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WO2025128981A1 - Compositions et procédés d'édition génomique - Google Patents

Compositions et procédés d'édition génomique Download PDF

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Publication number
WO2025128981A1
WO2025128981A1 PCT/US2024/060012 US2024060012W WO2025128981A1 WO 2025128981 A1 WO2025128981 A1 WO 2025128981A1 US 2024060012 W US2024060012 W US 2024060012W WO 2025128981 A1 WO2025128981 A1 WO 2025128981A1
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Prior art keywords
protein
composition
genome editing
cell
graphene
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David Aguilar
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Sohm Inc
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Sohm Inc
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • 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)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • Methods and compositions include modifying a genome editing protein by joining the genome editing protein to a material that modulates the function of the genome editing protein in response to one or more stimuli.
  • kinases and phosphatases are methods and compositions for fine control of genome targeting and/or editing that can respond functionally to stimuli. Also disclosed are methods of targeting kinases and phosphatases to areas of DNA that can affect the phosphorylation and dephosphorylation of transcription factors, other DNA binding proteins and proteins that have close association with mitochondrial or genomic DNA.
  • a composition comprising a DNA binding protein selected from a meganuclease, a zinc finger protein, a TALEN, a cas DNA binding protein, a DNA binding-reverse transcriptase fusion protein, or a DNA bindingintegrase fusion protein joined to a material comprising a graphene, graphene oxide, carbon nanotube, fullerene, nanodiamond, or any combination thereof.
  • the DNA binding protein is nuclease inactive, defective, or inhibited e.g., a DNA binding protein comprising one or more mutations that inactivate a nuclease associated with the DNA binding protein.
  • the DNA binding protein can be selected from the group consisting of a cas DNA binding protein, a cas DNA binding-reverse transcriptase fusion protein, and a cas DNA binding protein-integrase fusion protein and the aforementioned DNA binding proteins optionally have an inactivated nuclease domain such as can be generated with one or more inactivating mutations.
  • the material can comprise graphene or graphene oxide.
  • the composition can further comprise a guide RNA.
  • the composition can further comprise an enzyme, a drug or a diagnostic agent or any combination thereof joined to the material.
  • a method for detecting a selected nucleotide sequence in a genome of a subject or a cell comprising administering a composition to the subject or contacting the cell with the composition; and detecting the selected nucleotide sequence, such as by using colorimetry, fluorescence detection, immunodetection, or rf reflectrometry.
  • the composition can be for use in detecting a selected sequence in a genome of a subject or a cell.
  • a method is providing for providing a drug to a selected nucleotide sequence in a genome of a subject or a cell comprising administering a composition to the subject or the cell; and, optionally, determining or measuring the presence of the drug in the subject or cell and/or selecting a subject or a cell to receive the drug.
  • the composition can be for use in providing a drug to a subject or a cell.
  • a composition comprising a cas protein joined to a protein comprising a kinase or phosphatase.
  • the protein comprising the kinase can comprise a MAP kinase, Src, a nuclear kinase, a serine/threonine kinase or a tyrosine kinase.
  • the protein comprising the phosphatase can comprise a Shp, Shp2 or a nuclear phosphatase.
  • the cas protein has an inactivated nuclease domain such as can be generated with one or more inactivating mutations.
  • a method of modifying the phosphorylation state of a selected protein associated with a polynucleotide comprises administering a composition to a cell or a subject; and, optionally, determining or measuring the modification of the phosphorylation of the selected protein and/or selecting a protein associated with a polynucleotide for modification of its phosphorylation state and/or selecting a cell or a subject to receive an agent that modifies the phosphorylation state of the selected protein.
  • the subject or cell can have a disease or disorder.
  • the disease can be a viral disease, a microbial disease, cancer or a cardiovascular disease.
  • the composition can be for use in treating a viral disease, a microbial disease, cancer or a cardiovascular disease.
  • a method of detecting a selected nucleotide sequence in a subject, a cell, or an isolated population of polynucleotides or oligonucleotides comprising contacting a composition with the subject, the cell, or the isolated population of polynucleotides or nucleotides and performing a diagnostic evaluation.
  • the diagnostic evaluation can comprise colorimetry, fluorescence detection, immunodetection, or rf reflectometry.
  • a method of genome editing comprising: modifying a genome editing protein by joining the genome editing protein to a material that modulates the function of the genome editing protein in response to one or more stimuli; and providing one or more stimuli to the modified genome editing protein, which is joined to the material.
  • response of the modified genome editing protein to the one or more stimuli can modulate genome editing in a target.
  • the genome editing protein can comprises a zinc finger endonuclease, TALEN, CRISPR/cas9, Cpfl, meganuclease, Cas3, CaslO, or an ABBIE system.
  • the modified genome editing protein can be joined to at least one other protein, which comprises a recombinase, reverse transcriptase, or topoisomerase.
  • the material that modifies the genome editing protein can comprise a graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate or carbon nanotube.
  • the material that modifies the genome editing protein can comprise silicon, silica or a silicone polymer.
  • modifying the genome editing protein can comprise joining the genome editing protein to a protein comprising a kinase.
  • the protein comprising the kinase can comprise a tyrosine kinase, serine/threonine kinase, ERK, ERK1, ERK2, p38, ERK5, JNK, MEKK1, MEKK2, MEKK3, MEKK4, MEKK5, a nuclear kinase, MEK1, MEK2, MEK3, EGFR, Erbl, Erb2, Erb3, Src, Cyclin-dependent kinase (CDK), WEE1, polo-like kinasel (PLK1), casein kinase II (CK2), ATM, CHKs, DNA-PK, ATR, MAPK, MAPKK, MAP3K, MAP4K, a G-protein coupled receptor, a nuclear protein kinase, or EGFR.
  • CDK Cyclin-dependent kinase
  • WEE1 polo-like kinasel
  • CK2 casein kinase II
  • ATM
  • modifying the genome editing protein can further comprise joining the genome editing protein to a protein comprising a phosphatase.
  • the protein comprising the phosphatase can comprise a Shp phosphatase, Shp2, Shpl, PPM phosphatase, PP2C phosphatase, a nuclear phosphatase or a cytoplasmic phosphatase.
  • the material that modifies the genome editing protein can be joined to the genome editing protein via one or more ionic interactions.
  • the material that modifies the genome editing protein can be joined to the genome editing protein via one or more functional groups or functional linkers.
  • the material that modifies the genome editing protein can be joined to the genome editing protein via one or more covalent bonds.
  • the one or more stimuli can be endogenous to a cell.
  • the one or more stimuli can be exogenous to a cell.
  • the one or more stimuli can comprise light, temperature, pH, calcium, ion concentration, or electrical stimuli.
  • the one or more stimuli can comprise electromagnetic radiation.
  • the one or more stimuli can induce a cleavage of a group in the modified genome editing protein, which alters its activity.
  • the one or more stimuli can induce a conformational change in the modified genome editing protein, which alters its activity.
  • the one or more stimuli can induce activation of the modified genome editing protein. In some embodiments, the one or more stimuli can induce deactivation of the modified genome editing protein. In some embodiments, the one or more stimuli can induce the production of a detectable signal or marker. In some embodiments, modifying the genome editing protein can further comprise joining the genome editing protein to an additional material.
  • the additional material can comprise a metal (e.g., gold, silver, iron, iron oxide, titanium dioxide, lanthanide oxide, transition metal, or a transition metal oxide), graphene-like materials (e.g., molybdenum disulfide, tungsten disulfide, niobium diselenide, or boron nitride), a semiconductor, silica, or a polymer, or any combination thereof.
  • a metal e.g., gold, silver, iron, iron oxide, titanium dioxide, lanthanide oxide, transition metal, or a transition metal oxide
  • graphene-like materials e.g., molybdenum disulfide, tungsten disulfide, niobium diselenide, or boron nitride
  • the material modifying the genome editing protein can be additionally joined to a polymer comprising a component of extracellular matrix (e.g., collagen, fibronectin, or laminin) or a synthetic polymers (e.g., polyaniline, polypyrrole, or polythiophene).
  • the external dimensions of the material modifying a genome editing protein in the X-Y plane can be nano scaled (e.g., one nano-sized graphene flake) or macro scaled.
  • the minimal dimensions of the material modifying a genome editing protein in the X-Y plane can be 1 nm by 1 nm.
  • the external dimensions of the material modifying a genome editing protein along the Z-axis can be directly proportional to the number of layers of material incorporated.
  • the minimal dimension along the Z-axis can be defined by the thickness of a non-functionalized graphene monolayer, e.g., 0.34 nm.
  • maximum dimension of a particle of the material in the material modifying a genome editing protein can be from 0.00000001 nm to 1000 nm, .0001 nm to 100 nm, 0.1 nm to 50 nm, 0.5 nm to 10 nm, or any integer found between these ranges.
  • binding of the modified DNA binding protein to DNA can be directed by use of a guide RNA.
  • the target can be a subject in need of a therapy for a disease, or a cell.
  • the subject or cell can have a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.
  • a composition comprising: a genome editing protein joined to a material that modulates the function of the genome editing protein in response to one or more stimuli.
  • response of the modified genome editing protein to the one or more stimuli can modulate genome editing in a target.
  • the genome editing protein can comprise a zinc finger endonuclease, TALEN, CRISPR/cas9, Cpfl, a meganuclease, Cas3, CaslO, or the ABBIE system.
  • the genome editing protein can be joined to a recombinase, reverse transcriptase, or a topoisomerase.
  • the material can comprise a graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate, a polymer, or a carbon nanotube.
  • the material can comprise silicon, silica or a silicone polymer.
  • the genome editing protein can be joined to a protein comprising a kinase.
  • the protein comprising the kinase can comprise a tyrosine kinase, a serine/threonine kinase, ERK, ERK1, ERK2, p38, ERK5, JNK, MEKK1, MEKK2, MEKK3, MEKK4, MEKK5, a nuclear kinase, MEK1, MEK2, MEK3, EGFR, Erbl, Erb2, Erb3, Src, Cyclin-dependent kinase (CDK), WEE1, polo-like kinasel (PLK1), casein kinase II (CK2), ATM, CHKs, DNA-PK, ATR, MAPK, MAPKK, MAP3K, MAP4K, a G-protein coupled receptor, a nuclear protein kinase, or EGFR, or any combination thereof.
  • CDK Cyclin-dependent kinase
  • WEE1 polo-like kinasel
  • CK2 casein kin
  • the genome editing protein can be joined to a protein comprising a phosphatase.
  • the protein comprising the phosphatase can comprise a Shp phosphatase, Shp2, Shpl, a PPM phosphatase, a PP2C phosphatase, a nuclear phosphatase or a cytoplasmic phosphatase.
  • the material can be joined to the genome editing protein via one or more ionic interactions.
  • the material can be joined to the genome editing protein via one or more functional groups or functional linkers.
  • the material can be joined to the genome editing protein via one or more covalent bonds.
