Compositions and methods for genome editing

Abstract

Disclosed herein 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.

Description

COMPOSITIONS AND METHODS FOR GENOME EDITINGINCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] Any and all applications for which a foreign or domestic priority claim is identified in the PCT Request as filed with the present application are hereby incorporated by reference under 37 CFR §1.57. The present Application claims priority to U.S. Provisional Application No. 63 / 610343, filed December 14, 2023. The contents of this provisional application is hereby incorporated by reference.REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided in a file entitled SOHM.004WOseqlist, which was created on December 10, 2024, and is 270,069 bytes in size. The information in the electronic Sequence Listing is hereby expressly incorporated by reference in its entirety.BACKGROUND OF THE INVENTIONField of the Invention

[0003] Aspects of the present disclosure relate generally to methods and compositions for fine genome editing and / or targeting. 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.SUMMARY

[0004] Disclosed herein 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.

[0005] In some embodiments, a composition is provided 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the material can comprise graphene or graphene oxide. In some embodiments, the composition can further comprise a guide RNA. In some embodiments, the composition can further comprise an enzyme, a drug or a diagnostic agent or any combination thereof joined to the material. In some embodiments, a method is provided 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. In some embodiments, the composition can be for use in detecting a selected sequence in a genome of a subject or a cell. In some embodiments, 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. In some embodiments, the composition can be for use in providing a drug to a subject or a cell.

[0006] In some embodiments, a composition comprising a cas protein joined to a protein comprising a kinase or phosphatase is provided. In some embodiments, the protein comprising the kinase can comprise a MAP kinase, Src, a nuclear kinase, a serine / threonine kinase or a tyrosine kinase. In some embodiments, the protein comprising the phosphatase can comprise a Shp, Shp2 or a nuclear phosphatase. In some embodiments, the cas proteinhas an inactivated nuclease domain such as can be generated with one or more inactivating mutations.

[0007] In some embodiments, 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. In some embodiments, the subject or cell can have a disease or disorder. In some embodiments, the disease can be a viral disease, a microbial disease, cancer or a cardiovascular disease. In some embodiments, the composition can be for use in treating a viral disease, a microbial disease, cancer or a cardiovascular disease. In some embodiments, a method of detecting a selected nucleotide sequence in a subject, a cell, or an isolated population of polynucleotides or oligonucleotides is provided, comprising contacting a composition with the subject, the cell, or the isolated population of polynucleotides or nucleotides and performing a diagnostic evaluation. In some embodiments, the diagnostic evaluation can comprise colorimetry, fluorescence detection, immunodetection, or rf reflectometry.

[0008] In some embodiments, a method of genome editing is provided 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. In some embodiments, response of the modified genome editing protein to the one or more stimuli can modulate genome editing in a target. In some embodiments, the genome editing protein can comprises a zinc finger endonuclease, TALEN, CRISPR / cas9, Cpfl, meganuclease, Cas3, CaslO, or an ABBIE system. In some embodiments, the modified genome editing protein can be joined to at least one other protein, which comprises a recombinase, reverse transcriptase, or topoisomerase. In some embodiments, the material that modifies the genome editing protein can comprise a graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate or carbon nanotube. In some embodiments, the material that modifies the genome editing protein can comprise silicon, silica or a silicone polymer. In someembodiments, modifying the genome editing protein can comprise joining the genome editing protein to a protein comprising a kinase. In some embodiments, 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. In some embodiments, modifying the genome editing protein can further comprise joining the genome editing protein to a protein comprising a phosphatase. In some embodiments, the protein comprising the phosphatase can comprise a Shp phosphatase, Shp2, Shpl, PPM phosphatase, PP2C phosphatase, a nuclear phosphatase or a cytoplasmic phosphatase. In some embodiments, the material that modifies the genome editing protein can be joined to the genome editing protein via one or more ionic interactions. In some embodiments, 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. In some embodiments, the material that modifies the genome editing protein can be joined to the genome editing protein via one or more covalent bonds. In some embodiments, 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. 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 modified genome editing protein, which alters its activity. In some embodiments, the one or more stimuli can induce a conformational change in the modified genome editing protein, which alters its activity. In some embodiments, 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. In some embodiments, the additional material can comprise a metal (e.g., gold, silver, iron, iron oxide, titanium dioxide, lanthanide oxide, transition metal, or atransition 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. In some embodiments, 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). In some embodiments, 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. In some embodiments, the minimal dimensions of the material modifying a genome editing protein in the X-Y plane can be 1 nm by 1 nm. In some embodiments, 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. In some embodiments, when the material is graphene, the minimal dimension along the Z-axis can be defined by the thickness of a non-functionalized graphene monolayer, e.g., 0.34 nm. In some embodiments, 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. In some embodiments, binding of the modified DNA binding protein to DNA can be directed by use of a guide RNA. In some embodiments, the target can be a subject in need of a therapy for a disease, or a cell. In some embodiments, the subject or cell can have a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.

[0009] In some embodiments, a composition is provided 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. In some embodiments, response of the modified genome editing protein to the one or more stimuli can modulate genome editing in a target. In some embodiments, the genome editing protein can comprise a zinc finger endonuclease, TALEN, CRISPR / cas9, Cpfl, a meganuclease, Cas3, CaslO, or the ABBIE system. In some embodiments, the genome editing protein can be joined to a recombinase, reverse transcriptase, or a topoisomerase. In some embodiments, the material can comprise a graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate, a polymer, or a carbon nanotube. In some embodiments, the material can comprise silicon, silica or a silicone polymer. In some embodiments, the genome editingprotein can be joined to a protein comprising a kinase. In some embodiments, 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. In some embodiments, the genome editing protein can be joined to a protein comprising a phosphatase. In some embodiments, 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. In some embodiments, the material can be joined to the genome editing protein via one or more ionic interactions. In some embodiments, the material can be joined to the genome editing protein via one or more functional groups or functional linkers. In some embodiments, the material can be joined to the genome editing protein via one or more covalent bonds. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the material can be joined to a component of extracellularmatrix (e.g., collagen, fibronectin, or laminin) or a synthetic polymer (e.g., polyaniline, polypyrrole, or polythiophene). In some embodiments, external dimensions of the material in the X-Y plane can be nano scaled (e.g., one nano-sized graphene flake) or macro scaled. In some embodiments, the minimal dimensions of the material modifying a genome editing protein in the X-Y plane can be 1 nm by 1 nm. In some embodiments, 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. In some embodiments, when the material comprises graphene, the minimal dimension along the Z-axis can be defined by the thickness of a non-functionalized graphene monolayer, e.g., 0.34 nm. In some embodiments, 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. In some embodiments, binding of the modified DNA binding protein to DNA can be directed by use of a guide RNA. In some embodiments, 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. In some embodiments, the target can be a subject in need of a therapy, or a cell. In some embodiments, the subject or cell can have a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease. In some embodiments, a method of treating or inhibiting a disease or disorder comprising administration of one or more of the aforementioned compositions is provided. In some embodiments, 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. In some embodiments, use of any one or more of the compositions described herein as a medicament is provided. In some embodiments, 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. In some embodiments, 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.

[0010] In some embodiments, a method of genome editing is provided comprising: modifying a donor polynucleotide by joining the donor polynucleotide to amaterial 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. In some embodiments, 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. In some embodiments, the material can comprise a graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate, a polymer, or a carbon nanotube. In some embodiments, the material can comprise silicon, silica or a silicone polymer. In some embodiments, the material can be joined to the donor polynucleotide via one or more ionic interactions. In some embodiments, the material can be joined to the donor polynucleotide via one or more functional groups or functional linkers. In some embodiments, the material can be joined to the donor polynucleotide via one or more covalent bonds. In some embodiments, 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, ionic concentration, or electrical stimuli. In some embodiments, the one or more stimuli can comprise electromagnetic radiation. In some embodiments, the one or more stimuli can induce the modified donor polynucleotide to produce a detectable signal. In some embodiments, external dimensions of the material in the X-Y plane can be nano scaled (e.g., one nano-sized graphene flake) or macro scaled. In some embodiments, the minimal dimensions of the material modifying the donor polynucleotide in the X-Y plane can be 1 nm by 1 nm. In some embodiments, the external dimensions of the material modifying a donor polynucleotide along the Z-axis can relate to the number of layers of materials incorporated. In some embodiments, when the material comprises graphene, the minimal dimension along the Z-axis can be defined by the thickness of a non-functionalized graphene monolayer, e.g., 0.34 nm. In some embodiments, 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. In some embodiments, the genome editing of the incorporated modified donor polynucleotide further can comprise a guide RNA. In some embodiments, the target can be a subject in need of therapy, or a cell. In someembodiments, the subject or cell can have a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.

[0011] In some embodiments, a composition is provided comprising: a donor polynucleotide joined to a material that can modulate editing of the donor polynucleotide in response to one or more stimuli. In some embodiments, the modified donor polynucleotide can be incorporated into a genomic DNA sequence of a target. In some embodiments, providing the one or more stimuli can modulate genome editing of the incorporated modified donor polynucleotide. In some embodiments, 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. In some embodiments, the material can comprise graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate, a polymer, or a carbon nanotube. In some embodiments, the material can comprise silicon, silica or a silicone polymer. In some embodiments, the material can be joined to the donor polynucleotide via one or more ionic interactions. In some embodiments, the material can be joined to the donor polynucleotide via one or more functional groups or functional linkers. In some embodiments, the material can be joined to the donor polynucleotide via one or more covalent bonds. In some embodiments, 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, ionic concentration, or electrical stimuli. In some embodiments, the one or more stimuli can comprise electromagnetic radiation. In some embodiments, the one or more stimuli can induce the modified donor polynucleotide to produce a detectable signal or marker. In some embodiments, 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. In some embodiments, the minimal dimensions of the material modifying the donor polynucleotide in the X-Y plane can be 1 nm by 1 nm. In some embodiments, the external dimensions of the material along the Z-axis can depend on the number of layers of materials incorporated. In some embodiments, when the material comprises graphene, the minimal dimension along the Z-axis can be defined by the thickness of a non-functionalized graphene monolayer, e.g., 0.34 nm. In some embodiments, maximum dimension of a particle of the material can be from 0.00000001 nm to 1000 nm, .0001 nm to100 nm, 0.1 nm to 50 nm, 0.5 nm to 10 nm, or any integer found between these ranges. In some embodiments, the genome editing of the incorporated modified donor polynucleotide further can comprise a guide RNA. In some embodiments, the target can be a subject in need of a therapy or a cell. In some embodiments, the subject or cell can have a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease. In some embodiments, a method of treating a disease or disorder is provided comprising administration of a composition. In some embodiments, 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. In some embodiments, the composition is provided for use as a medicament. In some embodiments, a method of detecting a disease or disorder in a subject or a cell is provided, such as a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease 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.

[0012] Embodiments provided herein are further described in the following enumerated aspects:

[0013] 1. 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.

[0014] 2. The 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.

[0015] 3. The composition of aspect 1 or 2, wherein the material comprises graphene or graphene oxide.

[0016] 4. The composition of any one of aspects 1-3, further comprising a guideRNA.

[0017] 5. The composition of any one of aspects 1-4, further comprising an enzyme, a drug or a diagnostic agent or any combination thereof joined to the material.

[0018] 6. 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.

[0019] 7. The composition of any one of aspects 1-5 for use in detecting a selected sequence in a genome of a subject or a cell.

[0020] 8. 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.

[0021] 9. The composition of aspect 5 for use in providing a drug to a subject or a cell.

[0022] 10. 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.

[0023] 11. The 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.

[0024] 12. The composition of aspect 10, wherein the protein comprising the phosphatase comprises a Shp, Shp2 or a nuclear phosphatase.

[0025] 13. 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.

[0026] 14. The method of aspect 13, wherein the subject or cell has a disease or disorder.

[0027] 15. The method of aspect 14, wherein the disease is a viral disease, a microbial disease, cancer or a cardiovascular disease.

[0028] 16. The composition of any one of aspects 10-12 for use in treating a viral disease, a microbial disease, cancer or a cardiovascular disease.

[0029] 17. 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.

[0030] 18. The method of aspect 14, wherein the diagnostic evaluation comprises colorimetry, fluorescence detection, immunodetection, or rf reflectometry.

[0031] 19. 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.

[0032] 20. The method of aspect 19, wherein the genome editing protein comprises a zinc finger endonuclease, TALEN, CRISPR / cas9, Cpfl, meganuclease, Cas3, CaslO, or an ABBIE system.

[0033] 21. The method of any one of aspects 19 or 20, wherein the modified genome editing protein is joined to at least one other protein, which comprises a recombinase, reverse transcriptase, or topoisomerase.

[0034] 22. The method of any one of aspects 19-21, wherein, the material that modifies the genome editing protein comprises a graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate or carbon nanotube.

[0035] 23. The method of any one of aspects 19-22, wherein, the material that modifies the genome editing protein comprises silicon, silica or a silicone polymer.

[0036] 24. The method of any one of aspects 19-23, wherein modifying the genome editing protein comprises joining the genome editing protein to a protein comprising a kinase.

[0037] 25. The method of aspect 24, wherein 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.

[0038] 26. The method of any one of aspects 19-25, wherein modifying the genome editing protein further comprises joining the genome editing protein to a protein comprising a phosphatase.

[0039] 27. The method of aspect 26, wherein the protein comprising the phosphatase comprises a Shp phosphatase, Shp2, Shpl, PPM phosphatase, PP2C phosphatase, a nuclear phosphatase or a cytoplasmic phosphatase.

[0040] 28. The method of any one of aspects 19-27, wherein the material that modifies the genome editing protein is joined to the genome editing protein via one or more ionic interactions.

[0041] 29. The method of any one of aspects 19-28, wherein the material that modifies the genome editing protein is joined to the genome editing protein via one or more functional groups or functional linkers.

[0042] 30. The method of any one of aspects 19-29, wherein the material that modifies the genome editing protein is joined to the genome editing protein via one or more covalent bonds.

[0043] 31. The method of any one of aspects 19-30, wherein the one or more stimuli are endogenous to a cell.

[0044] 32. The method of any one of aspects 19-30, wherein the one or more stimuli are exogenous to a cell.

[0045] 33. The method of any one of aspects 19-30, wherein the one or more stimuli comprise light, temperature, pH, calcium, ion concentration, or electrical stimuli.

[0046] 34. The method of any one of aspects 19-30, wherein the one or more stimuli comprise electromagnetic radiation.

[0047] 35. The method of any one of aspects 19-34, wherein the one or more stimuli induce a cleavage of a group in the modified genome editing protein, which alters its activity.

[0048] 36. The method of any one of aspects 19-35, wherein the one or more stimuli induce a conformational change in the modified genome editing protein, which alters its activity.

[0049] 37. The method of any one of aspects 19-36, wherein the one or more stimuli induce activation of the modified genome editing protein.

[0050] 38. The method of any one of aspects 19-37, wherein the one or more stimuli induce deactivation of the modified genome editing protein.

[0051] 39. The method of any one of aspects 19-38, wherein the one or more stimuli induce the production of a detectable signal or marker.

[0052] 40. The method of any one of aspects 19-39, wherein modifying the genome editing protein further comprises joining the genome editing protein to an additional material.

[0053] 41. The method of aspect 40, wherein 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.

[0054] 42. The method of any one of aspects 19-41, wherein the material modifying the genome editing protein is additionally joined to a polymer comprising a component of extracellular matrix (e.g., collagen, fibronectin, or laminin) or a synthetic polymer (e.g., polyaniline, polypyrrole, or polythiophene).

[0055] 43. The method of any one of aspects 19-42, wherein the external dimensions of the material modifying a genome editing protein in the X-Y plane is nano scaled (e.g., one nano-sized graphene flake) or macro scaled.

[0056] 44. The method of aspect 43, wherein the minimal dimensions of the material modifying a genome editing protein in the X-Y plane are 1 nm by 1 nm.

[0057] 45. The method of any one of aspects 19-44, wherein the external dimensions of the material modifying a genome editing protein along the Z-axis are directly proportional to the number of layers of material incorporated.

[0058] 46. The method of aspect 45, wherein, when the material is graphene, the minimal dimension along the Z-axis is defined by the thickness of a non-functionalized graphene monolayer, e.g., 0.34 nm.

[0059] 47. The method of aspect 45, wherein maximum dimension of a particle of the material in the material modifying a genome editing protein 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.

[0060] 48. The method of any one of aspects 19-47, wherein binding of the modified DNA binding protein to DNA is directed by use of a guide RNA.

