Cre recombination into the Rosa26 locus with different Bxbl recognition sites. 7A shows the expression and quantification of mScarlct in cells that have an “open” attP site downstream of the PuroR gene while Bxbl+Cre are expressed (Rx 3.2). 7B shows the expression and quantification of mScarlet in cells that have a “locked"’ attL site downstream of the PuroR gene while Bxb 1+Cre are expressed (Rx 4.2). Epifluorescence microscopy images were taken at 2-, 4-, and 7-days post nucleofection. mScarlet expression was measured in 10,000 cells by flow cytometry at 2-, 4-, and 7-days post nucleofection.

[0060] Figure 8 shows a graph comparing tire expression of mScarlet at 2-, 4-, and 7-days post nucleofection, in percentage, of the recombination reactions illustrated in Figure 7. The dashed line shows that the integration of transgenic elements that is mediated through Cre / loxP recombination is non-stable, and 7 days post nucleofection there are less than 1% of cells expressing mScarlet. The continuous line shows that the integration of transgenic elements that are mediated through both recombination reactions Cre / loxP & Bxbl / attP / attB is more stable. Y-axis describes the mScarlet+ cells in percentage. X-axis describes the days after the induction of the recombination reactions. This data suggests that it is possible to integrate transgenic elements through reversible / bidirectional and irreversible / unidirectional recombination reactions, i.e.: a Cre / loxP reversible / bidirectional recombination and a Bxbl / attP / attB irreversible / unidirectional recombination.

[0061] Figure 9 shows a table simplifying the data from figure 1 to figure 8. Reactions 1.1 to 2.3 demonstrates that attP / attB recombination is necessary to have irreversible / unidirectional integration through Bxbl. Reactions 3.1 to 4.3 suggests that it is possible to have Bimodal Recombinase-Mediated Cassette Exchange (biRMCE), i.e.: two modes of recombination: a Cre / loxP reversible / bidirectional recombination and a Bxbl / attP / attB irreversible / unidirectional recombination.

[0062] Figures 10A-10D show a diagram of the strategy to compare whole plasmid integration versus recombinase-mediated cassette exchange. 10A show s the representation of tire “landing pad” 2inl- loxP-attP-TagBFP2-nls-FRT into the Rosa26 locus. 10B show s the pDonor-2inl-loxP-attB-mScarlet-FRT carrying different recombination sites. 10C show s the Rosa26 locus after the recombination of the pDonor- 2inl-loxP-attB-mScarlet-FRT mediated by the expression of the different recombinases. 10D shows the Rosa26 locus after Cre / loxP recombination.

[0063] Figures 11A-11F shows DNA Cassette Exchange Validation via biRMCE. Panels 11A- 1 ID show the expression of a cytoplasmic red fluorescent protein (mScarlct) and / or nuclear blue fluorescent protein (TagBFP2). Expression of nuclear TagBFP2 reveals the cells that previously integrated a transgenic mScarlet through the integration of the whole plasmid instead of the DNA cassette exchange. Expressionof TagBFP2 is dependent on the Cre / loxP excision of the mScarlet cassette. Panel 11A shows several TagBFP2+ cells revealing the previous whole plasmid integration via Bxbl. Panels 1 IB-11C show barely any TagBFP+ cells demonstrating the previous DNA cassette exchange via biRMCE using Bxbl-FlpO or FlpO-Bxbl, respectively. Panel 11D shows a few TagBFP+ cells indicating the previous DNA cassette exchange via dRMCE using FlpO-Cre. Epifluorescence microscopy images were taken 48h post expression of Cre. Panel HE shows a graph with the percentage of cells expressing TagBFP2 from panels A to D confirming that expression of Bxbl by itself induces whole plasmid integration instead of cassette exchange. Panel 1 IF shows a graph with the percentage of cells expressing mScarlet from panels B to D. This shows that both biRMCE and dRMCE integrate transgenic elements through DNA cassette exchange with high efficiency (>95%). Flow cytometry was performed to quantify cells expressing the fluorescent reporters at 48h post nucleofection. These data demonstrate that biRMCE and dRMCE integrate transgenic elements into a defined locus through DNA cassette exchange.

[0064] Figure 12 shows that biRMCE is more stable and efficient than dRMCE to integrate transgenic elements into recipient genomic DNA. Graph showing the efficiency of the expression of transgenic elements into the Rosa26 locus at different time points. Y-axis describes the percentage of mScarlet+ cells. X-axis describes the days after the induction of the recombination. The dashed line shows the kinetics of dRMCE. Tire continuous line shows the kinetics of biRMCE. This result demonstrates that biRMCE is a stable reaction to integrate transgenic elements into a defined locus.

[0065] Figure 13 shows a diagram of the strategy to validate the lock-in of DNA cassette exchanges through biRMCE. A colorless cell line with one landing pad is used to compare dRMCE & biRMCE side by side. The cell line has into the Rosa26 locus a CAG promoter followed by a loxP site, an attP site, a puromycin resistance gene (PuroR), and a FRT site. Hie cell line is named Rosa26-2inl-loxP- attP-PuroR-FRT. For dRMCE three plasmids were used ( 1 : Promoter-less donor vector-Zox -EGFP-nls- FRT; 2: Promoter-less donor vector- / oxP-Scarlet-nls-FRT; 3: pCag-FlpO-Cre). For biRMCE three plasmids were used (1: Promoter-less donor vector-affB-EGFP-nls-FRT; 2: Promoter-less donor vector- oftB-Scarlct-nls-FRT; 3: pCag-Bxbl-FlpO).

[0066] Figure 14 shows that biRMCE locks the exchange of DNA cassettes in the landing pad of the Rosa26 locus at an early time. The top panels show cells expressing nuclear mScarlet and / or nuclear EGFP via dRMCE (A) or biRMCE (B). Arrowheads indicate cells expressing both mScarlet and EGFP. Lower panels show flow cytometry' quantification of cells expressing nuclear mScarlet and / or nuclear EGFP via dRMCE (C) or biRMCE (D), yellow cells are double positives to mScarlet and EGFP. Pictures were taken two days post-nucleofection with an epifluorescence microscope. Flow cytometry was perforated twodays post-nucleofection. These data demonstrate that biRMCE locks the DNA cassette exchanges since early time points.

[0067] Figure 15A-15D shows that biRMCE keeps the lock-in of the exchange of DNA cassettes in the landing pad of the Rosa26 locus at a late time. The top panels show cells expressing nuclear mScarlet and / or nuclear EGFP via dRMCE (A) or biRMCE (B). Arrowheads indicate cells expressing both mScarlet and EGFP. Lower panels show flow' cytometry quantification of cells expressing nuclear mScarlet and / or nuclear EGFP via dRMCE (C) or biRMCE (D), yellow cells are double positives to mScarlet and EGFP. Note that there are fewer color cells in the dRMCE condition indicating that the integration is not as stable as biRMCE. Pictures were taken eight days post-nucleofection with an cpifluorcsccncc microscope. Flow' cytometry was performed eight days post-nucleofection. These data demonstrate that biRMCE keeps the lock-in of DNA cassette exchanges.

[0068] Figure 16A-16H shows the strategy and the validation of a genetic landing containing genetic elements to be compatible with both dRMCE / MADR and intronic biRMCE. Figure 16A show's a diagram of the mTmG landing pad of the Rosa26 locus and a promoter-less donor vector ready to insert its flanked DNA cassette through dRMCE / MADR. Figure 16B shows the new landing pad of tire Rosa26 locus containing an intron, with an attP site on it, between the first ATG and the rest of the open reading frame (ATG-less TagBFP-nls). This new landing pad has all the elements to be compatible with both dRMCE / MADR and intronic biRMCE. Panels C-E show the expression, by fluorescence microscopy and flow' cytometry, of the nuclear Tag-BFP-WPRE after dRMCE / MADR reaction in heterozygous mTmG neural stem cells. Panels F-H show' the expression, by fluorescence microscopy and flow' cytometry, of the nuclear Tag-BFP without the WPRE sequence after dRMCE / MADR reaction in heterozygous mTmG neural stem cells. Arrowheads indicate some cells expressing nuclear Tag-BFP. Pictures were taken two days post-nucleofection with an epifluorescence microscope. Flow cytometry was performed two days post- nucleofection.