  • the one or more stimuli can be endogenous to a cell. In some embodiments, the one or more stimuli can be exogenous to a cell. In some embodiments, the one or more stimuli can comprise light, temperature, pH, calcium, ion concentration, or electrical stimuli or any combination thereof. In some embodiments, the one or more stimuli can comprise electromagnetic radiation. In some embodiments, the one or more stimuli can induce a cleavage of a group in the genome editing protein, which alters its activity. In some embodiments, the one or more stimuli can induce a conformational change in the genome editing protein, which alters its activity. In some embodiments, the one or more stimuli can induce activation of the genome editing protein.
  • the one or more stimuli can induce a deactivation of the genome editing protein. In some embodiments, the one or more stimuli can induce the genome editing protein to provide a detectable signal or marker. In some embodiments, the genome editing protein can be joined to an additional material.
  • the additional material can comprise a metal (e.g., gold, silver, iron, iron oxide, titanium dioxide, lanthanide oxide, transition metal, or transition metal oxide), a graphene-like material (e.g., molybdenum disulfide, tungsten disulfide, niobium diselenide, or boron nitride), semiconductor, silica, a polymer, or any combinations thereof.
  • the material can be joined to a component of extracellular matrix (e.g., collagen, fibronectin, or laminin) or a synthetic polymer (e.g., polyaniline, polypyrrole, or polythiophene).
  • extracellular matrix e.g., collagen, fibronectin, or laminin
  • synthetic polymer e.g., polyaniline, polypyrrole, or polythiophene.
  • external dimensions of the material in the X-Y plane can be nano scaled (e.g., one nano-sized graphene flake) or macro scaled.
  • the minimal dimensions of the material modifying a genome editing protein in the X-Y plane can be 1 nm by 1 nm.
  • the external dimensions of the material modifying a genome editing protein along the Z-axis can be related to the number of layers of materials incorporated.
  • the minimal dimension along the Z-axis can be defined by the thickness of a non-functionalized graphene monolayer, e.g., 0.34 nm.
  • maximum dimension of a particle of the material can be from 0.00000001 nm to 1000 nm, .0001 nm to 100 nm, 0.1 nm to 50 nm, 0.5 nm to 10 nm, or any integer found between these ranges.
  • binding of the modified DNA binding protein to DNA can be directed by use of a guide RNA.
  • the composition can further comprise donor DNA modified with the material that modulates the function of the genome editing protein in response to one or more stimuli, such that the material becomes incorporated into genomic DNA.
  • the target can be a subject in need of a therapy, or a cell.
  • the subject or cell can have a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.
  • a method of treating or inhibiting a disease or disorder comprising administration of one or more of the aforementioned compositions is provided.
  • a composition is provided for use in treating or inhibiting a disease or disorder in a subject, such as a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.
  • a method of detecting a disease or disorder comprising administering a composition to a cell or a subject and detecting a nucleotide sequence indicative of the disease or disorder, such as by using colorimetry, fluorescence detection, immunodetection, or rf reflectometry.
  • a composition is provided for use in detecting a disease or disorder in a subject or cell, optionally, wherein the detection is performed by using colorimetry, fluorescence detection, immunodetection, or rf reflectometry.
  • a method of genome editing comprising: modifying a donor polynucleotide by joining the donor polynucleotide to a material that modulates editing of the donor polynucleotide in response to one or more stimuli; incorporating the modified donor polynucleotide into a genomic DNA sequence of a target; and providing the one or more stimuli to modulate genome editing of the incorporated modified donor polynucleotide.
  • the genome editing is performed with a protein comprising a zinc finger endonuclease, TALEN, CRISPR/cas9, Cpfl, a meganuclease, Cas3, CaslO, or the ABBIE system.
  • the material can comprise a graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate, a polymer, or a carbon nanotube.
  • the material can comprise silicon, silica or a silicone polymer.
  • the material can be joined to the donor polynucleotide via one or more ionic interactions.
  • the material can be joined to the donor polynucleotide via one or more functional groups or functional linkers.
  • the material can be joined to the donor polynucleotide via one or more covalent bonds.
  • the one or more stimuli can be endogenous to a cell.
  • the one or more stimuli can be exogenous to a cell.
  • the one or more stimuli can comprise light, temperature, pH, calcium, ionic concentration, or electrical stimuli.
  • the one or more stimuli can comprise electromagnetic radiation.
  • the one or more stimuli can induce the modified donor polynucleotide to produce a detectable signal.
  • external dimensions of the material in the X-Y plane can be nano scaled (e.g., one nano-sized graphene flake) or macro scaled.
  • the minimal dimensions of the material modifying the donor polynucleotide in the X-Y plane can be 1 nm by 1 nm.
  • the external dimensions of the material modifying a donor polynucleotide along the Z-axis can relate to the number of layers of materials incorporated.
  • the minimal dimension along the Z-axis can be defined by the thickness of a non-functionalized graphene monolayer, e.g., 0.34 nm.
  • maximum dimension of a particle of the material can be from 0.00000001 nm to 1000 nm, .0001 nm to 100 nm, 0.1 nm to 50 nm, 0.5 nm to 10 nm, or any integer found between these ranges.
  • the genome editing of the incorporated modified donor polynucleotide further can comprise a guide RNA.
  • the target can be a subject in need of therapy, or a cell.
  • the subject or cell can have a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.
  • a composition comprising: a donor polynucleotide joined to a material that can modulate editing of the donor polynucleotide in response to one or more stimuli.
  • the modified donor polynucleotide can be incorporated into a genomic DNA sequence of a target.
  • providing the one or more stimuli can modulate genome editing of the incorporated modified donor polynucleotide.
  • the genome editing can be performed with a protein comprising a zinc finger endonuclease, TALEN, CRISPR/cas9, Cpfl, a meganuclease, Cas3, CaslO, or the ABBIE system.
  • the material can comprise graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate, a polymer, or a carbon nanotube.
  • the material can comprise silicon, silica or a silicone polymer.
  • the material can be joined to the donor polynucleotide via one or more ionic interactions.
  • the material can be joined to the donor polynucleotide via one or more functional groups or functional linkers.
  • the material can be joined to the donor polynucleotide via one or more covalent bonds.
  • the one or more stimuli can be endogenous to a cell.
  • the one or more stimuli can be exogenous to a cell.
  • the one or more stimuli can comprise light, temperature, pH, calcium, ionic concentration, or electrical stimuli.
  • the one or more stimuli can comprise electromagnetic radiation.
  • the one or more stimuli can induce the modified donor polynucleotide to produce a detectable signal or marker.
  • external dimensions of the material modifying a donor polynucleotide in the X-Y plane can be nanoscaled (e.g., one nano-sized graphene flake) or macro scaled.
  • the minimal dimensions of the material modifying the donor polynucleotide in the X-Y plane can be 1 nm by 1 nm.
  • the external dimensions of the material along the Z-axis can depend on the number of layers of materials incorporated.
  • the minimal dimension along the Z-axis can be defined by the thickness of a non-functionalized graphene monolayer, e.g., 0.34 nm.
  • maximum dimension of a particle of the material can be from 0.00000001 nm to 1000 nm, .0001 nm to 100 nm, 0.1 nm to 50 nm, 0.5 nm to 10 nm, or any integer found between these ranges.
  • the genome editing of the incorporated modified donor polynucleotide further can comprise a guide RNA.
  • the target can be a subject in need of a therapy or a cell.
  • the subject or cell can have a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.
  • a method of treating a disease or disorder comprising administration of a composition.
  • the composition is provided for use in treating or inhibiting a disease such as a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.
  • a disease such as a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.
  • the composition is provided for use as a medicament.
  • a method of detecting a disease or disorder in a subject or a cell comprising administration of a composition to the subject or cell and detecting the presence of a marker or nucleotide sequence indicative of the disease or disorder e.g., by using colorimetry, fluorescence detection, immunodetection, or rf reflectrometry.
  • a composition comprising a DNA binding protein selected from a meganuclease, a zinc finger protein, a TALEN, a cas DNA binding protein, a DNA bindingreverse transcriptase fusion protein, or a DNA binding-integrase fusion protein, which may optionally have an inactivated nuclease domain, such as obtained by mutation of one or more residues that abrogate, inhibit or attenuate the nuclease domain, joined to a material comprising a graphene, graphene oxide, carbon nanotube, fullerene, nanodiamond, or any combination thereof.
  • composition of aspect 1, wherein the DNA binding protein is selected from the group consisting of a cas DNA binding protein, a cas DNA binding-reverse transcriptase fusion protein, and a cas DNA binding protein-integrase fusion protein.
  • composition of aspect 1 or 2, wherein the material comprises graphene or graphene oxide.
  • RNA RNA.
  • a method for detecting a selected nucleotide sequence in a genome of a subject or a cell comprising administering the composition of any one of aspects 1-5 to the subject or contacting the cell with the composition of any one of aspects 1-5; and detecting the selected nucleotide sequence, such as by using colorimetry, fluorescence detection, immunodetection, or rf reflectometry.
  • a method of providing a drug to a selected nucleotide sequence in a genome of a subject or a cell comprising administering the composition of aspect 5 to the subject or the cell; and, optionally, determining or measuring the presence of the drug in the subject or cell and/or selecting a subject or a cell to receive the drug.
  • composition of aspect 5 for use in providing a drug to a subject or a cell.
  • a composition comprising a cas protein joined to a protein comprising a kinase or phosphatase, optionally wherein the cas protein has an inactivated nuclease domain such as can be obtained with one or more mutations, which abrogate, inhibit, or attenuate the nuclease.
  • composition of aspect 10, wherein the protein comprising the kinase comprises a MAP kinase, Src, a nuclear kinase, a serine/threonine kinase or a tyrosine kinase.
  • composition of aspect 10, wherein the protein comprising the phosphatase comprises a Shp, Shp2 or a nuclear phosphatase.
  • a method of modifying the phosphorylation state of a selected protein associated with a polynucleotide comprising administering the composition of any one of aspects 10-12 to a cell or a subject; and, optionally, determining or measuring the modification of the phosphorylation of the selected protein and/or selecting a protein associated with a polynucleotide for modification of its phosphorylation state and/or selecting a cell or a subject to receive an agent that modifies the phosphorylation state of the selected protein.
  • a method of detecting a selected nucleotide sequence in a subject, a cell, or an isolated population of polynucleotides or oligonucleotides comprising contacting the composition of any one of aspects 1-5 with the subject, the cell, or the isolated population of polynucleotides or nucleotides and performing a diagnostic evaluation.
  • a method of genome editing comprising: modifying a genome editing protein by joining the genome editing protein to a material that modulates the function of the genome editing protein in response to one or more stimuli; and providing one or more stimuli to the modified genome editing protein, which is joined to the material; wherein response of the modified genome editing protein to the one or more stimuli modulates genome editing in a target.
  • the genome editing protein comprises a zinc finger endonuclease, TALEN, CRISPR/cas9, Cpfl, meganuclease, Cas3, CaslO, or an ABBIE system.
  • the material that modifies the genome editing protein comprises a graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate or carbon nanotube.
  • the material that modifies the genome editing protein comprises silicon, silica or a silicone polymer.
  • modifying the genome editing protein comprises joining the genome editing protein to a protein comprising a kinase.