[0061] 49. The method of any one of aspects 19-48, wherein the target is a subject in need of a therapy for a disease, or a cell.

[0062] 50. The method of aspect 46, wherein the subject or cell has a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.

[0063] 51. 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.

[0064] 52. The composition of aspect 51, wherein the genome editing protein comprises a zinc finger endonuclease, TALEN, CRISPR / cas9, Cpfl, a meganuclease, Cas3, CaslO, or the ABBIE system.

[0065] 53. The composition of any one of aspects 51 or 52, wherein the genome editing protein is joined to a recombinase, reverse transcriptase, or a topoisomerase.

[0066] 54. The composition of any one of aspects 51-53, wherein, the material comprises a graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate, a polymer, or a carbon nanotube.

[0067] 55. The composition of any one of aspects 51-54, wherein, the material comprises silicon, silica or a silicone polymer.

[0068] 56. The composition of any one of aspects 51-55, wherein the genome editing protein is joined to a protein comprising a kinase.

[0069] 57. The 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.

[0070] 58. The composition of any one of aspects 51-57, wherein the genome editing protein is joined to a protein comprising a phosphatase.

[0071] 59. The 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.

[0072] 60. The composition of any one of aspects 51-59, wherein the material is joined to the genome editing protein via one or more ionic interactions.

[0073] 61. The composition of any one of aspects 51-60, wherein the material is joined to the genome editing protein via one or more functional groups or functional linkers.

[0074] 62. The composition of any one of aspects 51-61, wherein the material is joined to the genome editing protein via one or more covalent bonds.

[0075] 63. The composition of any one of aspects 51-62, wherein the one or more stimuli are endogenous to a cell.

[0076] 64. The composition of any one of aspects 51-62, wherein the one or more stimuli are exogenous to a cell.

[0077] 65. The 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.

[0078] 66. The composition of any one of aspects 51-64, wherein the one or more stimuli comprise electromagnetic radiation.

[0079] 67. The 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.

[0080] 68. The 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.

[0081] 69. The composition of any one of aspects 51-68, wherein the one or more stimuli induce activation of the genome editing protein.

[0082] 70. The composition of any one of aspects 51-69, wherein the one or more stimuli induce a deactivation of the genome editing protein.

[0083] 71. The composition of any one of aspects 51-70, wherein the one or more stimuli induce the genome editing protein to provide a detectable signal or marker.

[0084] 72. The composition of any one of aspects 51-71, wherein the genome editing protein is joined to an additional material.

[0085] 73. The 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.

[0086] 74. The composition of any one of aspects 51-73, wherein the material is joined to a component of extracellular matrix (e.g., collagen, fibronectin, or laminin) or a synthetic polymer (e.g., polyaniline, polypyrrole, or polythiophene).

[0087] 75. The 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.

[0088] 76. The 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.

[0089] 77. The composition of any one of aspects 51-76, wherein the external dimensions of the material modifying a genome editing protein along the Z-axis is related to the number of layers of materials incorporated.

[0090] 78. The 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.

[0091] 79. The composition of aspect 78, 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.

[0092] 80. The 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.

[0093] 81. The composition of any one of aspects 51-80, wherein the composition further comprises 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.

[0094] 82. The composition of any one of aspects 51-81, wherein the target is a subject in need of a therapy, or a cell.

[0095] 83. The composition of aspect 82, wherein the subject or cell has a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.

[0096] 84. A method of treating or inhibiting a disease or disorder comprising administration of the composition of any one of aspects 51-83

[0097] 85. The 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

[0098] 86. Use of the composition of anyone of aspects 51-83 as a medicament.

[0099] 87. 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.

[0100] 88. The 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.

[0101] 89. 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; andproviding the one or more stimuli to modulate genome editing of the incorporated modified donor polynucleotide.

[0102] 90. The method of aspect 89, wherein 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.

[0103] 91. The method of any one of aspects 89 or 90, wherein, the material comprises a graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate, a polymer, or a carbon nanotube.

[0104] 92. The method of any one of aspects 89-91, wherein, the material comprises silicon, silica or a silicone polymer.

[0105] 93. The method of any one of aspects 89-92, wherein the material is joined to the donor polynucleotide via one or more ionic interactions.

[0106] 94. The method of any one of aspects 89-93, wherein the material is joined to the donor polynucleotide via one or more functional groups or functional linkers.

[0107] 95. The method of any one of aspects 89-94, wherein the material is joined to the donor polynucleotide via one or more covalent bonds.

[0108] 96. The method of any one of aspects 89-95, wherein the one or more stimuli are endogenous to a cell.

[0109] 97. The method of any one of aspects 89-96, wherein the one or more stimuli are exogenous to a cell.

[0110] 98. The method of any one of aspects 89-97, wherein the one or more stimuli comprise light, temperature, pH, calcium, ionic concentration, or electrical stimuli.

[0111] 99. The method of aspect 98, wherein the one or more stimuli comprise electromagnetic radiation.

[0112] 100. The method of any one of aspects 89-99, wherein the one or more stimuli induce the modified donor polynucleotide to produce a detectable signal.

[0113] 101. The method of any one of aspects 89-100, wherein external dimensions of the material in the X-Y plane is nano scaled (e.g., one nano-sized graphene flake) or macro scaled.

[0114] 102. The method of aspect 101, wherein the minimal dimensions of the material modifying the donor polynucleotide in the X-Y plane are 1 nm by 1 nm.

[0115] 103. The method of any one of aspects 89-102, wherein the external dimensions of the material modifying a donor polynucleotide along the Z-axis relates to the number of layers of materials incorporated.

[0116] 104. The method of aspect 103, wherein, when the material comprises graphene, the minimal dimension along the Z-axis is defined by the thickness of a nonfunctionalized graphene monolayer, e.g., 0.34 nm.

[0117] 105. The method of aspect 104, wherein maximum dimension of a particle of the material is less than 5000 nm, 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.

[0118] 106. The method of any one of aspects 89-105, wherein the genome editing of the incorporated modified donor polynucleotide further comprises a guide RNA.

[0119] 107. The method of any one of aspects 83-100, wherein the target is a subject in need of therapy, or a cell.

[0120] 108. The method of aspect 107, wherein the subject or cell has a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.

[0121] 109. 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.

[0122] 110. The composition of aspect 109, wherein 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.

[0123] 111. The 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.

[0124] 112. The 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.

[0126] 114. The composition of any one of aspects 109-113, wherein the material is joined to the donor polynucleotide via one or more functional groups or functional linkers.

[0127] 115. The composition of any one of aspects 109-113, wherein the material is joined to the donor polynucleotide via one or more covalent bonds.

[0128] 116. The composition of any one of aspects 109-115, wherein the one or more stimuli are endogenous to a cell.

[0129] 117. The composition of any one of aspects 109-115, wherein the one or more stimuli are exogenous to a cell.

[0130] 118. The composition of any one of aspects 109-115, wherein the one or more stimuli comprise light, temperature, pH, calcium, ionic concentration, or electrical stimuli.

[0131] 119. The composition of aspect 118, wherein the one or more stimuli comprise electromagnetic radiation.

[0132] 120. The composition of any one of aspects 109-119, wherein the one or more stimuli induce the modified donor polynucleotide to produce a detectable signal or marker.

[0133] 121. The composition of any one of aspects 109-120, wherein external dimensions of the material modifying a donor polynucleotide in the X-Y plane is nanoscaled (e.g., one nano-sized graphene flake) or macro scaled.

[0134] 122. The composition of aspect 121, wherein the minimal dimensions of the material modifying the donor polynucleotide in the X-Y plane are 1 nm by 1 nm.

[0135] 123. The composition of any one of aspects 109-122, wherein the external dimensions of the material along the Z-axis depends on the number of layers of materials incorporated.

[0136] 124. The 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.

[0137] 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.

[0138] 126. The composition of any one of aspects 109-125, wherein the genome editing of the incorporated modified donor polynucleotide further comprises a guide RNA.

[0139] 127. The composition of any one of aspects 109-126, wherein the target is a subject in need of a therapy or a cell.

[0140] 128. The composition of aspect 127, wherein the subject or cell has a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.

[0141] 129. A method of treating a disease or disorder comprising administration of the composition of any one of aspects 109-128.

[0142] 130. The 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.

[0143] 131. The composition of any one of aspects 109-128 for use as a medicament.

[0144] 132. A method of detecting a disease or disorder in a subject or a cell, such as a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease 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.

[0145] 133. 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 unintegrateddonor 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.

[0146] 134. 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.

[0147] 135. The method of any one of aspects 133 or 134, wherein the carbon nanomaterial particles are labeled prior to incubation.

[0148] 136. The method of any one of aspects 133-135, wherein the donor integrated gDNA sample is amplified prior to confirming the integration of donor sequences.

[0149] 137. The method of claims any one of aspects 133-136, wherein the carbon nanomaterial particles comprise graphene.

[0150] 138. The method of any one of aspects 133-137, wherein the carbon nanomaterial is crosslinked to its binding partner.

[0151] 139. The method of any one of aspects 133-138, wherein the genome editing protein comprises an ABBIE system.

[0152] 140. The method of any one of aspects 133-138, wherein the genome editing protein comprises a viral integrase-dCas9 complex.

[0153] 141. The method of any one of aspects 133-140, wherein 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.

[0154] 142. The method of any one of aspects 133-141, wherein the detector or sensor uses electrostatic, luminescent, or fluorescent detection.

[0155] 143. 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.

[0156] 144. The methods or compositions of any of the proceeding aspects, further comprising a microfluidic chip with channels for sample input, mixing, and reaction zones.

[0157] 145. The composition or methods of any of the preceding aspects, wherein 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.

[0158] 146. The composition or methods of any of the preceding aspects, wherein the polynucleotides, or oligonucleotides are biotinylated.BRIEF DESCRIPTION OF THE DRAWINGS

[0159] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description, appended claims and accompanying figures where:

[0160] 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.

[0161] FIG. 2 shows a DNA plasmid system comprising, a vector comprising a catalytically inactive Cas9 / integrase fusion protein, a vector comprising a DNA sequence ofinterest, 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.

[0162] FIG. 3 shows detection of ABBIE 1 protein after isolation and purification from E coli. Coomassie stained gel.

[0163] FIG. 4 illustrates an example of a DNA binding protein joined to graphene oxide.

[0164] FIGs. 5A and 5B depict an experiment where viability of ovarian cancer cells was assessed following coincubation with ABBIE-engineered T cells.

[0165] 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.

[0166] FIG. 7 depicts a cell modified to integrate a biosensor directly within the genetic material.

[0167] 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.

[0168] FIGs. 9A and FIG. 9B show overviews of Homing remote control gene editing (ReCoG) and External ReCoG respectively.

[0169] 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. Optionally when active ABBIE is used, 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.

[0170] FIG. 11 describes embodiments for in vitro (ex vivo) detection of targetDNA.DETAILED DESCRIPTION

[0171] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0172] The following detailed description is provided to aid those skilled in the art in practicing the present disclosure. Even so, this detailed description should not be construed to unduly limit the present disclosure as modifications and variations in the embodiments discussed herein can be made by those of ordinary skill in the art without departing from the spirit or scope of the present discovery.Definitions

[0173] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein are expressly incorporated by reference in their entireties unless stated otherwise. In the event that there are a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

[0174] As used in this disclosure and the appended claims, the singular forms “a”, “an” and “the” include a plural reference unless the context clearly dictates otherwise. As used in this disclosure and the appended claims, the term “or” can be singular or inclusive. For example, A or B, can be A and B.

[0175] The terms “about” or “around” as used herein refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0176] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0177] By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0178] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. If there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise. The practice of the present disclosure will employ, unless indicated specifically to the contrary, conventional methods of molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration.

[0179] 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.

[0180] Where a range of values is provided, it is understood that the upper and lower limit, and each intervening value between the upper and lower limit of the range is encompassed within the embodiments.

[0181] The term “% 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. The term “% 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.

[0182] 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.

[0183] Endogenous

[0184] 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.

[0185] Exogenous

[0186] An “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.

[0187] Knockout

[0188] 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.

[0189] Upon knocking out a 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.

[0190] Upon knockout out of a gene, 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 least30%, 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%.

[0191] Modified

[0192] A “modified” organism is an organism that is different than an unmodified organism. For example, 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.

[0193] A “modified” nucleic acid sequence or amino acid sequence is different than the unmodified nucleic acid sequence or amino acid sequence. For example, a nucleic acid sequence can have one or more nucleic acids inserted, deleted, or added. For example, an amino acid sequence can have one or more amino acids inserted, deleted, or added.

[0194] Operably Linked

[0195] In some embodiments, 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. For example, 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.

[0196] Host Cell or Host Organism

[0197] A “host cell” can contain a polynucleotide encoding a polypeptide of the present disclosure. In some embodiments, a host cell is part of a multicellular organism. In other embodiments, a host cell is cultured as a unicellular organism.

[0198] ‘Host organisms” can include any suitable host, for example; a microorganism. Microorganisms which are useful for the methods described herein include, for example, bacteria (e.g., E. coli), yeast (e.g., Saccharomyces cerevisiae), and plants. The organism can be prokaryotic or eukaryotic. The organism can be unicellular or multicellular.

[0199] The host cell can be prokaryotic. Suitable prokaryotic cells include, but are not limited to, any of a variety of laboratory strains of Escherichia coli, Lactobacillus sp., Salmonella sp., and Shigella sp. (for example, as described in Carrier et al. (1992) J. Immunol. 148:1176-1181; U.S. Pat. No. 6,447,784; and Sizemore et al. (1995) Science 270:299-302). Examples of Salmonella strains which can be employed in the present disclosure include, but are not limited to, Salmonella typhi and S. typhimurium. Suitable Shigella strains include, but are not limited to, Shigella flexneri, Shigella sonnei, and Shigella disenteriae. Typically, the laboratory strain is one that is non-pathogenic. Non-limiting examples of other suitable bacteria include, but are not limited to, Pseudomonas pudila, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, and Rhodococcus sp.

[0200] In some embodiments, the host organism is eukaryotic. Suitable eukaryotic host cells include, but are not limited to, yeast cells, insect cells, plant cells, fungal cells, and algal cells.

[0201] Polynucleotides and Polypeptides [Nucleic Acids and Proteins]

[0202] The proteins of the present disclosure can be made by any method known in the art. The protein may be synthesized using either solid-phase peptide synthesis or by classical solution peptide synthesis also known as liquid-phase peptide synthesis. Using Val- Pro-Pro, Enalapril and Lisinopril as starting templates, several series of peptide analogs such as X-Pro-Pro, X- Ala-Pro, and X-Lys-Pro, wherein X represents any amino acid residue, may be synthesized using solid-phase or liquid-phase peptide synthesis. Methods for carrying out liquid phase synthesis of libraries of peptides and oligonucleotides coupled to a soluble oligomeric support have also been described. Bayer, Ernst and Mutter, Manfred, Nature 237:512-513 (1972); Bayer, Ernst, et al., J. Am. Chem. Soc. 96:7333-7336 (1974); Bonora, Gian Maria, et al., Nucleic Acids Res. 18:3155-3159 (1990). Liquid phase synthetic methods have the advantage over solid phase synthetic methods in that liquid phase synthesis methods do not require a structure present on a first reactant which is suitable for attaching the reactant to the solid phase. Also, liquid phase synthesis methods do not require avoiding chemical conditions which may cleave the bond between the solid phase and the first reactant (or intermediate product). In addition, reactions in a homogeneous solution may give betteryields and more complete reactions than those obtained in heterogeneous solid phase / liquid phase systems such as those present in solid phase synthesis.

[0203] In oligomer-supported liquid phase synthesis the growing product is attached to a large soluble polymeric group. The product from each step of the synthesis can then be separated from unreacted reactants based on the large difference in size between the relatively large polymer-attached product and the unreacted reactants. This permits reactions to take place in homogeneous solutions, and eliminates tedious purification steps associated with traditional liquid phase synthesis. Oligomer-supported liquid phase synthesis has also been adapted to automatic liquid phase synthesis of peptides. Bayer, Ernst, et al., Peptides: Chemistry, Structure, Biology, 426-432.