[0069] Figure 17A-H shows the strategy and the validation of a genetic landing pad that is compatible with both dRMCE / MADR and intronic biRMCE. Figure 17A show s a diagram of the “2inl- loxP-ATG-in-attP-TRON-(ATG-less-TagBFP-nls)-FRT” landing pad of the Rosa26 locus and a couple of promoter-less donor vectors ready to insert its flanked DNA cassette through dRMCE / MADR or intronic biRMCE. Figure 17B shows the new landing pads of the Rosa26 locus after dRMCE / MADR or intronic biRMCE. Figure 17C-17E show the expression, by fluorescence microscopy and flow' cytometry, of the nuclear miRFP-670 after dRMCE / MADR reaction in heterozygous TagBFP neural stem cells. Figure 17F- 17H show the expression, by fluorescence microscopy and flow cytometry, of the nuclear miRFP-670 after intronic biRMCE reaction in heterozygous TagBFP neural stem cells. Arrowheads indicate cells expressingnuclear miRFP-670. Pictures were taken two days post-nucleofection with an epifluorescence microscope. Flow cytometry was performed two days post-nucleofection.

[0070] Figure 18 shows a diagram of the strategy to validate the lock-in of DNA cassette exchanges through intronic biRMCE. Intronization of both the landing pad of the Rosa 26 locus and donor plasmids are used to validate the lock-in of the intronic biRMCE. The landing pad has a CAG promoter followed by a loxP site, an ATG start codon, an intron with an attP embedded on it, any ATG-less genetic element, and an FRT site. The landing pad is named Rosa26-2inl-loxP-in-attP-TRON-(ATG-less-Gene)- FRT. Promoter-less donor plasmids contain genetic elements, without a start codon ATG, flanked by an attB site with a part of an intron and an FRT site. Bxbl and Flp recombinases arc necessary to perform intronic biRMCE.

[0071] Figure 19 (panels A-F) shows a diagram of the strategy to validate the minimal recognition site for biRMCE. A) shows the representation of the minimal recognition site for biRMCE in which the “irreversible” recognition site is immediately upstream of the “reversible” recognition site. B) shows tire representation of the “landing pad” 3inl-loxP-(attP / VloxP)-TagBFP2-nls-FRT into the Rosa26 locus. C) shows a heterozygous neural stem cell line expressing nuclear TagBFP2, the cell line carries the landing pad described in figure B. D) shows the pDonor-2inl-loxP-attB-mScarlet-VloxP carrying different specific recombination sites. E) shows the Rosa26 locus after recombining the pDonor-2inl-loxP-attB- m Scarlet- VI oxP via Bxbl. F) shows the Rosa26 locus after recombining the pDonor-2inl-loxP-attB- mScarlet-VloxP via Bxbl and VCre.

[0072] Figure 20 (panels A-D) shows DNA Cassette Exchange Validation using the minimal recognition site for biRMCE. A) shows a heterozygous mScarlet cell line carrying a landing pad obtained after Bxb 1 recombination. B) shows a heterozygous mScarlet cell line carrying a landing pad obtained after Bxbl and VCre recombination. C) shows the mScarlet cell line, described in figure A, expressing nuclear TagBFP2 after Cre recombination. Flow cytometry quantification indicates that 98% of the cells are TagBFP2 positive. The expected landing pad is shown at the bottom of the panel. D) shows the mScarlet cell line, described in figure B, expressing nuclear TagBFP2 after Cre recombination. Flow cytometry quantification indicates that 3.5% of the cells are TagBFP2 positive. The expected landing pad is shown at the bottom of the panel. Pictures of C and D were taken with an epifluorescence microscope two days postinduction. Flow cytometry7was performed two days post-induction.

[0073] Figure 21 (panels A-D) shows the knock-in of the MADR and biRMCE-compatible elements into tire human GAPDH locus. A) shows a diagram, spanning from exon six to nine of the endogenous human GAPDEI locus. B) shows a diagram after tire knock-in of the human GAPDH locus. The edited locus carries a TagBFP2 under the GAPDH promoter and a secondary cistron containing a CAGpromoter upstream of a flanked miRFP670 by MADR and biRMCE specific recombination sites. C) shows the HEK [GAPDH-TagBFP2nls-Cag-2inl(loxP-attP)-miRFP67nls-FRT] cell line after the proper knock- in and some rounds of purification. D) shows the genotyping of HEK cells before and after the knock-in. PCR fragments indicates left homology arm, right homology arm, Cag-miRFP670 cistron, and the nonedited GAPDH locus. WT = wild-type non-edited HEK cells, K.I. = Knock-In edited HEK cells. Color strands indicate the size of the respective highlighted PCR fragments.

[0074] Figure 22 (panels A-D) shows the validation and efficiency of MADR and biRMCE in the human GAPDH locus. A) shows a diagram of the strategy for validation and comparison of MADR and biRMCE in the human GAPDH locus using a mScarlct donor vector. B) shows a diagram of the expected human GAPDH locus after the mScarlet cassette exchange via MADR or biRMCE. C) shows mScarlet expression in tire HEK [GAPDH-TagBFP2nls-Cag-2inl(loxP-attP)-miRFP67nls-FRT] cell line after MADR or biRMCE. )D shows flow cytometry quantification of cells expressing mScarlet after MADR or biRMCE. Pictures were taken with an epifluorescence microscope ten days post-induction. Flow cytometry was performed ten days post-induction.

[0075] Figure 23, alike figure 13, shows a diagram of the strategy to validate the lock-in of DNA cassette exchanges through biRMCE. A colorless cell line with one landing pad is used to compare dRMCE / MADR & biRMCE side by side. The cell line has into the Rosa26 locus a CAG promoter, a loxP site, an attP site, a puromycin resistance gene (PuroR), and a FRT site. The cell line is named Rosa26-2inl- loxP-attP-PuroR-FRT. For dRMCE / MADR three plasmids were used ( 1 : Promoter-less donor vector- / ox - miRFP670-nls-FRT; 2: Promotcr-lcss donor vcctor-ZoxP-BFP-nls-FRT; 3: pCag-FlpO-Crc). For biRMCE three plasmids were used (4: Promoter-less donor vector-al / ,8- miRFP670-nls-FRT; 5: Promoter-less donor vector-attS-BFP-nls-FRT: 6: pCag-Bxbl-FlpO).

[0076] Figure 24 (panels A-D), alike figure 14, shows that biRMCE locks the exchange of DNA cassettes in the landing pad of the Rosa26 locus at an early time. Top panels show cells expressing nuclear miRFP670 and / or nuclear BFP via dRMCE / MADR (A) or biRMCE (B) two days post-induction. Arrowheads indicate cells expressing both miRFP670 and BFP. Lower panels show flow cytometry quantification of cells expressing nuclear miRFP670 and / or nuclear BFP via dRMCE / MADR (C) or biRMCE (D) two days post-induction: purple dots indicate miRFP670 positive cells, blue dots indicate BFP positive cells, red dots indicate double positives cells to miRFP670 and BFP. Pictures were taken with an epifluorescence microscope two days post-nucleofection. Flow cytometry was performed two days postinduction. These data demonstrate that biRMCE locks the DNA cassette exchanges since early time points.

[0077] Figure 25 (panels A-D), alike figure 15, shows that biRMCE keeps the lock-in of the exchange of DNA cassettes in tire landing pad of the Rosa26 locus at later time points. Top panels showflow cytometry’ quantification of cells expressing nuclear miRFP670 and / or nuclear BFP via dRMCE / MADR (A) or biRMCE (B) four days post-induction. Lower panels show flow cytometry’ quantification of cells expressing nuclear miRFP670 or nuclear BFP via dRMCE / MADR (C) or biRMCE (D) eight days post-induction. Purple dots indicate miRFP670 positive cells, blue dots indicate BFP positive cells. Note that there are fewer color cells eight days post-induction in the dRMCE / MADR condition indicating that the integration is not as stable as biRMCE. Flow cytometry was performed four- and eight- days post-induction. These data demonstrate that biRMCE keeps the lock-in of DNA cassette exchanges at later time points.DESCRIPTION OF THE INVENTION

[0078] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, 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 belongs. Singleton et al.. Dictionary of Microbiology and Molecular Biology 3rded., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7thed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4thed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application.

