Methods for enhancing homologous recombination and compositions thereof

Abstract

The present disclosure relates to methods, kits and compositions for improving the efficiency of homologous recombination. Specifically, the invention provides a method. The present disclosure relates to cloning DNA molecules directly into the genome and / or binding one or more DNA binding agents (TAL effector domain to which Cas9 binds or truncated guide RNA) to specific sites by using promoter capture in combination with short homologous arm, nuclear localization signals, methods whereby replacement or reconstitution of chromatin at a target locus and / or to thereby enhance the accessibility of a target locus for further enzymatic modification. The methods and compositions provided herein are particularly useful for genome editing and enhancing the enzymatic processes involved therein.

Description

[0001] This application is a divisional application of an invention patent application with an application date of September 7, 2018, application number 201880071907.3, and invention name “Methods and compositions for improving homologous recombination”. Technical Field

[0002] The present disclosure relates to methods, kits and compositions for improving the efficiency of homologous recombination. Specifically, the present disclosure relates to methods for cloning DNA molecules directly into the genome and / or binding one or more DNA binders (TAL effector domains bound by Cas9 or truncated guide RNAs) to specific sites by combining promoter capture with short homology arms and nuclear localization signals, thereby replacing or reconstructing the chromatin at the target locus and / or enhancing the accessibility of the target locus for further enzymatic modification. The methods and compositions provided herein are particularly useful for genome editing and enhancing the enzymatic processes involved therein. Background Art

[0003] Recent advances in TALEN or CRISPR-mediated genome editing tools have enabled researchers to efficiently introduce double-strand breaks (DSBs) into mammalian genomes. Most DSBs are then repaired by the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway. In mammalian cells, the NHEJ pathway is dominant and error-prone. However, the HDR pathway allows precise genome editing by using sister chromatids or exogenous DNA molecules. Many attempts have been made to improve HDR efficiency, but the efficiency is still low. For example, simultaneous blocking of KU70 and DNA ligase IV gene expression using siRNA increased HDR efficiency by 4 to 5 times. See Chu et al., Nat. Biotechnol. 33: 543-548 (2015). The use of Cas9 nickase and a long DNA donor template resulted in a 5% HDR efficiency in human embryonic stem cells (hESCs). See Rong et al., “Homologous recombination in human embryonic stem cells using CRISPR / Cas9nickase and a long DNA donor template,” Protein Cell 5:258-260 (2014). A recent report showed that the combined use of the CRISPR system and in utero electroporation resulted in an EGFP integration efficiency of about 2% for the β-actin gene in brain neurons. See Uemura et al., “Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR / Cas9-mediated knock-in and in utero electroporation techniques,” Sci Rep. 6:35861 (2016). Double loss of human POLQ and LIG4 showed that random integration was eliminated. However, a large number of undefined insertions were also observed.See Saito et al., “Dual loss of human POLQ and LIG4 abolishes random integration,” Nat. Commun. 8:16112 (2017). An adeno-associated virus (AAV) system using an infection multiplicity of 106 can integrate a chimeric antigen receptor (CAR) into the TRAC locus with an efficiency of approximately 40%. See Eyquem et al., “Targeting a CAR to the TRAC locus with CRISPR / Cas9 enhances tumor rejection,” Nature 543:113-117 (2017). Although recombinant AAV systems are considered safe for the treatment of serious human diseases, the production of GMP-grade AAV requires the establishment of a strict quality control system.

[0004] Traditionally, long homology arms (500bp to 2kb) are used to integrate relatively large DNA fragments into the mammalian genome. Due to low efficiency and random integration, it is necessary to construct a targeting vector and screen a large number of single cell colonies. Therefore, this method is usually slow (about 4 to 6 months) and monotonous, which hinders the use of mammalian cells to express recombinant proteins. In order to accelerate the protein production process, transient gene expression is often used to exclude the colony screening step. Although transient expression causes high-level protein production, transgenics are only expressed in a limited time period. Therefore, it becomes expensive to produce recombinant proteins using mammalian systems. In order to meet the demand for recombinant proteins for biopharmaceutical purposes in the future market, it is necessary to use a cost-effective method for quickly and efficiently selecting high-productivity clones.

[0005] The present disclosure relates in part to compositions and methods for editing nucleic acid molecules. There is a real need for efficient systems and techniques for modifying genomes. This article and related advantages address this need. Some embodiments provide a method for cloning relatively large DNA molecules directly into mammalian genomes by combining promoter capture with short homology arms. Due to high efficiency and specificity, people can bypass the cloning cell isolation step and use stable cell pools to produce recombinant proteins. Summary of the invention

[0006] The compositions and methods described herein are intended to improve gene editing. As described elsewhere herein, many compositions and methods have been identified that increase the efficiency of gene editing.

[0007] Described herein is a method for performing homologous recombination in an initial nucleic acid molecule, the method comprising: generating a double-strand break in the initial nucleic acid molecule to produce a cleaved nucleic acid molecule, and contacting the cleaved nucleic acid molecule with a donor nucleic acid molecule, wherein the initial nucleic acid molecule comprises a promoter and a gene, wherein the donor nucleic acid molecule comprises: (i) matching ends at the 5' and 3' ends of 12 bp to 250 bp in length, (ii) a promoterless selection marker, (iii) a reporter gene, (iv) a self-cleaving peptide that connects the promoterless selection marker and the reporter gene or LoxP located on either side of the promoterless selection marker, and (iv) optionally a linker between the promoterless selection marker and the reporter gene.

[0008] In some embodiments, the double-strand break in the nucleic acid molecule is: (i) less than or equal to 250 bp from the ATG start codon, for N-terminal labeling of the cleaved nucleic acid molecule; or (ii) less than or equal to 250 bp from the stop codon, for C-terminal labeling of the cleaved nucleic acid molecule.

[0009] In some embodiments, double strand breaks are induced by at least one nucleic acid cleavage entity or electroporation. In some embodiments, at least one nucleic acid cleavage entity comprises a nuclease containing one or more zinc finger proteins, one or more transcription activator-like effectors (TALEs), one or more CRISPR complexes, one or more argonaute-nucleic acid complexes, or one or more meganucleases. In some embodiments, at least one nucleic acid cleavage entity is administered using an expression vector, plasmid, ribonucleoprotein complex (RNC) or mRNA.

[0010] In some embodiments, the promoterless selection marker comprises a protein, an antibiotic resistance selection marker, a cell surface marker, a cell surface protein, a metabolite, or an active fragment thereof. In some embodiments, the promoterless selection marker is a protein. In some embodiments, the protein is focal adhesion kinase (FAK), angiopoietin-related growth factor (AGF) receptor, or epidermal growth factor receptor (EGFR).

[0011] In some embodiments, the promoter-less selection marker is an antibiotic resistance selection marker. In some embodiments, the antibiotic resistance selection marker is a recombinant antibody. In some embodiments, the antibiotic resistance selection marker is a human IgG antibody.

[0012] In some embodiments, the reporter gene comprises a fluorescent protein reporter. In some embodiments, the fluorescent protein reporter is an emerald green fluorescent protein (EmGFP) reporter or an orange fluorescent protein (OFP) reporter.

[0013] In some embodiments, the promoterless selectable marker is: (i) linked to the 5' end of the reporter gene for N-terminal labeling of the cleaved nucleic acid molecules; or (ii) linked to the 3' end of the reporter gene for C-terminal labeling of the cleaved nucleic acid molecules.

[0014] In some embodiments, the donor nucleic acid molecule comprises a joint between a promoterless selection marker and a reporter gene. In some embodiments, the distance between the promoterless selection marker and the reporter gene is less than or equal to 300nt, 240nt, 180nt, 150nt, 120nt, 90nt, 60nt, 30nt, 15nt, 12nt or 9nt. In some embodiments, the distance is 6 nucleotides. In some embodiments, the joint is a polyglycine joint (e.g., about 2 to about 5 glycine residues).

[0015] In some embodiments, the self-cleaving peptide is a self-cleaving 2A peptide.

[0016] In some embodiments, matched ends are added to the 5' and 3' ends of the donor nucleic acid molecules by PCR amplification.

[0017] In some embodiments, the matching ends share greater than or equal to 95% sequence identity.

[0018] In some embodiments, the matching ends comprise single-stranded DNA or double-stranded DNA.

[0019] In some embodiments, the matching ends on the 5' and 3' ends of the donor nucleic acid molecules have a length of 12 bp to 200 bp, 12 bp to 150 bp, 12 bp to 100 bp, 12 bp to 50 bp, or 12 bp to 40 bp. In some embodiments, the matching ends have a length of 35 base pairs (bp).

[0020] In some embodiments, the original nucleic acid molecule is present in a cell or a plasmid.

[0021] In some embodiments, the donor nucleic acid molecule comprises a length less than or equal to 1 kb, 2 kb, 3 kb, 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, or 30 kb.

[0022] In some embodiments, the donor nucleic acid molecule is integrated into the cleaved nucleic acid molecule by homology directed repair (HDR). In some embodiments, wherein HDR is greater than or equal to 10%, 25%, 50%, 75%, 90%, 95%, 98%, 99% or 100%. In some embodiments, HDR is 100%.