[0204] For solid-phase peptide synthesis, 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. Usually, the carboxyl terminus of the peptide is linked to a polymer from which it can be liberated upon treatment with a cleavage reagent. In a common method, 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). In an automated solidphase method, peptides are synthesized by loading the carboxy-terminal amino acid onto an organic linker (e.g., PAM, 4-oxymethylphenylacetamidomethyl), which is covalently attached to an insoluble polystyrene resin cross-linked with divinyl benzene. The terminal amine may be protected by blocking with t-butyloxycarbonyl. Hydroxyl- and carboxylgroups are commonly protected by blocking with O-benzyl groups. Synthesis is accomplished in an automated peptide synthesizer, such as that available from Applied Biosystems (Foster City, Calif). Following synthesis, the product may be removed from the resin. The blocking groups are removed by using hydrofluoric acid or trifluoromethyl sulfonic acid according to established methods. A routine synthesis may produce 0.5 mmole of peptide resin. Following cleavage and purification, a yield of approximately 60 to 70% is typically produced. Purification of the product peptides is accomplished by, for example, crystallizing the peptide from an organic solvent such as methyl-butyl ether, then dissolving in distilled water, and using dialysis (if the molecular weight of the subject peptide is greaterthan about 500 daltons) or reverse high pressure liquid chromatography (e.g., using a Cl 8 column with 0.1% trifluoroacetic acid and acetonitrile as solvents) if the molecular weight of the peptide is less than 500 daltons. Purified peptide may be lyophilized and stored in a dry state until use. Analysis of the resulting peptides may be accomplished using the common methods of analytical high pressure liquid chromatography (HPLC) and electrospray mass spectrometry (ES-MS).

[0205] In other cases, a protein, for example, a protein is produced by recombinant methods. For production of any of the proteins described herein, host cells transformed with an expression vector containing the polynucleotide encoding such a protein can be used. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell such as a yeast, or the host can be a prokaryotic cell such as a bacterial cell. 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.

[0206] Codon Optimization

[0207] One or more codons of an encoding polynucleotide can be “biased” or “optimized” to reflect the codon usage of the host organism. For example, one or more codons of an encoding polynucleotide can be “biased” or “optimized” to reflect chloroplast codon usage or nuclear codon usage. Most amino acids are encoded by two or more different (degenerate) codons, and it is well recognized that various organisms utilize certain codons in preference to others. “Biased” or codon “optimized” can be used interchangeably throughout the specification. Codon bias can be variously skewed in different plants, including, for example, in alga as compared to tobacco. Generally, the codon bias selected reflects codon usage of the plant (or organelle therein) which is being transformed with the nucleic acids of the present disclosure.

[0208] A polynucleotide that is biased for a particular codon usage can be synthesized de novo, or can be genetically modified using routine recombinant DNAtechniques, for example, by a site directed mutagenesis method, to change one or more codons such that they are biased for chloroplast codon usage.

[0209] Percent Sequence Identity

[0210] One example of an algorithm that is suitable for determining percent sequence identity or sequence similarity between nucleic acid or polypeptide sequences is the BLAST algorithm, which is described, e.g., in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAS TN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=4, and a comparison of both strands. For amino acid sequences, 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). In addition to calculating percent sequence identity, 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)). 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. For example, 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.

[0211] 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. Alternatively, the viral integrase (or a bacterial or phage recombinase) is covalently linked to a TALE protein or zinc finger proteins where these proteins are designed to target a specific sequence of DNA in a genome.

[0212] This may be provided in an expression vector or as a purified protein. B) A gene of interest (or DNA sequence of interest) with or without homology arms to be incorporated into the desired genome. The GOI or DNA sequence of interest may bemodified to be recognized by the viral integrase as needed. For example, the viral att sites can be added to the ends of the DNA sequence. C) Other reagents needed for polynucleotide transfection and / or introduction of protein into cells.

[0213] Nucleic Acid

[0214] The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably in this disclosure. They refer 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. The following are non-limiting examples of 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. 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.

[0215] Guide RNA

[0216] In aspects of the disclosure the terms “chimeric RNA”, “chimeric guide RNA”, “guide RNA”, “single guide RNA” and “synthetic guide RNA” are used interchangeably and refer to the polynucleotide sequence comprising the guide sequence, the tracr sequence and the tracr mate sequence. The term “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”. The term “tracr mate sequence” may also be used interchangeably with the term “direct repeat(s)”.

[0217] Wild Type

[0218] As used herein the term “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.

[0219] Variant

[0220] As used herein the terms “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.

[0221] Engineered

[0222] The terms “non-naturally occurring” or “engineered” are used interchangeably and indicate the involvement of man-made technology. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.

[0223] Complementary

[0224] “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%, or percentages in between over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0225] Amino Acids

[0226] Full Name, Three-Letter Code, One-Letter Code

[0227] Aspartic Acid Asp D

[0228] Glutamic Acid Glu E

[0229] Lysine Lys K

[0230] Arginine Arg R

[0247] The expression “amino acid” as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids. “Standard amino acid” means any of the twenty standard L-amino acids commonly found in naturally occurring proteins / peptides. “Non-standard amino acid residue” means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source. As used herein, “synthetic amino acid” encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within the peptides of the present disclosure, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the peptide's circulating half-life without adversely affecting their activity. Additionally, a disulfide link may be present or absent in the peptides.

[0248] Amino acids may be classified into seven groups on the basis of the side chain R: (1) aliphatic side chains; (2) side chains containing a hydroxyl (OH) group; (3) side chains containing sulfur atoms; (4) side chains containing an acidic or amide group; (5) sidechains containing a basic group; (6) side chains containing an aromatic ring; and (7) proline, an imino acid in which the side chain is fused to the amino group.

[0249] As used herein, the term “conservative amino acid substitution” is defined herein as exchanges within one of the following five groups:

[0250] I. Small Aliphatic, Nonpolar or Slightly Polar Residues:

[0251] Ala, Ser, Thr, Pro, Gly;

[0252] II. Polar, Negatively Charged Residues and their Amides:

[0253] Asp, Asn, Glu, Gin;

[0254] III. Polar, Positively Charged Residues:

[0255] His, Arg, Lys;

[0256] IV. Large, Aliphatic, Nonpolar Residues:

[0257] Met Leu, He, Vai, Cys (He; autocorrect is not literate)

[0258] V. Large, Aromatic Residues:

[0259] Phe, Tyr, Tip (Trp, likewise)

[0260] The present disclosure utilizes, unless otherwise provided, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, Fritsch and Mamatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc ): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

[0261] Vectors

[0262] Gene expression vectors (DNA-based or viral) will be used to express the fusion integrases in cells or tissues as well as to provide the DNA sequence (or gene) of interest with the appropriate sites needed for the integrase or recombinase to integrate that DNA (or gene) into the genome of the host species or cell. A number of gene expression vectors are known in the art. Vectors will be use for the gene of interest (or DNA sequence of interest). Vectors may be cut with a number of restriction enzymes known in the art.

[0263] CRISPR / Cas9

[0264] CRISPR / Cas9 is described in U.S. Pat. No. 8,697,359, U.S. Pat. No. 8,889,356 and Ran et al (Nature Protocols, 2013, volume 8, pages 2281-2308). Cas9 protein utilizes RNA guides in order to bind specific sequences of DNA in a genome. The RNA guides (guide RNAs) may be designed to be from 10 to 40, from 12 to 35, from 15 to 30, or for example, from 18 to 22, or 20 nucleotides in length. See Hsu et al, Nature Biotechnology, September 2013, volume 31, pages 827-832, which uses Cas9 from Streptococcus pyogenes. Another key Cas9 is from Staphylococcus Aureus (a smaller Cas9 than that of S pyogenes). The Cas9 protein utilizes guide RNAs to bind specific regions of a DNA sequence.

[0265] 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.

[0266] Cas9 is also available in its natural wt form, and also a human optimized codon form for better expression of Cas9 constructs in cells, (see Mali et al, Science, 2013, volume 339, pages 823-826). Codon optimization of Cas9 may be conducted dependent on the species for its expression. Depending on whether one produces a protein form of the Integrase / Cas9 fusion protein (also known as ABBIE1) or a nucleotide expression vector form, the optimized or non-optimized (wt) form may be used.

[0267] RNA guides toward a specific DNA sequence can be designed by various computer-based tools.

[0268] CRISPR / Cpfl

[0269] Cpfl is another protein, which uses a guide RNA in order to bind a specific sequence in genomic DNA. Cpfl also cuts DNA making a staggered cut. Cpfl may be made to be catalytically inactive for cutting ability.

[0270] Other CRISPR Proteins

[0271] These are proteins that utilize a guide RNA to target a specific DNA sequence and whether they have the ability to cut DNA or not. Some of these proteins may naturally have other enzymatic / catalytic functions.

[0272] TALEN

[0273] Transcription Activator-Like Effector Nucleases (TALENs) are fusion proteins with restriction enzymes generated by fusing the TAL effector DNA binding domain to a DNA cleavage domain. These reagents enable efficient, programmable, and specific DNA cleavage and represent powerful tools for genome editing in situ. Transcription activator-like effectors (TALEs) can be quickly engineered to bind practically any DNA sequence. The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that are engineered to work together to cleave DNA at the same site. TALENs that work together may be referred to as a left-TALEN and a right-TALEN, which references the handedness of DNA. See U.S. Pat. No. 8,440,432.

[0274] TAL effectors are proteins secreted by Xanthomonas bacteria. The DNA binding domain contains a highly conserved 33-34 amino acid sequence with the exception of the 12th and 13th amino acids. These two locations are highly variable (Repeat Variable Diresidues (RVD)) and show a strong correlation with specific nucleotide recognition. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.

[0275] 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 DNAtargeting 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.

[0276] The relationship between amino acid sequence and DNA recognition of the TALEN binding domain allows for designable proteins. In this case artificial gene synthesis is problematic because of improper annealing of the repetitive sequence found in the TALE binding domain. One solution to this is to use a publicly available software program named DNAWorks to find oligonucleotides suitable for assembly in a two-step PCR; oligonucleotide assembly followed by whole gene amplification. A number of modular assembly methods for generating engineered TALE constructs have also been reported in the art.

[0277] 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.

[0278] Zinc Finger Proteins

[0279] 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.

[0280] Zinc finger protein endonucleases have been well-established.

[0281] Zinc finger proteins (ZFPs) 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 twohistidines. This class of ZFPs is also known as C2H2 ZFPs. Additional classes of ZFPs have also been suggested. See, e.g., Jiang et al. (1996) J. Biol. Chem. 271 :10723-10730 for a discussion of Cys-Cys-His-Cys (C3H) ZFPs. To date, over 10,000 zinc finger sequences have been identified in several thousand known or putative transcription factors. Zinc finger domains are involved not only in DNA recognition, but also in RNA binding and in proteinprotein binding. Current estimates are that this class of molecules will constitute about 2% of all human genes.

[0282] Many zinc finger proteins have conserved cysteine and histidine residues that tetrahedrally-coordinate the single zinc atom in each finger domain. In particular, 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 metal coordinating ligands bind to the zinc ion and, in the case of zif268-type zinc fingers, the short amphipathic a-helix binds in the major groove of DNA. In addition, the structure of the zinc finger is stabilized by certain conserved hydrophobic amino acid residues (e.g., the residue directly preceding the first conserved Cys and the residue at position +4 of the helical segment of the finger) and by zinc coordination through the conserved cysteine and histidine residues.

[0283] Other DNA Binding Proteins that May Bind Specific Target Sequences in Genomic DNA

[0284] 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.

[0285] Integrases

[0286] 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 (oractive) 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.

[0287] 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). 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). Along with the subterminal CA located at the outermost positions 3 and 4 in retroviral DNA ends (positions 1 and 2 being the 3' end processed nucleotides, these 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). Longer substrates are used more efficiently than shorter ones by HIV-1 IN which indicates that binding interactions extend at least 14-21 bp inward from the viral DNA end. Brin and Leis (2002a) analysed the specific features of the HIV-1 LTRs and concluded that both the U3 and U5 LTR recognition sequences are required for IN-catalysed concerted DNA integration, even though the U5 LTRs are more efficient substrates for IN processing in vitro (Bushman & Craigie, 1991; Sherman et al., 1992). The positions 17-20 of the IN recognition sequences are needed for a concerted DNA integration mechanism, but the HIV-1 IN tolerates considerable variation in both the U3 and U5 termini extending from the invariant subterminal CA dinucleotide (Brin & Leis, 2002b). The instant disclosure includes a DNA vector that contains viral (retroviral or HIV) LTR regions at the 5' and 3' ends of a location to house the DNA sequence or gene of interest to be integrated into the genome. The LTR regions do not have to be the full length LTRs as long as they function to interact with the integrase for proper integration. The LTR regions may be modified to contain detectable (e.g., fluorescent), PCR detection, or selectable markers (e.g. antibiotic resistance). The vector is designed to be cut and linearized so that the LTR regions are at the 5' and 3' ends of the DNA fragment (via designed restriction sites to restriction endonuclease).

[0288] 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. In one aspect of the disclosure, 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. As full activity of integrase has also been observed to involve tetramers of integrase, fusion proteins may be designed with 1, 2, 3, 4 integrase proteins linked by flexible linkers that may be 1 to 20 amino acids in length or 4-12 amino acids in length.

[0289] Recombinases

[0290] Recombinases including Cre, Flp, R, Dre, Kw, and Gin recombinase are described in U.S. Pat. No. 8,816,153 and US 2004 / 0003420 hereby expressly incorporated by reference in their entireties. Recombinases such as Cre recombinase use LoxP sites in order to excise a sequence from the genome. Recombinases can be modified to become constitutively active for their recombination activity and also become less site specific. Thus, it is possible to target such constitutively active recombinase proteins with no sequence specificity to specific sequences of DNA in a genome by incorporating them into fusion proteins of the instant disclosure. In this manner, the CRISPR / Cas9, TALE or zinc finger protein domain specifies the DNA sequence where the recombinase will contribute its recombination activity. Such recombinase proteins may be wild-type, constitutively active or dead for recombinase activity. A Cas9-recombinase such as Cas9-Gin or Cas9-Cre may be produced by use of a linker sequence or by direct fusion.

[0291] Nuclear Localization Signal Sequence (NLS) for Fusion Proteins

[0292] The signal peptide domain (also referred to as “NLS”) is, for example, derived from yeast GAL4, SKI3, L29 or histone H2B proteins, polyoma virus large Tprotein, VP1 or VP2 capsid protein, SV40 VP1 or VP2 capsid protein, Adenovirus Ela or DBP protein, influenza virus NS1 protein, hepatitis virus core antigen or the mammalian lamin, c-myc, max, c-myb, p53, c-erbA, jun, Tax, steroid receptor or Mx proteins (see Boulikas, Crit. Rev. Eucar. Gene Expression, 3, 193-227 (1993)), simian virus 40 (“SV40”) T-antigen (Kalderon et. al, Cell, 39, 499-509 (1984)) or other proteins with known nuclear localization. The NLS is, for example, derived from the SV40 T-antigen, but may be other NLS sequences known in the art. Tandem NLS sequences may be used.

[0293] Linker Regions

[0294] The various 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.

[0295] Expression Vectors

[0296] Many nucleic acids may be introduced into cells to lead to expression of a gene. As used herein, the term 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. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone. 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).

[0297] 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.

[0298] 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.

[0299] 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 Nucleofection™ may also be used.

[0300] 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 setof 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.

[0301] Many different types of vectors are known in the art. For example, plasmids and viral vectors, including retroviral vectors, are known. 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).

[0302] 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.

[0303] A pooled population of edited cells are considered a mixture of cells that have received a gene edit and cells that have not.

[0304] Exemplary Abbiel In Vitro Assay

[0305] 1. Incubate ABBIE 1 protein with guide RNA;

[0306] 2. Incubate ABBIE 1 / guide RNA with donor DNA having partial LTRs to form pre-initiation complex;

[0307] 3. Incubate pre-initiation complex with plasmid containing gene to be edited (e.g. CXCR4); and

[0308] 4. PCR and DNA sequencing confirmations for donor DNA integration.

[0309] Cas9protocols are described in, for example, Gagnon et al., 2014, http: / / labs.mcb.harvard.edu / schierNertEmbryo / Cas9_Protocols.pdf.

[0310] Assays for integrase activity are described in, for example, Merkel et al., Methods, 2009, volume 47, pages 243-248.

[0311] A family of graphene-based or graphene-related materials (here, the terms “graphene-based” and “graphene-related” are used interchangeably) 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 groups, porphyrin, pyridine, polymers and combinations thereof). Specific examples of graphene-related materials include graphene oxide (GO), graphite oxide, and reduced graphene oxide (rGO). Carbon nanomaterials comprise graphene, carbon nanostructures including nano-dendrites, nano-hexacones, nanospheres, nano-donuts, nanoparticles and nanorods, or combination thereof.