[0079] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0080] As used herein the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 5% of that referenced numeric indication, unless otherwise specifically provided for herein. For example, the language “about 50%” covers the range of 45% to 55%. In various embodiments, the term “about” when used in connection with a referenced numeric indication can mean the referenced numeric indication plus or minus up to 4%, 3%, 2%, 1%. 0.5%, or 0.25% of that referenced numeric indication, ( / ’specifically provided for in the claims.

[0081] “Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote aparticular age or sex. Thus adult and newborn subjects, whether male or female, are intended to be including within the scope of this term. In some embodiments, the subject is a human.

[0082] Embodiments of the present invention describe Bimodal Rccombinasc-Mcdiatcd CassetteExchange (biRMCE). BiRMCE takes advantage of a simultaneous unidirectional and bidirectional recombination reactions in a system and solves many issues related to the sole use of bidirectional or unidirectional recombination reactions for the integration of genetic elements. First, there is no cross recombination between bidirectional and unidirectional recombinase recognition sites assuring the specificity of the system. Second, it assures the insertion of the genetic cassette that is flanked by the unidirectional and the bidirectional recombinase recognition sites avoiding the integration of the whole genetic vector. Because there is one irreversible reaction and one reversible reaction, this ensures the lock of the system by not allowing extra integrations of different genetic cassettes. As an analogy, BiRMCE can be described as the sperm competition for the egg where only one sperm can enter the egg, thus, the genetic cassettes are the “sperms,” and the targeting locus is the “egg.” Besides, it is possible to perform BiRMCE and dRMCE in the same locus giving more flexibility to the previous M ADR technology.

[0083] Strengths of the Bi-RMCE include but are not limited to: (1) It is a one-step reaction to insert flanked DNA cassettes. (2) Tire system is locked allowing high-throughput insertion of DNA payloads and the immediate downstream analysis and / or applications, e.g.: it is much faster to select the cells that integrate the DNA cassettes, this is mainly because the invader DNA cassettes do not cany' promoters. Downstream applications can be performed a few hours later of biRMCE. (3) There is no risk of cross recombination between recombinase recognition sites because the process depends on different recombination reactions. (4) It ensures that only one DNA cassette embedded in a plasmid is inserted in a host genome because it depends on irreversible and reversible reactions. Insertion of DNA cassettes via two unidirectional / irreversible reactions, like heterotypic sites for an integrase, can produce two plasmid integrations if the “TRANS” reaction overcomes the “CIS” reaction. (5) It is more robust and stable than reactions depending only on reversible recombination reactions.

[0084] Additionally, it is a more robust, stable, and multiplexed than dRMCE such as those described in Osterwalder et al. Dual RMCE for efficient re-engineering of mouse mutant alleles. Nat Methods 7, 893-895 (2010). It is faster and easier than DROID and STRAIT-IN technologies (see e.g.. Neil et al., Assembly of large mobilizable genetic cargo by double recombinase operated insertion of DNA (DROID), Plasmid, Volume 104, 2019; Blanch-Asensio et al., STRAIGHT-IN enables high-throughput targeting of large DNA payloads in human pluripotent stem cells, Cell Reports Methods, Volume 2, Issue 10, 2022). Further, it is more reliable than using two heterotypic INTEGRASE recognition sites to exchange cassettes. (See e.g., Inniss et al. A novel Bxbl integrase RMCE system for high fidelity site-specificintegration of mAb expression cassette in CHO Cells. Biotechnology and Bioengineering. Vol. 114, Issue 8, August 2017, pages 1837-1846; Low et al. Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxbl integrase. Sci Rep 12, 5424 (2022).

[0085] As discussed herein, tire lock-in properties of biRMCE are better than dRMCE / MADR at 2- and 4-days post-induction. (See Figure 13-15 and 23-25.) In the data shown in figures 23-25, the experiments were performed with a changed in the “color’ of the genetic reporters to be those that are far away between them in the color spectrum. This facilitated the analysis, had a better resolution, and output. In the experiments resulting in the data in figures 13-15, red and green genetic reporters were used which were closer in the spectrum.

[0086] Also shown herein (e.g., figures 19 and 20) only about ~80bp of DNA is needed to obtain biRMCE. This small number of nucleotides is enough because we demonstrated that biRMCE can be performed if the specific recombination sites are attached close to each other without other DNA sequences between them. This is of key importance because it is going to be far easier and safer to integrate only ~80bp of DNA, for example, using prime editing to insert the ~80bp of DNA. Therefore, the method inserts ~80 bp of DNA and later adds, via biRMCE, large amounts of DNA including entire synthetic chromosomes. It will also be helpful when adding very small DNA sequences flanked by the biRMCE recombination sites, e.g., adding a small sequence that encodes a sgRNA.

[0087] Further shown (e.g., figures 21 and 22) is that that biRMCE is functional in the human genome. To demonstrate this, we inserted the biRMCE recombination sites in tire GAPDH locus of the human HEK-293T cell line.

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

[0089] Various embodiments provide for a system for biRMCE. The system can comprise various compositions, as such, the system can be viewed as a combination of compositions.

[0090] Various embodiments provide for a system, comprising: (a) a donor vector, comprising: (i) one or more polyadenylation signals or transcription stop element upstream from a transgene or a nucleic acid encoding an RNA, (ii) tire transgene or the nucleic acid encoding the RNA, and (iii) recombinase recognition sites comprising at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site; and (b) two recombinases specific to the recombinaserecognition sites. In various embodiments, the system further comprises an intron, or part 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 tin rd recombinase 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 10threcombinase recognition site, and the system further comprises a 4th, 5th, 6th, 7th, 8th, 9th, or 10threcombinase specific to the at 4th, 5th, 6th, 7th, 8th, 9th, or 10threcombinase recognition site, respectively.

[0093] In various embodiments, the system further comprises a mammalian cell comprising a locus targeted by the donor vector and the two recombinases, and optionally the at least third recombinase. In various embodiments, the mammalian cell is within a mammal.

[0094] In various embodiments, the two recombinases are provided by(i) one expression vector, comprising two genes encoding recombinases specific to the recombinase recognition sites, or(ii) two expression vectors, a first expression vector comprising one gene encoding a first recombinase that is specific to the unidirectional recombinase recognition site, and a second expression vector comprising one gene encoding a second recombinase that is specific to the bidirectional recombinase recognition site, or(iii) one mRNA encoding the two recombinases, or(iv) two mRNA, a first mRNA encoding a first recombinase that is specific to the unidirectional recombinase recognition site, and the second mRNA encoding a second recombinase that is specific to the bidirectional recombinase recognition site, or(v) one viral vector comprising two genes encoding recombinases specific to the recombinase recognition sites, or(vi) two viral vectors, a first viral vector comprising one gene encoding a first recombinase that is specific to the unidirectional recombinase recognition site, and a second viral vector comprising one gene encoding a second recombinase that is specific to the bidirectional recombinase recognition site, or(vii) one recombinant protein comprising the unidirectional recombinase and the bidirectional 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.

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

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

[0097] In various embodiments, the one or more proteins other than the recombinase can be a nuclease. In various embodiments, the one or more proteins other than tire recombinase can be a reverse transcriptase. The one or more proteins other than the recombinase can be a polymerase. In various embodiments, the one or more proteins other than the recombinase can be a transposase.

[0098] In still other embodiments, the one or more proteins can be a combination of any two or three of a nuclease, reverse transcriptase, polymerase, and transposase. In still other embodiments, the one or more proteins can be a combination of a nuclease, reverse transcriptase, polymerase, and transposase. A nonlimiting example include a nuclease (e.g.. Cas9, nickase like dCas9) fused to a reverse transcriptase, fused to a unidirectional recombinase, fused to a bidirectional recombinase.

[0099] In various embodiments, the one or more proteins other than the recombinase can be a therapeutic protein. 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. Pegfilgrastim, Ranibizumab, Darbepoetin alfa, Interferon betala (Avonex), Interferon beta-la (Rebif), Insulin aspart, Rhu insulin. Octocog alfa. Insulin lispro. Cetuximab, Peginterferon alfa-2a, Interferon beta- lb, Eptacog alfa, Insulin aspart, OnabotulinumtoxinA, Epoetin beta, Rec antihemophilic factor, Filgrastin, Insulin detemir, Natalizumab, Insulin (humulin), and Palivizumab.