  • the protein comprising the kinase comprises a tyrosine kinase, serine/threonine kinase, ERK, ERK1, ERK2, p38, ERK5, TNK, MEKK1, MEKK2, MEKK3, MEKK4, MEKK5, a nuclear kinase, MEK1, MEK2, MEK3, EGFR, Erbl, Erb2, Erb3, Src, Cyclin-dependent kinase (CDK), WEE1, polo-like kinasei (PLK1), casein kinase II (CK2), ATM, CHKs, DNA-PK, ATR, MAPK, MAPKK, MAP3K, MAP4K, a G-protein coupled receptor, a nuclear protein kinase, or EGFR.
  • CDK Cyclin-dependent kinase
  • WEE1 polo-like kinasei
  • CK2 casein kinase II
  • ATM CH
  • modifying the genome editing protein further comprises joining the genome editing protein to a protein comprising a phosphatase.
  • the protein comprising the phosphatase comprises a Shp phosphatase, Shp2, Shpl, PPM phosphatase, PP2C phosphatase, a nuclear phosphatase or a cytoplasmic phosphatase.
  • modifying the genome editing protein further comprises joining the genome editing protein to an additional material.
  • the additional material comprises a metal (e.g., gold, silver, iron, iron oxide, titanium dioxide, lanthanide oxide, transition metal, or a transition metal oxide), graphene-like materials (e.g., molybdenum disulfide, tungsten disulfide, niobium diselenide, or boron nitride), a semiconductor, silica, or a polymer, or any combination thereof.
  • a metal e.g., gold, silver, iron, iron oxide, titanium dioxide, lanthanide oxide, transition metal, or a transition metal oxide
  • graphene-like materials e.g., molybdenum disulfide, tungsten disulfide, niobium diselenide, or boron nitride
  • a composition comprising: a genome editing protein joined to a material that modulates the function of the genome editing protein in response to one or more stimuli; wherein response of the modified genome editing protein to the one or more stimuli modulates genome editing in a target.
  • the material comprises a graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate, a polymer, or a carbon nanotube.
  • composition of aspect 56, wherein the protein comprising the kinase comprises a tyrosine kinase, a serine/threonine kinase, ERK, ERK1, ERK2, p38, ERK5, JNK, MEKK1, MEKK2, MEKK3, MEKK4, MEKK5, a nuclear kinase, MEK1, MEK2, MEK3, EGFR, Erbl, Erb2, Erb3, Src, Cyclin-dependent kinase (CDK), WEE1, polo- like kinasel (PLK1), casein kinase II (CK2), ATM, CHKs, DNA-PK, ATR, MAPK, MAPKK, MAP3K, MAP4K, a G-protein coupled receptor, a nuclear protein kinase, or EGFR, or any combination thereof.
  • CDK Cyclin-dependent kinase
  • WEE1 polo- like kinasel
  • composition of aspect 58, wherein the protein comprising the phosphatase comprises a Shp phosphatase, Shp2, Shpl, a PPM phosphatase, a PP2C phosphatase, a nuclear phosphatase or a cytoplasmic phosphatase.
  • composition of any one of aspects 51-64, wherein the one or more stimuli comprise light, temperature, pH, calcium, ion concentration, or electrical stimuli or any combination thereof.
  • composition of any one of aspects 51-66, wherein the one or more stimuli induce a cleavage of a group in the genome editing protein, which alters its activity.
  • composition of any one of aspects 51-67, wherein the one or more stimuli induce a conformational change in the genome editing protein, which alters its activity.
  • composition of aspect 72, wherein the additional material comprises a metal (e.g., gold, silver, iron, iron oxide, titanium dioxide, lanthanide oxide, transition metal, or transition metal oxide), a graphene-like material (e.g., molybdenum disulfide, tungsten disulfide, niobium diselenide, or boron nitride), semiconductor, silica, a polymer, or any combinations thereof.
  • a metal e.g., gold, silver, iron, iron oxide, titanium dioxide, lanthanide oxide, transition metal, or transition metal oxide
  • a graphene-like material e.g., molybdenum disulfide, tungsten disulfide, niobium diselenide, or boron nitride
  • semiconductor silica
  • silica silica
  • polymer or any combinations thereof.
  • a component of extracellular matrix e.g., collagen, fibronectin, or laminin
  • a synthetic polymer e.g., polyaniline, polypyrrole, or polythiophene
  • composition of any one of aspects 51-74, wherein external dimensions of the material in the X-Y plane is nano scaled (e.g., one nano-sized graphene flake) or macro scaled.
  • composition of aspect 75 wherein the minimal dimensions of the material modifying a genome editing protein in the X-Y plane are 1 nm by 1 nm.
  • composition of aspect 77 wherein, when the material comprises graphene, the minimal dimension along the Z-axis is defined by the thickness of a nonfunctionalized graphene monolayer, 0.001nm-0.75 nm, e.g., 0.34 nm.
  • maximum dimension of a particle of the material is from 0.00000001 nm to 1000 nm, .0001 nm to 100 nm, 0.1 nm to 50 nm, 0.5 nm to 10 nm, or any integer found between these ranges.
  • composition of any one of aspects 51-79, wherein binding of the modified DNA binding protein to DNA is directed by use of a guide RNA.
  • composition of aspect 82, wherein the subject or cell has a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.
  • composition of any one of aspects 51-83 for use in treating or inhibiting a disease or disorder in a subject such as a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease
  • a method of detecting a disease or disorder comprising administering the composition of any one of aspects 51-83 to a cell or a subject and detecting a nucleotide sequence indicative of the disease or disorder, such as by using colorimetry, fluorescence detection, immunodetection, or rf reflectometry.
  • composition of any one of aspects 51-83 for use in detecting a disease or disorder in a subject or cell optionally, wherein the detection is performed by using colorimetry, fluorescence detection, immunodetection, or rf reflectometry.
  • a method of genome editing comprising: modifying a donor polynucleotide by joining the donor polynucleotide to a material that modulates editing of the donor polynucleotide in response to one or more stimuli; incorporating the modified donor polynucleotide into a genomic DNA sequence of a target; and providing the one or more stimuli to modulate genome editing of the incorporated modified donor polynucleotide.
  • the material comprises a graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate, a polymer, or a carbon nanotube.
  • a composition comprising: a donor polynucleotide joined to a material that modulates editing of the donor polynucleotide in response to one or more stimuli; wherein the modified donor polynucleotide is incorporated into a genomic DNA sequence of a target; and wherein providing the one or more stimuli modulates genome editing of the incorporated modified donor polynucleotide.
  • composition of any one of aspects 109 or 110, wherein, the material comprises graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate, a polymer, or a carbon nanotube.
  • composition of any one of aspects 109 -111, wherein, the material comprises silicon, silica or a silicone polymer. [0125] 113. The composition of any one of aspects 109-112, wherein the material is joined to the donor polynucleotide via one or more ionic interactions.
  • composition of aspect 118, wherein the one or more stimuli comprise electromagnetic radiation.
  • composition of aspect 123 wherein, when the material comprises graphene, the minimal dimension along the Z-axis is defined by the thickness of a non-functionalized graphene monolayer, e.g., 0.34 nm.
  • 125 The composition of aspect 124, wherein maximum dimension of a particle of the material is from 0.00000001 nm to 1000 nm, .0001 nm to 100 nm, 0.1 nm to 50 nm, 0.5 nm to 10 nm, or any integer found between these ranges.
  • composition of any one of aspects 109-125, wherein the genome editing of the incorporated modified donor polynucleotide further comprises a guide RNA.
  • composition of aspect 127, wherein the subject or cell has a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.
  • composition of any one of aspects 109-128 for use in treating or inhibiting a disease such as a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.
  • composition of any one of aspects 109-128 for use as a medicament is provided.
  • a method of detecting a disease or disorder in a subject or a cell comprising administration of the composition of any one of aspects 109-128 to the subject or cell and detecting the presence of a marker or nucleotide sequence indicative of the disease or disorder e.g., by using colorimetry, fluorescence detection, immunodetection, or rf reflectrometry.
  • a method of genome editing comprising incubating a donor polynucleotide with carbon nanomaterial particles to allow for intercalation of the donor polynucleotide with carbon nanomaterial particles; washing the carbon nanomaterial bound donor polynucleotide to remove unbound carbon nanomaterial particles to leave substantially pure carbon nanomaterial particle-donor polynucleotide; incubating the carbon nanomaterial particle-donor polynucleotide with a genome editing protein and one or more appropriate targeting guide RNAs (gRNA) to form a preintegration complex with guide RNA (PICg); incubating the PICg with target genomic DNA (gDNA); integrating the carbon nanomaterial particle-donor polynucleotide of the PICg into the gDNA; washing to remove unintegrated donor polynucleotide; confirming the integration of donor sequences by passing the carbon nanomaterial particle donor integrated gDNA sample through a detector or sensor and comparing to control with scrambled guide RNA and/or no donor.
  • gRNA targeting guide
  • a method of genome editing comprising; conjugating a genome editing protein to carbon nanomaterial particles using covalent bonding or TT-TT stacking interaction; coating a surface comprising a plate, microplate, well, column or gel with the carbon nanomaterial particle-bound genome editing protein; introducing a target genomic polynucleotide sample into the setup; washing the surface with a buffer solution to remove unbound polynucleotides, retaining only the specifically bound target polynucleotide; adding a donor DNA sequence that is complementary to the target genomic polynucleotide region; introducing a buffer system that activates the genome editing protein, enabling it to integrate the donor DNA into the target genomic sequence; confirming the integration of donor sequences by passing the donor integrated gDNA sample through a detector or sensor and comparing to control with scrambled guide RNA and/or no donor.
  • the target genomic gDNA is selected from a cancer cell, from a mutant cell, from a diseased cell, or from a sample with viral or microbial DNA.
  • a method of modifying the acetylation state of a selected protein associated with a polynucleotide comprising administering the composition of any one of aspects 10-12 to a cell or a subject; and, optionally, determining or measuring the modification of the acetylation of the selected protein and/or selecting a protein associated with a polynucleotide for modification of its acetylation state and/or selecting a cell or a subject to receive an agent that modifies the acetylation state of the selected protein.
  • FIG. 1 shows a) an exemplary catalytically inactive Cas9/HIVl integrase fusion protein, b) an exemplary TALE/HIV1 integrase fusion protein, c) an exemplary zinc finger protein/HIVl integrase fusion protein, and d) an exemplary Cas9/HIVl integrase fusion protein designed to opposite sides of the DNA at the targeted site.
  • Each of the fusion proteins binds to a specific target sequence of DNA.
  • ZnFn is a Zinc finger protein.
  • “Integrase” represents one integrase unit or two integrase units linked, for example, by a short amino acid linker. In some embodiments, the integrase may be replaced by a recombinase.
  • Cas9 may be catalytically active or inactive.
  • FIG. 2 shows a DNA plasmid system comprising, a vector comprising a catalytically inactive Cas9/integrase fusion protein, a vector comprising a DNA sequence of interest, and a vector comprising a reverse transcriptase.
  • a guide RNA (gRNA) or RNAs may be provided separately.
  • Another vector can be used to express a gRNA.
  • “1 or 2” refers to one integrase or two integrases linked by, for example, an amino acid linker.
  • FIG. 3 shows detection of ABBIE 1 protein after isolation and purification from E coli. Coomassie stained gel.