[0312] 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)).

[0313] 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. In one embodiment, CNTs are subjected to treatment with acid prior to protein adsorption. Recent studies have demonstrated that acid treatment of SWNTs induces defects on thesurface of the nanotubes (Hu, et al., Jour. Phys. Chem. B, 107: 13838-42 (2003)), as well as promote de-bundling (Liang, et al., Nano Lett., 4: 1257-60 (2004)), which can be correlated with an increase in surface area (Hemraj -Benny, et al., Jour. Coll. Interf Sci., 317(2):375-82 (2008)). In one embodiment, 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)). In one embodiment, the CNTs are reduced following acid treatment. For example, following nitric 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. The examples below demonstrate that treatment of CNTs with 3M HN03 significantly increases surface area of SWNTs, which is further increased by subsequent treatment with LiBH4.

[0314] In addition to non-specific adsorption, 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. 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. Biol., 9(6):674-9 (2005); Pantarotto, et al., J. Am. Chem. Soc., 125(20):6160-4 (2003); Williams, et al., Nature, 420(6917):761 (2002); Pamtarotto, et al., Chem. Commun., 1: 16-7 (2004).

[0315] A functionalized graphene comprising at least one functional group can be covalently bonded directly to an edge of a graphene plane, the functional group being selected from the group consisting of — NO2, — NH2, — SO3H, halide, — N3, — MgBr and — SH, the process comprising:

[0316] a) preparing a sample of graphene to be functionalized,

[0317] b) contacting the graphene with one or more reagents suitable for introducing at least one functional group selected from the group consisting of — NO2, —NH2, — S03H, halide, — N3, — MgBr and — SH to any available edge of the sample of graphene, and

[0318] c) isolating the functionalised graphene bearing only the at least one functional group selected from the group consisting of — NO2, — NH2, — SO3H, halide, — N3, —MgBr and — SH.

[0319] In one embodiment, when at least one functional group is NO2, — NH2, — SO3H or — SH, at least a portion of functional group forms part of the graphene conjugate.

[0320] In one embodiment, the at least one organic or inorganic moiety is covalently attached via a coupling reaction between the organic or inorganic moiety (or a precursor thereof), and the at least one functional group.

[0321] In another embodiment, the at least one organic or inorganic moiety is selected from the group consisting of amino acids, proteins, antibodies, carbohydrates, polymers, nanoparticles, metals, chelators, nucleic acids, oligonucleotides, aptamers, fluorophores, metal oxides, biologically active compounds or biomarkers. In another embodiment, the at least one organic or inorganic moiety is a chelator. Edge-modified graphene directly coupled to a chelator for metal ion or metal may find use in end applications such as metal sequestration and recovery. For example, a chelator graphene may be added to a mixture of metal ions. Chelators selectively bind target metal ions to remove them from the mixture. The graphene-chelator complex can then be removed by filtration. The metal can be released electrochemically, and the graphene reused. Chelators that fluoresce (e.g., chelated lanthanides) highlight edge regions (including defects in the basal plane, i.e., internal edges) and are useful for quantifying defects that will impede carrier mobility. Chelated metals on graphene can also be used for medical imaging. Examples of chelated metals for this purpose include, but are not limited to, indium-i l l in di ethylene triamine pentaacetic acid (DTP A) for single-photon emission computed tomography and gadolinium(III) in l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA) in MRI imaging. Edge-modified graphene linked to chelators such as these may be used to follow the distribution of graphene in biology, or may be used as a proxy for the position of agents such as drug molecules that are loaded on the basal plane of the graphene.

[0322] Kinases and phosphatases are known to be involved in signaling in the cell. A number of kinases and phosphatases alter proteins that associate with DNA or RNA.In some embodiments, kinases, phosphatases or the active domains thereof (e.g. kinase or phosphatase active domain) can be bound to a DNA binding protein as provided above with or without the added material (e.g. graphene or other carbon-based material).

[0323] In some embodiments, compounds may be bound to the material (e.g. graphene or carbon-based material as stated above). In some embodiments, these compounds may be diagnostic, pharmaceutical, biological, peptides, small organic compounds, fluorescent or other detectable compounds, colorimetric compounds.

[0324] In some embodiments, kinases can include Erk, p38, JNK and other MAPK, MAPKK, MAP3K and MAP4K proteins or kinase domains thereof. In some embodiments, a kinase may be Src kinase or a nuclear protein kinase. In some embodiments, a kinase may be a kinase domain of receptor kinase like EGFR or Erb2 or other kinase. In some embodiments, kinases may be serine threonine receptor kinase, tyrosine receptor kinases, G-protein coupled receptors or kinase domains thereof. In some embodiments, phosphatases include Shp phosphatases like Shp2 and nuclear phosphatases. In some embodiments, the kinases and phosphatases can be of human, mouse, rat, primate, pig, bacteria, microbes, plants or other origin. In some embodiments, the kinases or phosphatases or kinase or phosphatase domains thereof are attached to the DNA binding protein or Cas protein by peptide or non-peptide linkers or by direct attachment.

[0325] Novel technological tools are needed for remote stimulation of cells. Optimal physiological stimulation tools should be able to change the membrane potential quickly, reversibly, and repeatedly; to control the amplitude, duration, and direction of these changes; to be minimally intrusive and compatible with non-invasive detection methods.

[0326] To satisfy these requirements, novel materials with suitable properties can be considered. The family of graphene-based or graphene-related materials (here, the terms “graphene-based” and “graphene-related” are used interchangeably) recently stepped into the spotlight after the 2010 Nobel Prize in Physics, and subsequent explosion in development of numerous applications for these materials 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 multi-layered), graphite, polycyclic aromatic hydrocarbons, carbon nanotubes,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 groups, porphyrin, pyridine, polymers and combinations thereof). Specific examples of graphene-related materials include graphene oxide (GO), graphite oxide, and reduced graphene oxide (rGO).

[0327] Graphene and graphene-related materials exhibit extraordinary electronic, mechanical, and optical properties, which could make them invaluable in various biomedical applications, including construction of a biocompatible interface for remote stimulation of cells using electromagnetic radiation. Graphene is highly inert and chemically stable, which results in excellent biocompatibility. Its unique properties include high electrical conductivity, high mobilities of charge carriers, exceptional mechanical properties (e.g., high stiffness, strength, and elasticity), high thermal conductivity, broadband absorption and high transparency over the visible spectrum. Moreover, it is possible to tune electronic and optical properties of graphene for a specific application by pursuing one of many routes such as changing the stacking pattern of graphene sheets, changing the shape of graphene structures, substituting carbon atoms in a graphene lattice, and functionalizing graphene.

[0328] Graphene has a zero bandgap, leading to a broadband absorption of incident light with the energy below '3.5 eV. It means that graphene having the high optical absorption coefficient (7 / 105cm-1) can efficiently detect wavelengths of light ranging from 300 to 2500 nm, which includes the entire visible spectrum, infrared and even terahertz regions.

[0329] 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. When 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 opticalabsorption 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%.

[0330] GO is a highly oxidized graphene-related material with numerous oxygencontaining functional groups. In contrast to graphene with its zero-band gap and high electronic conductivity, GO is a wide- bandgap semiconductor with a bandgap >3.5 eV, and very poor electronic conductivity. The removal of the oxygen-containing functional groups leads to the decrease in the optical bandgap from >3.5 eV to <1 eV, the increase in the optical absorption, and the restoration of the electrical conductivity. This reduction process results in another graphene-related material known as rGO or chemically converted graphene. While graphene and other non-functionalized graphene-related materials (e.g., graphite, carbon nanotubes and fullerenes), are hydrophobic, 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.

[0331] Among current biomedical applications for graphene and its derivatives are their utilization for tissue engineering, antibacterial treatment, drug and gene delivery, and as contrast agents for bioimaging. For example, 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. These applications utilized not only pure graphene and its derivatives, but also their hybrid nanocomposites with diverse nanostructures (e.g., semiconductor quantum dots, carbon nanotubes), proteins (e.g., chitosan), polymers (e.g., polypropylene carbonate)), or other chemical entities (e.g., PEG). It was suggested that positive effects of graphene-related materials on creating favorable cell microenvironment are based on their mechanical properties, micro-scale topographic features, and surface chemistry characteristics.

[0332] All existing biomedical applications take advantage of passive steady-state properties of graphene and its derivatives. Currently, there are no applications that utilize external stimuli to actively change physicochemical properties of graphene-relatedbiointerfaces, and subsequently to change the functional state of cells interacting with these interfaces.

[0333] 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. In some embodiments, 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.

[0334] In some embodiments, 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).

[0335] In some embodiments, 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.

[0336] In some embodiments, 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. In some embodiments, the minimal dimensions in the X-Y plane can be 1 nm by 1 nm. In some embodiments, external dimensions along the Z-axis can depend on the number of layers of graphene-related materials incorporated into a G-biointerface. In some embodiments, the minimal dimension along the Z-axis can be defined by the thickness of a non-functionalized graphene monolayer, 0.34 nm.

[0337] In some embodiments, the target can be one or more intact cells, one or more cellular fractions, or one or more artificial membrane structures. In some embodiments, examples of cellular fractions include any luminal organelles such as nucleus, ribosomes, mitochondria, endoplasmic reticulum, Golgi apparatus, vacuoles, synaptic vesicles and lysosomes. In some embodiments, examples of artificial membrane structures can include phospholipid micelles, micro- and nanocapsules, and semi-liquid films on supportive structures.

[0338] In some embodiments, the one or more cells can generally be any type of cells which have a membrane and membrane potential. In some embodiments, 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. In some embodiments, the methods can also be used on non-cell materials such as artificial membranes, liposomes, and phospholipid bilayers. In some embodiments, examples of primary mammalian cells can comprise human, mouse, rat, dog, cat, bear, moose, cow, horse, pig, or Chinese hamster ovary (“CHO”) cells. In some embodiments, 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.

[0339] In some embodiments, 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.

[0340] In some embodiments, graphene, carbon nanotubes and other materials can be incorporated in various sizes. In some embodiments, 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.

[0341] 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. In some embodiments, kinase,phosphatases or the active domains thereof (e.g. kinase or phosphatase active domain) can be bound to a DNA binding protein as provided above with or with the added material (e.g. graphene). In some embodiments, these fusion proteins can also be bound with materials as described above (e.g. graphene oxide, graphene, carbon nanotubes).

[0342] In some embodiments, kinases can include Map kinases. In some embodiments, kinases can include receptor tyrosine kinases and receptor serine / threonine kinases. In some embodiments, kinases can be tyrosine kinases or serine / threonine kinases. In some embodiments, kinases can include ERK, ERK1, ERK2, p38, ERK5, JNK, MEKK1, MEKK2, MEKK3, MEKK4, MEKK5, nuclear kinases, MEK1, MEK2, MEK3, and others. In some embodiments, receptor kinases can include EGFR, Erbl, Erb2, Erb3 and others. In some embodiments, Src can be used for the invention. In some embodiments, cyclin- dependent kinases (CDKs), WEE1, polo-like kinasei (PLK1), and casein kinase II (CK2), ATM, CHKs, DNA-PK or ATR can be kinases that target DNA processes. In some embodiments, kinase domains of any of the mentioned kinases can also be attached to a DNA binding protein / domain as mentioned above. In some embodiments, the DNA binding can be controlled by use of a guide RNA. In some embodiments, 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.

[0343] In some embodiments, phosphatases (dephosphorylases) can include nuclear phosphatases and cytoplasmic phosphatases. In some embodiments, phosphatases can include Shp phosphatases, Shp2, Shpl, and others. In some embodiments, phosphatases can include those that regulate transcription factors and / or other nuclear proteins. In some embodiments, phosphatases can include PPM phosphatases and PP2C phosphatase. In some embodiments, phosphatase domains of any of the mentioned kinases can also be attached to a DNA binding protein / domain as mentioned above. In some embodiments, the DNA binding can be controlled by use of a guide RNA. In some embodiments, these may be human, mammalian, mouse, rat, primate, microbial, bacterial, fungal, yeast, plant or of other origins.

[0344] In some embodiments, 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 theappropriate 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. In some embodiments, methods can include activating and / or detecting a signal that emits from the material bound to the protein. In some embodiments, 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. In some embodiments, methods can include targeting disease genes. In some embodiments, methods can include targeting safe harbor loci in the cell’s DNA. In some embodiments, methods can include attaching the material or protein to other molecules like those that promote detection, drugs, vitamins or such. In some embodiments, 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). In some embodiments, the subject or cell for administration can be human, primate, mammalian, bacterial, fungal, yeast, fish, amphibian, reptile, plant or others.

[0345] 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. In some embodiments, integration can be performed by providing the integration cas9 system, guide RNA and graphene oxide bound donor DNA to cells. In some embodiments, after incubating the cells to allow integration to proceed, cells can be harvested and DNA can be extracted. In some embodiments, 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. In some embodiments, sequencing can be performed to find where the Donor DNA was incorporated in the genomic DNA. In some embodiments, the extracted DNA can be fragmented by DNases or other enzymes prior to binding of graphene oxide to the provided graphene oxide binding surface. In some embodiments, thesurface 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. In some embodiments, this system can be used on mitochondrial or genomic DNA. In some embodiments, 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.

[0346] 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). In some embodiments, detection as discussed above can be performed in this instance to find which cells, the modified protein material has entered or even which chromosome it has bound. In some embodiments, detection can be done with techniques including microscopy, flow cytometry, fluorescent microscopy, and the like. In some embodiments, detection is also possible by a high speed electrical readout with radiofrequency (rf) reflectometry.

[0347] Another method is to control the outcome of a gene editing event. In some embodiments, the graphene oxide or graphene (or other material) bound DNA binding protein (e.g. integrase fused to cas9 or reverse transcriptase bound to cas9 or cas9) can be provided to cells and then manipulated by electrical pulses from outside the cell. In some embodiments, this can cause the bound DNA binding protein to activate or deactivate based on a cleavage, molecular blocking, protein or molecular interactional change, or conformational change that occurs within the DNA binding protein that is stimulated by the electrical impulse. In some embodiments, the electrical impulse can be controlled via the user for the electrical potential, appropriate voltage and / or current. In some embodiments, this has been shown to reduce the oxygen groups on graphene oxide, which lowers electrical resistance of graphene oxide materials.

[0348] Another method is using a kinase or phosphatase bound cas9 (or other DNA binding protein) to import into cells to target a promoter region of DNA and alter the phosphorylation state of a transcription factor, chromatin or other DNA associated protein. In some embodiments, targeting a transcription factor like p53, Sox, Elkl, FoxA or Myc and increasing or decreasing its phosphorylation can change its transcriptional activity, thus, decreasing or increasing expression of a gene. In some embodiments, changingphosphorylation of a DNA repair enzyme can increase or decrease its ability to repair a given site (e.g. phosphorylation state of BRCA, ATM or ATR). In some embodiments, this can be used as a transcription (or DNA repair or DNA modification) modifying therapeutic agent for diseases and disorders.

[0349] Graphene oxide (GO) has been produced as a matrix for protein immobilization in different technical applications including fluorescence, or electrochemical sensors, labeling and imaging, therapeutic purposes, and targeted delivery (Simsikova et al, March 2017, journal of Nanomedicine Research, volume 5, pages 1-4). In some embodiments, GO can be used in nano particle or micro particle form to be bound to molecules such as protein or DNA.

[0350] In some embodiments, graphene or graphene oxide that has been functionalized by oxygen- containing groups such as epoxide, hydroxyl, sulfonate, phosphate, amino acid, and carboxylic groups or amine groups can be reacted with an amino acid, peptide, nucleic acid, oligonucleotide, polynucleotide or polyethylene glycol linker. In some embodiments, the linker may be further functionalized before the reaction. In some embodiments, no linker is needed when the graphene or graphene oxide is functionalized. In some embodiments, the link is non-covalent such as ionic or hydrogen bonding. As stated in previously in the disclosure, in some embodiments, line graphene or graphene oxide can be linked to detectable moieties such as fluorophores, gold nanoparticles, or antibodies in addition to the peptide, protein, oligonucleotide or polynucleotide compound.