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

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

[0102] In various embodiments, the one or more proteins other than the recombinase can be a nuclease. In various embodiments, the one or more proteins other than the recombinase can be a reverse transcriptase. The one or more proteins other than the recombinase can be a polymerase. In various embodiments, the one or more proteins other than the recombinase can be atransposase.

[0103] In still other embodiments, the one or more proteins can be a combination of any two or three of a nuclease, reverse transcriptase, polymerase and transposase. In still other embodiments, the one or more proteins can be a combination of a nuclease, reverse transcriptase, polymerase and transposase. A nonlimiting example include a nuclease (e.g.. Cas9, nickase like dCas9) fused to a reverse transcriptase, fused to a unidirectional recombinase, fused to a bidirectional recombinase.

[0104] In various embodiments, the one or more proteins other than the third recombinase is a therapeutic protein. Nonlimiting examples of therapeutic protein are as provide herein.

[0105] In various embodiments, the unidirectional recombinase recognition site is upstream from the bidirectional recombinase recognition site. In various embodiments, the unidirectional recombinase recognition site is downstream to a promoter.

[0106] In various embodiments, the unidirectional recombinase is Bxbl, or any mutant thereof. In various embodiments, the unidirectional recombinase is Bxbl and tire bidirectional recombinase is Flp. In various embodiments, the unidirectional recombinase is Bxbl and the bidirectional recombinase is Cre, VCre, SCre, Nigri, Panto, Vika, or a mutant thereof.

[0107] In various embodiments, the unidirectional recombinase is PhiC31, PhiBTl, PhiCl, MR11, R4, TP901-1, Al 18. FC1, PhiRV, TGI, Phi370.I, W , BL3, SPBc, K38, or any mutant thereof.

[0108] In various embodiments, the third recombinase is selected from Bxbl, Phic31, PhiBTl, PhiCl, MR11, R4, TP901-1, Al 18, FC1, PhiRV, TGI, Phi370.1, W|3, BL3, SPBc, K38, FLp, Cre, VCre, SCre, Nigri, Panto, Vika, or a mutant thereof.

[0109] In various embodiments, the first recombinase recognition site of tire recombinase recognition sites is attB. or any mutant thereof. Hie first recombinase recognition site of the recombinase recognition sites can alternately be attP. or any mutant thereof.

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

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

[0112] In various embodiments, one or both of the recombinase recognition sites comprise a mutation.

[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 flippase recognition target (FRT), loxP, VloxP, SloxP, nox, or pox. In various embodiments, the third recombinase recognition site is modified loxP, flippase recognition target (FRT), VloxP, SloxP, nox, or pox. In various embodiments, the third recombinase recognition site are attB and attP. In various embodiments, the third recombinase recognition site comprises a mutation.

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

[0115] In various embodiments, the donor vector is selected from the group consisting of plasmid, linear DNA (e.g., PCR fragment, synthetic linear DNA), minicircle, viral vector, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), and human artificial chromosome (HAC). A nonlimiting example of a viral vector is an adcno-associatcd viral (AAV) vector. In various embodiments the donor vector is selected from the group consisting of linear single-stranded DNA, close-ended doublestranded DNA, circular single-stranded DNA, circular double -stranded DNA, and RNA.

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

[0117] In other embodiments, the donor vector comprises an intron, or part of an intron upstream from the transgene or nucleic acid encoding the RNA.

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

[0119] In various embodiments, the expression vector comprising recombinases are under tissuespecific promoters.

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

[0121] In various embodiments, the transgene or the RNA comprises disease associated mutations. In various embodiments, the transgene or the RNA comprise a gain-of-function (GOF) gene mutation, loss- of-fiinction (LOF) gene mutation, or both.

[0122] In various embodiments, the mammalian cell is a human cell, and the locus is an AAVS1 locus, Hl l locus, HPRT1 locus, Rogil locus, Rogi2 locus, GAPDH locus, TATA-Box Binding Protein (TBP) locus, Kinesin Family Member (KIF11) Locus, TRAC locus, ZAP-70 locus. Linker of Activation ofT cells (LAT) locus, or Lymphocyte Cytosolic Protein 2 (LCP2 ) locus, and the method is an in vitro, ex vivo, or in vivo method. While examples are listed, any human genomic locus can be used in accordance with various embodiments of the invention.

[0123] In various embodiments, the locus comprises a first polynucleotide encoding a first protein, a secondary cistron comprising a promoter, recombinase recognition sites recognized by a recombinases in the system, a second polynucleotide encoding open reading frame for a first protein. In various embodiments, a first polynucleotide encoding a second protein is downstream of a gene of the locus.

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

[0125] In various embodiments, the first protein or 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, AmCyanl, Midori-Ishi Cyan, TagCFP, mTFPl (Teal), EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellowl, mBanana, Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed. DsRed2. DsRed-Express (Tl), DsRed-Monomer, mTangerine, mRuby, mApple, mStrawberry, AsRed2, mRFPI, JRed, mCherry, HcRedl, mRaspberry, dKeima-Tandem, HcRed- Tandem, mPlum, and AQ143.

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

[0127] In various embodiments, the mammalian cell is a mouse cell, and the locus is ROSA26 locus, Hippl 1 locus, Tigre locus, ColAl locus, Hprt locus, GAPDH locus, TATA-Box Binding Protein (TBP) locus, Kinesin Family Member (KIF11) Locus, TRAC locus, Zap-70 locus, Linker of Activation of T cells (LAT) locus, or Lymphocyte Cytosolic Protein 2 (LCP2 ) locus, and the method is an in vitro, ex vivo, or in vivo method. While examples are listed, any murine genomic locus can be used in accordance with various embodiments of the invention.

[0128] In various embodiments, the locus in the mouse cell comprises a first polymucleotide encoding a first protein, a secondary cistron comprising a promoter, a recombinase recognition sites recognized by a recombinases in the system, a second polynucleotide encoding open reading frame for a second protein. In various embodiments, the first protein or the second protein, or both are fluorescent proteins. Examples of fluorescent proteins, promoters, and recombinase recognition sites are as described herein.Methods of genetic manipulation

[0129] Various embodiments of the invention provide for a method of genetic manipulation of a mammalian cell, comprising: transfecting or transducing the mammalian cell with any one of the systems of the invention as described herein.

[0130] In various embodiments, the system targets a locus and the locus comprises recombinase recognition sites comprising at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site. In various embodiments, a unidirectional recombination is upstream from a bidirectional recombination on the locus.

[0131] In various embodiments, the locus comprises a third recombinase recognition site. In various embodiments, locus comprises an additional recombinase recognition site; for example a 4th, 5th, 6th, 7th, 8th, 9th, or 10threcombinase recognition site.

[0132] In various embodiments, the mammalian cell is a human cell, the system targets anAAVS1 locus. Hl l locus, or HPRTl locus, Rogil locus, Rogi2 locus, GAPDH locus, TATA-Box Binding Protein (TBP) locus, Kinesin Family Member (KIF11) Locus, TRAC locus, ZAP-70 locus, LAT (Linker of Activation of T cells) locus, or LCP2 (Lymphocyte Cytosolic 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 cell is a mouse cell, and the system targets a ROSA26 locus, Hippl 1 locus, Tigre locus, ColAl locus, Hprt locus, GAPDH locus, TATA-Box Binding Protein (TBP) locus, Kinesin Family Member (KIF11) Locus, Trac locus, Zap-70 locus, Lat (Linker for activation of T cells) locus, or Lcp2 (Lymphocyte cytosolic protein 2) locus, and the method is an in vitro, ex vivo, or in vivo method.

[0133] In various embodiments, the method further comprises administering to the cell one or more recombinase enzymes.

[0134] In various embodiments, tire one or more recombinase enzymes comprise, a Bxbl recombinase, a Cre recombinase, a flippase recombinase, a Nigri recombinase, a Panto recombinase, a Vika recombinase, VCre recombinase, or SCre recombinase

[0135] In various embodiments, the mammalian cell comprises a blood cell, a tumor cell, a nontumor cell, an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a tissue precursor cell.Non-human animal models & method of generating the non-human animal models

[0136] Various embodiments provide for anon-human animal model, comprising: the non-human animal comprising any one of the systems of the present invention as described herein.

[0137] In various embodiments, the non-human animal model is 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.

