[go: up one dir, main page]

WO2025006709A1 - Procédé pour l'introduction spécifique de sites d'éléments génétiques dans des loci modifiés par échange de cassettes médié par la recombinase bimodale (birmce) - Google Patents

Procédé pour l'introduction spécifique de sites d'éléments génétiques dans des loci modifiés par échange de cassettes médié par la recombinase bimodale (birmce) Download PDF

Info

Publication number
WO2025006709A1
WO2025006709A1 PCT/US2024/035759 US2024035759W WO2025006709A1 WO 2025006709 A1 WO2025006709 A1 WO 2025006709A1 US 2024035759 W US2024035759 W US 2024035759W WO 2025006709 A1 WO2025006709 A1 WO 2025006709A1
Authority
WO
WIPO (PCT)
Prior art keywords
recombinase
locus
recognition site
specific
tire
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/035759
Other languages
English (en)
Inventor
Joshua BREUNIG
Alberto AYALA-SARMIENTO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cedars Sinai Medical Center
Original Assignee
Cedars Sinai Medical Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cedars Sinai Medical Center filed Critical Cedars Sinai Medical Center
Publication of WO2025006709A1 publication Critical patent/WO2025006709A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/40—Systems of functionally co-operating vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/70—Vectors containing special elements for cloning, e.g. topoisomerase, adaptor sites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • This invention relates to genetic manipulation: for example, in cells, organ models, and non-human animal models.
  • 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.
  • 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 be cross 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.
  • 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.
  • Various embodiments provide for a system, comprising:
  • a donor vector comprising:
  • the donor vector can further comprise at least a third recombinase recognition site
  • the system can further comprise at least a third recombinase specific to the at least third recombinase recognition site.
  • 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.
  • the two recombinases can be provided by
  • 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
  • the encoded recombinases 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.
  • 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.
  • the at least a third recombinases can be provided by
  • one expression vector comprising a gene encoding the at least third recombinase specific to the third recombinase recognition site, or
  • the expression vector 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.
  • the mRNA 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the bidirectional recombinase can be Flp.
  • 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.
  • 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.
  • the unidirectional recombinase recognition site can be attB. In various embodiments, the unidirectional recombinase recognition site can be attP.
  • the bidirectional recombinase recognition site can be flippase recognition target (FRT), loxP, VloxP, SloxP, nox, or pox.
  • FRT flippase recognition target
  • loxP flippase recognition target
  • VloxP VloxP
  • SloxP SloxP
  • nox or pox.
  • one or both of the recombinase recognition sites can comprise a mutation.
  • 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).
  • the viral vector can be an adeno-associated viral (AAV) vector.
  • the donor vector can comprise at least four polyadenylation signals upstream from the transgene or nucleic acid encoding tire RNA.
  • 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.
  • the donor vector can further comprise a post- transcriptional regulatory element.
  • the donor vector can further comprise a polyadenylation signal downstream from the transgene or nucleic acid encoding the RNA.
  • the donor vector can further comprise an open reading frame (ORF) that begins with a splice acceptor.
  • the donor vector can further comprise a fluorescent reporter.
  • the expression vector comprising recombinases can be under tissue-specific promoters.
  • the RNA can be siRNA, shRNA, sgRNA, crRNA, pegRNA, IncRNA or miRNA.
  • the transgene or the RNA can comprise disease associated mutations.
  • the transgene or the RNA can comprise a gain-of-function (GOF) gene mutation, loss-of-fiinction (LOF) gene mutation, or both.
  • GAF gain-of-function
  • LEF loss-of-fiinction
  • the mammalian cell can be a human cell
  • 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.
  • the locus can comprise 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 second protein.
  • the first protein or the second protein or both can be fluorescent proteins.
  • the first polynucleotide encoding the protein can be downstream of a gene of the locus.
  • 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.
  • TBP TATA-Box Binding Protein
  • KIF11 Kinesin Family Member
  • LAT Linker of Activation of T cells
  • LCP2 Lymphocyte Cytosolic Protein 2
  • 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.
  • 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.
  • 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.