  • FIG. 4 illustrates an example of a DNA binding protein joined to graphene oxide.
  • FIGs. 5A and 5B depict an experiment where viability of ovarian cancer cells was assessed following coincubation with ABBIE-engineered T cells.
  • FIGs. 6A-6D depict mechanisms of gene editing.
  • FIG. 6A depicts a key for the images used in FIGs 6B-6D.
  • FIG. 6B depicts an overview of the activity of various DNA editing tools involving a break, a processing event, followed by a change in the DNA code.
  • FIG. 6C depicts an overview of the DNA editing activity of the Abbie 1 system.
  • FIG. 6D depicts an overview of the use of the Abbie 1 system to introduce a biosensor directly within the genetic material of an organism.
  • FIG. 7 depicts a cell modified to integrate a biosensor directly within the genetic material.
  • FIG. 8 depicts a chip design comprising a central processing unit further comprising a control unit and arithmetic/logic unit.
  • the chip further comprises a memory unit.
  • FIGs. 9A and FIG. 9B show overviews of Homing remote control gene editing (ReCoG) and External ReCoG respectively.
  • FIG. 10 depicts embodiments of ABBIE (or DNA or RNA editing) protein bound to graphene (or other carbon nano material) bound to a surface of a plate, tube, gel or column.
  • Guide RNA is bound to the Cas DNA binding portion of the protein. Incubation with a sample having genomic DNA allows binding of target gDNA where appropriate guide RNA is present.
  • a donor DNA is integrated into the genomic DNA on the surface at the site of the bound proteins. Unbound materials are washed out and integration is assessed by passing through a detector or sensor for an electrostatic, luminescent or fluorescent signal. Difference in signals detected will denote integration of donor DNA.
  • FIG. 11 describes embodiments for in vitro (ex vivo) detection of target
  • the terms “individual”, “subject”, or “patient” as used herein, means a human or a non- human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate, or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate.
  • % w/w or “% wt/wt” as used herein has its ordinary meaning as understood in light of the specification and refers to a percentage expressed in terms of the weight of the ingredient or agent over the total weight of the composition multiplied by 100.
  • % v/v or “% vol/vol” as used herein has its ordinary meaning as understood in the light of the specification and refers to a percentage expressed in terms of the liquid volume of the compound, substance, ingredient, or agent over the total liquid volume of the composition multiplied by 100.
  • the invention is generally disclosed herein using affirmative language to describe the numerous embodiments.
  • the invention also includes embodiments in which subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures.
  • An “endogenous” nucleic acid, nucleotide, polypeptide, or protein as described herein is defined in relationship to the host organism.
  • An endogenous nucleic acid, nucleotide, polypeptide, or protein is one that naturally occurs in the host organism.
  • exogenous nucleic acid, nucleotide, polypeptide, or protein as described herein is defined in relationship to the host organism.
  • An exogenous nucleic acid, nucleotide, polypeptide, or protein is one that does not naturally occur in the host organism or is a different location in the host organism.
  • a gene is considered “knocked out” when an exogenous nucleic acid is transformed into a host organism (e.g. by random insertion or homologous recombination) resulting in the disruption (e.g. by deletion, insertion) of the gene.
  • the activity of the corresponding protein can be decreased. For example, by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, or 100%, as compared to the activity of the same protein wherein the gene has not been knocked out.
  • the transcription of the gene can be decreased, as compared to a gene that has not been knocked out, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, or 100%.
  • a “modified” organism is an organism that is different than an unmodified organism.
  • a modified organism can comprise a fusion protein of the disclosure that results in a knockout of a targeted gene sequence.
  • a modified organism can have a modified genome.
  • a “modified” nucleic acid sequence or amino acid sequence is different than the unmodified nucleic acid sequence or amino acid sequence.
  • a nucleic acid sequence can have one or more nucleic acids inserted, deleted, or added.
  • an amino acid sequence can have one or more amino acids inserted, deleted, or added.
  • a vector comprises a polynucleotide “operably linked” to one or more control elements, such as a promoter and/or a transcription terminator.
  • a nucleic acid sequence is operably linked when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operatively linked to DNA for a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide;
  • a promoter is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • Operably linked sequences can be contiguous and, in the case of a secretory leader, contiguous and in reading phase.
  • the procedure entails the sequential assembly of the appropriate amino acids into a peptide of a desired sequence while the end of the growing peptide is linked to an insoluble support.
  • the carboxyl terminus of the peptide is linked to a polymer from which it can be liberated upon treatment with a cleavage reagent.
  • an amino acid is bound to a resin particle, and the peptide generated in a stepwise manner by successive additions of protected amino acids to produce a chain of amino acids. Modifications of the technique described by Merrifield are commonly used. See, e.g., Merrifield, J. Am. Chem. Soc. 96: 2989-93 (1964).
  • Introduction of the expression vector into the host cell can be accomplished by a variety of methods including calcium phosphate transfection, DEAE-dextran mediated transfection, polybrene, protoplast fusion, liposomes, direct microinjection into the nuclei, scrape loading, biolistic transformation and electroporation.
  • Large scale production of proteins from recombinant organisms is a well-established process practiced on a commercial scale and well within the capabilities of one skilled in the art.
  • a polynucleotide that is biased for a particular codon usage can be synthesized de novo, or can be genetically modified using routine recombinant DNA techniques, for example, by a site directed mutagenesis method, to change one or more codons such that they are biased for chloroplast codon usage.
  • the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (as described, for example, in Henikoff & Henikoff (1989) Proc. Natl. Acad Sci. USA, 89: 10915).
  • W word length
  • E expectation
  • BLOSUM62 scoring matrix as described, for example, in Henikoff & Henikoff (1989) Proc. Natl. Acad Sci. USA, 89: 10915.
  • the BLAST algorithm also can perform a statistical analysis of the similarity between two sequences (for example, as described in Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787 (1993)).
  • BLAST algorithm One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, less than about 0.01, or less than about 0.001.
  • the instant disclosure comprises a system comprising: A) A viral integrase (or a recombinase) covalently linked to a Cas protein (e.g. Cas9) that is, for example, inactive for DNA cutting ability or nuclease activity.
  • a viral integrase or a recombinase covalently linked to a Cas protein (e.g. Cas9) that is, for example, inactive for DNA cutting ability or nuclease activity.
  • the viral integrase or a bacterial or phage recombinase
  • TALE protein zinc finger proteins where these proteins are designed to target a specific sequence of DNA in a genome.
  • This may be provided in an expression vector or as a purified protein.
  • a gene of interest or DNA sequence of interest
  • the GOI or DNA sequence of interest may be modified to be recognized by the viral integrase as needed.
  • the viral att sites can be added to the ends of the DNA sequence.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • loci locus defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched poly
  • a polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • chimeric RNA refers to the polynucleotide sequence comprising the guide sequence, the tracr sequence and the tracr mate sequence.
  • guide sequence refers to the about 20 bp (12-30 bp) sequence within the guide RNA that specifies the target site and may be used interchangeably with the terms “guide” or “spacer”.
  • tracr mate sequence may also be used interchangeably with the term “direct repeat(s)”.
  • wild type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
  • variant or “mutant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature. In relation to the genes, these terms indicate a number of changes in a gene that make it different from the wild-type gene including single nucleotide polymorphisms (SNPs), insertions, deletions, gene shifts among others.
  • SNPs single nucleotide polymorphisms
  • a catalytically inactive form of Cas9 is described in Guilinger et al, Fusion of catalytically inactive Cas9 to Fold nuclease improves the specificity of genome modification, Nature Biotechnology, Apr. 25, 2014, volume 32, pages 577-582.
  • Guilinger et al attached the catalytically inactive Cas9 to a Fokl enzyme to achieve greater specificity in making cuts in genomic DNA.
  • This catalytically inactive Cas9 allows for Cas9 to use RNA guides for binding of genomic DNA, while not being able to cut the DNA.
  • RNA guides toward a specific DNA sequence can be designed by various computer-based tools.
  • the integrase or recombinase can be used to construct hybrid integrase or recombinase that are active in a yeast or cell assay. These reagents are also active in plant cells and in animal cells.
  • TALEN studies used the wild- type Fokl cleavage domain, but some subsequent TALEN studies also used Fokl cleavage domain variants with mutations designed to improve cleavage specificity and cleavage activity. Both the number of amino acid residues between the TALEN DNA binding domain and the integrase or recombinase domain and the number of bases between the two individual TALEN binding sites are parameters for achieving high levels of activity.
  • the number of amino acid residues between the TALEN DNA binding domain and the integrase or recombinase domain may be modified by introduction of a spacer (distinct from the spacer sequence) between the plurality of TAL effector repeat sequences and the integrase or recombinase domain.
  • the spacer sequence may be 6 to 102 or 9 to 30 nucleotides or 15 to 21 nucleotides. These spacers will usually not provide other activity to the hybrid protein besides providing a link between the DNA targeting protein (Cas9, TALE or zinc finger protein) and the integrase or recombinase.
  • the amino acids for the spacers and for other uses in the instant disclosure are as listed above.
  • TALEN genes Once the TALEN genes have been assembled together they are inserted into plasmids; the plasmids are then used to transfect the target cell where the gene products are expressed and enter the nucleus to access the genome.
  • TALENs can be used to edit genomes by inducing double-strand breaks (DSB), which cells respond to with DNA repair, however, the instant disclosure seeks to use the power of viral integrases or bacterial or phage recombinases to insert DNA sequences of interest into targeted sites in the genome. See disclosure of WO 2014134412 and U.S. Pat. No. 8,748,134, hereby expressly incorporated by reference in their entireties.
  • Zinc finger proteins for binding DNA and their design are described in U.S. Pat. No. 7,928,195, US 2009/0111188, and U.S. Pat. No. 7,951,925 hereby expressly incorporated by reference in their entireties.
  • Zinc finger proteins utilize a number of linked zinc finger domains in a specified order to bind to a specific sequence of DNA.
  • Zinc finger protein endonucleases have been well-established.
  • Zinc finger proteins are proteins that can bind to DNA in a sequence-specific manner. Zinc fingers were first identified in the transcription factor TFIIIA from the oocytes of the African clawed toad, Xenopus laevis. A single zinc finger domain of this class of ZFPs is about 30 amino acids in length, and several structural studies have demonstrated that it contains a beta turn (containing two conserved cysteine residues) and an alpha helix (containing two conserved histidine residues), which are held in a particular conformation through coordination of a zinc atom by the two cysteines and the two histidines. This class of ZFPs is also known as C2H2 ZFPs. Additional classes of ZFPs have also been suggested.
  • ZFPs Many zinc finger proteins have conserved cysteine and histidine residues that tetrahedrally-coordinate the single zinc atom in each finger domain.
  • most ZFPs are characterized by finger components of the general sequence: -Cys-(X)2-4-Cys- (X)12-His-(X)3-5-His- (SEQ ID NO:49, in which X represents any amino acid (the C2H2 ZFPs).
  • the zinc-coordinating sequences of this most widely represented class contain two cysteines and two histidines with particular spacings.
  • the folded structure of each finger contains an antiparallel 0-turn, a fingertip region and a short amphipathic a-helix.