[0023] In some embodiments, the integration efficiency of the donor nucleic acid molecule is greater than or equal to 50%, 75%, 90%, 95%, 98%, 99% or 100%. In some embodiments, the integration efficiency of the donor nucleic acid molecule is 100%.

[0024] In some embodiments, the method further comprises modifying the donor nucleic acid molecule at the 5' end, the 3' end, or the 5' and 3' ends. In some embodiments, the donor nucleic acid molecule is modified at the 5' and 3' ends. In some embodiments, the donor nucleic acid molecule is modified by one or more nuclease resistance groups in at least one chain at at least one end. In some embodiments, the one or more nuclease resistance groups include one or more phosphorothioate groups, one or more amino groups, 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxy nucleotides, 5-C-methyl nucleotides, or a combination thereof.

[0025] In some embodiments, the method further comprises treating the donor nucleic acid molecule with at least one non-homologous end joining (NHEJ) inhibitor. In some embodiments, at least one NHEJ inhibitor is DNA-dependent protein kinase (DNA-PK), DNA ligase IV, DNA polymerase 1 or 2 (PARP-1 or PARP-2), or a combination thereof. In some embodiments, the DNA-PK inhibitor is Nu7206 (2-(4-morpholinyl)-4H-naphtho[1,2-b]pyran-4-one), Nu7441 (8-(4-dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one), Ku-0060648 (4-ethyl-N-[4-[2-(4-morpholinyl)-4-oxo-4H-1-benzopyran-8-yl]-1-dibenzothienyl]-1-piperazineacetamide), Compound 401 (2-(4-morpholinyl)-4H-pyrimido[2,1-a]isoquinolin-4-one), DMNB (4,5-dimethoxy-2-nitrobenzaldehyde), ETP 45658 (3-[1-methyl-4-(4-morpholinyl)-1H-pyrazolo[3,4-d]pyrimidin-6-ylphenol), LTURM 34 (8-(4-dibenzothienyl)-2-(4-morpholinyl)-4H-1,3-benzoxazin-4-one), or P1 103 hydrochloride (3-[4-(4-morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol hydrochloride).

[0026] In some embodiments, the mammal is a human, a mammalian laboratory animal, a mammalian farm animal, a mammalian sports animal, or a mammalian pet. In some embodiments, the mammal is a human.

[0027] In some embodiments, the cell or plasmid is prepared by any homologous recombination method described herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell.

[0028] Also described herein is a method of cell therapy comprising administering an effective amount of any of the cells described herein to a subject in need thereof.

[0029] In some embodiments, the cell is a T cell and the promoter-less selectable marker is a chimeric antigen receptor (CAR).

[0030] Also described herein is a method of producing a promoterless selectable marker, the method comprising activating a promoter of a cell or plasmid prepared by any of the homologous recombination methods described herein to produce the promoterless selectable marker.

[0031] Also described herein is a composition comprising a promoterless selectable marker produced by any of the methods for producing a promoterless selectable marker described herein.

[0032] Also described herein is a method of treating a subject in need thereof, the method comprising administering an effective amount of a promoterless selectable marker produced by any of the methods described herein for producing a promoterless selectable marker.

[0033] Also described herein are drug screening assays comprising a promoterless selectable marker produced by any of the methods described herein for producing a promoterless selectable marker.

[0034] Also described herein is a kit for producing a promoterless selection marker, comprising a promoterless selection marker connected to a reporter gene by a self-cleaving peptide or a LoxP located on either side of the selection marker. In some embodiments, the reporter gene is GFP or OFP. In some embodiments, the kit further comprises at least one nucleic acid cleavage entity. In some embodiments, the kit further comprises at least one NHEJ inhibitor. In some embodiments, the kit further comprises one or more nuclease resistance groups.

[0035] Also described herein is a recombinant antibody expression cassette comprising: matching ends at the 5' and 3' ends of the cassette, wherein the length of the matching ends is less than or equal to 250 bp; a promoterless selection marker; a reporter gene; a self-cleaving peptide connecting the promoterless selection marker and the reporter gene; and optionally a linker present between the promoterless selection marker and the reporter gene, wherein the promoterless selection marker is linked to the 5' end of the reporter gene for N-terminal labeling of the cleaved nucleic acid molecule, or is linked to the 3' end of the reporter gene for C-terminal labeling of the cleaved nucleic acid molecule.

[0036] Also described herein are compositions and methods for altering endogenous nucleic acid molecules present in a cell, the methods comprising introducing a donor nucleic acid molecule (e.g., a donor DNA molecule) into the cell, wherein the donor nucleic acid molecule is operably linked to one or more intracellular targeting moieties that are capable of localizing the donor nucleic acid molecule to a location in the cell where the endogenous nucleic acid molecule is located.

[0037] In some embodiments, the location of the endogenous nucleic acid molecule in the cell is in the nucleus, mitochondria, or chloroplasts.

[0038] In some aspects, gene editing proteins and related methods are provided so that intracellular nucleic acid molecules undergo effective site-specific cleavage, even when introduced into cells in small amounts. Therefore, compositions and methods are provided that allow high levels of site-specific cleavage even when present at low concentrations. Many factors may affect the amount of intracellular nucleic acid cleavage that occurs. Such factors include (1) the amount of active gene editing reagents contacted with the locus intended for cleavage; (2) the level of cleavage activity exhibited by the gene editing reagents; and (3) the amount of donor nucleic acid immediately adjacent to the cleavage site. More generally, the amount of editing that occurs at a particular intracellular locus in a cell population is determined based on the percentage of cells in which at least one locus undergoes cleavage relative to diploid cells.

[0039] In some embodiments, the one or more intracellular targeting moieties are nuclear localization signals. In some embodiments, the nuclear localization signal is operably linked to the 5' end of the donor nucleic acid molecule.

[0040] In some embodiments, the donor nucleic acid molecule is operably linked to at least one nucleic acid cleavage entity. In some embodiments, the at least one nucleic acid cleavage entity comprises a nuclease containing one or more zinc finger proteins, one or more transcription activator-like effectors (TALEs), one or more CRISPR complexes, one or more Agg protein-nucleic acid complexes, one or more meganucleases, or one or more meganucleases.

[0041] In some embodiments, the donor DNA molecule is not linked to a nucleic acid cleavage entity.

[0042] In some embodiments, the length of the donor nucleic acid molecule (e.g., donor DNA molecule) is about 25 to about 8,000 nucleotides (e.g., about 25 to about 8,000 nucleotides, about 25 to about 5,000 nucleotides, about 25 to about 3,000 nucleotides, about 25 to about 2,000 nucleotides, about 25 to about 1,500 nucleotides, about 30 to about 100 nucleotides, about 30 to about 200 nucleotides, about 50 to about 500 nucleotides, about 50 to about 2,000 nucleotides, about 50 to about 8,000 nucleotides, about 75 to about 2,000 nucleotides, about 250 to about 5,000 nucleotides, etc.). An example in which a short donor nucleic acid molecule may be needed is SNP insertion or correction. For example, in this case, the donor nucleic acid molecule may have two homology arms of 15 nucleotides each and a single nucleotide for changing the target locus.

[0043] Furthermore, the donor nucleic acid molecule can be single-stranded, double-stranded, linear or circular.

[0044] In addition, the donor nucleic acid molecule can have one or more nuclease resistance groups within 50 nucleotides of at least one end. These nuclease resistance groups can be thiophosphate groups. In addition, two thiophosphate groups can be positioned within 50 nucleotides of at least one end.

[0045] In some embodiments, the donor nucleic acid molecule comprises a positive selection marker and / or a negative selection marker. In addition, the negative selection marker can be herpes simplex virus thymidine kinase.

[0046] In certain embodiments, the donor nucleic acid molecule has two regions of target locus sequence complementation present in the cell. In addition, the positive selection marker, when present, can be positioned between two regions of the sequence complementation of the donor nucleic acid molecule. In addition, the negative selection marker, when present, can not be positioned between two regions of the sequence complementation of the donor nucleic acid molecule. In other words, the negative selection marker can be positioned outside two regions of the sequence complementation.

[0047] In some embodiments, donor nucleic acid molecules operably linked to one or more intracellular targeting moieties that enable the donor DNA molecule to localize to the location of the endogenous nucleic acid molecule in the cell can be used in conjunction with other compositions and methods described herein. Thus, further provided herein are methods of contacting a cell with one or more of the following: (1) one or more nucleic acid cleavage entities, (2) one or more nucleic acid molecules encoding at least one component of a nucleic acid cleavage entity, (3) one or more DNA binding modulator enhancers, (4) one or more nucleic acid molecules encoding at least one component of a DNA binding modulator enhancer, or (5) one or more non-homologous end joining (NHEJ) inhibitors.

[0048] As described elsewhere herein, it has been found that the use of non-homologous end joining (NHEJ) inhibitors can enhance the efficiency of homologous recombination. Therefore, further provided herein are methods of contacting a cell with one or more non-homologous end joining (NHEJ) inhibitors, particularly wherein the one or more non-homologous end joining (NHEJ) inhibitors are DNA-dependent protein kinase inhibitors. Other non-homologous end joining (NHEJ) inhibitors that can be used include one or more compounds selected from the group consisting of: (1) Nu7206, (2) Nu7441, (3) Ku-0060648, (4) DMNB, (5) ETP 45658, (6) LTURM34, and (7) P1103 hydrochloride.