  • a unidirectional recombination can be upstream from a bidirectional recombination on tire locus.
  • 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.
  • 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.
  • TBP TATA-Box Binding Protein
  • KIF11 Kinesin Family ⁇ Member
  • LAT Linker of Activation of T cells
  • LCP2 Lymphocyte Cytosolic Protein 2
  • the method can further comprise administering to the cell one or more recombinase enzymes.
  • 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.
  • 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.
  • a non-human animal model comprising: a non-human animal comprising a system of the present invention.
  • 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.
  • 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.
  • 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.
  • LEF loss-of-function
  • GAF gain-of-function
  • 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.
  • GAF gain of function mutation
  • LEF loss of function mutation
  • 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.
  • the unidirectional recombination is upstream from the bidirectional recombination on the locus.
  • 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.
  • 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.
  • Various embodiments provide for a mammalian cell comprising the system of the present invention.
  • FIGS 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • biRMCE Bimodal Recombinase-Mediated Cassette Exchange
  • 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.
  • FIGS 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).
  • mScarlct cytoplasmic red fluorescent protein
  • TagBFP2 nuclear blue fluorescent protein
  • 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.
  • Expression of 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.
  • FIG. 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.
  • FIG. 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.
  • dRMCE 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).
  • 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).
  • FIG 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 two days post-nucleofection. These data demonstrate that biRMCE locks the DNA cassette exchanges since early time points.
  • FIG. 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.
  • 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.
  • 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 expressing nuclear miRFP-670. Pictures were taken two days post-nucleofection with an epifluorescence microscope. Flow cytometry was performed two days post-nucleofection.
  • FIG 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.
  • FIG. 19 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.
  • Figure 20 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 cytometry 7 was performed two days post-induction.
  • Figure 21 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 CAG promoter 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.
  • FIG. 22 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.
  • FIG 23 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.
  • 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).
  • 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).
  • FIG. 24 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.
  • FIG. 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 show flow cytometry’ quantification of cells expressing nuclear miRFP670 and/or nuclear BFP via dRMCE/MADR (A) or biRMCE (B) four days post-induction.
  • 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.
  • the language “about 50%” covers the range of 45% to 55%.
  • 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.
  • “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 a particular age or sex. Thus adult and newborn subjects, whether male or female, are intended to be including within the scope of this term.
  • the subject is a human.
  • Embodiments of the present invention describe Bimodal Rccombinasc-Mcdiatcd Cassette
  • 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.
  • 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.
  • 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.
  • biRMCE is functional in the human genome.
  • 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.
  • 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 recombinase recognition sites.
  • the system further comprises an intron, or part of an intron, or at least one splice acceptor site.
  • 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.
  • 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.
  • the donor vector comprises a 4 th , 5 th , 6 th , 7 th , 8 th , 9 th , or 10 th recombinase recognition site
  • the system further comprises a 4 th , 5 th , 6 th , 7 th , 8 th , 9 th , or 10 th recombinase specific to the at 4 th , 5 th , 6 th , 7 th , 8 th , 9 th , or 10 th recombinase recognition site, respectively.
  • 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.
  • the mammalian cell is within a mammal.
  • the two recombinases are provided by
  • 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.
  • the encoded recombinases are fused together.
  • the encoded two recombinases are fused together.
  • the encoded recombinases are fused together.