[0351] In some embodiments, the graphene or graphene oxide that is functionalized and / or bound to linker can be bound to peptides, proteins, RNA, DNA, oligonucleotides or polynucleotides via a reaction to form ester, amino ester, phospho ester, or ether groups between the functionalized or linker bound graphene or graphene oxide and the hydroxyl, amino, carboxyl or phosphate groups of the peptides, proteins, RNA, DNA, oligonucleotides or polynucleotides. In some embodiments, this process can create graphene or graphene oxide (G or GO) linked compounds including peptides, proteins, RNA, DNA, oligonucleotides or polynucleotides. In some embodiments, the proteins can be dCas9, Cas9, a Cas protein, ABBIE, Cpfl or any of those mentioned in this disclosure. In some embodiments, the DNA can be a donor DNA to be integrated into genomic DNA. The graphene, graphene oxide or fullerene can be incorporated into the donor DNA.

[0352] In some embodiments, the graphene or graphene oxide linked compounds indicated above can be provided into a cell by standard techniques such as electroporation, lipid based transfection, surfactant based transfection. In some embodiments, when a Cas protein or ABBIE protein is used a ribonucleotide protein complex (RNP) can be formed with guide RNA before providing into the cell.

[0353] In some embodiments, detection can be performed using techniques including fluorescence, x-ray, and / or reflectometry depending on the molecules present in the linked compounds.

[0354] In some embodiments, microbes to be detected and / or treated in an animal body, plant or area, can include coronavirus, influenza, plant viruses, HIV, Herpes simplex Virus and other viruses, E. coli, Salmonella, Staphylococcus, Streptococcus, Shigella, Aspergillus, Pseudomonas and other pathogenic bacteria, yeasts like Saccharomyces spp and Candida spp., fungi, protozoa and others. In some embodiments, bacteria can be gram positive or gram negative or antibiotic-resistant strains. In some embodiments, viruses can be RNA viruses or DNA viruses.

[0355] In some embodiments, cancer cells and cancer genes can be detected and / or treated including breast cancer, lung cancer, liver cancer, prostate cancer, colon cancer, melanoma, renal cancer and others.

[0356] In some embodiments, genes targeted can include BRCA, p53, and other oncogenes.

[0357] Detection methods

[0358] In some embodiments, the detection methods can be fluorescent microscopy, immunodetection, column separation and isolation, flow cytometry and electrical sensors.

[0359] In some embodiments, an electrochemical aptasensor based on a reduced graphene oxide-titanium dioxide nanocomposite can detect bacteria and cells and the optimized aptasensor has shown high sensitivity with a wide detection range (10-108 CFU / mL), and also a low (limit of detection) LOD of 10 CFU / mL.

[0360] In some embodiments, a microfluidic immunochip can apply biofunctionalized graphene oxide for bacteria and cell detection with the LOD as low as 0.376 CFU / mL.

[0361] In some embodiments, an ultrasound-assisted self-assembly of monolayer graphene oxide can be used with a high affinity for bacteria with LOD as low as 10 CFU / mL.

[0362] In some embodiments, a graphene oxide and graphene dot-based fluorescence resonance energy transfer biosensor for immunosensing can be used that shows a LOD for these bacteria of about 10 CFU / mL.

[0363] In some embodiments, a graphene-based electrical biosensor can be used for the detection of bacteria which shows sensitivity as low as 10-100 cell / mL.

[0364] In some embodiments, there is a potentiometric biosensor for living bacterium detection based on graphene (Pourmadadi et al, Food Technology and Biotechnology, 2021, volume 59, pages 496-506).

[0365] An example of performing detection involves a graphene oxide linked dcas9 with guide RNA to a mutated sequence in the nrf2 or BRCA gene being electroporated into cells. In some embodiments, the cells are then subjected to detection with a graphene dot-based fluorescence resonance energy transfer (FRET) biosensor. In some embodiments, if the mutation is present and the GO linked Dcas9 has bound the genomic DNA it can be detected on the genomic DNA. In some embodiments, controls can include scrambled guide RNA, no guide RNA and standard dcas9.

[0366] Another example involves GO linked to donor DNA. In some embodiments, the donor DNA can be integrated via the ABBIE protein using guide RNA. In some embodiments, the genomic DNA can be isolated by known techniques and subjected to column or bead isolation of graphene oxide or functionalized graphene oxide bound DNA. In some embodiments, amplification and sequencing can be performed on the DNA fragments captured to see whether specific binding to the expected target site is achieved or not.

[0367] In some embodiments, incorporation of nanomaterials such as graphene based transmitters / receivers is provided herein. In some embodiments, integration of graphene-based biosensors directly within the genetic material of an organism is provided. In some embodiments, graphene-based biosensors can be directly connected to the ongoing cellular activities of reading and copying of nucleic acids.

[0368] In some embodiments, graphene-based biosensors, activated by binding with other molecules or specific DNA sequences, could trigger external communications through physical (color change) or electronic detectors. In some embodiments, multiple waysof interfacing intercalated biosensors with power sources or fields are provided herein, either internal or external to the body, including wireless kill signals, data acquisition of living materials (pH, current, output), programmable Cells, or origami-DNA can be used as programmable nanobots for drug delivery.

[0369] In some embodiments, methods for transmitting and receiving a signal within a cell are provided. In some embodiments, a wireless remote signal can be provided to a cell and the cell responds with an output. In some embodiments, the signal is transmitted to a target cell nucleus. In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell nucleus is modified with a Nano-transmiXer and receiver.

[0370] In some embodiments, a chip is further provided herein. In some embodiments, the chip can comprise a central processing unit further comprising a control unit and arithmetic / logic unit. In some embodiments the chip further comprises a memory unit. In some embodiments, the chip can process thousands of bits of code rapidly. In some embodiments, the activity of the chip is between an input device and output device.

[0371] In some embodiments, methods of Remote-Control Genome Editing (ReCoG editing) are provided. In some embodiments, ReCoG editing allows one to more precisely define where the protein will target. In some embodiments, ReCoG editing can improve specificity of edits in the genome. In some embodiments, ReCoG editing can provide a higher resolution of processing living code within a living organism. In some embodiments, methods of Homing ReCoG editing and External ReCoG editing are provided.

[0372] In some embodiments, Homing ReCog editing can use a wireless signal to direct the processor to target code. In some embodiments, Homing ReCog editing can include two-part signal transmission where a protein binds specifically to DNA and completes a circuit to bring editing protein to desired site for activity.

[0373] In some embodiments, 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). In some embodiments, External ReCog editing can allow a user to remotely activate the proteins that are in the desired locales (only proteins that transmit a signal). For example, 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 instruction may cause the signal tochange the conformation or cause a cleavage of a protein that receives the signal to increase or decrease its activity.

[0374] In some embodiments, the nano remote devices can be modified to attach to a protein, non-toxic to cells and either biodegradable and / or easily removed by the body. In some embodiments, modified nano graphene (e.g. NGO— PEGylated) exhibits little toxicity as compared to NGO without modification.

[0375] In some embodiments, the systems provided herein can be applied to novel therapeutics. In some embodiments, the systems provided herein can be applied to use of wireless (remote) drug delivery. In some embodiments, provided herein can be applied to incorporation into DNA origami nanobots. In some embodiments, provided herein can be applied to increase drug efficacy. In some embodiments, the systems provided herein can be applied to decrease off target toxicity. In some embodiments, the systems provided herein can be applied to fields including, but not limited to biofuels, agriculture, pharmaceuticals, and diagnostics.

[0376] Biotinylation alternative or addition

[0377] Binding biotin to proteins, polynucleotides, or oligonucleotides can be as effective as binding to graphene particles for detection purposes due to several factors related to specificity, sensitivity, and the versatility of biotin-streptavidin interactions.

[0378] 1. High Affinity Binding:

[0379] Biotin exhibits an exceptionally high affinity for streptavidin or avidin, with a dissociation constant in the picomolar range. This strong binding affinity ensures that even low concentrations of biotinylated molecules are detected effectively. When proteins, polynucleotides, or oligonucleotides are labeled with biotin, the resulting complex is detected with high sensitivity, similar to the functionalization of graphene particles.

[0380] 2. Versatile Functionalization:

[0381] Biotin is easily incorporated into various biomolecules, enabling the functionalization of proteins, nucleotides, or oligonucleotides for specific binding applications. This versatility allows for the design of tailored detection systems depending on the target, whether it be proteins, DNA, or RNA, mirroring the adaptability seen with graphene-based biosensors.

[0382] 3. Scalable Detection Systems:

[0383] Biotinylated proteins and nucleotides are coupled with various detection methods, such as enzyme-linked assays (e.g., ELISA), fluorescence, or chemiluminescence. These techniques can be integrated into microfluidic devices or other platforms, enabling scalable and multiplexed detection similar to the use of graphene nanomaterials in biosensing applications.

[0384] 4. Minimal Background Noise:

[0385] The use of biotin-streptavidin interactions typically results in minimal background noise in detection assays. This facilitates achieving a high signal-to-noise ratio, which is desirable for sensitive detection. In contrast, while graphene enhances electrical signals, it may also introduce noise depending on the complexity of the sample matrix.

[0386] 5. Biocompatibility:

[0387] Biotinylated biomolecules are generally more biocompatible than various graphene-based materials, which may require additional surface modifications to reduce cytotoxicity. Biocompatibility allows for the application of biotin-based detection in clinical and environmental samples without adverse reactions.

[0388] 6. Cost-Effectiveness:

[0389] Producing biotin-labeled proteins or nucleotides is often more cost- effective than synthesizing and functionalizing graphene nanoparticles. This cost efficiency facilitates the development of accessible diagnostic tools and assays, which are preferred for widespread adoption in clinical and research settings.

[0390] 7. Broad Range of Applications:

[0391] Biotinylated proteins, polynucleotides, and oligonucleotides are employed in a wide array of applications, including diagnostics, therapeutics, or research. This broad applicability allows for the development of diverse detection systems tailored to various targets, similar to the versatility offered by graphene-based sensors.

[0392] While graphene particles offer unique benefits in terms of electrical conductivity and surface area for biosensing applications, biotinylation of proteins, polynucleotides, or oligonucleotides presents a powerful and effective alternative for detection. The high affinity and specificity of biotin-streptavidin interactions, coupled with versatility, cost-effectiveness, and biocompatibility, make biotin-based detection methods a valuable tool in biosensing and diagnostic technologies.

[0393] In some embodiments, 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. In some embodiments, the proteins, polynucleotides, or oligonucleotides described herein are biotinylated.

[0394] Additionally, kinases or phosphatases that regulate base editing such as through adenine base editors are envisioned.

[0395] 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. Such kinases and phosphatases bound DNA binding proteins like Cas proteins are envisioned to further activate and deactivate HR at a given site. In some embodiments, additional HR proteins or other DNA associated proteins are added or provided to the cells as necessary.

[0396] 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.

[0397] Acetylases and deacetylase proteins are envisioned to be linked to DNA binding domains like those that use guide RNA for targeting. These can regulate Cas (e.g. Cas9).

[0398] Cas9 can be acetylated by enzymes that utilize acetyl-phosphate or acyl- CoA metabolites as acetyl donors, including enzymes present within the host cell where Cas9 is used for genome editing; this acetylation can occur both in vitro and in vivo, potentially affecting Cas9's activity by modifying lysine residues on the protein. Thus, an acetylase or deacetylase enzyme linked to dcas9 through an amino acid or other linker is envisioned. This can be used to regulate (on and off) Cas9 or other Cas activity as well as other proteins regulated by this mechanism.

[0399] Kinases and phosphatases that control helicase, integrases and Cas proteins- Two kinases that help trigger eukaryote helicase activation: Cdc7 kinase and S phase cyclin-dependent kinases (S-CDKs).

[0400] Cdc7 directly phosphorylates the Mcm2-7 complex and is activated by the cell-cycle-regulated accessory subunit Dbf4 or the related Drfl. These kinases can be bound to dcas in these systems.

[0401] In some embodiments, a method of genome editing is provided, 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.

[0402] In some embodiments, a method of genome editing is provided, 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.

[0403] In some embodiments, the carbon nanomaterial particles are labeled prior to incubation.

[0404] In some embodiments, the donor integrated gDNA sample is amplified prior to confirming the integration of donor sequences.

[0405] In some embodiments, the carbon nanomaterial particles comprise graphene.

[0406] In some embodiments, the carbon nanomaterial is crosslinked to its binding partner.

[0407] In some embodiments, the genome editing protein comprises an ABBIE system.

[0408] In some embodiments, the genome editing protein comprises a viral integrase- dCas9 complex.

[0409] In some embodiments, 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.

[0410] In some embodiments, the detector or sensor uses electrostatic, luminescent, or fluorescent detection.

[0411] In some embodiments, a method of modifying the acetylation state of a selected protein associated with a polynucleotide is provided, 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.

[0412] In some embodiments, a microfluidic chip with channels for sample input, mixing, and reaction zones is further provided.

[0413] In some embodiments, 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.

[0414] In some embodiments, the polynucleotides, or oligonucleotides described herein are biotinylated.EXAMPLES

[0415] The following examples are intended to provide illustrations of the application of the present disclosure. The following examples are not intended to completely define or otherwise limit the scope of the disclosure. One of skill in the art will appreciate that many other methods known in the art may be substituted in lieu of the ones specifically described or referenced herein.Example 1: DNA Vectors for Expression Cas9-Integrase Fusion Proteins

[0416] 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. In other experiments, 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 (or other sequence useful for protein purification) 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) is commonly used for expression vectors for mammalian cells. The U6 promoter is also commonly used. A T7 promoter may be used for in vitro transcription in certain embodiments.Example 2: DNA Vector for Expression of the DNA Sequence of Interest (Gene of Interest)

[0417] 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). When using a recombinase, the integrase recognition sites may not be included. Markers, such as drugresistance 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.Example 3: DNA Vector for Reverse Transcriptase Expression

[0418] 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.Example 4: Co-Expression of DNA Targeting-Integrases (or Recombinases) with DNA Sequence of Interest

[0419] Cells were electroporated for the vectors described above along with the Cas9 RNA guides required for the target site in the genome. In some experiments, 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). In other experiments, 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.Example 5: Test Experiments and Assaying for Off-Target Insertions

[0420] Cells missing expression of a particular gene, such as mouse embryonic fibroblasts from a knockout mouse model or cells genetically engineered to be knockouts for a given 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. Limiteddilution 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.Example 6: Cas9 Linked Integrase Protein Expression and Isolation

[0421] 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. 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. After sufficient time for expression has occurred, 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

[0422] 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

[0423] Incubate Abbiel isolated protein (other specific DNA sequence binding protein linked to retroviral integrase) with insertable / integratable DNA having viral LTR regions in a suitable buffer, (for formation of tetramer or other multimer depending on the instance). Alternatively, a premade composition of isolated Abbiel protein with guide RNA may be combined with the insertable DNA sequence. Include guide RNA and incubate to incorporate guide RNA. Transfect or electroporate (or other technique of providing protein to cells) Abbiel / DNA preparation into cells. Allow time for genome / DNA editing to take place. Check for insertion of designed insertable DNA sequence into the specific site of the genomic DNA of the cell. Check for non-specific insertions by PCR and DNA sequencing.

[0424] As currently planned, 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. 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.Example 9: Design of Constructs for Fusion Proteins

[0425] 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.) One may produce a recombinant fusion protein with E coli as the source. Isolate the protein bystandard 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.

[0426] 1. Mix Donor DNA with appropriate LTR domains and insertable sequence, and fusion protein and incubate for 10 minutes, (alternatively add Donor DNA after the RNP complex formation)

[0427] 2. Resuspend each RNA oligo (crRNA and tracrRNA) in Nuclease-FreeIDTE Buffer. For example, use a final concentration of 100 pM.

[0428] 3. Mix the two RNA oligos in equimolar concentrations in a sterile microcentrifuge tube. For example, create a final duplex concentration of 3 M using the following table: Component Amount 100 pM crRNA 3 pL 100 pM tracrRNA 3 pL Nuclease-Free Duplex Buffer 94 pL Final volume 100 pL

[0429] 4. Heat at 95° C. for 5 mm.

[0430] 5. Remove from heat and allow to cool to room temperature (15-25° C.) on your bench top.

[0431] 6. If needed, dilute duplexed RNA to a working concentration (for example, 3 pM) in Nuclease-Free Duplex Buffer.

[0432] 7. Dilute fusion protein to a working concentration (for example, 5 pM) inWorking Buffer (20 mM HEPES, 150 mM KCI, 5% Glycerol, 1 mM DTT, pH 7.5).

[0433] 8. For each transfection, combine 1.5 pmol of duplexed RNA oligos (StepA5) with 1.5 pmol of fusion protein (Step A6) in Opti-MEM Media to a final volume of 12.5 pL.