[0049] In addition, donor nucleic acid molecules operably linked to one or more intracellular targeting moieties can be introduced into cells using gene editing reagents designed to cut intracellular DNA at a target locus. Thus, at least one of the one or more nucleic acid cleavage entities can be selected from the group consisting of: (1) zinc finger nucleases, (2) TAL effector nucleases, and (3) CRISPR complexes. Similarly, the present invention includes the use of at least one of the one or more DNA binding regulatory enhancers, which are selected from the group consisting of: (1) zinc finger nucleases, (2) TAL effector nucleases, and (3) CRISPR complexes. In addition, at least one of the one or more DNA binding regulatory enhancers, when used, can be designed to bind within 50 nucleotides of the target locus.

[0050] The invention further includes, in part, methods for performing homologous recombination in eukaryotic cells, the methods comprising contacting the cells with (1) a donor nucleic acid molecule (e.g., a donor DNA molecule) and (2) (i) a nucleic acid cleavage entity, (ii) a nucleic acid encoding a nucleic acid cleavage entity, or (iii) at least one component of a nucleic acid cleavage entity and a nucleic acid encoding at least one component of a nucleic acid cleavage entity, wherein the donor nucleic acid molecule is associated with an intracellular targeting moiety capable of localizing the donor nucleic acid molecule to a location in the cell where an endogenous nucleic acid molecule is located.

[0051] Such methods further comprise contacting the cell with one or more of the following: (1) one or more non-homologous end joining (NHEJ) inhibitors, (2) one or more DNA binding regulatory enhancers, (3) one or more nucleic acids encoding DNA binding regulatory enhancers, and (4) at least one component of the one or more DNA binding regulatory enhancers and nucleic acids encoding at least one component of the one or more DNA binding regulatory enhancers.

[0052] The present invention also includes, in part, a composition comprising a nucleic acid molecule (e.g., a DNA molecule), wherein the nucleic acid molecule is covalently linked to one or more intracellular targeting moieties, and wherein the nucleic acid molecule is about 25 nucleotides to about 8,000 nucleotides in length (e.g., about 25 to about 8,000 nucleotides, about 25 to about 5,000 nucleotides, about 25 to about 3,000 nucleotides, about 25 to about 2,000 nucleotides, about 25 to about 1,500 nucleotides, about 30 to about 100 nucleotides, about 30 to about 200 nucleotides, about 50 to about 500 nucleotides, about 50 to about 2,000 nucleotides, about 50 to about 8,000 nucleotides, about 75 to about 2,000 nucleotides, about 250 to about 5,000 nucleotides, etc.). In some cases, the nucleic acid molecule is a donor nucleic acid molecule (e.g., a donor DNA molecule). In some cases, one or more intracellular targeting moieties are nuclear localization signals. In other cases, two or more intracellular targeting moieties (eg, nuclear localization signals, chloroplast targeting signals, mitochondrial targeting signals, etc.) are covalently linked to the nucleic acid molecule.

[0053] In one aspect, a method for increasing the accessibility of a target locus in a cell is provided. The method comprises: (1) introducing a first DNA binding regulatory enhancer into a cell comprising a nucleic acid encoding the target locus, wherein the first DNA binding regulatory enhancer is not endogenous to the cell; and (2) allowing the first DNA binding regulatory enhancer to bind to a first enhancer binding sequence of the target locus, thereby enhancing the accessibility of the target locus relative to the absence of the first DNA binding regulatory enhancer.

[0054] In one aspect, a method for replacing chromatin of a target locus in a cell is provided. The method comprises: (1) introducing a first DNA binding regulatory enhancer into a cell comprising a nucleic acid encoding the target locus, wherein the first DNA binding regulatory enhancer is not endogenous to the cell; and (2) allowing the first DNA binding regulatory enhancer to bind to a first enhancer binding sequence of the target locus, thereby replacing the chromatin of the target locus.

[0055] In one aspect, a method for reconstructing chromatin of a target locus in a cell is provided. The method comprises: (1) introducing a first DNA binding regulatory enhancer into a cell comprising a nucleic acid encoding the target locus, wherein the first DNA binding regulatory enhancer is not endogenous to the cell; and (2) allowing the first DNA binding regulatory enhancer to bind to a first enhancer binding sequence of the target locus, thereby reconstructing the chromatin of the target locus.

[0056] In one aspect, a method for increasing the accessibility of a target locus in a cell is provided. The method comprises (1) introducing into a cell comprising a nucleic acid encoding a target locus: (i) a first DNA binding regulatory enhancer, wherein the first DNA binding regulatory enhancer is not endogenous to the cell; and (ii) a second DNA binding regulatory enhancer, wherein the second DNA binding regulatory enhancer is not endogenous to the cell. (2) allowing the first DNA binding regulatory enhancer to bind to a first enhancer binding sequence of the target locus; and (3) allowing the second DNA binding regulatory enhancer to bind to a second enhancer binding sequence of the target locus, thereby enhancing the accessibility of the target locus relative to the lack of the first DNA binding regulatory enhancer or the second DNA binding regulatory enhancer.

[0057] In one aspect, a method for replacing chromatin of a target locus in a cell is provided. The method comprises: (1) introducing into a cell comprising a nucleic acid encoding the target locus: (i) a first DNA binding regulatory enhancer, wherein the first DNA binding regulatory enhancer is not endogenous to the cell; and (ii) a second DNA binding regulatory enhancer, wherein the second DNA binding regulatory enhancer is not endogenous to the cell. (2) allowing the first DNA binding regulatory enhancer to bind to a first enhancer binding sequence of the target locus; and (3) allowing the second DNA binding regulatory enhancer to bind to a second enhancer binding sequence of the target locus, thereby replacing chromatin of the target locus.

[0058] In one aspect, a method for reconstructing chromatin of a target locus in a cell is provided. The method comprises: (1) introducing into a cell comprising a nucleic acid encoding the target locus: (i) a first DNA binding regulatory enhancer, wherein the first DNA binding regulatory enhancer is not endogenous to the cell; and (ii) a second DNA binding regulatory enhancer, wherein the second DNA binding regulatory enhancer is not endogenous to the cell. (2) allowing the first DNA binding regulatory enhancer to bind to a first enhancer binding sequence of the target locus; and (3) allowing the second DNA binding regulatory enhancer to bind to a second enhancer binding sequence of the target locus, thereby reconstructing the chromatin of the target locus.

[0059] In one aspect, a method for enhancing the activity of a regulatory protein or a regulatory complex at a target locus in a cell is provided. The method comprises (1) introducing into a cell comprising a nucleic acid encoding a target locus: (i) a first regulatory protein or a first regulatory complex capable of binding to a regulator binding sequence of the target locus, wherein the regulator binding sequence comprises a regulatory site; and (ii) a first DNA binding regulatory enhancer capable of binding to a first enhancer binding sequence of the target locus. And (2) binding the first DNA binding regulatory enhancer to the first enhancer binding sequence, thereby enhancing the activity of the first regulatory protein or the first regulatory complex at the target locus in the cell.

[0060] In one aspect, a method for regulating a target locus in a cell is provided. The method comprises (1) introducing into a cell comprising a nucleic acid encoding the target locus: (i) a first regulatory protein or a first regulatory complex capable of binding to a regulator binding sequence of the target locus, wherein the regulator binding sequence comprises a regulatory site; and (ii) a first DNA binding regulatory enhancer capable of binding to a first enhancer binding sequence of the target locus. And (2) causing the first regulatory protein or the first regulatory complex to regulate the regulatory site, thereby regulating the target locus in the cell.

[0061] In embodiments, the method comprises introducing a second DNA binding modulator capable of binding a second enhancer binding sequence of the target locus.

[0062] In embodiments, the first regulatory protein or first regulatory complex is not endogenous to the cell.

[0063] In embodiments, the homologous recombination rate at the target locus is increased relative to the absence of the first DNA binding regulatory enhancer.

[0064] In embodiments, the second enhancer binding sequence is linked to the first enhancer binding sequence via a regulator binding sequence.

[0065] In embodiments, the method further comprises introducing a second regulatory protein or a second regulatory complex capable of binding to the regulator binding sequence.

[0066] In an embodiment, the first regulatory protein or the second regulatory protein comprises a DNA binding protein or a DNA regulatory enzyme. In an embodiment, the DNA binding protein is a transcription repressor or a transcription activator. In an embodiment, the DNA regulatory enzyme is a nuclease, a deaminase, a methylase or a demethylase.

[0067] In embodiments, the first regulatory protein or the second regulatory protein comprises a histone regulatory enzyme. In embodiments, the histone regulatory enzyme is a deacetylase or an acetylase.

[0068] In an embodiment, the first regulatory protein is a first DNA-binding nuclease conjugate. In an embodiment, the second regulatory protein is a second DNA-binding nuclease conjugate. In an embodiment, the first DNA-binding nuclease conjugate includes a first nuclease, and the second DNA-binding nuclease conjugate includes a second nuclease. In an embodiment, the first nuclease and the second nuclease form a dimer. In an embodiment, the first nuclease and the second nuclease are independently transcription activator-like effector nucleases (TALENs).