  • the proteins include those unrelated to the zinc finger proteins, TALEN and CRISPR proteins that may bind to specific sequences in genomic DNA of various organisms. These may include transcription factors, transcriptional repressors, meganucleases, endonuclease DNA binding domains and others.
  • Integrases and endonuclease fusion proteins thereof are described in US 2009/0011509. Integrases introduced are lentiviral integrase and HIV1 (human immunodeficiency virus 1) integrase.
  • the instant disclosure fuses a catalytically inactive (or active) Cas9, TALE or Zinc finger protein to an integrase to target the integrase to a specific region of DNA in the genome that is chosen by the user.
  • the HIV-1 integrase like other retroviral integrases, is able to recognize special features at the ends of the viral DNA located in the U3 and U5 regions of the long terminal repeats (LTRs) (Brown, 1997).
  • LTRs long terminal repeats
  • the LTR termini are the only viral sequences thought to be required in cis for recognition by the integration machinery of retroviruses. Short imperfect inverted repeats are present at the outer edges of the LTRs in both murine and avian retroviruses (reviewed by Reicin et al., 1995).
  • sequences are both necessary and sufficient for correct proviral integration in vitro and in vivo. Sequences internal to the CA dinucleotide appear to be important for optimal integrase activity (Brin & Leis, 2002a; Brin & Leis, 2002b; Brown, 1997). The terminal 15 bp of the HIV-1 LTRs have been shown to be crucial for correct 3' end processing and strand transfer reactions in vitro (Reicin et al., 1995; Brown, 1997).
  • Integrases consist of three domains connected by flexible linkers. These domains are an N-terminal HH-CC zinc-binding domain, a catalytic core domain and a C- terminal DNA binding domain (Lodi et al, Biochemistry, 1995, volume 34, pages 9826- 9833). In some aspects of the disclosure the integrase bound to the Cas9 (or other DNA binding molecule) will not have the C-terminal binding domain.
  • two different fusion proteins will be produced where one has catalytically inactive Cas9 (or TALE or zinc finger protein) fused with the N-terminal zinc binding domain of an integrase and the other has catalytically inactive Cas9 (or TALE or zinc finger protein) fused with the catalytic core domain of the integrase.
  • the two different fusion proteins will be designed to bind to opposite strands of the genomic DNA as seen with TALE-Fokl or Zinc finger-Fokl systems. In this manner, when the N-terminal domain and the catalytic core come in contact, at the site on the genomic DNA, it will exhibit integrase activity.
  • linkers used between fusion proteins/peptides being synthesized will be composed of amino acids. At the DNA level, these are represented by 3 base pair (bp) codons as known in the genetic code. Linkers may be from 1 to 1000 amino acids in length and any integer in between. For example, linkers are from 1 to 200 amino acids in length, 5 to 30 amino acids, or linkers are from 1 to 20 amino acids in length.
  • nucleic acid includes DNA, RNA, and nucleic acid analogs, and nucleic acids that are double-stranded or single-stranded (i.e., a sense or an antisense single strand).
  • Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine and 5-bromo-2'-doxycytidine for deoxycytidine.
  • Modifications of the sugar moiety include modification of the 2' hydroxyl of the ribose sugar to form 2'-0- methyl or 2'-0-allyl sugars.
  • the deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7(3): 187; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5.
  • nucleic acid sequences can be operably linked to a regulatory region such as a promoter. Regulatory regions can be from any species. As used herein, operably linked refers to positioning of a regulatory region relative to a nucleic acid sequence in such a way as to permit or facilitate transcription of the target nucleic acid. Any type of promoter can be operably linked to a nucleic acid sequence. Examples of promoters include, without limitation, tissue-specific promoters, constitutive promoters, and promoters responsive or unresponsive to a particular stimulus (e.g., inducible promoters).
  • Additional regions that may be useful in nucleic acid constructs include, but are not limited to, polyadenylation sequences, translation control sequences (e.g., an internal ribosome entry segment, IRES), enhancers, inducible elements, or introns.
  • Such regulatory regions may not be necessary, although they may increase expression by affecting transcription, stability of the mRNA, translational efficiency, or the like.
  • Such regulatory regions can be included in a nucleic acid construct as desired to obtain optimal expression of the nucleic acids in the cell(s). Sufficient expression can sometimes be obtained without such additional elements.
  • a nucleic acid construct may be used that encodes signal peptides or selectable markers.
  • Signaling (marker) peptides can be used such that an encoded polypeptide is directed to a particular cellular location (e.g., the cell surface).
  • Non-limiting examples of such selectable markers include puromycin, ganciclovir, adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR), hygromycin-B-phosphtransferase, thymidine kinase (TK), and xanthin-guanine phosphoribosyltransferase (XGPRT). These markers are useful for selecting stable transformants in culture.
  • Other selectable markers include fluorescent polypeptides, such as green fluorescent protein, red fluorescent, or yellow fluorescent protein.
  • Nucleic acid constructs can be introduced into cells of any type using a variety of biological techniques known in the art. Non-limiting examples of these techniques would include the use of transposon systems, recombinant viruses that can infect cells, or liposomes or other non-viral methods such as electroporation, microinjection, or calcium phosphate precipitation, that are capable of delivering nucleic acids to cells. A system called NucleofectionTM may also be used.
  • Nucleic acids can be incorporated into vectors.
  • a vector is a broad term that includes any specific DNA segment that is designed to move from a carrier into a target DNA.
  • a vector may be referred to as an expression vector, or a vector system, which is a set of components needed to bring about DNA insertion into a genome or other targeted DNA sequence such as an episome, plasmid, or even virus/phage DNA segment.
  • Vectors most often contain one or more expression cassettes that comprise one or more expression control sequences, wherein an expression control sequence is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence or mRNA, respectively.
  • Plasmids and viral vectors including retroviral vectors
  • Mammalian expression plasmids typically have an origin of replication, a suitable promoter and optional enhancer, and also any necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences.
  • Such vectors include plasmids (which may also be a carrier of another type of vector), adenovirus, adeno-associated virus (AAV), lentivirus (e.g., modified HIV-1, SIV or FIV), retrovirus (e.g., ASV, ALV or MoMLV), and transposons (P-elements, Tol-2, Frog Prince, piggyBac or others).
  • plasmids which may also be a carrier of another type of vector
  • adenovirus e.g., adeno-associated virus (AAV)
  • lentivirus e.g., modified HIV-1, SIV or FIV
  • retrovirus e.g., ASV, ALV or MoMLV
  • transposons P-elements, Tol-2, Frog Prince, piggyBac or others.
  • Bacterial and viral genes and proteins for use in the disclosure are listed below in the section entitled “SEQUENCES OF THE DISCLOSURE”.
  • Other viral integrases for example, those from mouse mammary tumor virus (MMTV) and adenovirus can also be used in the methods and compositions disclosed herein.
  • a pooled population of edited cells are considered a mixture of cells that have received a gene edit and cells that have not.
  • graphene-based or graphene-related materials are used in development of numerous applications in energy, electronics, sensors, light processing, medicine, and environmental fields.
  • Graphene the “founding” member of this family, is a two-dimensional material made of sp2-hybridized carbon atoms arranged in a hexagonal honeycomb lattice.
  • the extended family of graphene-related materials includes graphene (single- and multilayered), graphite, polycyclic aromatic hydrocarbons, carbon nanotubes, fullerenes, functionalized fullerenes, various graphene nanostructures of different dimensionalities (e.g., graphene nanoparticles, or graphene quantum dots: graphene nanoribbons: graphene nanomeshes; graphene nanodisks; graphene foams; graphene nanopillars), any combinations of other graphene-related materials, substituted graphene- related materials (e.g., the substitution of carbon atoms with N, B, P, S, Si, or others), and graphene-related materials functionalized with reactive functional groups (e.g., carboxyl groups, esters, amides, thiols, hydroxyl groups, diol groups, ketone groups, sulfonate groups, carbonyl groups, aryl groups, epoxy groups, phenol groups, phosphonic acids, amine
  • Proteins may be attached to CNTs (carbon nanotubes) covalently through reaction with the functionalized CNT surface or non-covalently by non-specific adsorption (Kam, et al., J. Am. Chem. Soc., 126(22):6850-l (2004); Karajanagi, et al., Langmuir, 20: 11594-9 (2004)).
  • CNTs have a high capacity for protein adsorption due to their high surface area.
  • the surface area of CNTs available for protein adsorption may also be adjusted by altering the surface chemistry of the CNT. In this way, accessible surfaces that are a priori not available for protein adsorption may be made accessible through chemical treatment.
  • CNTs are subjected to treatment with acid prior to protein adsorption. Recent studies have demonstrated that acid treatment of SWNTs induces defects on the surface of the nanotubes (Hu, et al., Jour. Phys. Chem.
  • CNTs are treated with nitric acid prior to protein adsorption, which introduces carboxylic acid groups at the open ends leading to sites of defects and hence increasing the capacity for protein adsorption (Hu, et al., Jour. Phys. Chem. B, 107:13838-42 (2003)).
  • the CNTs are reduced following acid treatment.
  • CNTs may be treated with lithium borohydride to preferentially reduce the oxygenated groups created by the acid treatment, favoring the dispersion of the CNTs in solution (U.S. Published Application No. 2004/0232073, herein expressly incorporated by reference in its entirety) and further increasing the surface area available for protein adsorption.
  • LiBH4 lithium borohydride
  • proteins can also be attached to CNTs through covalent interactions through various functional groups.
  • Functionality refers to conjugation of a molecule to the surface of the CNT via a functional chemical group (carboxylic acids, aldehydes, amines, sulfhydryls and hydroxyls) present on the CNT and present on the molecule to be attached.
  • a functional chemical group carboxylic acids, aldehydes, amines, sulfhydryls and hydroxyls
  • Biochemical functionalization of CNTs using various proteins for potential applications in biological systems are described by Kam, et al., J. Am. Chem. Soc., 126(22):6850-l (2004); Bianco, et al., Curr. Opin. Chem.
  • kinases can include Erk, p38, JNK and other MAPK, MAPKK, MAP3K and MAP4K proteins or kinase domains thereof.
  • a kinase may be Src kinase or a nuclear protein kinase.
  • a kinase may be a kinase domain of receptor kinase like EGFR or Erb2 or other kinase.
  • kinases may be serine threonine receptor kinase, tyrosine receptor kinases, G-protein coupled receptors or kinase domains thereof.
  • the single layer of graphene is highly transparent with absorption of 2.3% across the visible spectrum and beyond with an absorption peak of '10% in the ultraviolet.
  • graphene absorbs photons, it transforms their energy into electrical current by creating photo-generated excitons via photoelectric and/or photo-thermoelectric mechanisms.
  • the photoresponsivity of graphene is somewhat low ( ⁇ 10 mAW-1) due to the low optical absorption in monolayer graphene and the short recombination lifetime (on the scale of a picosecond) of the photo-generated carriers, leading to a low internal quantum efficiency of '6- 16%.
  • GO is hydrophilic due to the presence of oxygen-containing functional groups.
  • rGO is intermediate in hydrophilicity because the number of remaining oxygen-containing functional groups in rGO is lower than in the highly oxidized GO.