[0434] 9. Incubate at room temperature for 5 min to assemble the RNP complexes.Example 10: Reverse Transfect gRNA-Fusion Protein in a 96-Well Plate

[0435] 1. Incubate the following at room temperature for 20 min to form transfection complexes: Component Amount RNP (Step A8) 12.5 pL Lipofectamine® RNAiMAX Transfection Reagent 1.2 pL Opti-MEM® Media 11.3 pL Total volume 25.0 pL

[0436] 2. During incubation (Step Bl), dilute cultured cells to 400,000 cells / mL using complete media without antibiotics.

[0437] 3. When incubation is complete, add 25 pL of transfection complexes(from Step Bl) to a 96- well tissue culture plate.

[0438] 4. Add 125 pL of diluted cells (from Step B2) to the 96- well tissue culture plate (50,000 cells / well; final concentration of RNP will be 10 nM).

[0439] 5. Incubate the plate containing the transfection complexes and cells in a tissue culture incubator (37° C., 5% CO2) for 48 hr. To detect on-target mutations, use PCR with appropriate primers (primers within donor sequence and primers surrounding the target insertion site).Example 11: Protocol for Testing the Specificity of CRISPR / Cas9

[0440] Produce dCas9 (DNA cutting inactive Cas9) linked to biotin (dCas9- biotin). Cas9 (s pyogenes, s aureus, etc.). Biotinylation methods are described below.

[0441] 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.

[0442] Biotinylation method #2.1: biotin functionalized with succinimidyl-ester can be incorporated at surface-exposed lysines residues (no enzymatic reaction required). For proteins as big as Cas9, this can be a viable option.

[0443] Biotinylation method #2.2: along the same line, biotin-maleimide is commercially available, and they can be conjugated at surface-exposed cysteines (no enzyme).

[0444] Testing will be accomplished to characterize the biotinylated Cas9 does in terms of DNA-binding and cleavage. Streptavidin-coated 96-well plates are commercially available, but may also be produced in-house. Bind dCas9-biotin to plastic plates (96-well, 24- well, 384- well, etc.). Provide designed guide RNAs to each well. Allow time for guide RNAs to interact with Cas9 protein. Provide genomic DNA to each well or DNA with targeted sequence. Allow time for Cas9 binding to DNA. Wash wells with appropriate buffer. Provide an adapter (DNA oligomer). Allow time to bind. Restriction-digest the genomicDNA to make it more tractable and easier to ligate the adapter. Wash wells. Perform DNA sequencing to identify sites of binding (on target vs. off target).Example 12: Transfection for the Knock-in Experiment at the Nrf2 Locus Using Abbiel

[0445] Note: 500 ng protein and 120 ng sgRNA are used for a single reaction. The amount of DNA depends on the size of the donor constructs. Donor DNA (DNA with LTR sequences) may be incubated with ABBIE1 before, during, or after providing / transfecting / electroporating to the cells. All reactions are prepared in sterile biosafety cabinet.

[0446] 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.

[0447] Day 2: ABBIE1 Preparation:

[0448] Tube l:

[0449] Purified 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. Add the sgRNA to the 1.3-fold molar excess (approximately 120 ng) to the protein / DNA complex and continue the incubation for additional 10 minutes at room temperature. The volume of this mixture is 25 pL.

[0450] Tube 2:

[0451] 2 pL of transfection reagent (RNAiMAX, Life Technologies) was added to 23 pL of OptiMEM. And allowed to incubate for 10 minutes at room temperature.

[0452] Combined Tube 1 and Tube 2 (50 ml final volume) and incubated for 15 minutes at room temperature.

[0453] Added the entire 50 pL transfection mixture to the well.

[0454] Half of the pooled edited cells were harvested 48 hours after transfection for the verification of the genomic DNA editing in a pooled population. Verification of edited genome was performed by polymerase chain reaction (PCR). We performed PCR against the targeted region as described below (See PCR protocol) the remainder was seeded onto 6 cm culture dishes (Corning) and allowed to recover for 48 hours.

[0455] Day 5: Screening of Phenotypic Changes Via Western Blotting.

[0456] Standard western blot analysis was performed for NrF2 isoforms using primary antibodies targeting 55 kD isoform (Santa Cruz Biotechnology, sc-722) as well as 98 kD isoform (Abeam, ab-62352). GAPDH (Santa Cruz Biotechnology, sc-51907).Example 13: PCR Conditions for Detection of Gene Editing Using Abbiel for Nrf2 and CXCR4 Locus

[0457] Accession number for human Nrf2, Uniprot: QI 6236, Ensembl gene ID: ENSG00000116044

[0458] Editing target sequences and PAMs for Nrf2 (exon 2): Used for sgRNA design 1-3.

[0459] GCGACGGAAAGAGTATGAGC TGG (SEQ ID NO: 275)

[0460] TATTTGACTTCAGTCAGCGA CGG (SEQ ID NO: 276)

[0461] TGGAGGCAAGATATAGATCT TGG (SEQ ID NO: 277)

[0462] Primer Key for Detection of Integration at Nrf2 Target

[0463] Primer Set 1: Primer 1 : 5'-GTGTTAATTTCAAACATCAGCAGC-3' (SEQ ID NO: 278), Primer 2: 5'-GACAAGACATCCTTGATTTG-3' (SEQ ID NO: 279)

[0464] Primer Set 2: Primer 1 : 5'-GAGGTTGACTGTGTAAATG-3' (SEQID NO: 280), Primer 2: 5'-GATACCAGAGTCACACAACAG-3' (SEQ ID NO: 281)

[0465] Primer Set 3: Primer 1: 5'-TCTACATTAATTCTCTTGTGC-3' (SEQ ID NO: 282), Primer 2: 5'-GAT ACC AGAGTCACACAAC AG-3' (SEQ ID NO: 283)

[0466] Accession number for human CXCR4, Uniprot P61073, Ensembl gene ID: ENSG00000121966

[0467] Editing target sequence and PAM for CXCR4 (Exon 2): Used for sgRNA design 1.

[0468] GGGCAATGGATTGGTCATCC TGG (SEQ ID NO: 284)

[0469] Primer Key for Detection of Integration at CXCR4 Target

[0470] Primer Set 1: Primer 1 : 5'-TCTACATTAATTCTCTTGTGC-3'(SEQ ID NO: 285), Primer 2: 5'-GACAAGACATCCTTGATTTG-3' (SEQ ID NO: 286)

[0471] Primer Set 2: Primer 1: 5'-TCTACATTAATTCTCTTGTGC-3'(SEQ ID NO: 287), Primer 2: 5'-GATACC AGAGTCACACAAC AG-3' (SEQ ID NO: 288)

[0472] Primer Set 3: Primer 1 : 5'-GAGGTTGACTGTGTAAATG-3' (SEQ ID NO: 289), Primer 2: 5'-GACAAGACATCCTTGATTTG-3' (SEQ ID NO: 290)

[0473] Primer Set 4: Primer 1 : 5'-GAGGTTGACTGTGTAAATG-3' (SEQ ID NO: 291), Primer 2: 5'-GATACCAGAGTCACACAACAG-3' (SEQ ID NO: 292)

[0474] PCR Cycling conditions used for detection of integrated donor DNA

[0475] *Note annealing temperatures will vary depending on primer sequence

[0476] 1. Initial denaturation: 4 min 94° C.

[0477] 2. denaturation: 30 sec 94° C.

[0478] 3. annealing: 30 sec 55° C.

[0479] 4. extension: 30 sec 72° C.

[0480] 5. go to step 2: 40 cycles

[0481] 6. final extension: 4 min 72° C.

[0482] 7. final hold: oo 4° C.

[0483] Avi-tagged Cas9 for biotinylation

[0484] Sequence of the avi-tag used for Cas9 biotinylation

[0485] Ammo Acid Sequence: G G D L E G S GL N D I F E A Q K I E W H E* (SEQ ID NO: 293)

[0486] Nucleic Acid Sequence:GAAAATTGAATGGCATGAATAA (SEQ ID NO: 294)

[0487] First Underlined section=Cas9 C-terminus

[0488] Italicized section=restriction site / linker

[0489] Second underlined section=avi-tag (biotinylation site highlighted)Example 14: Expression Protocol for Abbiel Fusion Protein

[0490] 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

[0492] Inoculate a 1 liter flask containing LB and antibiotic. Allow bacterial culture to grow until 0.6 OD is achieved then induce with Isopropyl 0-D-1- thiogalactopyranoside (IPTG) at a 1 mM final concentration. Allow the culture to expand for 6-8 hours and centrifuge the suspended bacterial culture at a minimum of two thousand G force for 10 minutes. Freeze pellet at -80 C for further processing at a later time.

[0493] Protein Preparation and Purification

[0494] All steps are performed at room temperature. Lyse the cells by 2 cycles of freeze-thaw in 20 mM Tris pH8.0, 300 mM NaCl, 0.1 mg / ml chicken egg white lysozyme. Centrifuge at 6,000 g for 15 minutes and retain the supernatant. Load the supernatant onto a Ni-IDA agarose column equilibrated in 20 mM Tris pH8.0, 300 mM sodium chloride. Elute the protein with a 0-to-200 mM gradient of imidazole. Identify the fractions containing the fusion protein by a 7% SDS-PAGE. Pool the fractions and dilute with 20 mM Tris pH8.0 so that the final NaCl concentration is 50 mM. Load onto a Q-sepharose column and elute with a 0-to-500 mM gradient of sodium chloride. Identify the fractions containing the fusion protein by a 7% SDS-PAGE. Pool the fractions and dilute with 20 mM Tris pH8.0 so that the final NaCl concentration is 100 mM. Load onto an SP-sepharose column and elute with a 0- to-500 mM gradient of sodium chloride. Identify the fractions containing the fusion protein by a 7% SDS-PAGE. Pool the fractions, measure the concentration by its UV absorbance at 280 nm, and concentrate by a centrifugal filter to the final concentration of 400 pg / ml. Add glycerol to the final concentration of 50%. Store at -20° C. Notable examples (made using procedures discussed above with variety of linkages)-ERK5-dcas9, ERK2-dcas9, EGFR- kinase domain EGFR-cas9, Shp2-dcas9, cas9 nickase-p38, cas9-integrase-graphene oxide, cas9-graphene, cas9-carbon nanotube.Example 15: ABBIE-Engineered T cells

[0495] 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 ashort amino acid linker. 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.

[0496] As depicted in FIG. 5A, 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. As depicted in FIG. 5B, 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.Example 16: Smart Gene Editing

[0497] 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.

[0498] As shown in FIG. 7, 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.

[0499] 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.

[0500] 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). For example, 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. Alternatively, 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 canbe 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

[0501] 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, 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. In some cases, 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.

[0502] Example 18: Microfluidics Technique to Detect Target Genomic DNA Using Graphene-Bound Donor DN A

[0503] Overview: Steps for some embodiments of this technique are summarized in FIG. 11.

[0504] 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,

[0505] Involved in the Technique:

[0506] 1. Preparation of Graphene-Bound Donor DNA:

[0507] Synthesis of Graphene Particles: Prepare graphene oxide (GO) or reduced graphene oxide (rGO) particles, which will serve as the platform for attaching donor DNA.

[0508] Functionalization: Conjugate donor DNA sequences that are complementary to the target genomic DNA region with the graphene particles. This can be achieved through covalent bonding or %-x stacking interactions.

[0509] 2, Microfluidic Device Design:

[0510] Channel Fabrication: Design a microfluidic chip with channels for sample input, mixing, and reaction zones. The channels should be optimized for efficient fluid flow and binding interactions.

[0511] Inlet for Target Sample: The chip should have an inlet for introducing the genomic sample (e.g., from microbial or viral DNA sources).

[0512] 3. Integration of Donor DNA:

[0513] Application of viral integrase-dCas9: Introduce the viral integrase-dCas9 complex into the microfluidic system. The dCas9 protein, guided by a specific RNA sequence, recognizes the target genomic DNA. The integrase enzyme then facilitates the integration of the graphene-bound donor DNA at the designated target site.

[0514] Flow Dynamics: Optimize the flow rate to allow'' sufficient time for binding and integration while maintaining the effective concentration of all components in solution.

[0515] 4. Detection of Integrated DNA:

[0516] Amplification and Detection: After integration, apply PCR or another amplification technique to amplify the integrated DNA followed by detection methods. Options may include:

[0517] Fluorescent Probes: Design probes that hybridize to the integrated DNA and emit a fluorescent signal upon binding.

[0518] Electrochemical Detection: Utilize the conductive properties of graphene to measure changes in electrical conductivity when the target DNA is present.

[0519] Readout: Use fluorescence microscopy or electrochemical sensors integrated into the microfluidic device to read the results.

[0520] Applications:

[0521] 1. Detection of Microbial and Viral DNA:

[0522] Pathogen Identification: This technique can be applied to detect specific bacterial or viral infections by targeting unique genomic sequences associated with pathogens. For example, integrating donor DNA that targets the genomic regions of viruses like HIV or SARS-CoV-2 could confirm the presence of these pathogens in clinical samples.

[0523] Antibiotic Resistance Genes: The method can also be tailored to identify specific antibiotic resistance genes in bacterial strains, aiding in the treatment of infections.

[0524] 2. Identification of Disease Mutations:

[0525] Targeted .Mutation Analysis: The technique can be adapted to detect specific genetic mutations associated with diseases, such as cancer. By designing donor DNA that targets known mutations in oncogenes or tumor suppressor genes, the presence of these mutations can be confirmed through integration and subsequent amplification.

[0526] Benefits of Using Viral Integrase-dCas9:

[0527] Precision: The combination of dCas9 and viral integrase allows for precise targeting of specific genomic regions, minimizing off-target effects and improving specificity in detection.

[0528] Versatility: This approach can be adapted to various genomic targets, including microbial, viral, or human DNA, making it a flexible tool for diagnostics. Efficiency: The integration of donor DNA directly into the genome can lead to long-lasting effects, allowing for sustained detection of target sequences.

[0529] This microfluidics technique employing graphene-bound donor DNA and viral integrase-dCas9 presents a powerful method for detecting and identifying specific genomic sequences associated with microbial infections or disease mutations. The combination of precise targeting, efficient integration, and flexible detection strategies makes it a promising tool for advancing molecular diagnostics in clinical and research settings.

[0530] Example 19: Technique for Isolating Target DNA or RNA Using Graphene-Bound Integrase-dCas9 Protein

[0531] Overview: Aspects for some embodiments of this technique are summarized in FIG, 10.

[0532] This technique leverages graphene-bound viral or microbial integrase- dCas9 proteins to isolate target DNA or RNA from genomic samples. The graphene's properties allow it to bind to surfaces or gels, facilitating the capture of nucleic acids. When a donor DNA sequence is introduced, and the integrase-dCas9 is activated in an appropriate buffer, in situ integration of the donor DNA into the target genomic DNA occurs. This method enhances the specificity and efficiency of nucleic acid isolation and detection.

[0533] Protocol

[0534] 1. Preparation of Graphene-Bound Integrase-dCas9:

[0535] Synthesis of Graphene Nanoparticles: Create graphene oxide (GO) or reduced graphene oxide (rGO) nanoparticles that will serve as the substrate for the integrase- dCas9 protein.

[0536] Functionalization: Conjugate the integrase-dCas9 protein to the graphene surface using covalent bonding or rr-rr stacking interactions. This can be done through reactive groups on the protein (e.g., amine groups) and functional groups on the graphene surface.

[0537] 2. Surface or Gel Binding:

[0538] Surface Coating: Coat a microplate, well, or gel with the graphenebound integrase-dCas9. This can be achieved by incubating the surface with the functionalized graphene solution, allowing the graphene to adhere to the surface.

[0539] Gel Matrix Integration: For gel applications, incorporate the graphenebound integrase-dCas9 into the gel matrix during polymerization, ensuring that the protein is evenly distributed throughout the gel.

[0540] 3. Isolation of Target DNA or RNA:

[0541] Sample Introduction: Introduce the genomic DNA or RNA sample into the setup. The target nucleic acids wall interact with the integrase-dCas9 bound on the graphene, allowing for selective capture.

[0542] Washing Steps: After a suitable incubation period (to allow binding), wash the surface or gel with a buffer solution to remove unbound nucleic acids, retaining only the specifically bound target DNA or RNA.

[0543] 4. In Situ Integration:

[0544] Donor DNA Addition: Add a donor DNA sequence that is complementary to the target genomic DNA region. This donor DNA should be designed to include sequences that facilitate integration.