[0069] In an embodiment, the first DNA-binding nuclease conjugate comprises a first transcription activator-like (TAL) effector domain operably linked to a first nuclease (TALEN). In an embodiment, the first DNA-binding nuclease conjugate comprises a first TAL effector domain operably linked to a first FokI nuclease. In an embodiment, the second DNA-binding nuclease conjugate comprises a second TAL effector domain operably linked to a second nuclease (TALEN). In an embodiment, the second DNA-binding nuclease conjugate comprises a second TAL effector domain operably linked to a second FokI nuclease. In an embodiment, the first DNA-binding nuclease conjugate comprises a first zinc finger nuclease. In an embodiment, the second DNA-binding nuclease conjugate comprises a first zinc finger nuclease.

[0070] In an embodiment, the first regulatory complex is a first ribonucleoprotein complex. In an embodiment, the second regulatory complex is a second ribonucleoprotein complex. In an embodiment, the first ribonucleoprotein complex includes a CRISPR-associated protein 9 (Cas9) domain bound to a gRNA or an Agg protein domain bound to a guide DNA (gDNA). In an embodiment, the second ribonucleoprotein complex includes a CRISPR-associated protein 9 (Cas9) domain bound to a gRNA or an Agg protein domain bound to a guide DNA (gDNA).

[0071] In embodiments, the first regulatory protein, the first regulatory complex, the second regulatory protein, or the second regulatory complex is not endogenous to the cell. In embodiments, the first regulatory protein and the second regulatory protein are not endogenous to the cell. In embodiments, the first regulatory complex and the second regulatory complex are not endogenous to the cell. In embodiments, the first DNA binding regulatory enhancer or the second DNA binding regulatory enhancer is not endogenous to the cell. In embodiments, the first DNA binding regulatory enhancer and the second DNA binding regulatory enhancer are not endogenous to the cell.

[0072] In embodiments, the first DNA binding modulator enhancer is a first DNA binding protein or a first DNA binding nucleic acid. In embodiments, the first DNA binding modulator enhancer is a first transcription activator-like (TAL) effector protein or a truncated first guide RNA (gRNA).

[0073] In an embodiment, the second DNA binding modulator enhancer is a second DNA binding protein or a second DNA binding nucleic acid. In an embodiment, the second DNA binding modulator enhancer is a TAL effector protein or a truncated gRNA.

[0074] In embodiments, the first DNA binding regulatory enhancer is a first TAL effector protein and the second DNA binding regulatory enhancer is a second TAL effector protein. In embodiments, the first DNA binding regulatory enhancer is a TAL effector protein and the second DNA binding regulatory enhancer is a truncated gRNA. In embodiments, the first DNA binding regulatory enhancer is a truncated first gRNA and the second DNA binding regulatory enhancer is a truncated second gRNA. In embodiments, the first DNA binding regulatory enhancer is a truncated gRNA and the second DNA binding regulatory enhancer is a TAL effector protein.

[0075] In an embodiment, the first regulatory protein is a first DNA-binding nuclease conjugate and the second regulatory protein is a second DNA-binding nuclease conjugate. In an embodiment, the first regulatory protein is a DNA-binding nuclease conjugate and the second regulatory complex is a ribonucleoprotein complex. In an embodiment, the first regulatory complex is a first ribonucleoprotein complex and the second regulatory complex is a second ribonucleoprotein complex. In an embodiment, the first regulatory complex is a ribonucleoprotein complex and the second regulatory protein is a DNA-binding nuclease conjugate.

[0076] In an embodiment, the first enhancer binding sequence and / or the second enhancer binding sequence are independently separated from the regulator binding sequence by less than 200 nucleotides (e.g., about 5 to about 180, about 10 to about 180, about 20 to about 180, about 5 to about 90, about 5 to about 70, about 5 to about 60, about 5 to about 50, about 5 to about 40, about 5 to about 30, about 15 to about 80, about 15 to about 60, about 15 to about 50, about 15 to about 40, about 20 to about 40, about 20 to about 40 nucleotides, etc.). In an embodiment, the first enhancer binding sequence is independently separated from the regulator binding sequence by less than 150 nucleotides. In an embodiment, the first enhancer binding sequence and / or the second enhancer binding sequence are separated from the regulator binding sequence by less than 100 nucleotides. In an embodiment, the first enhancer binding sequence and / or the second enhancer binding sequence are independently separated from the regulator binding sequence by less than 50 nucleotides. In an embodiment, the first enhancer binding sequence and / or the second enhancer binding sequence are independently separated from the regulator binding sequence by 4 to 30 nucleotides. In an embodiment, the first enhancer binding sequence and / or the second enhancer binding sequence are independently separated from the regulator binding sequence by 7 to 30 nucleotides. In an embodiment, the first enhancer binding sequence and / or the second enhancer binding sequence are independently separated from the regulator binding sequence by 4 nucleotides, 7 nucleotides, 12 nucleotides, 20 nucleotides or 30 nucleotides.

[0077] In an embodiment, the first enhancer binding sequence and / or the second enhancer binding sequence are independently separated from the regulatory site by 10 to 40 nucleotides. In an embodiment, the first enhancer binding sequence and / or the second enhancer binding sequence are independently separated from the regulatory site by 33 nucleotides.

[0078] In an embodiment, the first enhancer binding sequence has a sequence of SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. In an embodiment, the second enhancer binding sequence has a sequence of SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, or SEQ ID NO: 41.

[0079] In embodiments, the first DNA binding regulatory enhancer or the second DNA binding regulatory enhancer enhances the activity of the first regulatory protein, the first regulatory complex, the second regulatory protein, or the second regulatory complex at the regulatory site.

[0080] In one aspect, a cell is provided, comprising a nucleic acid encoding a target locus regulatory complex. The complex comprises: (i) a target locus comprising a first enhancer binding sequence and a regulator binding sequence comprising a regulatory site; (ii) a first regulatory protein or a first regulatory complex bound to the regulator binding sequence; and (iii) a first DNA-binding regulatory enhancer bound to the first enhancer binding sequence.

[0081] In embodiments, the target locus further comprises a second enhancer binding sequence connected to the first enhancer binding sequence via a regulator binding sequence.

[0082] In embodiments, the cell comprises a second DNA binding modulator-enhancer bound to a second enhancer binding sequence.

[0083] In one aspect, a cell comprising a nucleic acid encoding a target locus complex is provided. The complex includes (i) a target locus comprising a first enhancer binding sequence; and (ii) a first DNA binding regulatory enhancer bound to the first enhancer binding sequence, wherein the first DNA binding regulatory enhancer is not endogenous to the cell, and wherein the first DNA binding regulatory enhancer is capable of enhancing the accessibility of the target locus relative to the absence of the first DNA binding regulatory enhancer.

[0084] In one aspect, a cell comprising a nucleic acid encoding a target locus complex is provided. The complex comprises (1) a target locus comprising: (i) a first enhancer binding sequence; and (ii) a second enhancer binding sequence. (2) a first DNA binding regulatory enhancer that binds to the first enhancer binding sequence of the target locus, wherein the first DNA binding regulatory enhancer is not endogenous to the cell; and (3) a second DNA binding regulatory enhancer that binds to the second enhancer binding sequence of the target locus, wherein the second DNA binding regulatory enhancer is not endogenous to the cell, wherein the first DNA binding regulatory enhancer and the second DNA binding regulatory enhancer are capable of enhancing the accessibility of the target locus relative to the absence of the first DNA binding regulatory enhancer and the second DNA binding regulatory enhancer.

[0085] In certain aspects, kits are provided. Kits provided herein may include one or more of the following: (i) a first regulatory protein, a first regulatory complex; (ii) a first DNA binding regulatory enhancer; (iii) one or more nucleic acid molecules; (iv) one or more intracellular targeting moieties; and (v) one or more non-homologous end joining inhibitors.

[0086] Also provided herein are gene editing reagents, such as Cas9 proteins, and nucleic acids encoding such reagents, such reagents comprising two or more (e.g., about two to about twelve, about three to about twelve, about four to about twelve, about five to about twelve, about two to about seven, about three to about seven, etc.) nuclear localization signals (NLS) (e.g., non-classical, monotyping and / or bityping NLS). Exemplary Cas9 proteins are those proteins comprising two or more bityping nuclear localization signals (NLS). In addition, all or some of the two or more bityping nuclear localization signals may be located within twenty amino acids of at least one end, such as the N-terminus and / or C-terminus of the Cas9 protein. The position here refers to the portion closest to the end in the NLS. Therefore, if the C-terminal amino acid of the NLS is followed by ten other amino acids, and the last amino acid is the C-terminus of the protein, the position of the NLS is eleven amino acids from the C-terminus. In other words, the position count is determined by the last amino acid of the NLS.