  • the encoded recombinases arc fused together.
  • the two recombinant proteins arc fused together.
  • 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.
  • 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.
  • the one or more proteins can be a combination of any two or three of a nuclease, reverse transcriptase, polymerase, and transposase.
  • 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.
  • the one or more proteins other than the recombinase can be a therapeutic protein.
  • 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.
  • 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
  • the expression vector 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.
  • 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.
  • 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
  • the encoded recombinase is further fused to the encoded one or more proteins.
  • 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.
  • 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.
  • 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.
  • the one or more proteins other than the third recombinase is a therapeutic protein.
  • therapeutic protein are as provide herein.
  • 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.
  • 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.
  • 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.
  • the second recombinase recognition site of the recombinase recognition sites is flippase recognition target (FRT), loxP, VloxP, SloxP, nox, or pox.
  • the second recombinase recognition site of the recombinase recognition sites is modified loxP, flippase recognition target (FRT), VloxP, SloxP, nox, or pox.
  • the unidirectional recombinase is PhiC31 and the recombinase recognition sites are attB and attP.
  • one or both of the recombinase recognition sites comprise a mutation.
  • the third recombinase recognition site is attB, or any mutant thereof, attP, or any mutant thereof.
  • the third recombinase recognition site is flippase recognition target (FRT), loxP, VloxP, SloxP, nox, or pox.
  • the third recombinase recognition site is modified loxP, flippase recognition target (FRT), VloxP, SloxP, nox, or pox.
  • the third recombinase recognition site are attB and attP.
  • the third recombinase recognition site comprises a mutation.
  • the at least one additional recombinase recognition site is attB, or any mutant thereof, attP, or any mutant thereof.
  • the at least one additional recombinase recognition site is flippase recognition target (FRT), loxP, VloxP, SloxP, nox, or pox.
  • the at least one additional recombinase recognition site is modified loxP, flippase recognition target (FRT), VloxP, SloxP, nox, or pox.
  • the at least one additional recombinase recognition site are attB and attP.
  • the at least one additional recombinase recognition site comprises a mutation.
  • the at least one additional recombinase recognition site is for example, the 4 th , 5 th , 6 th , 7 th , 8 th , 9 th or 10 th recombinase recognition site.
  • 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.
  • 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.
  • 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.
  • the donor vector comprises an intron, or part of an intron upstream from the transgene or nucleic acid encoding the RNA.
  • the donor vector further comprises a post-transcriptional regulatory element.
  • the donor vector further comprises a polyadenylation signal downstream from the transgene or nucleic acid encoding the RNA.
  • the donor vector further comprises an open reading frame (ORF) that begins with a splice acceptor.
  • the donor vector further comprises a fluorescent reporter.
  • the expression vector comprising recombinases are under tissuespecific promoters.
  • the RNA is siRNA, shRNA. sgRNA, crRNA, pegRNA, IncRNA or miRNA.
  • the transgene or the RNA comprises disease associated mutations.
  • the transgene or the RNA comprise a gain-of-function (GOF) gene mutation, loss- of-fiinction (LOF) gene mutation, or both.
  • GAF gain-of-function
  • LEF loss- of-fiinction
  • the mammalian cell is a human cell
  • 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.
  • 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.
  • a first polynucleotide encoding a second protein is downstream of a gene of the locus.
  • promotors include but are not limited to CAG, CMV, EFla, PGK, TRE, U6, and UAS.
  • the first protein or the second protein, or both are fluorescent proteins.
  • 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, D
  • DsRed2 DsRed2.
  • DsRed-Express (Tl) DsRed-Monomer, mTangerine, mRuby, mApple, mStrawberry, AsRed2, mRFPI, JRed, mCherry, HcRedl, mRaspberry, dKeima-Tandem, HcRed- Tandem, mPlum, and AQ143.
  • the first fluorescent protein is TagBFP2
  • the promoter is a CAG promotor
  • the second fluorescent protein is miRFP670.
  • the mammalian cell is a mouse cell
  • 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
  • TBP TATA-Box Binding Protein
  • KIF11 Kinesin Family Member
  • LAT Linker of Activation of T cells
  • LCP2 Lymphocyte Cytosolic Protein 2
  • 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.
  • 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.