[0545] Activation of Integrase-dCas9: Introduce an appropriate buffer system that activates the integrase-dCas9 complex, enabling it to integrate the donor DNA into the target genomic sequence. This buffer preferably provides optimal pH, ionic strength, and necessary cofactors (if any) required for integrase activity.

[0546] 5. Detection of Integrated DNA / RNA:

[0547] After integration, there are several methods to detect the incorporated sequences:

[0548] Polymerase Chain Reaction (PCR): Use specific primers targeting the integrated region for amplification. The presence of PCR products indicates successful integration.

[0549] Fluorescent Probes: Employ fluorescently labeled probes that hybridize to the integrated DNA. Detection can be performed using fluorescence microscopy or flow' cytometry.

[0550] Electrochemical Detection: Utilize the conductive properties of graphene to detect changes in electrical signals when the target DNA or RNA is present, providing a rapid and sensitive detection method.

[0551] .Applications of This System

[0552] 1. Pathogen Detection:

[0553] This technique can be employed to isolate and identify specific viral or microbial DNA or RNA from clinical samples. For example, targeting sequences from pathogens like SARS-CoV-2 or antibiotic-resistant bacteria can facilitate rapid diagnosis.

[0554] 2. Genetic Mutation Analysis:

[0555] The system can be adapted for detecting specific mutations associated with diseases, such as cancer. By integrating donor DNA that carries the mutation, the presence of disease-associated sequences can be confirmed,

[0556] 3. Environmental Monitoring:

[0557] The technique can be applied in environmental microbiology to isolate and detect specific microbial strains from water or soil samples, aiding in biodiversity assessments and pollution monitoring.

[0558] 4. Synthetic Biology:

[0559] In research setings, this method can be used for the precise integration of synthetic DNA constructs into target genomes for engineering purposes, such as creating genetically modified organisms.

[0560] This innovative technique utilizing graphene-bound integrase-dCas9 proteins offers a novel approach for isolating target DNA or RNA from complex genomic samples. The combination of selective binding, in situ integration capabilities, and sensitive detection methods enhances the potential applications in diagnostics, environmental monitoring, and synthetic biology, paving the way for more efficient and accurate nucleic acid analysis.

[0561] Sequences of the Disclosure

[0562] For each sequence provided below, the following information is provided: type of sequence (nucleic acid or amino acid), source (e.g. E. coli), length, and identification number (if available).

[0563] A first polynucleotide of the disclosure can encode, for example, a Cas9, Cpfl, TALE, or ZnFn protein. A second polynucleotide of the disclosure can encode, for example, an integrase, transposase, or recombinase. Listed below are exemplary first and second polynucleotide sequences and protein sequences, along with exemplary linker sequences, that can be used in the compositions (constructs, fusion proteins) and methods described herein. Other 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. For example, SEQ ID NO: 49, SEQ ID NO: 57, SEQ ID NO: 58, and / or portions thereof.

[0564] A linker can be any length, for example, 3 to 300 nucleotides in length, 6 to 60 nucleotides in length, or any length that will allow the first and second polynucleotide to be fused. A polypeptide can be made by an organism, e.g. E. coli or be made synthetically, or a combination of both.

[0565] Exemplary 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.

[0566] Exemplary amino acid sequences: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 48, 50, 52, 58, 63, 65, 67, 69, 71, 72-78, and 80.

[0567] TABLE 1 : FIRST PROTEIN, SECOND PROTEIN, OR LINKER

[0568] TABLE 2: PARTIAL LIST OF SEQUENCESADDITIONAL SEQUENCESSEQ ID NO: 1NAME: S.thermophilus Csnl cds HQ712120.1SEQUENCE:SEQ ID NO: 5NAME: S.mutans Cas9SEQUENCE:ATGAAAAAACCTTACTCTATTGGACTTGATATTGGAACCAATTCTGTTGGTTGGGCTGTTGTGACAGATGACTACAAAGTTCCTGCTAAGAAGATGAAGGTTCTGGGAAATACAGATAAAAGTCATATCGAGAAAAATTTGCTTGGCGCTTTATTATTTGATAGCGGGAATACTGCAGAAGACAGACGGTTAAAGAGAACTGCTCGCCGTCGTTACACACGTCGCAGAAATCGTATTTTATATTTGCAAGAGATTTTTTCAGAAGAAATGGGCAAGGTAGATGATAGTTTCTTTCATCGTTTAGAGGATTCTTTTCTTGTTACTGAGGATAAACGAGGAGAGCGCCATCCCATTTTTGGGAATCTTGAAGAAGAAGTTAAGTATCATGAAAATTTTCCAACCATTTATCATTTGCGGCAATATCTTGCGGATAATCCAGAAAAAGTTGATTTGCGTTTAGTTTATTTGGCTTTGGCACATATAATTAAGTTTAGAGGTCATTTTTTAATTGAAGGAAAGTTTGATACACGCAATAATGATGTACAAAGACTGTTTCAAGAATTTTTAGCAGTCTATGATAATACTTTTGAGAATAGTTCGCTTCAGGAGCAAAATGTTCAAGTTGAAGAAATTCTGACTGATAAAATCAGTAAATCTGCTAAGAAAGATAGAGTTTTGAAACTTTTTCCTAATGAAAAGTCTAATGGCCGCTTTGCAGAATTTCTAAAACTAATTGTTGGTAATCAAGCTGATTTTAAAAAGCATTTTGAATTAGAAGAGAAAGCACCATTGCAATTTTCTAAAGATACTTATGAAGAAGAGTTAGAAGTACTATTAGCTCAAATTGGAGATAATTACGCAGAGCTCTTTTTATCAGCAAAGAAACTGTATGATAGTATCCTTTTATCAGGGATTTTAACAGTTACTGATGTTGGTACCAAAGCGCCTTTATCTGCTTCGATGATTCAGCGATATAATGAACATCAGATGGATTTAGCTCAGCTTAAACAATTCATTCGTCAGAAATTATCAGATAAATATAACGAAGTTTTTTCTGATGTTTCAAAAGACGGCTATGCGGGTTATATTGATGGGAAAACAAATCAAGAAGCTTTTTATAAATACCTTAAAGGTCTATTAAATAAGATTGAGGGAAGTGGCTATTTCCTTGATAAAATTGAGCGTGAAGATTTTCTAAGAAAGCAACGTACCTTTGACAATGGCTCTATTCCACATCAGATTCATCTTCAAGAAATGCGTGCTATCATTCGTAGACAGGCTGAATTTTATCCGTTTTTAGCAGACAATCAAGATAGGATTGAGAAATTATTGACTTTCCGTATTCCCTACTATGTTGGTCCATTAGCGCGCGGAAAAAGTGATTTTGCTTGGTTAAGTCGGAAATCGGCTGATAAAATTACACCATGGAATTTTGATGAAATCGTTGATAAAGAATCCTCTGCAGAAGCTTTTATCAATCGTATGACAAATTATGATTTGTACTTGCCAAATCAAAAAGTTCTTCCTAAACATAGTTTATTATACGAAAAATTTACTGTTTACAATGAATTAACAAAGGTTAAATATAAAACAGAGCAAGGAAAAACAGCATTTTTTGATGCCAATATGAAGCAAGAAATCTTSEQ ID NO: 9SEQUENCE:TTTATTTCAGAGGGATTGAGAGACTATCAATTCCTTGATAGTGGGCAAAAGAAGGCCGAGATATGTATACTGAAGATCCACTTGATATTAATCGTTTAAGTGATTATGATATCGACCATATTATTCCACAATCTTTTATAAAAGATGACTCTATTGACAATAAGGTTCTGGTTTCATCAGCTAAAAACCGTGGGAAATCGGATAATGTACCGAGTGAAGATGTTGTCAATAGGATGAGACCGTTTTGGAATAAATTATTGAGCTGTGGATTGATTTCTCAACGGAAATACAGCAATCTAACCAAAAAAGAATTAAAACCAGATGATAAGGCTGGTTTCATCAAACGTCAATTGGTTGAGACAAGACAAATTACAAAGCATGTTGCACAAATTTTAGACGCTCGTTTTAATACAAAACGTGATGAAAATAAAAAAGTAATTCGTGATGTCAAAATTATCACTTTAAAATCTAATTTAGTTTCACAATTTCGTAAAGACTTTAAATTTTACAAAGTACGTGAGATTAATGATTACCATCATGCGCATGACGCTTATCTTAATGCAGTTATAGGAAAAGCTTTATTAGATGTTTATCCGCAGTTAGAGCCCGAATTTGTTTATGGTGAGTACCCTCATTTTCATGGATATAAAGAAAATAAAGCAACTGCTAAGAAATTTTTCTATTCAAATATTATGAATTTTTTTAAGAAAGATGATATCCGTACCGATGAAAATGGTGAGATTGTTTGGAAAAAAGATGAGCATATTTCTAATATTAAAAGGGTGCTTTCCTATCCCCAAGTTAATATTGTTAAGAAAGTAGAAATACAGACTGTTGGACAAAATGGGGGACTTTTTGACGATAATCCTAAATCACCATTAGAGGTTACACCTAGTAAACTTGTTCCACTAAAAAAAGAATTAAACCCTAAAAAATATGGAGGATATCAAAAACCGACGACAGCTTATCCTGTTTTACTGATAACAGATACTAAACAGCTAATTCCAATCTCAGTAATGAATAAGAAGCAATTTGAACAAAATCCGGTTAAATTTTTAAGAGATAGAGGCTATCAACAGGTAGGAAAGAATGACTTTATTAAATTACCCAAATATACCCTAGTTGATATCGGTGATGGGATTAAACGCCTATGGGCTAGTTCGAAAGAAATACATAAAGGAAATCAATTAGTTGTATCTAAAAAATCTCAAATTTTGCTTTATCATGCACATCACTTAGATAGTGATTTGAGTAATGATTATCTTCAAAATCATAATCAACAATTCGATGTTTTATTTAATGAAATTATTTCTTTTTCTAAAAAATGTAAATTGGGAAAAGAACATATTCAGAAAATTGAAAATGTTTACTCCAATAAGAAGAATAGTGCATCAATAGAAGAATTAGCAGAGAGTTTTATTAAATTATTAGGATTTACACAATTAGGTGCAACTTCCCCATTTAATTTTTTAGGGGTAAAACTAAATCAAAAACAATATAAAGGTAAAAAAGATTATATTTTACCGTGTACAGAGGGGACCCTTATCCGCCAATCTATCACTGGTCTTTACGAAACACGAGTTGATCTTAGTAAAATAGGAGAAGACTAASEQ ID NO: 12SEQ ID NO: 16NAME: gi|l 50381362|gb|ABR68182.11 integrase, partial [Human immunodeficiency virus 1] SEQUENCE:SEQ ID NO: 17NAME: gi|459980|gb|L20651.1|STLKIAPOL Simian T-cell lymphotropic virus type I integrase (pol) gene, partial cds SEQUENCE:SEQ ID NO: 18NAME: gi|459981|gb|AAA47841.1| integrase, partial [Simian T-lymphotropic virus 1]SEQUENCE:LVERSNGILI<TLLYI<YFTDI<PDLPMDNALSIALWTINHLNVLTHCHSEQ ID NO: 19NAME: gi|321156784:1-1509 Streptococcus pneumoniae integrative and conjugative element ICESpnl 1930, strain 11930 SEQUENCE:TTCATTGSNSMDIYTDDAKDPFYPSSN aSGAC TSEQ ID NO: 22NAME: gi|43091|emb|CAA41325.1| integrase, partial (plasmid) [Escherichia coli]SEQUENCE:SEQ ID NO: 23>gi|397912605:40372-41898 Thermoanaerobacterium phage THSA-485A, complete genome - recombinaseSEQ ID NOS: 27-46These are exemplary sequences of polynucleotides encoding the TALE repeat modules for use in linking to integrases or recombinases as described in this invention.SEQ ID NO: 27SEQ ID NO: 49>gb|AYLT01000127.1|: 11804-12046 Staphylococcus aureus subsp. aureus SKI 585 contig000127, whole genome shotgun sequenceSEQ ID NO: 50>gi|669035130|gb|KFD30483.1| hypothetical protein D484_02234 [Staphylococcus aureus subsp. aureus SKI 585] - s aureus cas9MENFNYNNIKKLVLENVDIDKVKEVYKDYELLNYTIKNQTLYMNDYEVAKVSEKHLNENINNLRGTVNLDEKCILSLTYLSEQ ID NO: 51NAME: dna of linker2SEQUENCE: agcggcagcgaaaccccgggcaccagcgaaagcgcgaccccggaaagcSEQ ID NO: 52NAME: dCas9 proteinSEQUENCE:MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFSEQ ID NO: 53NAME: NLS nucleotide with ATGNLS-linkerl-Integrase-linker2-dCas9, or Integrase-linker l-NLS-linker2-dCas9 or Integrase- linker2-dCas9-linkerl-NLS or Integrase-linker2-dCas9-NLSLinker 1 = GGSSEQ ID NO: 61NAME: Linker 2SEQUENCE:SGSETPGTSESATPESSEQ ID NO: 62NAME: MMTV integrase cDNA, gb| AF071010.11: 16-1113 Mouse mammary tumor virus putative integrase, env polyprotein, and superantigen mRNA, complete cdsSEQUENCE:NAME: gb|AXUN02000059.1|:5116-8850 Youngiibacter fragilis 232.1 contig_151, whole genome shotgun sequence - recombinase SEQUENCE:SEQ ID NO: 67NAME: gi|571264559|emb|CDF47133.1| transposase [Peptoclostridium difficile}SEQUENCE:MSRTRRNFSAKFKSELVIELLKGEKDLNTTATENNIQPNLLRNWKKEFLDKASVVFNDTREDNLKEKLALERKEKAEYAKKVGQLTMQVDWLKKKSEETLGPDYESKFSPKPFEDSEQ ID NO: 68NAME: gb|CP009444.1|:1317724-1320543 Francisella philomiragia strain GA01-2801, complete genome CpflSEQUENCE:SEQ ID NO: 75SEQUENCE:SEQ ID NO: 81 a protein domain that characterizes zinc finger proteinsCX(2-4)CX(12)HX(3-5)H (X(2-4) means XX or XXX or XXXX for example)SEQ ID NO: 82AAGAAAACAACAGATTGTTCCGTTTGTTCCGTTGGGGACTTTCCAGGAGACGTGGCCTGAGTGATAAGCCGCTGGGGACTTTCCGAAGAGGCGTGACGGGACTTTCCAAGGCGACGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATGCTGCATATAAGCAGCTGCTTTCTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTATCTAGAFirst 5’LTR is underlined, plain text is neo, and 3’LTR is bolded (1179 bp)SEQ ID NO: 101An abbreviated version of 5’LTR and 3’LTR with neo sequence within (224 bp)First 5’LTR is underlined, plain text is neo, and 3’LTR is boldedGACAAGACATCCTTGATTTGTGGGTCTATAACACACAAGGCTTCTTCCCTGATTGGCAAAACTACACACCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTATCTAGARegarding SEQ ID NO: 72Genbank Protein ID: WP_021736722.1NCBI protein GI from NR Database or local GI (for proteins originated from WGS database): 545612232Contig ID in WGS database: AWUR01000016.1Contig description: Acidaminococcus sp. BV3L6 contig00028, whole genome shotgun sequenceProtein completeness: CompleteProteins analyzed experimentally: 8Non-redundant set: nrOrganism: Acidaminococcus sp B V3L6Taxonomy:Bacteria ,Firmicutes, Negativicutes, Selenomonadales, Acidaminococcaceae, Acidaminococcus,Acidaminococcus sp. BV3L6Regarding SEQ ID NO: 73Genbank Protein ID: WP 044919442.1NCBI protein GI from NR Database or local GI (for proteins originated from WGS database): 769142322Contig ID in WGS database: JQKK01000008.1Contig description: Lachnospiraceae bacterium MA2020 T348DRAFT_scaffold00007.7_C, whole genome shotgun sequenceProtein completeness: CompleteProteins analyzed experimentally: 9Non-redundant set: nrOrganism: Lachnospiraceae_bacterium_MA2020Taxonomy: 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.

[0570] Additional nucleic acid sequences and protein sequences that can be used in the disclosed compositions and methods - Cfpl human cleaving proteins alignment. SEQ ID NO: 86 (first row) and SEQ ID NO: 90 (second row).