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

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

[0140] In various embodiments, the transgene or RNA is selected from the group consisting of an oncogene, loss-of-function (LOF) mutation of a tumor suppressor gene, gain-of-function (GOF) mutation of a proto-oncogene, pseudogene, siRNA, shRNA, sgRNA, pegRNA, crRNA, IncRNA, miRNA, epigenetic modification, non-coding genetic or epigenetic abnormality associated with human disease, and combinations thereof.

[0141] In various embodiments, the system targets a locus in the non-human animal model and the locus comprises recombinase recognition sites comprising at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site.

[0142] In various embodiments, the unidirectional recombination is upstream from the bidirectional recombination on tire locus.

[0143] Examples of the non-human animals include mouse, rat, dog, guinea pig, rabbit, hamster, swine, sheep, and non-human primates (e.g., monkey (e.g., macaque, rhesus monkey) and ape).

[0144] Various embodiments of the invention provide for a method of generating a non-human animal model of the present invention by transfecting or transducing the non-human animal model with any one of the systems of the present invention.

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

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

[0147] Various embodiments provide for a mammalian cell comprising the system of the present invention.

[0148] Various embodiments provide for a method of assessing the effects of a drag candidate, comprising: providing a mammalian cell comprising the system of the present invention; contacting the drug candidate to the mammalian cell; and assessing the effects of the drug candidate on tire mammalian cell.EXAMPLES

[0149] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from tire scope of the invention.Example 1 Experimental proceduresMice

[0150] All mice used were maintained and euthanized according to the Cedars- Sinai Institutional Animal Care and Use committee. mT / mG (Gt(ROSA)26Sortm4(ACTB-tdTomato.-EGFP)Luo / J) mice (Muzumdar, Tasic, Miyamichi, Li, & Luo, 2007) were bred with C57BL / 6J mice to generate heterozygous mice. Male and female pups between postnatal day (P) 0 and P2 were used for downstream experiments.Plasmid cloning

[0151] The pDonor plasmids were derived from MADR-pDonors using NEBuilder HiFi DNA Assembly Master Mix (NEB) in combination with standard restriction digestion techniques (Kim et al., 2019). Briefly, specific recombination sites were created by oligo synthesis and inserted into the MADR- pDonor. Expression recombinase vectors were derived from pCag-FlpO-2A-Cre EV (Addgene 129419) that were previously validated (Kim et al., 2019). The pCag-NLS-HA-Bxbl (Addgene 51271) plasmid was used as a template for Bxbl PCR. Downstream generation of plasmids were done by removing the existing ORF and adding a new cassette using HiFi DNA Assembly. PCR was done using a standard protocol with KAPA HiFi PCR reagents.Cell lines generation

[0152] Heterozygous P0-P2 mT / mG pup brains were dissociated to establish the polyclonal mouse neural stem cell line (mNSC) used in tire study. Sex was not noted due to the lack of reliable visual methods. The cell lines were generated and maintained as previously described (Breunig et al., 2015). Cells were cultured in a CELLstart CTS (Thermo Fisher Scientific, Waltham, MA) treated flasks. Cells were grown in media containing Neurobasal-A Medium (Life Technologies 10888-022) supplemented with B-27without vitamin A (Life Technologies 12587-010), GlutaMAX (Life Technologies 35050), Antibiotic-Antimycotic (Life Technologies 15240), human epidermal growth factor (hEGF) (Sigma E9644), heparin (Sigma H3393), and basic fibroblast growth factor (bFGF) (Milliporc GF003). De novo generation of recipient cell lines were made by targeting the ROSA26 locus of the mTmG cells via dual recombinase-mediated cassette exchange (Osterwalder et al., 2010) nucleofecting tire pCag-FlpO-2A-Cre EV plasmid and the respective MADR-pDonors (Kim et aL, 2019). Selection and purification of the cell lines were done by flow cytometry.Cell nucleofection

[0153] mNSC nucleofection was achieved using tire Nucleofector 2b device and the Mouse Neural Stem Cell Kit according to tire manufacturer’s recommendations (Lonza AG). The nucleofection mix contained plasmids or mRNA with a total quantity of lOpg or Ipg, respectively.Epifluorescence microscopy

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

[0155] Cells were collected as previously described (Kim et al., 2019). Cells were dissociated using Accutase (Millipore), pelleted at 800 g for 3 min, and resuspended in EDTA. FACS was done on a Beckman Coulter MoFlo at the Cedars-Sinai Flow Cytometry Core.Example 2 ResultsIndirect biRMCE reaction validation

[0156] To test biRMCE indirectly we sought first to confirm that Bxbl / attB / attP recombination is irrcvcrsiblc / unidircctional (Ghosh, Wasil, & Hatfull, 2006; Merrick, Zhao, & Rosser, 2018). To confirm this, we created tw o heterozygous new recipient cell lines carrying in the Rosa26 locus an attP or an attR site betw een the Cag promoter and the puromycin resistance gene (PuroR) followed by an FRT site (Figure1A-B). To make the cell lines, we performed dRMCE / MADR using the respective pDonors to swap the mTmG cassette for the new ones. Next, we develop a promoter-less donor vector containing a reporter gene, mScarlet, flanked by an attB site and an FRT site (pDonor-attB-mScarlet-FRT). Then, we performed nucleofection of the pDonor-attB-mScarlet-FRT with or without plasmids that express the recombinases, Bxb 1 or FlpO, (Figure 1 A-B, reactions 1. 1 -2.3) . The cells carrying the “open” attP sites expressed mScarlet in the presence of Bxb 1 or Bxbl and FlpO together (Figure A, reactions 1.1 & 1.2). There was barely any expression of mScarlet in the cells that have the “locked” attR site in the presence of Bxbl or Bxbl and FlpO together (Figure 2B, reactions 2.1 & 2.2). There was no mScarlet expression in the absence of the recombinases (Figure 2A-B, reaction 1.3 & 2.3). Scarlet expression quantification comparison in reactions1.1 versus 1.2 and 2.1 versus 2.2 clearly show an increase or decrease in the recombination that depends on the attP or attR sites (Figure 3 A-B). These results confirm that an attR site, the product of the recombination between an attP and an attB mediated by Bxbl, is barely prone to allow de novo recombination.

[0157] We followed a similar strategy to visualize if it is possible to have recombinase mediated cassette exchange instead of whole plasmid integration. To confirm this, we created two heterozygous new recipient cell lines carrying in the Rosa26 locus an attP or an attR site between the polyadenylation signal of the PuroR gene and the FRT site (Figure 4A-B). To make the cell lines we performed dRMCE / MADR using the respective pDonors to swap the mTmG cassette for the new ones. Next, we develop a promoterless donor vector containing a reporter gene, mScarlet, flanked by a loxP site and an attB site (pDonor- loxP-mScarlet-attB). Then, we performed nucleofection of the pDonor-loxP-mScarlet-attB with or without plasmids that express the recombinases, Bxbl or Cre, (Figure 4A-B, reactions 3.1-4.3). There was barely any expression of mScarlet in the cells that express exclusively Bxbl, either carrying the “open” attP or “locked” attR site (Figure 5A-B. reactions 3.1 & 4. 1). This is possible because Bxb 1 by itself allows whole plasmid integration, but mScarlet is not expressed due to the pDonor vector carries several polyadenylation signals upstream of the open reading frame. Interestingly, there was mScarlet expression in both cell lines when Bxbl and Cre are expressed (Figure 5A-B, reactions 3.2 & 4.2), suggesting that integration of the pDonors are mediated by Cre / loxP recombination. There was no mScarlet expression in the absence of the recombinases (Figure 5A-B, reaction 3.3 & 4.3). Comparison of mScarlet expression in reaction 3.1 versus4.1 clearly show barely expression of mScarlet when the attP or attR are downstream the polyadenylation signal and only Bxbl is expressed (Figure 6A). Curiously, comparison of mScarlet expression between reaction 3.2 versus 4.2 show more mScarlet positive cells in the cell line that contains the “open” attP site (Figure 6B). Thus, we compared reaction 3.2 versus reaction 4.2 at different time points. After two- and seven-days post nucleofection, we observed a decrease of the mScarlet signal over the time in both reactions (Figure 7A-B). Nevertheless, when comparing the quantification of mScarlet in both reactions over time isclear that cells carrying the “locked” attR site are not expressing mScarlet anymore at day seven post nucleofection demonstrating that all the initial mScarlet expression is due to integration and excision of the whole plasmid mediated by Cre / loxP recombination (Figure 8, dashed line). On the other hand, there are still some remaining cells expressing mScarlet, at day seven post nucleofection, in the cell line that carries the “open” attP site (Figure 8, continuous line). These data suggest that the integration of the mScarlet transgene is mediated by to two different modes of recombination, a reversible / bidirectional recombination through Cre / loxP and an irreversible / unidirectional recombination through Bxbl / attP / attB, named bimodal recombinase-mediated cassette exchange (biRMCE). All previous strategies, data, and results for indirect biRMCE reaction validation are simplified in the table of figure 9. Note that all 12 reactions highly suggest that there is biRMCE in tire Rosa26 locus. biRMCE confirmation