  • 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.
  • 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.
  • a unidirectional recombination is upstream from a bidirectional recombination on the locus.
  • the locus comprises a third recombinase recognition site.
  • locus comprises an additional recombinase recognition site; for example a 4 th , 5 th , 6 th , 7 th , 8 th , 9 th , or 10 th recombinase recognition site.
  • the mammalian cell is a human cell
  • the system targets an
  • AAVS1 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.
  • TBP TATA-Box Binding Protein
  • KIF11 Kinesin Family Member
  • Locus TRAC locus
  • ZAP-70 locus ZAP-70 locus
  • LAT Linker of Activation of T cells locus
  • LCP2 Lymphocyte Cytosolic Protein 2, also known as SLP-76 locus
  • the mammalian cell is a mouse cell
  • 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.
  • the method further comprises administering to the cell one or more recombinase enzymes.
  • 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
  • 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]
  • anon-human animal model comprising: the non-human animal comprising any one of the systems of the present invention as described herein.
  • 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.
  • 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.
  • the non-human animal model comprises a gain of function mutation (GOF), a loss of function mutation (LOF), or both.
  • GAF gain of function mutation
  • LEF loss of function mutation
  • 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.
  • LEF loss-of-function
  • GAF gain-of-function
  • 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.
  • the unidirectional recombination is upstream from the bidirectional recombination on tire locus.
  • non-human animals examples include mouse, rat, dog, guinea pig, rabbit, hamster, swine, sheep, and non-human primates (e.g., monkey (e.g., macaque, rhesus monkey) and ape).
  • non-human primates e.g., monkey (e.g., macaque, rhesus monkey) and ape).
  • 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.
  • Various embodiments of the invention provide for a non-human animal model generated by any one of the methods of the present invention.
  • 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 drug candidate to the non-human animal model; and assessing the effects of the drug candidate on the non-human animal model.
  • Various embodiments provide for a mammalian cell comprising the system of the present invention.
  • 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.
  • 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.
  • 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
  • mNSC polyclonal mouse neural stem cell line
  • 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).
  • Neurobasal-A Medium Life Technologies 10888-022
  • B-27without vitamin A Life Technologies 12587-010
  • GlutaMAX Life Technologies 35050
  • Antibiotic-Antimycotic Life Technologies 15240
  • human epidermal growth factor hEGF
  • heparin Sigma H3393
  • bFGF basic fibroblast growth factor
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • dRMCE dRMCE
  • biRMCE when plasmids expressing two recombinases in the same cistron were used.
  • 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).
  • biRMCE is mediated by two different modes of recombination, an irreversible/unidirectional recombination, and a reversible/bidirectional recombination.
  • 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.
  • biRMCE and dRMCE because the latter is based on two reversible/bidirectional recombination reactions.
  • Each pDonor is flanked by specific recombination sites to be recognized only by the respective recombinases ( Figure 13).
  • 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 while being reliable to lock the system avoiding extra trans recombination between the genomic landing pad and the exogenous invader pDonors.
  • 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).
  • ATG an attP site
  • TagBFP-nls sequence without the first ATG
  • FRT site Figure 16B.
  • 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 (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS, GlutaMAX (Life Technologies 35050) and penicillin-streptomycin-amphotericin (Thermo Fisher Scientific, Waltham, MA).
  • 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 and sgRNA were acquired from IDT. Cas9 protein and sgRNAs were complexed at a 1 : 1 ratio to make RNP complexes.
  • 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.
  • 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.
  • biRMCE Lock-in of DNA integration via biRMCE
  • biRMCE is mediated by two different modes of recombination, an irreversible/unidirectional recombination, and a reversible/bidirectional recombination.
  • 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.
  • biRMCE and dRMCE because the latter is based on two reversible/bidirectional recombination reactions.
  • 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.
  • wc obtained the cell line with the new Rosa26 locus ( Figure 19C).
  • 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).
  • biRMCE 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.
  • 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).