[0087] In addition, the gene editing reagent (e.g., Cas9 protein) may include an NLS having different amino acid sequences or the same amino acid sequence. In addition, the gene editing reagent (e.g., Cas9 protein) may include one or more (e.g., about one to about five, about one to about four, etc.) affinity tags. The combination of gene editing reagents may include one or more of the following amino acid sequences: (A) KRTAD GSEFE SPKKK RKVE (SEQ ID NO: 48), (B) KRTAD GSEFE SPKKA RKVE (SEQ ID NO: 49), (C) KRTAD GSEFE SPKKK AKVE (SEQ ID NO: 50), (D) KRPAA TKKAG QAKKK K (SEQ ID NO: 51), and (E) KRTAD GSEFEP AAKRV KLDE (SEQ ID NO: 52). The NLS used in combination with the gene editing reagent may include one or more amino acid sequences, and the amino acid sequence belongs to the range of one or more of the following formulas: (A) KRX 5-15 KKN1N2KV(SEQ ID NO:53), (B)KRX (5-15) K(K / R)(K / R) 1-2 (SEQ ID NO:54), (C) KRX (5-15) K(K / R)X(K / R) 1-2 (SEQ ID NO:55), wherein X is an amino acid sequence of 5 to 15 amino acids in length and wherein N1 is L or A, and wherein N2 is L, A or R. In addition, the claimed specific Cas9 proteins that can be used in the compositions and methods described herein include Fig.41 and Fig.42The amino acid sequence shown in .

[0088] In addition, provided herein are TALE proteins comprising one or more (e.g., about two to about six, about two to about five, about two to about four, about two to about three, about three to about five, etc.) heterologous nuclear localization signals (e.g., monopartite NLS, bipartite NLS, etc.). In some aspects, provided herein are TALE proteins comprising amino acids ( Fig.46 811-830 of ), wherein the amino acids at positions 815-816 and 824-825 are Gly-Ser or Gly-Gly; and a TALE protein comprising amino acids ( Fig.46 The TALE protein provided herein may comprise amino acids 810-1029 of the ... Fig.46 Amino acids 752-1021.

[0089] In some aspects, provided herein are amino acids ( Fig.47 The TALE protein provided herein may comprise amino acids ( Fig.47 The TALE protein may also comprise amino acids 821-840 of positions 827-828, wherein the amino acids at positions 827-828 are Gly-Ser or Gly-Gly. Fig.46 of amino acids.

[0090] The TAL proteins provided herein in various aspects can comprise a repeat region comprising 4 to 25 (e.g., about 5 to about 22, about 6 to about 22, about 8 to about 22, about 10 to about 22, about 12 to about 22, about 12 to about 26, about 13 to about 20, etc.) repeat units.

[0091] Also provided herein is a method for engineering intracellular nucleic acids in a cell, the method comprising introducing into the cell one or more TALE proteins (e.g., one or more TALE proteins mentioned above) or nucleic acids encoding the one or more TALE proteins, wherein the one or more TALE proteins are designed to bind to a target locus within the cell. In some aspects, such methods further comprise introducing into the cell one or more donor nucleic acid molecules, wherein the one or more donor nucleic acid molecules have a sequence homologous to a nucleic acid within 50 (e.g., about 0 to about 50, about 0 to about 40, about 0 to about 30, about 0 to about 20, about 6 to about 40, etc.) nucleotides of the target locus. One or more regions.

[0092] Further provided herein is a method for homologously recombinating intracellular nucleic acid molecules within a cell population at a cleavage site, the method comprising: (a) generating one or more double-stranded breaks in the intracellular nucleic acid molecules at the cleavage site to produce cleaved nucleic acid molecules; and (b) contacting the cleaved nucleic acid molecules with one or more donor nucleic acid molecules, wherein the one or more donor nucleic acid molecules have at least ten (e.g., about 10 to about 500, about ten to about 500, about 10 to about 400, about 10 to about 300, about 10 to about 250, about 20 to about 300, about 25 to about 300, about 30 to about 350, etc.) nucleotides or base pairs homologous to a nucleic acid within 100 base pairs on each side of the cleavage site, wherein at least 95% (e.g., about 95% to about 100%, about 95% to about 99%, about 96% to about 99%, about 95% to about 98%, about 96% to about 99%, etc.) of the cells in the cell population undergo homology-directed repair at the cleavage site in the presence of at least one of the one or more donor nucleic acid molecules. In some respects, one or more donor nucleic acid molecules contain one or more selection markers or one or more reporter genes, and after homology-directed repair, the selection marker or reporter gene are operably connected to a promoter present in the intracellular nucleic acid molecule. In addition, one or more donor nucleic acid molecules can be connected to one or more nuclear localization signals, and the nuclear localization signal allows the one or more donor nucleic acid molecules (donor nucleic acid molecules) to be located in the nucleus of the cell mass.

[0093] In some aspects, the cell population can be contacted with one or more of the following: (1) one or more nucleic acid cleavage entities, (2) one or more nucleic acid molecules encoding at least one component of the nucleic acid cleavage entity, (3) one or more DNA binding modulator enhancers, (4) one or more nucleic acid molecules encoding at least one component of the DNA binding modulator enhancer, and / or (5) one or more non-homologous end joining (NHEJ) inhibitors. In addition, one or more of the one or more donor nucleic acid molecules can be single-stranded.

[0094] In other aspects, the cell group can be contacted with one of the multiple nucleic acid molecules of one or more nucleic acid cutting entities or encoding one or more nucleic acid cutting entities, and then the cell group can be contacted with one or more donor nucleic acid molecules. In addition, the cell group can be contacted with one or more donor nucleic acid molecules, and then the cell group can be contacted with one or more nucleic acid cutting entities or one of the multiple nucleic acid molecules encoding one or more nucleic acid cutting entities. In addition, after the cell group is contacted with one of the multiple nucleic acid molecules of one or more nucleic acid cutting entities or encoding one or more nucleic acid cutting entities, the cell group can be contacted with one or more donor nucleic acid molecules for 1 to 60 minutes. On the contrary, after the cell group can be contacted with one or more donor nucleic acid molecules, the cell group can be contacted with one of the multiple nucleic acid molecules of one or more nucleic acid cutting entities or encoding one or more nucleic acid cutting entities for 1 to 60 minutes. In some cases, the cell group can be contacted with one or more nucleic acid cutting entities or one of the multiple nucleic acid molecules encoding one or more nucleic acid cutting entities and one or more donor nucleic acid molecules simultaneously.

[0095] In other aspects as described above, one or more nucleic acid cleavage entities or one of the multiple nucleic acid molecules encoding one or more nucleic acid cleavage entities and one or more donor nucleic acid molecules can be introduced into cells together or separately by electroporation. In addition, one or more nucleic acid cleavage entities or one of the multiple nucleic acid molecules encoding one or more nucleic acid cleavage entities can be first introduced into cells, followed by electroporation of one or more donor nucleic acid molecules, or one or more donor nucleic acid molecules can be first introduced into cells, followed by electroporation of one or more nucleic acid cleavage entities or one of the multiple nucleic acid molecules encoding one or more nucleic acid cleavage entities.

[0096] Other objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0097] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0098] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and, together with the description, serve to explain the principles described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] In order to more fully understand the principles and advantages disclosed herein, reference is now made to the following description in conjunction with the accompanying drawings, in which:

[0100] Figure 1A and 1B Protein labeling by promoter trapping and short homology arms is shown. Figure 1A N-terminal tags are shown. The promoter-free selection marker puromycin is linked to an emerald green fluorescent protein (EmGFP) reporter or an orange fluorescent protein (OFP) reporter gene via a self-cleaving 2A peptide, followed by PCR to add 35nt homology arms at the 5' and 3' ends. An endogenous promoter drives the expression of puromycin, the reporter gene, and the endogenous gene. TALEN or CRISPR induces double-strand breaks (DSBs) near the translation start site. Figure 1B C-terminal tagging is shown. The EmGFP or OFP reporter gene is linked to the promoterless selection marker puromycin via a self-cleaving 2A peptide followed by the addition of 35nt homology arms at the 5' and 3' ends. The endogenous promoter drives the expression of the endogenous gene, the reporter gene, and puromycin. TALEN or CRISPR induces DSBs near the translation termination site. The stop codon is eliminated between the endogenous gene and the reporter gene. Figure 1A and 1B In the PCR, the donor DNA is inserted into the genome by homologous recombination. The junctions at the 5' and 3' ends are analyzed by PCR using primer sets F1 / R1 and F2 / R2, respectively.

[0101] Figures 2A to 2D The effects of donor format and dosage as well as homology arm length on HDR efficiency are shown. Figure 2A In , Cas9RNP and different amounts of donor DNA with 35nt homology arms were delivered to 293FT cells by electroporation. Samples lacking gRNA were used as controls. 48 hours after transfection, cells were analyzed by flow cytometry to determine the percentage of OFP-positive cells that did not undergo puromycin selection (-). Alternatively, cells were treated with puromycin for 7 days before flow cytometric analysis (+). Figure 2B In the experiment, various homology arm lengths were added to the insertion cassette by PCR amplification and then co-transfected with Cas9RNP into 293FT cells. Figure 2A Cells were analyzed by flow cytometry as described. Figure 2C and 2D In the experiment, Cas9RNP and a donor plasmid with about 500 nt homology arms, or a single-stranded (ss) or double-stranded (ds) DNA donor with 35 nt homology arms were transfected into 293FT or human primary T cells by electroporation. 48 hours after transfection, cells were analyzed by flow cytometry.