[0158] We confirmed biRMCE by making some adjustments from the previous strategy. First, we made a heterozygous new recipient cell line carrying in the Rosa26 locus an attP site between the Cag promoter and the nuclear TagBFP2 gene followed by an FRT site (Figure 10A). To make the cell line we performed dRMCE / MADR using the respective pDonor-2inl-loxP-attP-TagBFP2-nls-FRT to swap the mTmG cassette for the new one. Next, we created a promoter-less donor vector containing a reporter gene, mScarlet, flanked upstream by loxP & attB sites and downstream by an FRT site named pDonor-2inl-loxP- attB-mScarlet-FRT (Figure 10B). Then, we developed tw o new plasmids each expressing two recombinases in the same plasmid under tire same promoter. The plasmids are pCag-Bxbl-FlpO and pCag- FlpO-Bxbl. For controls, we used pCag-FlpO-Cre and pCag-Bxbl (Figure 10C). Later, we induced Cre expression to observe the excision of the mScarlet cassette in case there are two loxP sites in the Rosa26 locus (Figure 10D). We proceeded to nucleofect the pDonor-2inl-loxP-attB-mScarlet-FRT into the heterozygous cell line 2inl-loxP-attP-TagBFP2-nls-FRT using four different plasmids encoding the respective recombinases. Because the loxP & attP sites are upstream of the nuclear TagBFP2 in the recipient cell line and the loxP & attB sites are upstream mScarlet in the pDonor we ensure to have an expression of mScarlet while losing nuclear TagBFP2 in those targeted cells no matter the type of recombinase plasmid used. We observed cells expressing exclusively mScarlet in the different conditions and proceed to purify the cells and generate four different cell lines. We believed that the nuclear TagBFP2 cassette should be still present in the Rosa26 locus when the pCag-Bxbl plasmid was used because it promotes whole plasmid integration, but the nuclear TagBFP2 cassette should not be present in the Rosa26 locus if there was dRMCE or biRMCE when plasmids expressing two recombinases in the same cistron were used. To confirm this, we nucleofected Cre mRNA into the different cell lines to observe Cre / loxP excision of the mScarlet cassette while expressing again nuclear TagBFP2. We observed many nuclear TagBFP2 cells in tire cell line that was inducedpreviously using pCag-Bxbl (Figure 11A), and few cells expressing nuclear TagBFP2 in the cell line that was induced previously via dRMCE using pCag-FlpO-Cre (Figure 1 ID). We also observed a few nuclear TagBFP2 in the cell lines that were induced previously using pCag-Bxbl-FlpO or pCag-FlpO-Bxb 1 indicating that the previous mScarlet integration was mediated by biRMCE (Figure 11 B-C). Quantification of nuclear TagBFP2 after Cre / loxP recombination demonstrates that pCag-Bxbl integrated the whole plasmid (Figure HE). Quantification of mScarlet after Cre / loxP recombination demonstrates that both biRMCE and dRMCE integrate the DNA that is flanked by their respective specific recombination sites (Figure HF).Stability and efficiency of biRMCE and dRMCE

[0159] After validation of 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, we nucleofected the cell line Rosa26-2inl-loxP-attP-TagBFP2-nls-FRT with the pDonor-2inl-loxP-attB-mScarlet-FRT along with pCag-FlpO-Cre or pCag-Bxbl-FlpO and followed the expression of mScarlet at different time points. At an early time point, two days post nucleofection, dRMCE is more efficient than biRMCE. Nevertheless, at mid and late time points, seven- and eleven-days post nucleofection biRMCE keeps its efficiency compared to dRMCE which drops considerably lower (Figure 12). This data demonstrates that biRMCE is more stable and thus more efficient than dRMCE to integrate transgenic elements.Lock-in of DNA integration via biRMCE

[0160] biRMCE is mediated by two different modes of recombination, an irreversible / unidirectional recombination, and a reversible / bidirectional recombination. Tirus, biRMCE should block tire integration of more DNA cassettes into the Rosa26 locus once the first recombination has happened, as long as the attP / attB recombination happens between the promoter and the open reading frame. To demonstrate this, we compared biRMCE and dRMCE because the latter is based on two reversible / bidirectional recombination reactions. We sought a strategy in which two different pDonors are used each containing a different reporter, nuclear mScarlet and nuclear EGFP. Each pDonor is flanked by specific recombination sites to be recognized only by the respective recombinases (Figure 13). At an early time point, after two days post-induction of dRMCE / MADR and biRMCE. it is clear that there are more double-positive cells expressing mScarlet and EGFP in dRMCE condition (Figure 14 A-B). Flow cytometry analysis confirmed that there are considerably fewer double-positive cells in the biRMCE condition (Figure 14 C-D). At a later time point, eight days post-induction, there are even fewer double-positive cells in the biRMCE condition (Figure 15). Uris demonstrates that biRMCE allows quick and stable transgenesis whilebeing reliable to lock the system avoiding extra trans recombination between the genomic landing pad and the exogenous invader pDonors.Intronic biRMCE validation

[0161] We developed an intronic version of biRMCE by adding an attP site embedding in an intro of the Rosa26 locus and by adding a part of the same intron downstream of the attB site of the pDonor. This strategy allows removing the Kozak sequence and the first ’ATG’’ of the open reading frame from the pDonors. To validate intronic biRMCE, we created a new cell line by targeting the endogenous Rosa26 locus of mT / mG cells via dRMCE / MADR and the pDonor-2inl-loxP-ATG-in-attP-TRON-(ATG-less- TagBFP-nls)-FRT (Figure 16A). After the insertion of the cassette, the resulting genetic “landing pad” of the Rosa26 locus should have a loxP site followed by an “ATG” sequence, an attP site (embedded in an intron), a TagBFP-nls sequence (without the first ATG), and a FRT site (Figure 16B). We proceed to nucloefect heterozygous mT / mG cells using the plasmid pCag-FlpO-Cre and the two versions of the pDonor-2inl-loxP-ATG-in-attP-TRON-(ATG-less-TagBFP-nls)-FRT. The different versions of the pDonor rely on that one has a WPRE sequence and the other does not. We observed several cells expressing nuclear Tag-BFP using the different versions of the pDonors (Figure 16C-H). These data indicate that an intron, with an attP site embedded on it, between the first “ATG” exon and an open reading frame without an “ATG” sequence allows the correct expression of the open reading frame (nuclear Tag-BPF). Because the resulting genetic “landing pad” of the Rosa26 locus has a loxP site, an attP site, and an FRT site it should be compatible with both dRMCE / MADR and intronic biRMCE by using the respective pDonors and recombinases (Figure 17A). After dRMCE / MADR or intronic biRMCE two different genetic “landing pads” can be developed (Figure 17B). To confinn this, we isolated the cells expressing nuclear Tag-BFP to make a new cell line. Then, we nucleofected tire cells using the respective plasmids to confirm dRMCE / MADR or intronic biRMCE. We observed several cells expressing nuclear miRFP-670 using the respective pDonors and recombinases (Figure 17C-H). This data confirms that the “2inl” genetic landing pad is compatible with both dRMCE / MADR and intronic biRMCE. It also confinns that pDonors without both a Kozak sequence and the first “ATG” of the open reading frame can be used to carry transgenes that can be correctly expressed via intronic biRMCE.Example 3 Experimental proceduresCommercial cell lines