  • graphene and its derivatives are their utilization for tissue engineering, antibacterial treatment, drug and gene delivery, and as contrast agents for bioimaging.
  • incorporation of graphene-related materials as structural elements both for planar or three-dimensional scaffolds for cell cultures greatly enhanced cell adhesion, improved the cell proliferation, accelerated the rate of cell maturation, enhanced the neurite sprouting and outgrowth, and supported the neuronal lineage during the stem cell differentiation.
  • Graphene-related materials can be combined with various supplementary materials in order to engineer specific properties in a G-biointerface. Such properties might include the increased absorption efficiency, the desired spectral activation profile, p- or n- type functionality, the speed of switch on-off, processability, durability, compatibility, electrical conductivity, possible chemical functionalization for creating specific biointerfaces, and a three-dimensional configuration.
  • examples of supplementary materials can include structures made of metal (e.g., gold, silver, iron, iron oxide, titanium dioxide, lanthanide oxide, transition metal, and transition metal oxide), graphene-like materials (e.g., molybdenum disulfide, tungsten disulfide, niobium diselenide, and boron nitride), semiconductor, silica, polymers, or combinations thereof.
  • metal e.g., gold, silver, iron, iron oxide, titanium dioxide, lanthanide oxide, transition metal, and transition metal oxide
  • graphene-like materials e.g., molybdenum disulfide, tungsten disulfide, niobium diselenide, and boron nitride
  • semiconductor e.g., silica, polymers, or combinations thereof.
  • graphene-related materials can be combined with polymers, including, but not limited to, the naturally occurring components of extracellular matrix (e.g., collagen, fibronectin, and laminin) or synthetic polymers (e.g., polyaniline, polypyrrole, and polythiophene).
  • polymers including, but not limited to, the naturally occurring components of extracellular matrix (e.g., collagen, fibronectin, and laminin) or synthetic polymers (e.g., polyaniline, polypyrrole, and polythiophene).
  • graphene-related materials can be used as standalone structures without any external structural support, or directly deposited on a substrate. In some embodiments, they can also be combined with other materials that can provide independent structural support. In some embodiments, specific spatial configurations of a G- biointerface can be achieved both by exploiting inherent dimensionality of various graphene- related materials and by incorporating appropriate supplementary materials.
  • external dimensions of a G-biointerface in the X-Y plane can range from nano dimensions (e.g., one nano-sized graphene flake) to macro dimensions limited only by manufacturing capabilities.
  • the minimal dimensions in the X-Y plane can be 1 nm by 1 nm.
  • external dimensions along the Z-axis can depend on the number of layers of graphene-related materials incorporated into a G-biointerface.
  • the minimal dimension along the Z-axis can be defined by the thickness of a non-functionalized graphene monolayer, 0.34 nm.
  • the target can be one or more intact cells, one or more cellular fractions, or one or more artificial membrane structures.
  • examples of cellular fractions include any luminal organelles such as nucleus, ribosomes, mitochondria, endoplasmic reticulum, Golgi apparatus, vacuoles, synaptic vesicles and lysosomes.
  • examples of artificial membrane structures can include phospholipid micelles, micro- and nanocapsules, and semi-liquid films on supportive structures.
  • the one or more cells can generally be any type of cells which have a membrane and membrane potential.
  • the cells can be bacterial (Gram-positive or Gram-negative), eucaryotic, procaryotic, fungal, insect, avian, reptilian, oocyte, fly, zebrafish, fish, nematode, amphibian, or mammalian cells.
  • the methods can also be used on non-cell materials such as artificial membranes, liposomes, and phospholipid bilayers.
  • examples of primary mammalian cells can comprise human, mouse, rat, dog, cat, bear, moose, cow, horse, pig, or Chinese hamster ovary (“CHO”) cells.
  • other examples of types of cells can comprise immune system cells (e.g., B-cells, T-cells), oocytes, red blood cells, white blood cells, neurons, CMs, epithelial, glia, fibroblast, stem cells, cancer cells, secretory cells, or immortalized cells.
  • cells in order to be activated via G-biointerfaces, cells have to contact or to be positioned in the close proximity to the surface of graphene-related materials. In some embodiments, to achieve such positioning, either cells can be added to G- biointerfaces, or G-biointerfaces can be added to cells. In some embodiments, other related items are described in US20170143762A1, herein expressly incorporated by reference in its entirety.
  • graphene, carbon nanotubes and other materials can be incorporated in various sizes.
  • a maximum dimension of a particle of the material may be from 0.00000001 nm to 1000 nm, .0001 nm to 100 nm, 0.1 nm to 50 nm, 0.5 nm to 10 nm, or any integer found between these ranges.
  • kinases and phosphatases are known to be involved in signaling in the cell and are involved in activating or deactivating transcription factors.
  • Several kinases and phosphatases alter proteins that associate with DNA or RNA.
  • kinase, phosphatases or the active domains thereof e.g. kinase or phosphatase active domain
  • these fusion proteins can also be bound with materials as described above (e.g. graphene oxide, graphene, carbon nanotubes).
  • kinases can include Map kinases.
  • kinases can include receptor tyrosine kinases and receptor serine/threonine kinases.
  • kinases can be tyrosine kinases or serine/threonine kinases.
  • kinases can include ERK, ERK1, ERK2, p38, ERK5, JNK, MEKK1, MEKK2, MEKK3, MEKK4, MEKK5, nuclear kinases, MEK1, MEK2, MEK3, and others.
  • receptor kinases can include EGFR, Erbl, Erb2, Erb3 and others.
  • Src can be used for the invention.
  • cyclin- dependent kinases CDKs
  • WEE1 polo-like kinasei
  • CK2 casein kinase II
  • ATM CHKs
  • DNA-PK or ATR can be kinases that target DNA processes.
  • kinase domains of any of the mentioned kinases can also be attached to a DNA binding protein/domain as mentioned above.
  • the DNA binding can be controlled by use of a guide RNA.
  • these can be human, mammalian, mouse, rat, primate, microbial, viral, bacterial, fungal, yeast, plant or of other origins. See MAP Kinase Pathways - PMC for details on MAPK pathways. See PMC6827047/pdf/cancers-l 1 -01618.pdf and PMC 10299397/pdf/ij ms-24-10212.pdf.
  • phosphatases can include nuclear phosphatases and cytoplasmic phosphatases.
  • phosphatases can include Shp phosphatases, Shp2, Shpl, and others.
  • phosphatases can include those that regulate transcription factors and/or other nuclear proteins.
  • phosphatases can include PPM phosphatases and PP2C phosphatase.
  • phosphatase domains of any of the mentioned kinases can also be attached to a DNA binding protein/domain as mentioned above.
  • the DNA binding can be controlled by use of a guide RNA.
  • these may be human, mammalian, mouse, rat, primate, microbial, bacterial, fungal, yeast, plant or of other origins.
  • methods can include first administering any of the aforementioned proteins, genome editing proteins, fusion proteins or molecules to a cell, tissue or organism. In some embodiments, methods can further include administering the appropriate guide RNA, donor DNA, and/or cofactors before, during or after the first administration. In some embodiments, methods can include incorporating the donor DNA polynucleotide into the genome of a cell via genome editing procedures. In some embodiments, methods can include controlling operation of the proteins via the material (e.g. graphene) bound to the protein. In some embodiments, methods can include control of genome editing functions such as integration, DNA cutting or nicking or deletions of genomic DNA.
  • methods can include activating and/or detecting a signal that emits from the material bound to the protein.
  • methods can include the material causing the protein to change conformation and/or promote a cleavage event (e.g. cleavage of kinase, phosphatase, integrase domain, linker or cas9 domain) that changes function of the genome editing protein to turn on or off DNA editing and/or integration.
  • methods can include targeting disease genes.
  • methods can include targeting safe harbor loci in the cell’s DNA.
  • methods can include attaching the material or protein to other molecules like those that promote detection, drugs, vitamins or such.
  • methods can include cancer drugs, cardiovascular drugs, neural drugs, and DNA altering drugs (drugs that act on enzymes affecting genomic DNA expression and/or repair for example).
  • the subject or cell for administration can be human, primate, mammalian, bacterial, fungal, yeast, fish, amphibian, reptile, plant or others.
  • An exemplary method involves using donor DNA bound to graphene oxide with an engineered integrase cas9 system for integration of the donor DNA with bound graphene oxide.
  • integration can be performed by providing the integration cas9 system, guide RNA and graphene oxide bound donor DNA to cells.
  • cells can be harvested and DNA can be extracted.
  • extracted DNA can be attached to a graphene oxide binding surface, the surface can be washed to maintain the attached DNA on the surface but to remove all other DNA, and then the bound DNA can be detached from the graphene oxide and collected.
  • sequencing can be performed to find where the Donor DNA was incorporated in the genomic DNA.
  • the extracted DNA can be fragmented by DNases or other enzymes prior to binding of graphene oxide to the provided graphene oxide binding surface.
  • the surface can be a sheet, a dish, a plate, a plate with wells, a column or other suitable surface coated with a component that binds graphene oxide.
  • this system can be used on mitochondrial or genomic DNA.
  • detection can be accomplished by detecting the small electrical pulses from graphene or detecting antibodies, antibodies linked to detectable moieties/compounds, fluor ophores, colorimetric enzymes or other diagnostic compounds that have been bound to the graphene.
  • Another exemplary method is using the modified protein-material that comprises cas9, reverse transcriptase cas9, or Integrase-cas9 that can be bound or linked to a material (e.g. graphene oxide, graphene, carbon nanotubes).
  • a material e.g. graphene oxide, graphene, carbon nanotubes.
  • detection can be performed in this instance to find which cells, the modified protein material has entered or even which chromosome it has bound.
  • detection can be done with techniques including microscopy, flow cytometry, fluorescent microscopy, and the like.
  • detection is also possible by a high speed electrical readout with radiofrequency (rf) reflectometry.
  • rf radiofrequency
  • biotin-streptavidin interactions are used in place of the carbon nanomaterial particles, or graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate, polymer, or carbon nanotube systems described herein.
  • the proteins, polynucleotides, or oligonucleotides described herein are biotinylated.
  • kinases or phosphatases that regulate base editing are envisioned.
  • kinases such as ATM and ATR are regulators of homologous recombination (HR) proteins by phosphorylating at specific sites, while phosphatases like EYA4 dephosphorylate these sites, effectively controlling the activity of HR proteins and influencing the DNA repair process.
  • HR homologous recombination
  • phosphatases like EYA4 dephosphorylate these sites, effectively controlling the activity of HR proteins and influencing the DNA repair process.
  • Such kinases and phosphatases bound DNA binding proteins like Cas proteins are envisioned to further activate and deactivate HR at a given site.
  • additional HR proteins or other DNA associated proteins are added or provided to the cells as necessary.
  • HIV integrase is phosphorylated, and this phosphorylation is considered necessary for activity, with studies showing that specific kinases like JNK and GCN2 can phosphorylate integrase, impacting its function during HIV infection; phosphorylation often occurs on the C-terminal domain of the protein and can influence its interaction with cellular cofactors, regulating its enzymatic activity.
  • the carbon nanomaterial particles are labeled prior to incubation.
  • the donor integrated gDNA sample is amplified prior to confirming the integration of donor sequences.