[0102] Figures 3A to 3ECharacterization of clonal cells with OFP integrated into the β-actin locus is shown. Cas9 RNP and donor DNA with 35 nt homology arms were delivered into 293FT cells by electroporation and clonal cells were isolated after puromycin selection. Clonal cells were analyzed by ligation PCR using one internal primer and one external primer or a pair of external primers. The resulting PCR products were analyzed by sequencing. Figure 3A and 3B The N-terminal and C-terminal junctions with either exact HDR (1) or HDR with indels (2) are shown, respectively. Figure 3A and 3B The precise HDR in (1) arrows indicate the junction between genomic DNA and donor DNA or Cas9 cleavage site. Figure 3A and 3B The sequence shown in bold in the figure indicates the 35 nt homology arm. The italic ATG indicates the start codon of β-actin. Figure 3A and 3B HDR with indels in (2) shows an example of indels forming around a junction. Figure 3A SEQ ID NOs 130-134 are disclosed respectively in order of appearance. Figure 3B SEQ ID NOs 135-139 are disclosed respectively in order of appearance. Figure 3C Characterization of the zygosity of the clonal cells is shown. Allele 1 has approximately 68% accurate HDR at both junctions, and 32% HDR with indels at the C or N termini or both. Allele 2 has an "A" insertion (㈡1ntA) in approximately 80% of the clones, and a deletion of more than 2nt (Δ>2nt) in 18% of the clones and 2% of the wild type (wt). Figure 3D and 3E N-terminal labeling of β-actin with OFP by Tal nuclease is shown. TALEN mRNA alone or together with donor DNA was transfected into HEK293FT cells by electroporation (Thermo Fisher Scientific, Cat. No. MPK5000). Figure 3D The genome editing efficiency (% indel) is shown and Figure 3E The percentage of OFP-positive cells (-) analyzed by flow cytometry and the percentage of OFP-positive cells (+) treated with puromycin are shown.

[0103] Figure 4A , 4B4C and 4C show N-terminal tagging of EmGFP against LRRK2 in A549 cells. Cas9RNP and donor DNA containing promoter-free puromycin-P2A-EmGFP fragment and about 35nt homology arms were delivered to cells by electroporation. 48 hours after transfection, cells were cloned. After amplification, clones were dissolved and the N-terminal ( Figure 4A ) or C-terminus ( Figure 4B ) and one outer primer, and analyzed by ligation PCR. Figure 4A SEQ ID NO: 140 is disclosed. Figure 4B SEQ ID NO: 141 is disclosed. Alternatively, a pair of external primers is used to analyze genomic modification of both alleles ( Figure 4C ). The obtained PCR products were analyzed by sequencing. Figure 4A and 4B The sequences in bold in the figure indicate homology arms. The arrows at the bottom indicate the Cas9 cleavage site or the junction between genomic DNA and donor DNA. Figure 4C 7nt_no HDR in indicates that HDR did not occur, but there is a 7nt deletion.

[0104] exist Figure 5A In 5B (SEQ ID NO: 56-62), 5B (SEQ ID NO: 63-69) and 5C, the C-terminus of FAK was labeled with EmGFP. Cas9RNP and donor DNA with short homology arms were transfected into 293FT by electroporation. After puromycin selection, cells were cloned. The junction was amplified by PCR, and then the N-terminal junction ( Figure 5A ) or C-terminal junction ( Figure 5B ) for sequencing analysis. Arrows indicate double-strand breaks (DSBs) or junctions between genomic DNA and donor DNA. In the case of precise HDR, short homology arms (bold) and stop codons (underlined) are also shown. Examples of HDR with Indels are also shown. Figure 5A and 5B middle. Figure 5C Analysis of genomic modifications on both alleles is shown.

[0105] In the picture Fig. 6A In 6B and 6C, EGFR was C-terminally tagged with EmGFP. A gRNA was designed to target the EGFR genomic locus near the stop codon. The Cas9RNP complex and donor DNA were delivered into 293FT cells by electroporation. Clonal cells were analyzed by conjugative PCR and sequencing. Fig. 6A N-terminal ligation analysis is shown (SEQ ID NO: 70), and Figure 6BC-terminal ligation analysis is shown (SEQ ID NO: 71). Figure 6C The genomic modification on each allele is shown. Figure 6C The 1ntA_no HDR in refers to one "A" insertion without an intervening sequence.

[0106] Fig. 7A The effect of end modification of the DNA donor on HDR efficiency is shown, and Figure 7B The effect of NHEJ inhibitors on HDR efficiency is shown. Fig. 7A In , DNA primers with modified ends were chemically synthesized and used to prepare donor DNA by PCR amplification. Cas9RNP and donor DNA were transfected into primary T cells by electroporation. 48 hours after transfection, the efficiency of the puromycin-P2A-OFP DNA fragment inserted into the β-actin locus was monitored by flow cytometry. Figure 7B In the present invention, the NHEJ inhibitor is added to the culture medium immediately after electroporation. "F" refers to the forward primer; "R" refers to the reverse primer; "PS" refers to phosphorothioate; "NH2" refers to amine modification, and "ssDNA" refers to single-stranded DNA.

[0107] Fig. 8A , 8B , 8C and 8D show the cloning and expression of recombinant antibodies in mammalian genomes. Fig. 8A The antibody expression cassette is shown, which contains a promoter-less puromycin selection marker followed by a self-cleaving 2A peptide (SEQ ID NO: 5). The expression of IgG heavy chain (HC) and light chain (LC) is driven by the CMV promoter. 35nt homology arms are added by PCR. Figure 8B (SEQ ID NO:72-76) and Figure 8C (SEQ ID NO: 77-82) N-terminal and C-terminal junction analysis are shown, respectively. Arrows indicate double-strand breaks (DSBs) and junctions between genomic DNA and donor DNA. The 35nt homology arms and some additional sequences are also highlighted in bold. WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) and stop codon are shown in Figure 8C middle. Fig.8D The relative percentages of clones producing antibodies (+) or not producing antibodies (-) as determined by ELISA analysis are shown.

[0108] Fig. 9 . Nuclear localization signal (NLS)-donor DNA design (SEQ ID NO: 83 and 127 and 84 and 128). The conjugation chemical used to attach the NLS peptide was succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) or

[0109] Fig.10 (SEQ ID NO8:85-92). HEK 293 strain modified with NLS donor DNA construct. On the left, the GFP gene is broken due to the deletion of 6 nucleotides that make up the fluorophore. Addition of a donor containing 6 bases restores GFP fluorescence. On the right is a similar break in the BFP gene. Addition of a donor with a single nucleotide polymorphism (SNP) converts the BFP coding sequence to the GFP coding sequence.

[0110] Fig.11 Dose response of phosphorothioate (PS) oligonucleotide donor DNA upon addition of a hexamer sequence to restore GFP activity compared to NLS-modified donor DNA. Ligated NLS-modified donor DNA, PS oligonucleotide donor DNA dose response.

[0111] Fig.12 Flow cytometric analysis of cells edited with equal concentrations of PS or NLS oligonucleotide donor DNA.

[0112] Fig.13 Dose response of PS oligonucleotide donor DNA compared to NLS-modified donor DNA to edit a single base to convert BFP-expressing cells to GFP-expressing cells. Dose response of PS oligonucleotide donor DNA compared to SMCC-ligated NLS-modified donor DNA.

[0113] Fig.14 .Figure shows a schematic diagram of exemplary architectures of TALEN and TAL-Buddy (nuclease-free) constructs. TAL-Buddy constructs are shown without a nuclease domain. In some cases, the nuclease domain may be present but may be disrupted (e.g., by indels and / or substitutions).

[0114] Fig.15 When a “TAL-Buddy” designed with a 7nt spacing relative to the TALEN binding sequence was added, indel formation at the CMPK1-C target was increased by approximately 2-fold.

[0115] Fig.16 .“TAL-Buddy” has been tested to improve TALEN cleavage when the spacing relative to the TALEN binding sequence is up to 100 nt.

[0116] Fig.17 .“TAL-Buddy” increased CRISPR-RNP indel formation by ≥20-fold at the UFSP2-SNP target when the spacing relative to the CRISPR sgRNA binding sequence was 20 nt.

[0117] Fig.18 .“TAL-Buddy” together with RNP formed by sgRNA and SpCas9-HF1 or eSpCas9 enhances indel formation.

[0118] Fig.19 . Schematic representation of the templates used to prepare sgRNA and "CR-PAL" gRNA.

[0119] Fig. 20 . Illustration of the function of "CR-PAL". Black represents "CR-PAL" with 15nt binding ability; gray represents sgRNA with 20nt binding ability.

[0120] Fig.21 When “CR-PAL” was used together with Cas9-RNP for the UFSP2-SNP target, indel formation increased more than 60-fold.

[0121] Fig. 22 .The figure shows the preparation of the No-FokI C-terminal fragment of "TAL-Buddy".