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des systèmes et des procédés de manipulation génétique de cellules de mammifère. L'invention concerne également des modèles animaux non humains utilisant ces systèmes, des procédés de génération de ces animaux non humains, et des procédés d'utilisation de ces animaux non humains.
PCT/US2024/035759 2023-06-27 2024-06-27 Procédé pour l'introduction spécifique de sites d'éléments génétiques dans des loci modifiés par échange de cassettes médié par la recombinase bimodale (birmce) Pending WO2025006709A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363523550P 2023-06-27 2023-06-27
US63/523,550 2023-06-27

Publications (1)

Publication Number Publication Date
WO2025006709A1 true WO2025006709A1 (fr) 2025-01-02

Family

ID=93939829

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/035759 Pending WO2025006709A1 (fr) 2023-06-27 2024-06-27 Procédé pour l'introduction spécifique de sites d'éléments génétiques dans des loci modifiés par échange de cassettes médié par la recombinase bimodale (birmce)

Country Status (1)

Country Link
WO (1) WO2025006709A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080313747A1 (en) * 2004-07-07 2008-12-18 Heidrun Kern Targeted Transgenesis of Short Hairpin Rna Expression Cassettes Using Recombinase Mediated Cassette Exchange
US20120124686A1 (en) * 2010-11-12 2012-05-17 Liqun Luo Site-Directed Integration of Transgenes in Mammals
US20120141441A1 (en) * 2010-12-03 2012-06-07 The Board Of Trustees Of The Leland Stanford Junior University Methods and Compositions for Treatment of Muscular Dystrophy

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080313747A1 (en) * 2004-07-07 2008-12-18 Heidrun Kern Targeted Transgenesis of Short Hairpin Rna Expression Cassettes Using Recombinase Mediated Cassette Exchange
US20120124686A1 (en) * 2010-11-12 2012-05-17 Liqun Luo Site-Directed Integration of Transgenes in Mammals
US20120141441A1 (en) * 2010-12-03 2012-06-07 The Board Of Trustees Of The Leland Stanford Junior University Methods and Compositions for Treatment of Muscular Dystrophy

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KANCA ET AL.: "An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms", ELIFE, vol. 8, no. e 51539, 1 November 2019 (2019-11-01), pages 1 - 22, XP055865491, DOI: 10.7554/eLife.51539 *

Similar Documents

Publication Publication Date Title
Hermann et al. Binary recombinase systems for high-resolution conditional mutagenesis
Miura et al. CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA
Masui et al. An efficient system to establish multiple embryonic stem cell lines carrying an inducible expression unit
Kazuki et al. Refined human artificial chromosome vectors for gene therapy and animal transgenesis
Schönig et al. Stringent doxycycline dependent control of CRE recombinase in vivo
Fenno et al. Targeting cells with single vectors using multiple-feature Boolean logic
US20220304286A1 (en) SYSTEMS AND METHODS FOR IN VIVO DUAL RECOMBINASE-MEDIATED CASSETTE EXCHANGE (dRMCE) AND DISEASE MODELS THEREOF
Chylinski et al. CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci
WO2014117050A2 (fr) Arnmi modifié en tant qu'échafaudage pour de l'arnsh
US11466290B2 (en) Systems and methods for in vivo dual recombinase-mediated cassette exchange (dRMCE) and disease models thereof
Mecozzi et al. Genetic tools for the stable overexpression of circular RNAs
Moriarity et al. Modular assembly of transposon integratable multigene vectors using RecWay assembly
Mao et al. An ES cell system for rapid, spatial and temporal analysis of gene function in vitro and in vivo
Abe et al. Construction of stable mouse artificial chromosome from native mouse chromosome 10 for generation of transchromosomic mice
Suzuki et al. Switchable genome editing via genetic code expansion
JP4293990B2 (ja) 哺乳類人工染色体
EP3152326B1 (fr) Système de biocapteur cellulaire à faible fuite
EP4141119A2 (fr) Vecteur d'adn linéaire fermé par covalence double brin auto-complémentaire à extrémité en boucle en épingle à cheveux, système et procédé de fabrication, et utilisations dudit vecteur d'adn ainsi obtenu
Fenno et al. INTRSECT: single-component targeting of cells using multiple-feature Boolean logic
Ubina et al. ExBoX–a simple Boolean exclusion strategy to drive expression in neurons
WO2025006709A1 (fr) Procédé pour l'introduction spécifique de sites d'éléments génétiques dans des loci modifiés par échange de cassettes médié par la recombinase bimodale (birmce)
Liu et al. Optimal tagging strategies for illuminating expression profiles of genes with different abundance in zebrafish
Robertson et al. Rapid modeling of an ultra-rare epilepsy variant in wild-type mice by in utero prime editing
Toth et al. Novel method to load multiple genes onto a mammalian artificial chromosome
Li et al. Gene and RNA editing: Revolutionary approaches to treating diseases

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24832907

Country of ref document: EP

Kind code of ref document: A1