  • the carbon nanomaterial particles comprise graphene.
  • the genome editing protein comprises an ABBIE system.
  • the genome editing protein comprises a viral integrase- dCas9 complex.
  • the detector or sensor uses electrostatic, luminescent, or fluorescent detection.
  • a method of modifying the acetylation state of a selected protein associated with a polynucleotide comprising administering any of the compositions described herein to a cell or a subject; and, optionally, determining or measuring the modification of the acetylation of the selected protein and/or selecting a protein associated with a polynucleotide for modification of its acetylation state and/or selecting a cell or a subject to receive an agent that modifies the acetylation state of the selected protein.
  • a microfluidic chip with channels for sample input, mixing, and reaction zones is further provided.
  • biotin-streptavidin interactions can be used in place of the carbon nanomaterial particles, or graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate, polymer, or carbon nanotube systems.
  • the polynucleotides, or oligonucleotides described herein are biotinylated.
  • the DNA sequence of catalytically inactive Cas9 is incorporated into an expression vector with a 12, 15, 18, 21, 24, 27 or 30 bp spacer (codes for 4, 5, 6, 7, 8, 9 or 10 amino acids as the linker between the Cas9 and the integrase) and the HIV1 integrase.
  • recombinases of bacterial or phage origin are used rather than integrases. These include Hin recombinase (SEQ ID NO: 25) and Cre recombinase (SEQ ID NO: 26) with or without mutations that allow them to recombine DNA at any other sites.
  • a His or cMyc tag may be included to isolate the fusion protein.
  • the expression vector uses a promoter that will be activated in the cells that will be provided with the vector.
  • the CMV cytomegalovirus promoter
  • the U6 promoter is also commonly used.
  • a T7 promoter may be used for in vitro transcription in certain embodiments.
  • the DNA sequence of interest will be inserted into the appropriate expression vector and sites will be appropriately added to the DNA sequence of interest so the HIV1 integrase will recognize the sequences for integration into the genome. These sites are termed att sites (U5 and U3 att sites) (see Masuda et al, Journal of Virology, 1998, volume 72, pages 8396-8402). Homology arms for the target site in the genome can be included in regions flanking the 5' and 3' ends of the DNA (gene) sequence of interest (see Ishii et al, PLOS ONE, Sep. 24, 2014, DOI: 10.1371/journal.pone.0108236).
  • the integrase recognition sites may not be included.
  • Markers such as drug resistance markers (e.g. blasticidin or puromycin), will be included in order to check for insertion of the DNA sequence of interest and to help assay for random insertions in the genome.
  • These resistance markers can be engineered in such a way to remove them from the targeted genome landing pad For example flanking the puromycin resistance gene with a LoxP sites and introducing exogenously expressed CRE would remove the internal sequence leaving a scar containing a LoxP site.
  • a reverse transcriptase may also be co-expressed in such systems as the designed DNA sequence (Gene) of interest in the vector will become expressed as RNA and will have to be converted back to DNA for integration by the integrase enzyme.
  • the reverse transcriptase may be viral in origin (e.g. a retrovirus such as HIV1). This may be incorporated within the same vector as the DNA sequence of interest.
  • Cells were electroporated for the vectors described above along with the Cas9 RNA guides required for the target site in the genome.
  • vectors were created that expressed all of the components (fusion Cas9-integrase (or recombinase), the Cas9 RNA guides, and the DNA sequence of interest with integrase recognition sites and with or without homology arms).
  • a reverse transcriptase may also be co-expressed in such systems as the designed DNA sequence (Gene) of interest in the vector will become expressed as RNA and will have to be converted back to DNA for integration by the integrase enzyme.
  • the reverse transcriptase may be viral in origin (e.g. a retrovirus such as HIV1).
  • the DNA sequence of interest in linearized before introduction to the cell.
  • the Cas9 RNA guide sequences and DNA sequence of interest had to be designed and inserted into the vector before use by standard molecular biology protocols.
  • Cells missing expression of a particular gene are transfected or electroporated with the above vectors where the gene of interest is included.
  • Chimeric primer sets designed to cover the inserted gene as well as flanking genomic sequence will be used to screen initial pools of edited cells.
  • Limited dilution cloning (LDC) and or FACS analysis is then performed to ensure monoclonality.
  • Next generation sequencing (NGS) or single nucleotide polymorphism (SNP) analysis is performed as a final quality control step to ensure isolated clones are homogenous for the designed edit.
  • Other mechanisms for screening can include but are not limited to qRT-PCR and western blotting with appropriate antibodies. If the protein is associated with a certain phenotype of the cells, the cells may be examined for rescue of that phenotype. The genomes of the cells are assayed for the specificity of the DNA insertion and to find the relative number of off-target insertions, if any.
  • Vectors designed for gene expression in E. coli or insect cells can be incorporated into E coli or insect cells and allowed to express for a given period of time. Several designs will be utilized to generate Cas9 (or inactive Cas9) linked integrase protein.
  • the vectors can also incorporate a tag that is not limited to a His or cMyc tag for eventual isolation of the protein with high purity and yield. Preparation of the chimeric protein can include but are not limited to standard chromatography techniques.
  • the protein may also be designed with one or more NLS (nuclear localization signal sequence) and/or a TAT sequence. The nuclear localization signal allows the protein to enter the nucleus.
  • NLS nuclear localization signal sequence
  • the TAT sequence allows for easier entry of a protein into a cell (it is a cell-penetrating peptide). Other cell penetrating peptides in the art may be considered.
  • protein lysate can be collected from the cells and purified in the appropriate column depending on the tag used. The purified protein can then be placed in the appropriate buffering solution and stored at either -20 or -80 degrees C.
  • Example 7 Using Cas9-Integrase to Incorporate Stop Codons Just Upstream of Transcription Start Site
  • the disclosure includes a method to create a knockout cell line or organism.
  • the above system is used with the DNA sequence of interest being 1, 3, 6, 10, 15 or 20 consecutive stop codons to be placed just after the ATG start site for the target gene. This will create an effective gene knockout as transcription/translation will be stopped when reaching the immediate stop codon after the ATG start site. Additional stop codons will help prevent possible run through of the transcriptase (if transcriptase by-passes the first stop codon).
  • Example 8 Using Abbiel (or Other Variations Having Other Specific DNA Binding Domains) as a Purified Protein to Edit the Genomes of Cells
  • the bacterial expression vector will be the pMAL- c5e, which is a discontinued product from NEB and one of the in-house cloning choices for Genscript. Codon-optimized Spy Cas9 is cloned with the his-tag and the TEV protease cleavage site in frame with the maltose-binding protein (MBP) tag.
  • MBP maltose-binding protein
  • the ORF is under the inducible Tac promoter, and the vector also codes for the lac repressor (LacI) for tighter regulation.
  • MBP will be used only as a stabilization tag and not a purification tag, for the amylose resin is quite expensive.
  • the soluble expressed material will be purified over the Ni- affinity chromatography, then Cas9 is released by the TEV protease from MBP, purified by cation exchange chromatography, and polished by gel filtration.
  • Design sequence specific Zinc finger domain, TALE, or guide RNA for CRISPR based approach toward a target DNA sequence Use on-line design software of choice. Produce DNA construct with coding sequences for integrase, transposase or recombinase; a suitable amino acid linker; the appropriate zinc finger, TALE or CRISPR protein (e.g. Cas9, Cpfl); and a nuclear localization signal (or mitochondrial localization signal) to form the site specific fusion integrase protein. These are envisioned in multiple arrangements.
  • a suitable tag may be included for protein isolation and purification if desired (e.g. maltose binding protein (MBP) or His tag).
  • DNA construct may utilize a mammalian cell promoter or a bacterial promoter common in the art (e.g. CMV, T7, etc.)
  • a mammalian cell promoter or a bacterial promoter common in the art (e.g. CMV, T7, etc.)
  • One may produce a recombinant fusion protein with E coli as the source. Isolate the protein by standard means in the art (e.g. MBP columns, nickel-sepharose columns, etc.).
  • Assemble the Donor-RNP complex duplex the RNA oligos and mix with fusion protein of the invention (when fusion protein has an endonuclease inactive CRISPR related protein for its DNA binding ability, e.g. ABBIE 1) — these steps of forming RNP are not necessary for Zinc finger domains and TALE.
  • IDTE Buffer For example, use a final concentration of 100 pM.
  • Step A5 with 1.5 pmol of fusion protein (Step A6) in Opti-MEM Media to a final volume of 12.5 pL.
  • dCas9 DNA cutting inactive Cas9 linked to biotin
  • Cas9 s pyogenes, s aureus, etc.
  • Biotinylation methods are described below.
  • Biotinylation or Graphene-lation method #1 engineer the avi-tag ( ⁇ 15 residues) at the N- or C-terminus, express and purify as the WT (un-tagged) protein. Use the E. coli biotin ligase (BirA) and biotin or chemically modified Graphene or GO to biotinylate or graphene-ylate the avi-tagged Cas9.
  • Biotinylation method #2.2 along the same line, biotin-maleimide is commercially available, and they can be conjugated at surface-exposed cysteines (no enzyme).
  • Donor DNA DNA with LTR sequences
  • All reactions are prepared in sterile biosafety cabinet.
  • Day 1 Human embryonic kidney (HEK 293 T) Cells were seeded into 24- well culture plate (Corning) at 200,000 HEK293T cells (ATCC) per well in 500 pL DMEM (Gibco) supplemented with 10% fetal bovine serum (Omega Scientific). Cells were allowed to recover for 24 hours.
  • HEK 293 T Human embryonic kidney
  • ABBIE1 protein (SEQ ID NO: 58) and donor DNA (SEQ ID NO: 101) in a reduced-serum transfection medium (OptiMEM, Life Technologies) at 1: 1 molar ratio for 10 minutes at room temperature.
  • OptiMEM reduced-serum transfection medium
  • the volume of this mixture is 25 pL.
  • RNAiMAX transfection reagent
  • PCR polymerase chain reaction
  • Primer Set 1 Primer 1 : 5'-GTGTTAATTTCAAACATCAGCAGC-
  • Primer Set 2 Primer 1 : 5'-GAGGTTGACTGTGTAAATG-3' (SEQ ID NO: 1
  • Primer 2 5'-GATACCAGAGTCACACAACAG-3' (SEQ ID NO: 281)
  • Primer Set 3 Primer 1: 5'-TCTACATTAATTCTCTTGTGC-3' (SEQ ID NO: 282), Primer 2: 5'-GAT ACC AGAGTCACACAAC AG-3' (SEQ ID NO: 283)
  • Primer Set 2 Primer 1: 5'-TCTACATTAATTCTCTTGTGC-3'
  • Primer Set 3 Primer 1 : 5'-GAGGTTGACTGTGTAAATG-3' (SEQ ID NO: 289), Primer 2: 5'-GACAAGACATCCTTGATTTG-3' (SEQ ID NO: 290)
  • Primer Set 4 Primer 1 : 5'-GAGGTTGACTGTGTAAATG-3' (SEQ ID NO: 291), Primer 2: 5'-GATACCAGAGTCACACAACAG-3' (SEQ ID NO: 292)
  • Transformation of expression construct containing full-length fusion protein Take competent E. coli cells from -80° C. freezer. Turn on water bath to 42° C. Put competent cells in a 1.5 ml tube (Eppendorf or similar). For transforming a DNA construct, use 50 ul of competent cells. Keep tubes on ice. Add 50 ng of circular DNA into E. coli cells. Incubate on ice for 10 min. to thaw competent cells. Put tube(s) with DNA and E. coli into water bath at 42° C. for 45 seconds. Put tubes back on ice for 2 minutes to reduce damage to the E. coli cells. Add 1 ml of LB (with no antibiotic added). Incubate tubes for 1 hour at 37° C. (Can incubate tubes for 30 minutes Spread about 100 ul of the resulting culture on LB plates with appropriate antibiotic Pick colonies about 12-16 hours later. [0491] Inoculation and Expansion
  • FIG. 1 shows a) an exemplary catalytically inactive Cas9/HIVl integrase fusion protein, b) an exemplary TALE/HIV1 integrase fusion protein, c) an exemplary zinc finger protein/HIVl integrase fusion protein, and d) an exemplary Cas9/HIVl integrase fusion protein designed to opposite sides of the DNA at the targeted site.