[0122] Fig.23 . The figure shows the test "Buddy TAL" (293FT). Target: CMPK1-C (SEQ ID NO: 19); TALEN mRNA: 100 ng each; TAL-Buddy: 7 nt spacing (SEQ ID NO: 18); 1300 / 20 / 2. Repeat.

[0123] Fig.24 .The figure shows the tested spacing of Buddy TAL on TALEN. Target: CMPK1; Cell: 293FT; 1300 / 20 / 2. Spacing is important, TAL cannot be directly adjacent to TALEN. Spacing (0, 4, 7, 20nt) indicates the distance between the 18 base recognition sequence of TAL and the nearest TALEN pair.

[0124] Fig.25 .Figure shows testing of Buddy TALs for improving TALEN and CRISPR efficiency. Spacing affects cutting efficiency. There is no difference between TALs (grey hexagons) 7nt and 20nt from TALENs (dark grey arrows). With TAL and CRISPR targets (black circle fragments), 20nt from TAL is better.

[0125] Fig.26.The image shows a repetitive TAL-buddy edited with CRISPR. 293FT cells; CRISPR target: USFP2; TAL-buddy: 20nt spacing; 1150 / 20 / 2. Repeat.

[0126] Fig. 27 .Figure shows testing the activity of TAL-Buddy-restored low-performance mutants against high-fidelity Cas9. Target: UFSP2; Cell: 293FT; 1150 / 20 / 2; TAL-Buddy: 20nt spacing. HF-cas9 has undetectable activity, and our analysis suggests it is more imperfect than eCas9. In the absence of TAL, eCas9 1.1 has undetectable activity. With TAL, wt activity levels are obtained. This is important because the high-fidelity activity you obtain is localized only to the desired target site (ultra-high fidelity).

[0127] Fig.28 .Figure shows testing CRISPR-PAL using standard active cas9 and truncated gRNA. Target: UFSP2; Cells: 293FT; 1150 / 20 / 2; CR-PAL: 15mer gRNA; CR_PAL-left spacing: 36nt; CR_PAL-right spacing: 15nt. Cas9 will bind to, but not cut, the truncated gRNA (15mer). (Church et al., 2014 Kiani et al., Cas9gRNA engineering for genome editing, activation and repression, Nat. Methods doi:10.1038 (September 7, 2015). Truncated gRNAs are used to bracket the cleavage site and open the DNA so that the standard gRNA (20mer) can cut better. 5% (alone) to >50% (15mer). Cas9v2+20mer gRNA+L / R 15mer gRNA.

[0128] Fig.29 .Figure shows the Buddy TAL Activator concept. Binding of TAL to an activation domain (e.g., VP64) promotes active gene expression, thereby opening the DNA for enhanced editing by nucleases (TALEN, Cas9, etc.).

[0129] Fig.30 .This figure shows HDR in U2OS (sequence verified). The donor has a HindIII site insertion; 1300 / 20 / 2.

[0130] Fig.31 .This figure shows the effect of small molecules / additives on TALEN editing in A549 cells. Target: HTR2A-N; Donor has HindIII site insertion; Conditions: 1200 / 20 / 4; medium change every 24 hours; HindIII cleavage as indicated. NU7441 (DNAPK inhibitor) and B18R (immune response repressor).

[0131] Fig.32 .This figure shows an example of the relative position of TALEN and TAL-Buddy. Then, the TALEN pair is separated by 8 bases on each side of the target site. In this example, the spacing between the TAL-buddy and the TALEN is 7nt. The upper strand is SEQ ID NO: 20, and the lower strand is SEQ ID NO: 21.

[0132] Fig.33 This figure shows the "TAL-Buddy" designed close to the CRISPR cleavage site in the UFSP2-SNP target. 100ng of Lt and Rt "TAL-Buddy" mRNA and CRISPR-RNP (1000ng of Cas9 protein and 200ng of sgRNA) were added to prepare the TAL-Buddy. Approximately 50,000 293 human embryonic kidney cells (293FT) were transfected using an electroporation device (Thermo Fisher Scientific, catalog number MPK5000) with a pulse voltage of 1150, a pulse width of 20, and a pulse number of 2. Cells were harvested and lysed 48 to 72 hours after transfection. TM The formation of indels was analyzed using the Genomic Cleavage Detection Kit (Thermo Fisher Scientific, catalog number A24372). The top strand is SEQ ID NO: 42, and the bottom strand is SEQ ID NO: 43.

[0133] Fig.34 This figure shows the "CR-PAL" designed adjacent to the CRISPR cleavage site in the UFSP2-SNP target. 200ng of CR-PAL_Lt and CR-PAL_Rt were incubated with wild-type Cas9-RNP and used Approximately 50,000 293 human embryonic kidney cells (293FT) were transfected using an electroporation device (Thermo Fisher Scientific, catalog number MPK5000) with a pulse voltage of 1150, a pulse width of 20, and a pulse number of 2. Cells were harvested 48 to 72 hours after transfection and lysed. TMIndel formation was analyzed using the "Genomic Cleavage Detection Kit" (Thermo Fisher Scientific, catalog number A24372). The top strand is SEQ ID NO: 44, and the bottom strand is SEQ ID NO: 45.

[0134] Fig.35 . This figure shows the test "Buddy TAL" (293FT). The top strand is SEQ ID NO:46 and the bottom strand is SEQ ID NO:47.

[0135] Fig.36 It is a schematic diagram of the use of a pair of TAL-Buddy (also referred to herein as the first and second DNA binding regulatory enhancers) in combination with a pair of TAL-FokI nuclease fusions (also referred to herein as the first and second DNA binding nuclease conjugates). There are indications on the right and left sides of the figure, with the left side being the left TAL-Buddy binding and the right side being the right TAL-Buddy binding. The long solid line represents a portion of an intracellular nucleic acid molecule (e.g., a chromosome; also referred to herein as a target locus). Region A (shown at the left and right ends) of the nucleic acid molecule shown is the binding site of the two TAL-Buddy proteins (also referred to herein as the first and second enhancer binding sequences). Region B represents the distance between the TAL-Buddy binding site (e.g., the first and second enhancer binding sequences) and the TAL-FokI fusion protein binding site (also referred to herein as the first and second binding sequences). Region D represents the nucleic acid segment between the two TAL-FokI fusion protein binding sites. The white box in region D represents the site where the TAL-FokI fusion protein cleaves the nucleic acid (also referred to herein as the regulatory site). Region E represents a portion of the nucleic acid molecule whose accessibility is potentially enhanced.

[0136] Fig.37 is similar to Fig.36 Schematic diagram of , with the exception that a single TAL-VP16 fusion (also referred to herein as a regulatory protein) is used instead of a pair of TAL-FokI nuclease fusions. The unlabeled circles represent components of the transcription complex recruited by VP16. In addition, since a single TAL-VP16 fusion is used, there is only one region C. Moreover, the region B formed is formed by the middle base pair between region A (also referred to herein as the first and second enhancer binding sequences) and region C (also referred to herein as the regulatory binding sequence).

[0137] Fig.38Many different forms of donor nucleic acid molecules that can be used in the various embodiments described herein are shown. The open circle at the end represents a nuclease resistance group. Two circles mean that there are two groups. The black area represents a region that is homologous / complementary to one or more locus sequences of another nucleic acid molecule (e.g., chromosomal DNA). The cross-hatched area represents the nucleic acid between the sequence homology / complementary regions in the nucleic acid fragment. This figure shows different variants of donor nucleic acid molecules that can be used in different aspects of the present invention.

[0138] Fig.39 Schematic diagram of an exemplary Cas9 form based on the Cas9 protein model Streptococcus pyogenes Cas9. This 1368 amino acid protein is represented by the solid top line in the figure. The Cas9 proteins labeled V1-V5 are fusion proteins with nuclear localization signals (NLS) as components. The dotted box represents a single-type NLS, and the open box represents a double-type NLS. The gray box represents an affinity tag (e.g., a six-histidine tag (SEQ ID NO: 129)).

[0139] Fig.40 is similar to Fig.36 , which adds a more detailed view of the TAL cleavage locus with a schematic of the donor DNA molecule. A linear schematic of the donor DNA is shown at the bottom left. The straight solid line represents the region of homology to the target locus. The circular dashed line represents the insertion box. The "X" symbol represents the sequence homology region. The upward and downward dashed arrows represent two thiophosphate bonds in the 5' and 3' strands of the donor DNA homology arms. After nuclease digestion, these thiophosphate bonds are positioned to produce a 5' overhang of ten nucleotides in length. The open boxes on the left and right represent the bipartite NLS. On the right side of the linear schematic are two examples of insertion boxes. The upper insertion box is designed to both destroy the function at the insertion site and express the puromycin resistance marker. The lower insertion box is similar to the upper insertion box, but is also designed to insert the gene of interest operably linked to the tissue-specific promoter into the locus.

[0140] Fig.41 The amino acid sequence of Cas9V1 is shown (SEQ ID NO: 93). NLS and His tags are labeled as such.

[0141] Fig.42 The amino acid sequence of Cas9V2 is shown (SEQ ID NO: 94). The NLS is labeled as such.