[0571] Additional nucleic acid sequences and protein sequences that can be used in the disclosed compositions and methods. Tables provided above taken from Haft, D., et al. PLoS Computational Biology, November 2005, Vol. 1, Issue 6, pp. 474-483. SEQ ID NOS: 200-253; in order from the top to the bottom of the chart.Table 4SEQ ID NO: 262Primer 2:5’- GATACCAGAGTCACACAACAG-3 ’Accession number for human CXCR4Uniprot P61073Nucleic acid sequence:SEQ ID NO: 273GGCGGCGACCTCGAGGGTAGCGGTCTGAACGATATTTTTGAAGCGCAGAAAATTGAATGGCATGAATAA

[0572] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0573] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0574] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if aspecific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or claims, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0575] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0576] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-rangesas discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0577] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0578] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. 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 optionally comprises an inactivated nuclease domain, such as obtained by one or more mutations that abrogate, inhibit, or attenuate nuclease activity, joined to a material comprising a graphene, graphene oxide, carbon nanotube, fullerene, nanodiamond, or any combination thereof.

2. The composition of claim 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.

3. The composition of claim 1 or 2, wherein the material comprises graphene or graphene oxide.

4. The composition of any one of claims 1-3, further comprising a guide RNA.

5. The composition of any one of claims 1-4, further comprising an enzyme, a drug or a diagnostic agent or any combination thereof joined to the material.

6. 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 claims 1-5 to the subject or contacting the cell with the composition of any one of claims 1-5; and detecting the selected nucleotide sequence, such as by using colorimetry, fluorescence detection, immunodetection, or rf reflectometry.

7. The composition of any one of claims 1-5 for use in detecting a selected sequence in a genome of a subject or a cell.

8. 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 claim 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.

9. The composition of claim 5 for use in providing a drug to a subject or a cell.

10. A composition comprising a cas protein joined to a protein comprising a kinase or phosphatase, optionally wherein the cas protein comprises an inactivated nucleasedomain, such as obtained by one or more mutations that abrogate, inhibit, or attenuate nuclease activity.

11. The composition of claim 10, wherein the protein comprising the kinase comprises a MAP kinase, Src, a nuclear kinase, a serine / threonine kinase or a tyrosine kinase.

12. The composition of claim 10, wherein the protein comprising the phosphatase comprises a Shp, Shp2 or a nuclear phosphatase.

13. A method of modifying the phosphorylation state of a selected protein associated with a polynucleotide comprising administering the composition of any one of claims 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.

14. The method of claim 13, wherein the subject or cell has a disease or disorder.

15. The method of claim 14, wherein the disease is a viral disease, a microbial disease, cancer or a cardiovascular disease.

16. The composition of any one of claims 10-12 for use in treating a viral disease, a microbial disease, cancer or a cardiovascular disease.

17. 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 claims 1-5 with the subject, the cell, or the isolated population of polynucleotides or nucleotides and performing a diagnostic evaluation.

18. The method of claim 14, wherein the diagnostic evaluation comprises colorimetry, fluorescence detection, immunodetection, or rf reflectometry.

19. 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.

20. The method of claim 19, wherein the genome editing protein comprises a zinc finger endonuclease, TALEN, CRISPR / cas9, Cpfl, meganuclease, Cas3, CaslO, or an ABBIE system.

21. The method of any one of claims 19 or 20, wherein the modified genome editing protein is joined to at least one other protein, which comprises a recombinase, reverse transcriptase, or topoisomerase.

22. The method of any one of claims 19-21, wherein, the material that modifies the genome editing protein comprises a graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate or carbon nanotube.

23. The method of any one of claims 19-22, wherein, the material that modifies the genome editing protein comprises silicon, silica or a silicone polymer.

24. The method of any one of claims 19-23, wherein modifying the genome editing protein comprises joining the genome editing protein to a protein comprising a kinase.

25. The method of claim 24, wherein 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.

26. The method of any one of claims 19-25, wherein modifying the genome editing protein further comprises joining the genome editing protein to a protein comprising a phosphatase.

27. The method of claim 26, wherein the protein comprising the phosphatase comprises a Shp phosphatase, Shp2, Shpl, PPM phosphatase, PP2C phosphatase, a nuclear phosphatase or a cytoplasmic phosphatase.

28. The method of any one of claims 19-27, wherein the material that modifies the genome editing protein is joined to the genome editing protein via one or more ionic interactions.

29. The method of any one of claims 19-28, wherein the material that modifies the genome editing protein is joined to the genome editing protein via one or more functional groups or functional linkers.

30. The method of any one of claims 19-29, wherein the material that modifies the genome editing protein is joined to the genome editing protein via one or more covalent bonds.

31. The method of any one of claims 19-30, wherein the one or more stimuli are endogenous to a cell.

32. The method of any one of claims 19-30, wherein the one or more stimuli are exogenous to a cell.

33. The method of any one of claims 19-30, wherein the one or more stimuli comprise light, temperature, pH, calcium, ion concentration, or electrical stimuli.

34. The method of any one of claims 19-30, wherein the one or more stimuli comprise electromagnetic radiation.

35. The method of any one of claims 19-34, wherein the one or more stimuli induce a cleavage of a group in the modified genome editing protein, which alters its activity.

36. The method of any one of claims 19-35, wherein the one or more stimuli induce a conformational change in the modified genome editing protein, which alters its activity.

37. The method of any one of claims 19-36, wherein the one or more stimuli induce activation of the modified genome editing protein.

38. The method of any one of claims 19-37, wherein the one or more stimuli induce deactivation of the modified genome editing protein.

39. The method of any one of claims 19-38, wherein the one or more stimuli induce the production of a detectable signal or marker.

40. The method of any one of claims 19-39, wherein modifying the genome editing protein further comprises joining the genome editing protein to an additional material.

41. The method of claim 40, wherein the additional material comprises a metal (e.g., gold, silver, iron, iron oxide, titanium dioxide, lanthanide oxide, transition metal, or a-ISO-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.

42. The method of any one of claims 19-41, wherein the material modifying the genome editing protein is 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).

43. The method of any one of claims 19-42, wherein the external dimensions of the material modifying a genome editing protein in the X-Y plane is nano scaled (e.g., one nano- sized graphene flake) or macro scaled.

44. The method of claim 43, wherein the minimal dimensions of the material modifying a genome editing protein in the X-Y plane are 1 nm by 1 nm.

45. The method of any one of claims 19-44, wherein the external dimensions of the material modifying a genome editing protein along the Z-axis are directly proportional to the number of layers of material incorporated.

46. The method of claim 45, wherein, when the material is graphene, the minimal dimension along the Z-axis is defined by the thickness of a non-functionalized graphene monolayer, e.g., 0.34 nm.

47. The method of claim 45, wherein maximum dimension of a particle of the material in the material modifying a genome editing protein 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.

48. The method of any one of claims 19-47, wherein binding of the modified DNA binding protein to DNA is directed by use of a guide RNA.

49. The method of any one of claims 19-48, wherein the target is a subject in need of a therapy for a disease, or a cell.

50. The method of claim 46, wherein the subject or cell has a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.

51. 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.

52. The composition of claim 51, wherein the genome editing protein comprises a zinc finger endonuclease, TALEN, CRISPR / cas9, Cpfl, a meganuclease, Cas3, CaslO, or the ABBIE system.

53. The composition of any one of claims 51 or 52, wherein the genome editing protein is joined to a recombinase, reverse transcriptase, or a topoisomerase.

54. The composition of any one of claims 51-53, wherein, the material comprises a graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate, a polymer, or a carbon nanotube.

55. The composition of any one of claims 51-54, wherein, the material comprises silicon, silica or a silicone polymer.

56. The composition of any one of claims 51-55, wherein the genome editing protein is joined to a protein comprising a kinase.

57. The composition of claim 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.

58. The composition of any one of claims 51-57, wherein the genome editing protein is joined to a protein comprising a phosphatase.

59. The composition of claim 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.

60. The composition of any one of claims 51-59, wherein the material is joined to the genome editing protein via one or more ionic interactions.

61. The composition of any one of claims 51-60, wherein the material is joined to the genome editing protein via one or more functional groups or functional linkers.

62. The composition of any one of claims 51-61, wherein the material is joined to the genome editing protein via one or more covalent bonds.

63. The composition of any one of claims 51-62, wherein the one or more stimuli are endogenous to a cell.

64. The composition of any one of claims 51 -62, wherein the one or more stimuli are exogenous to a cell.

65. The composition of any one of claims 51-64, wherein the one or more stimuli comprise light, temperature, pH, calcium, ion concentration, or electrical stimuli or any combination thereof.

66. The composition of any one of claims 51 -64, wherein the one or more stimuli comprise electromagnetic radiation.

67. The composition of any one of claims 51 -66, wherein the one or more stimuli induce a cleavage of a group in the genome editing protein, which alters its activity.

68. The composition of any one of claims 51-67, wherein the one or more stimuli induce a conformational change in the genome editing protein, which alters its activity.

69. The composition of any one of claims 51-68, wherein the one or more stimuli induce activation of the genome editing protein.

70. The composition of any one of claims 51-69, wherein the one or more stimuli induce a deactivation of the genome editing protein.

71. The composition of any one of claims 51-70, wherein the one or more stimuli induce the genome editing protein to provide a detectable signal or marker.

72. The composition of any one of claims 51-71, wherein the genome editing protein is joined to an additional material.

73. The composition of claim 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.

74. The composition of any one of claims 51-73, wherein the material is joined to a component of extracellular matrix (e.g., collagen, fibronectin, or laminin) or a synthetic polymer (e.g., polyaniline, polypyrrole, or polythiophene).

75. The composition of any one of claims 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.

76. The composition of claim 75, wherein the minimal dimensions of the material modifying a genome editing protein in the X-Y plane are 1 nm by 1 nm.

77. The composition of any one of claims 51-76, wherein the external dimensions of the material modifying a genome editing protein along the Z-axis is related to the number of layers of materials incorporated.

78. The composition of claim 77, 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.

79. The composition of claim 78, 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.

80. The composition of any one of claims 51-79, wherein binding of the modified DNA binding protein to DNA is directed by use of a guide RNA.

81. The composition of any one of claims 51-80, wherein the composition further comprises 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.

82. The composition of any one of claims 51-81, wherein the target is a subject in need of a therapy, or a cell.

83. The composition of claim 82, wherein the subject or cell has a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.

84. A method of treating or inhibiting a disease or disorder comprising administration of the composition of any one of claims 51-8385. The composition of any one of claims 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 disease86. Use of the composition of anyone of claims 51-83 as a medicament.

87. A method of detecting a disease or disorder comprising administering the composition of any one of claims 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.

88. The composition of any one of claims 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.

89. 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.

90. The method of claim 89, wherein 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.

91. The method of any one of claims 89 or 90, wherein, the material comprises a graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate, a polymer, or a carbon nanotube.

92. The method of any one of claims 89-91, wherein, the material comprises silicon, silica or a silicone polymer.

93. The method of any one of claims 89-92, wherein the material is joined to the donor polynucleotide via one or more ionic interactions.

94. The method of any one of claims 89-93, wherein the material is joined to the donor polynucleotide via one or more functional groups or functional linkers.

95. The method of any one of claims 89-94, wherein the material is joined to the donor polynucleotide via one or more covalent bonds.

96. The method of any one of claims 89-95, wherein the one or more stimuli are endogenous to a cell.

97. The method of any one of claims 89-96, wherein the one or more stimuli are exogenous to a cell.

98. The method of any one of claims 89-97, wherein the one or more stimuli comprise light, temperature, pH, calcium, ionic concentration, or electrical stimuli.

99. The method of claim 98, wherein the one or more stimuli comprise electromagnetic radiation.

100. The method of any one of claims 89-99, wherein the one or more stimuli induce the modified donor polynucleotide to produce a detectable signal.

101. The method of any one of claims 89-100, wherein external dimensions of the material in the X-Y plane is nano scaled (e.g., one nano-sized graphene flake) or macro scaled.

102. The method of claim 101, wherein the minimal dimensions of the material modifying the donor polynucleotide in the X-Y plane are 1 nm by 1 nm.

103. The method of any one of claims 89-102, wherein the external dimensions of the material modifying a donor polynucleotide along the Z-axis relates to the number of layers of materials incorporated.

104. The method of claim 103, 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.

105. The method of claim 104, 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.

106. The method of any one of claims 89-105, wherein the genome editing of the incorporated modified donor polynucleotide further comprises a guide RNA.

107. The method of any one of claims 83-100, wherein the target is a subject in need of therapy, or a cell.

108. The method of claim 107, wherein the subject or cell has a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.

109. 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.

110. The composition of claim 109, wherein 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.

111. The composition of any one of claims 109 or 110, wherein, the material comprises graphene, graphene oxide, nanodiamond, fullerene, functionalized fullerene, acrylate, methacrylate, a polymer, or a carbon nanotube.

112. The composition of any one of claims 109 -111, wherein, the material comprises silicon, silica or a silicone polymer.

113. The composition of any one of claims 109-112, wherein the material is joined to the donor polynucleotide via one or more ionic interactions.

114. The composition of any one of claims 109-113, wherein the material is joined to the donor polynucleotide via one or more functional groups or functional linkers.

115. The composition of any one of claims 109-113, wherein the material is joined to the donor polynucleotide via one or more covalent bonds.

116. The composition of any one of claims 109-115, wherein the one or more stimuli are endogenous to a cell.

117. The composition of any one of claims 109-115, wherein the one or more stimuli are exogenous to a cell.

118. The composition of any one of claims 109-115, wherein the one or more stimuli comprise light, temperature, pH, calcium, ionic concentration, or electrical stimuli.

119. The composition of claim 118, wherein the one or more stimuli comprise electromagnetic radiation.

120. The composition of any one of claims 109-119, wherein the one or more stimuli induce the modified donor polynucleotide to produce a detectable signal or marker.

121. The composition of any one of claims 109-120, wherein external dimensions of the material modifying a donor polynucleotide in the X-Y plane is nanoscaled (e.g., one nano-sized graphene flake) or macro scaled.

122. The composition of claim 121, wherein the minimal dimensions of the material modifying the donor polynucleotide in the X- Y plane are 1 nm by 1 nm.

123. The composition of any one of claims 109-122, wherein the external dimensions of the material along the Z-axis depends on the number of layers of materials incorporated.

124. The composition of claim 123, wherein, when the material comprises graphene, the minimal dimension along the Z-axis is defined by the thickness of a nonfunctionalized graphene monolayer, e.g., 0.34 nm.

125. The composition of claim 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.

126. The composition of any one of claims 109-125, wherein the genome editing of the incorporated modified donor polynucleotide further comprises a guide RNA.

127. The composition of any one of claims 109-126, wherein the target is a subject in need of a therapy or a cell.

128. The composition of claim 127, wherein the subject or cell has a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.

129. A method of treating a disease or disorder comprising administration of the composition of any one of claims 109-128.

130. The composition of any one of claims 109-128 for use in treating or inhibiting a disease such as a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease.

131. The composition of any one of claims 109-128 for use as a medicament.

132. A method of detecting a disease or disorder in a subject or a cell, such as a viral disease, a microbial disease, cancer, diabetes or a cardiovascular disease comprising administration of the composition of any one of claims 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 reflectometry.

133. 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; and 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.

134. 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; and 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.

135. The method of any one of claims 133 or 134, wherein the carbon nanomaterial particles are labeled prior to incubation.

136. The method of any one of claims 133-135, wherein the donor integrated gDNA sample is amplified prior to confirming the integration of donor sequences.

137. The method of claims any one of claims 133-136, wherein the carbon nanomaterial particles comprise graphene.

138. The method of any one of claims 133-137, wherein the carbon nanomaterial is crosslinked to its binding partner.

139. The method of any one of claims 133-138, wherein the genome editing protein comprises an ABBIE system.

140. The method of any one of claims 133-138, wherein the genome editing protein comprises a viral integrase-dCas9 complex.

141. The method of any one of claims 133-140, wherein 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.

142. The method of any one of claims 133-141, wherein the detector or sensor uses electrostatic, luminescent, or fluorescent detection.

143. A method of modifying the acetylation state of a selected protein associated with a polynucleotide comprising administering the composition of any one of claims 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.

144. The method of any of the proceeding method claims, further comprising a microfluidic chip with channels for sample input, mixing, and reaction zones.

145. The composition or method of any of the preceding claims, wherein biotinstreptavidin 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.

146. The composition or method of any of the preceding claims, wherein the polynucleotides, or oligonucleotides are biotinylated.

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