  • Each of the fusion proteins binds to a specific target sequence of DNA.
  • ZnFn is a Zinc finger protein.
  • “Integrase” represents one integrase unit or two integrase units linked, for example, by a short amino acid linker.
  • FIG. 2 shows a DNA plasmid system comprising, a vector comprising a catalytically inactive Cas9/integrase fusion protein, a vector comprising a DNA sequence of interest, and a vector comprising a reverse transcriptase.
  • a guide RNA (gRNA) or RNAs may be provided separately.
  • Another vector can be used to express a gRNA.
  • “1 or 2” refers to one integrase or two integrases linked by, for example, an amino acid linker.
  • FIG. 3 shows detection of ABBIE 1 protein after isolation and purification from E coli. Coomassie stained gel.
  • FIG. 4 illustrates an example of a DNA binding protein joined to graphene oxide.
  • ABBIE engineered T cells targeting ovarian cancer cells for killing were mixed in a E lratio with ovarian cancer cells expressing green fluorescent protein (GFP). Live ovarian cancer cells fluoresced green, and dead cells would lose fluorescence.
  • the y-axis of FIG. 5A represents measurement of the “green” color. Whereas the x-axis described the control and experimental conditions. Lane 1 is a control lane and show live NCI-GFP ovarian cancer cells. Lane 2 is a control lane showing the effects of electroporation alone.
  • Lane 3 is a control lane showing ABBIE-engineered T cells expressing chimeric PD1 (chPDl) with scrambled sgRNA targeting, controlling for random targeting in T cells.
  • Lanes 4 and 5 are the experimental conditions and show ABBIE- engineered T cells expressing chPDl targeting safe harbor landing pads aavsl (lane 4) and hprtl (lane 5) respectively.
  • viability of GFP expressing ovarian cancer cells was assessed in the presence of ABBIE-engineered T cells.
  • the control and experimental conditions are the same as those in FIG. 5A.
  • the decrease in green color suggests that the ovarian cancer cells are dying in the presence of the ABBIE-engineered T cells.
  • the experiment was performed in duplicate, and two plates are presented for each condition.
  • the phase contrast column allows for sharper resolution and quantification of green color as well as the position of the cells in the dish.
  • the green channel only column describes fluorescent proteins alone.
  • FIG. 6A-6D show mechanisms of gene editing.
  • FIG. 6A depicts a key for the images used in FIG.s 6B-6D.
  • FIG. 6B shows an overview of the activity of various DNA editing tools involving a break, a processing event, followed by a change in the DNA code.
  • FIG. 6C shows an overview of the DNA editing activity of the Abbie 1 system.
  • FIG. 6D shows an overview of the use of the Abbie 1 system to introduce a biosensor directly within the genetic material of an organism.
  • a cell modified to integrate a biosensor directly within the genetic material can respond to a wireless remote signal provided to the modified cell and produce an output in response.
  • the signal is transmitted to a target cell nucleus.
  • the target cell can be a cancer cell.
  • the target cell nucleus can be modified with a Nano- transmiXer and receiver.
  • FIG. 8 A design for a chip as provided herein is shown in FIG. 8.
  • the chip can comprise a central processing unit further comprising a control unit and arithmetic/logic unit.
  • the chip further comprises a memory unit.
  • the chip can process thousands of bits of code rapidly.
  • the activity of the chip can be between an input device and output device.
  • FIG. 9A and FIG. 9B show overviews of Homing remote control gene editing (ReCoG) and External ReCoG respectively.
  • ReCoG editing can allow one to more precisely define where the protein will target.
  • ReCoG editing can improve specificity of edits in the genome.
  • ReCoG editing can provide a higher resolution of processing living code within a living organism.
  • Homing ReCoG editing can use a wireless signal to direct the processor to target code.
  • Homing ReCoG editing can include two-part signal transmission where a protein binds specifically to DNA and completes a circuit to the bring editing protein to the desired site for activity.
  • External ReCog editing can include design/program editing proteins with attachments that relay a signal when they reach the desired area (certain cell type or position in the genome).
  • External ReCog editing can allow a user to remotely activate the proteins that are in the desired locales (only proteins that transmit a signal).
  • the editing protein can be instructed to signal in cell A, if the editing protein reaches cell A, it sends a signal, if the editing protein reaches cell B then no signal is sent.
  • the donor DNA can be modified with graphene or other materials above and assays for to detect signal in DNA can be performed.
  • An oligo with probe can be targeted near the target area of the DNA integration and provide a signal when interacting with the modified oligo interacts with the probe.
  • the oligo (DNA or RNA) probe may be modified to interact with the unmodified or modified graphene or GO in the integrated DNA.
  • the modifications can be fluorophores, quenchers, antigens, antibodies, biotin streptavidin, or other molecules that can be bound to other molecules including those bound to a column.
  • the assay may be a microarray or an immunoprecipitation experiment.
  • the assay may be an ELISA (Enzyme- linked Immunosorbent Assay) or quantitative PCR experiment.
  • Example 17 Integrating factors to engineer cell lines for custom exosomes with stem cell exosome characteristics
  • Cell lines to be modified can be HEK cells, HeLa cells, CHO cells or any laboratory cell line that can be cultured through over 10, over 20 or over 40 passages while retaining their characteristics.
  • the genome integration will be performed as mentioned or by other means such as with cas9 or cas9 fusions or homologous recombination.
  • the laboratory cell line may or may not be a stem cell line.
  • Donor DNA would be designed to include one or more expressable sequences and a suitable promoter (constitutive or inducible) where the expressable sequences can be ESCRT composed of ESCRT-0, EXCRT-I, ESCRT-II, ESCRT-III and associated Vps proteins: Rab proteins, a largest family of small GTPase proteins, Rab4; gene modified to produce protein with an amino acid sequence or ubiquitylation site recognized by an ESCRT, Rab protein or other exosome sorting protein.
  • the proteins to be modified this way include growth factors (e.g.
  • FGFs FGFs, WNTs, hedgehogs, SHH, CTGFs, BMPs, BMP2, TGFs, VEGFs), miRNAs, siRNAs, extracellular signaling factors, antibodies, antigens, vaccines and other proteins that would exert biological or pharmacological action. Incorporation of these sequences into cell genomes will increase exosome formation and alter the contents of exosomes. For example, BMP or USAG-1 siRNA concentrations can be increased in exosomes. The exosomes can be harvested after genome editing and suitable culture time. Exosomes can be tested for numbers and contents.
  • modified exosome can mirror the actions of dental stem cell exosomes, skin stem cells, neural stem cells, cardiac stem cells, papillary stem cells, renal stem cells, mesenchymal stem cells, pancreatic stem cells, or bone marrow cell exosomes.
  • Modified exosomes can be used in dental regeneration or tissue healing strategies including healing injuries to organs like skin, heart, brain, kidney, pancreas, dental tissue, intestines/GI tract or mucous membranes. In one embodiment, they can aid in new tooth growth in humans and mammals (third set of teeth) via exosomal Inclusion of appropriate growth factors or siRNA, antibody or other molecule that blocks the action of USAG-1 or its species analogs.
  • Example 18 Microfluidics Technique to Detect Target Genomic DNA Using Graphene-Bound Donor DN A
  • This microfluidics technique involves integrating graphene- bound donor DNA with target genomic DNA at specific sites, followed by detection of the integrated DNA to confirm the presence of target sequences.
  • the method utilizes viral integrase-dCas9 as the integrating molecule, allowing for precise targeting and integration of donor DNA into the genome. This approach can be particularly useful for detecting microbial or viral DNA, as well as identifying specific disease mutations,
  • polynucleotide sequences, protein sequences, or linker sequences may be provided in the disclosure that are not listed in Table 1 below, but can be used in the compositions (constructs, fusion proteins) and methods described herein.
  • SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO: 58, and/or portions thereof may be provided in the disclosure that are not listed in Table 1 below, but can be used in the compositions (constructs, fusion proteins) and methods described herein.
  • SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO: 58, and/or portions thereof may be provided in the disclosure that are not listed in Table 1 below, but can be used in the compositions (constructs, fusion proteins) and methods described herein.
  • SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO: 58, and/or portions thereof may be provided in the disclosure that are not listed in Table 1 below, but can be used in the compositions (constructs, fusion proteins
  • nucleic acid sequences 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 27-47, 49, 55, 56, 57, 62, 64, 66, 68, 70, 79, 82, and 83.
  • SEQ ID NO: 16 NAME: gi
  • NAME gi
  • NAME gi
  • NAME gi
  • TALE repeat modules are exemplary sequences of polynucleotides encoding the TALE repeat modules for use in linking to integrases or recombinases as described in this invention.
  • NAME MMTV integrase cDNA, gb
  • NAME gi
  • NAME gb
  • SEQ ID NO: 81 a protein domain that characterizes zinc finger proteins
  • First 5’LTR is underlined, plain text is neo, and 3’LTR is bolded (1179 bp)
  • Non-redundant set nr
  • Organism Acidaminococcus sp B V3L6
  • Bacteria Firmicutes, Negativicutes, Selenomonadales, Acidaminococcaceae, Acidaminococcus,
  • Non-redundant set nr
  • Organism Lachnospiraceae_bacterium_MA2020
  • Taxonomy Bacteria, Firmicutes, Clostridia, Clostridiales, Lachnospiraceae, unclassified Lachnospiraceae, Lachnospiraceae bacterium MA2020 [0569] Additional nucleic acid sequences and protein sequences that can be used in the disclosed compositions and methods - CPF 1 alignment. SEQ ID NOS: 86-92; in order from the top to the bottom of the chart.

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Abstract

Sont divulgués ici des procédés et des compositions pour le réglage fin du ciblage et/ou de l'édition du génome qui peuvent répondre fonctionnellement à des stimuli. Sont également divulgués ici des procédés de ciblage de kinases et de phosphatases vers des zones d'ADN qui peuvent affecter la phosphorylation et la déphosphorylation de facteurs de transcription, d'autres protéines de liaison à l'ADN et des protéines qui présentent une association étroite avec l'ADN mitochondrial ou génomique.
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