[0162] Human embryonic kidney derived HEK293T, purchased from ATCC, were used for MADR and biRMCE in vitro validation. The cell line was maintained in DMEM high glucose (ThermoFisher Scientific, Waltham, MA) supplemented with 10% FBS, GlutaMAX (Life Technologies 35050) and penicillin-streptomycin-amphotericin (Thermo Fisher Scientific, Waltham, MA).GAPDH-MADR / biRMCE human cell line generation

[0163] A GAPDH targeting vector was made through DNA synthesis. P2A-TagBFP2nls-Cag- loxP-attP-miRFP670nls-FRT was inserted into the GAPDH vector and used for transfection in human cells. The GAPDH targeting vector was designed to knock-in the locus through homology-directed repair (HDR) after induction of double-strand breaks (DSBs) via Cas9 / sgRNA complexes. The cells were selected through flow cytometry by choosing the double positive cells to TagbBFP2 and miRFP670. Tire selected stable cell line was transfected with a 2inl-MADR / biRMCE-m Scarlet donor vector along with pCag-FlpO- Cre or pCag-Bxbl-FlpO to induce MADR or biRMCE respectively.Cas9 protein, sgRNA and ribonucleoprotein (RNP) complexes

[0164] Cas9 and sgRNA were acquired from IDT. Cas9 protein and sgRNAs were complexed at a 1 : 1 ratio to make RNP complexes.Cell lipofection

[0165] Coated plates with 0.01% Poly-L-lysine were used for plating HEK cells. Cells were transfected using lipofectamine 3000 (Thermo Fisher Scientific) when the confluency was around 60-70%. The media was replaced 24 hours post-transfection and changed every 48 hours.Genotyping

[0166] PCR was used to verify the proper targeted integration at the GAPDH locus. PCR primers were designed to amplify the left side and right side of the knock-in site, tire endogenous non-edited GAPDH locus, and the second cistron spanning the CAG promoter and the open reading frame. PCR fragments were loaded into a 1% agarose gel for visualization. PCR fragments were extracted from the agarose gel and sent for Sanger sequencing for subsequent validation.Example 4 ResultsLock-in of DNA integration via biRMCE

[0167] biRMCE is mediated by two different modes of recombination, an irreversible / unidirectional recombination, and a reversible / bidirectional recombination. Thus, biRMCE should block the integration of more DNA cassettes into tire Rosa26 locus once the first recombination has happened, as long as the attP / attB recombination happens between the promoter and tire open reading frame. To demonstrate this, we compared biRMCE and dRMCE because the latter is based on two reversible / bidirectional recombination reactions. We sought a strategy in which two different pDonors are used each containing a different reporter, nuclear miRFP670 and nuclear BFP. Each pDonor is flanked by specific recombination sites to be recognized only by the respective recombinases (Figure 23). At an early time point, after two days post-induction of dRMCE / MADR and biRMCE, it is clear that there arc more double-positive cells expressing miRFP670 and BFP in the dRMCE condition (Figure 24 A-B). Flow cytometry analysis confirmed that there are considerably fewer double-positive cells in the biRMCE condition (Figure 24 C-D). At later time points, four- and eight-days post-induction, there are not doublepositive cells in the biRMCE condition (Figure 25). This demonstrates that biRMCE allows quick and stable transgenesis while being reliable to lock the system avoiding extra trans recombination between the genomic landing pad and the exogenous invader pDonors.Minimal recognition site for biRMCE

[0168] Previously, we validated and confirmed biRMCE using specific recombination sites flanking open reading frames that span hundreds to thousands of base pairs of DNA. Thus, we sought to elucidate the minimal recognition site for having biRMCE. That is, joining both specific recombination sites, the irreversible and the reversible sites, immediately one after the other with no other DNA sequence between them (Figure 19A). To demonstrate this, we made a heterozygous recipient cell line carrying in the Rosa26 locus four specific recombination sites. Among those, the attP recognition site is immediately upstream of the VloxP recognition site followed by TagBFP2 open reading frame and a FRT site. There is also a loxP site close to the CAG promoter that is useful for downstream analysis (Figure 19B). After some rounds of purification, wc obtained the cell line with the new Rosa26 locus (Figure 19C). Next, wc created a promoter-less donor vector containing a reporter gene, mScarlet, flanked upstream by loxP & attB sites and downstream by a VloxP site named pDonor-2inl-loxP-attB-mScarlet- VloxP (Figure 19D). Then, the pCag-Bxbl plasmid was used to integrate the entire mScarlet vector while the pCag-Bxbl-VCre plasmid was used to insert only the mScarlet cassette that is flanked by attB and VloxP. (Figure 19E-F). After the proper vector integration via Bxbl or the proper cassette exchange through Bxbl and VCre, we selected the mScarlet positive cells to make pure cell lines carrying the new recombined Rosa26 locus. It is expected to have two loxP sites flanking the mScarlet open reading frame if there is whole vector integration, whereasit should be only one loxP site upstream of the m Scarlet open reading frame if there is cassette exchange through biRMCE (Figure 20A-B). Finally, Cre recombinase was induced in both cell lines to validate whole plasmid integration or biRMCE (Figure 20C-D). There were several TagBFP2 positive cells (98%) in the mScarlet cell line that was made using Bxbl which integrated the whole pDonor vector, meaning that the mScarlet open reading frame is being excised and the TagBFP2 open reading frame was returned close to the CAG promoter for proper expression (Figure 20C). On the other hand, there were very few TagBFP2 positive cells (3.5%) in the cell line that was made using Bxb 1 and VCre, meaning that most of the mScarlet cells cany' only one loxP site in the Rosa26 locus because the cassette exchange avoids the incorporation of a second loxP site (Figure 20D). All these data demonstrated that it is possible to perform biRMCE with the specific recombination sites attached close to each other without other DNA sequences between them. biRMCE is compatible in the human genome

[0169] Previously we have validated biRMCE in the mouse genome, although it is a mammal genome there are several significant differences with the human genome. Thus, it is important to incorporate transgenic elements into the human genome for several purposes including therapeutics. Because of this, we decided to incorporate the biRMCE elements 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 knocked-in locus has several genetic elements to facilitate the selection ofthe cells and to test biRMCE. We added a TagBFP2 sequence downstream of exon nine of the GAPDFI gene, we also added a secondary cistron carry ing a CAG promoter, a loxP site, an attP site, a miRFP670 open reading frame, and an FRT site (Figure 2 IB). After selection of the double positive cells, nuclear TagBFP2 and nuclear miRFP670, we obtained the HEK [GAPDH-TagBFP2nls-Cag-2inl(loxP-attP)-miRFP67nls-FRT] cell line (Figure 21C). PCR genotyping confirmed the proper knock-in into the human GAPDH locus (Figure 21D). Because the second cistron carries the genetic elements to test MADR and biRMCE we decided to compare them side by side using the pDonor-2inl-loxP-attB-mScarlet-FRT which is compatible with MADR and biRMCE by using the specific recombinases (Figure 22A). After MADR or biRMCE the expected recombined GAPDH locus should express mScarlet in the second cistron (Figure 22B). The transfection of the pDonor-2inl-loxP-attB-mScarlet-FRT and the recombinases plasmids, PCag-FlpO-Cre or pCag-Bxbl-FlpO, showed that there were mScarlet positive cells in both conditions (Figure 22C). Quantification analysis through flow cytometry showed that biRMCE is more efficient than MADR by one order of magnitude (Figure 22D). These data demonstrated that biRMCE can be performed in the human genome and biRMCE is more efficient than MADR.

[0170] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0171] Tire foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve 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 as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0172] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Although the open-ended tenn “comprising.” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative tenns such as “consisting of’ or “consisting essentially of.”

[0173] Unless stated otherwise, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the application (especially in the context of claims) may be constmed to cover both the singular and the plural. Tire recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into thespecification as if it were individually recited herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary' language (for example, "such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on tire scope of the application otherwise claimed. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0174] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0175] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A system, comprising:(a) a donor vector, comprising:(i) one or more polyadenylation signals or transcription stop element upstream from a transgene or a nucleic acid encoding an RNA,(ii) the transgene or the nucleic acid encoding the RNA, and(iii) recombinase recognition sites comprising at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site;(b) two recombinases specific to the recombinase recognition sites.

2. The system of 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 specific to the at least third recombinase recognition site.

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

4. The system of any one of claims 1-3, wherein the two recombinases are provided by(i) one expression vector, comprising two genes encoding recombinases specific to their recognition sites, or(ii) two expression vectors, a first expression vector comprising one gene encoding a first recombinase that is specific to the unidirectional recombinase recognition site, and a second expression vector comprising one gene encoding a second recombinase that is specific to the bidirectional recombinase recognition site, or(iii) one mRNA encoding the two recombinases specific to their recognition sites, or(iv) two mRNA, a first mRNA encoding a first recombinase that is specific to the unidirectional recombinase recognition site, and the second mRNA encoding a second recombinase that is specific to the bidirectional recombinase recognition site, or(v) one viral vector comprising two genes encoding recombinases specific to their recognition sites, or(vi) two viral vectors, a first viral vector comprising one gene encoding a first recombinase that is specific to the unidirectional recombinase recognition site, and a second viral vector comprising one gene encoding a second recombinase that is specific to the bidirectional recombinase recognition site, or(vii) one recombinant protein comprising the unidirectional recombinase and the bidirectional 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 of claim 4, wherein in (i) the one expression vector comprising two genes encoding recombinases specific to their recognition sites, the encoded recombinases are fused together; or in (iii) the one mRNA encoding the two recombinases specific to their recognition sites, the encoded two recombinases are fused together; or in (v) the one viral vector comprising two genes encoding recombinases specific to their recognition sites, the encoded recombinases are fused together; or in (viii) the two recombinant proteins are fused together.

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

7. The system of claim 5, wherein any one of the two fused recombinases are further fused to one or more proteins other than tire recombinase.

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

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

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

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

12. Tire system of any one of claims 1-11, wherein the donor vector further comprises an intron, part of an intron, or at least one splice acceptor site, and optionally, the unidirectional recombinase recognition site is embedded into an intron or part of the intron.

13. The system of any one of claims 1-12, wherein the unidirectional recombinase is Bxbl.

14. The system of any one of claims 1-12, wherein the unidirectional recombinase is selected from Bxb 1 , Phic31 , PhiBT 1 , PhiC 1 , MR11 , R4, TP901 - 1 , A 118, FC 1 , PhiRV, TG 1 , Phi370.1 , Wp, BL3 , SPBc, K38, and any mutants thereof.

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

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

17. The system of any one of claims 2-12, wherein the third recombinase is selected from Bxb 1 , Phic31 , PhiBTl, PhiCl, MR11, R4, TP901-1, A118, FC1, PhiRV, TGI, Phi370.1, Wp, BL3, SPBc, K38, FLp, Cre, VCre. SCre, Nigri, Panto, Vika, or a mutant thereof.

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

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

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

21. Tire system of any one of claims 1-17, wherein one or both of the recombinase recognition sites comprise a mutation.

22. The system of any one of claims 1-21, wherein the donor vector is selected from the group consisting of plasmid, linear PCR. linear single-stranded DNA, close-ended double-stranded DNA, circular single-stranded DNA, circular double-stranded DNA, RNA, minicircle, viral vector, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), and human artificial chromosome (HAC).

23. Tire system of claim 22, wherein the viral vector is an adeno-associated viral (AAV) vector.

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

25. The system of any one of claims 1-24, wherein the donor vector comprises an intron, or part of an intron upstream and / or downstream from the transgene or nucleic acid encoding the RNA.

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

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

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

29. Tire system of any one of claims 1-28, wherein the donor vector further comprises a fluorescent reporter.

30. The system of any one of claims 4-29. wherein tire expression vector comprising recombinases are under tissue-specific promoters.

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

32. Tire system of any one of claims 1-30, wherein the transgene or the RNA comprises disease associated mutations.

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

34. The system of any one of claims 1-33, wherein the mammalian cell is a human cell, and the locus is an AAVS1 locus, Hl l locus, HPRT1 locus, Rogil locus, Rogi2 locus, GAPDH locus, TATA- Box Binding Protein (TBP) locus, Kincsin Family Member (KIF11) Locus, TRAC locus, ZAP-70locus. Linker of Activation of T cells (LAT) locus, or Lymphocyte Cytosolic Protein 2 (LCP2 ) locus, and the method is an in vitro, ex vivo, or in vivo method.

35. Tire system of claim 34, wherein tire locus comprises a first polynucleotide encoding a first protein, a secondary cistron comprising a promoter, recombinase recognition sites recognized by a recombinases in tire system, a second polynucleotide encoding open reading frame for a second protein.

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

37. Tire system of claim 35 or claim 36, wherein the first polynucleotide encoding the protein is downstream of a gene of the locus.

38. The system of any one of claims 1-37, wherein the mammalian cell is a mouse cell, and the locus is ROSA26 locus. Hippl 1 locus, Tigre locus, ColAl locus, Hprt locus, GAPDH locus. TATA-Box Binding Protein (TBP) locus, Kinesin Family Member (KIF 11) Locus, TRAC locus, Zap-70 locus. Linker of Activation of T cells (LAT) locus, or Lymphocyte Cytosolic Protein 2 (LCP2 ) locus, and the method is an in vitro, ex vivo, or in vivo method.

39. The system of claim 38, wherein the locus comprises a first polynucleotide encoding a first protein, a secondary cistron comprising a promoter, a recombinase recognition sites recognized by a recombinases in tire system, a second polynucleotide encoding open reading frame for a second protein.

40. A method of genetic manipulation of a mammalian cell, comprising: transfecting or transducing the mammalian cell with the system of any one of claims 1-39.

41. The method of claim 40, wherein the system targets a locus and the locus comprises recombinase recognition sites comprising at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site.

42. The method of claim 40 or claim 41, wherein a unidirectional recombination is upstream from a bidirectional recombination on the locus.

43. Tire method of any one of claims 40-42, wherein the mammalian cell is a human cell, the system targets an AAVS1 locus, Hll locus. HPRT1 locus, Rogil locus, Rogi2 locus, GAPDH locus, TATA-Box Binding Protein (TBP) locus. Kinesin Family Member (KIF11) Locus, TRAC locus, ZAP-70 locus, Linker of Activation of T cells (LAT) locus, or Lymphocyte Cytosolic Protein 2 (LCP2 ) locus, and the method is an in vitro, ex vivo, or in vivo method.

44. The method of any one of claims 40-42, wherein the mammalian cell is a mouse cell, and the system targets a ROSA26 locus, Hippl 1 locus, Tigre locus, ColAl locus, Hprt locus, GAPDH locus,TATA-Box Binding Protein (TBP) locus, Kinesin Family Member (KIF11) Locus, TRAC locus, Zap-70 locus, Linker of Activation of T cells (LAT) locus, or Lymphocyte Cytosolic Protein 2 (LCP2 ) locus, and the method is an in vitro, ex vivo, or in vivo method.

45. Tire method of any one of claims 40-44, further comprising administering to the cell one or more recombinase enzymes.

46. The method of any one of claims 40-45. wherein the one or more recombinase enzymes comprise, a Bxbl recombinase, a Cre recombinase, a flippase recombinase, a Nigri recombinase, a Panto recombinase, a Vika recombinase, VCre recombinase, or SCre recombinase.

47. Tire method of any one of claims 40-46, wherein the mammalian cell comprises a blood cell, a tumor cell, a non-tumor cell, an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a tissue precursor cell.

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

49. The non-human animal model of claim 48 , wherein the non-human animal model is a personalized non-human animal model for a human subject’s cancer and tire transgene or RNA is based on the human subject’s cancer.

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

51. The non-human animal model of claim 49 or 50, wherein the transgene or RNA is selected from the group consisting of an oncogene, loss-of-function (LOF) mutation of a tumor suppressor gene, gain-of-function (GOF) mutation of a proto-oncogene, pseudogene, siRNA, shRNA, sgRNA, pegRNA, crRNA. IncRNA. miRNA, epigenetic modification, non-coding genetic or epigenetic abnormality associated with human disease, and combinations thereof.

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

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

54. The non-human animal model of any one of claims 48-53, wherein the unidirectional recombination is upstream from the bidirectional recombination on the locus.

55. A method of generating the non-human animal model, comprising:transfecting or transducing the non-human animal model with a system of any one of claims 1-29.

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

57. A method of assessing the effects of a drug candidate, comprising: providing the non-human animal model of any one of claims 48-54 or claim 56; administering the drug candidate to the non-human animal model; and assessing the effects of the drug candidate on the non-human animal model.

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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