[0142] Fig.43 A series of Cas9-NLS fusion protein formats are shown. "NP" refers to nucleoplasmin NLS. Fig.43 "6his" is disclosed as SEQ ID NO:129.

[0143] Fig.44A and 44B Shown are GCD data using different Cas9-NLS combinations with two different cell types.

[0144] Fig.45 is a schematic diagram showing common TALE structural forms. Sites 1, 2, and 3 are located outside the TALE region believed to be involved in DNA recognition and binding.

[0145] Fig.46 Display the amino acid sequence of the TALEN protein (SEQ ID NO:95) . This format of TALEN is referred to herein as "TALEN V3". The N-terminal region contains a V5 antigenic determinant and a "GG" linker, followed by a 136 amino acid region, followed by a repeat region. The 136 amino acid region contains (1) a series of repeat units (labeled "R-3", "R-2", "R-1" and "R0") with certain sequence homology to the individual repeat sequences of the repeat region and (2) a "less T box", amino acid sequence "RGA", which can be changed to reduce the 5'T requirement of the nucleic acid bound by the TALEN. The repeat region contains sixteen repeats of thirty-four amino acids. The half-repeat sequence (labeled "R1 / 2") is adjacent to the C-terminus of the repeat region. Two nuclear localization signals (labeled "NLS") are positioned further toward the C-terminus of the protein before and after the FokI nuclease domain.

[0146] Fig.47 Display the amino acid sequence of the TALEN protein (SEQ ID NO: 96 and 97), However, the amino acid sequence of the repeat region has been removed to simplify the figure. In addition, the protein shown in this figure has three NLSs.

[0147] Fig.48 Shown are genomic cleavage detection data for three different genomic loci in three different cell types, generated as described in Example 8 below.

[0148] Fig.49 Shown are genomic cleavage detection and homology-directed repair data for three different genomic loci in two different cell types, generated as described in Example 8 below.

[0149] Fig.50 Shown are genomic cleavage detection data for three different genomic loci in A549 cells, generated as described in Example 8 below.

[0150] Fig.51is a schematic diagram of some of the uses of TAL for opening and maintaining open chromatin. The upper portion of the schematic diagram shows the intracellular nucleic acid region where the nucleic acid is bound to the histone octamer to form chromatin. Each octamer is wrapped around about 145 base pairs of DNA, about 1.6 turns. Histone H1 is not shown in this schematic diagram. The dashed arrows indicate the Buddy-TAL binding sites. The boxes marked with the vertical line "TBS" refer to the TAL binding sites. "RNA Pol" refers to the RNA polymerase molecule that transcribes nucleic acid "downstream" from the "promoter".

[0151] Fig.52 The amino acid sequence of Buddy-TAL (SEQ ID NO: 143) is shown, which is the expression product of the nucleotide sequence described in SEQ ID NO: 18. This Buddy-TAL has two NLSs (boxed), one at the N-terminus and one at the C-terminus of the protein. In addition, the transcriptional activation domain that is usually present near the C-terminus has been deleted. The central region of the protein with underline is the repeat region. Two linkers (GS and GG) are also shown in the box. DETAILED DESCRIPTION

[0152] Overview

[0153] The compositions and methods described herein are intended to improve gene editing. As examples, these improvements include the following:

[0154] i. inserting a nucleic acid molecule (eg, a donor DNA molecule) into an intracellular nucleic acid molecule, wherein the inserted nucleic acid molecule is operably linked to a promoter present in the intracellular nucleic acid molecule.

[0155] ii. Use of non-homologous end joining inhibitors to promote gene editing.

[0156] iii. Use of DNA binding molecules (eg, DNA binding proteins, DNA binding protein / nucleic acid complexes) that bind at or near a target locus within a cell, wherein the DNA binding protein promotes enhanced accessibility of the target locus to other DNA binding molecules.

[0157] iv. Donor DNA is delivered intracellularly to the gene editing locus, and other DNA molecules are delivered to different locations within the cell (e.g., a linear DNA molecule containing an open reading frame is operably linked to a promoter and delivered to mitochondria).

[0158] The above improvements may be used individually or in combination with the other methods listed above as well as additional methods.

[0159] The present disclosure relates, in part, to the discovery that the combined use of promoter capture for a selectable marker and short homology arms for recombination achieves nearly 100% integration efficiency and up to 100% precise HDR.

[0160] Unlike the traditional method of using a targeting vector with 0.5kb to 2kb homology arms, the use of short homology arms seems to minimize the incidence of random integration of the foreign DNA of interest into the genome. Most importantly, the use of promoter capture for selection markers can select species that are correctly integrated because promoter-free selection markers are only expressed when the DNA molecule is accurately inserted into the genomic locus. In some embodiments, end modification of donor DNA with, for example, phosphorothioate or amino groups and / or treatment with NHEJ inhibitors further improves the efficiency of HDR. The accuracy of donor DNA integration is sequence-dependent. At some loci, 100% integration efficiency and 100% accurate HDR can be achieved.

[0161] The present disclosure also relates, in part, to compositions and methods for enhancing the accessibility of intracellular nucleic acid regions to molecules or molecular complexes that interact with intracellular nucleic acids in these regions.

[0162] The present disclosure further relates, in part, to compositions and methods for intracellular localization of nucleic acid molecules.In some cases, the nucleic acid molecule is a donor DNA molecule.

[0163] The present disclosure also relates to various combinations of the above to facilitate processes such as gene editing, gene activation, gene repression, DNA methylation, etc.

[0164] The present invention relates in part to compositions and methods for enhancing gene editing. Many variables affect the efficiency of gene editing. With respect to homology-directed repair (HDR), these factors include:

[0165] (1) (i) donor DNA and (ii) the amount of site-specific nuclease located in the cell nucleus, and the amount of site-specific nuclease activity in the cell nucleus,

[0166] (2) Accessibility of the target locus to site-specific nucleases,

[0167] (3) Timing aspects related to the presence of the donor and the nuclease in the nucleus,

[0168] (4) Target locus cleavage efficiency,

[0169] (5) HDR efficiency (including HDR:NHEJ ratio), and

[0170] (6) Donor DNA structure and composition.

[0171] It is expected that in some cases, especially with respect to HDR, gene editing efficiencies approaching 100% can be achieved.

[0172] Localization of gene editing reagents in the nucleus: Since many factors that influence gene editing efficiency are thought to be based on concentration-dependent mechanisms, the higher the amount of site-specific nuclease activity (a combination of the level of nuclease activity and the number of nucleases present) and the higher the concentration of donor DNA in the nucleus, the more HDR is expected to dominate NHEJ.

[0173] Although nucleic acid molecules and proteins can be produced in cells (e.g., vector-based systems), in many cases, the components of the gene editing system (e.g., donor DNA, site-specific nucleases, DNA binding regulatory enhancers, etc.) are introduced into cells. Such cell introduction can be accomplished using methods such as transfection and electroporation.

[0174] After the gene editing system components are introduced into the cell, they are generally required to be effectively localized to the nucleus. This is done because it is believed that the effective localization of the gene editing system components to nucleases is at least partially related to cytoplasmic degradation (a combination of (i) degradation activity and (ii) the amount of time spent in the cytoplasm). In addition, many factors can affect the efficiency of nuclear localization, including (1) the binding of the gene editing system components to one or more NLSs, (2) the choice of the NLS used, and (3) chemical modification of one or more gene editing system components (e.g., donor DNA).

[0175] In many cases, the nucleic acid molecules used in the methods described herein can be chemically modified. Chemical modifications include nuclease resistance groups, such as phosphorothioate groups, amino groups, 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxy nucleotides, 5-C-methyl nucleotides and combinations thereof. For example, the three nucleotides on the 5' and 3' end gRNA molecules can contain phosphorothioate bonds and / or can be 2'-O-methyl nucleotides. It has also been found that amine-terminal modifications of donor DNA enhance HDR (see, e.g. Fig. 7A and 7B ). In both cases, it is believed that this is at least partly due to the stabilization of the donor DNA molecule in the cytoplasm. The gRNA is also believed to be stabilized by binding to the Cas9 protein. It is therefore believed that when the gRNA is bound to the Cas9 protein, the cytoplasmic half-life of the gRNA is increased.

[0176] The data show and it is believed that when the gene editing system components are stable in terms of cytoplasmic degradation and quickly "shuttle" to the nucleus through the cytoplasm, the gene editing efficiency is improved. The rapid movement of the gene editing system components through the cytoplasm has another effect, which will be beneficial in many cases. This makes the gene editing system component activity reach high concentrations in the nucleus instantaneously, and obtains a lower cytoplasmic gene editing system component pool. Therefore, once the high concentration of gene editing system component activity in the nucleus is exhausted, there is little or no cytoplasmic reserve for additional gene editing activity.

[0177] Site-specific target locus cleavage activity: The efficiency of target locus cleavage is determined by many factors, some of which are described above. These factors include: (1) the gene editing system cleavage activity, (2) the amount of gene editing system components that mediate cleavage present at or near the target locus, and (3) the accessibility of the target locus to the gene editing system cleavage activity, as described herein.

[0178] The accessibility of the gene editing system cleavage activity to the target locus can vary with natural action, as it is accessible or inaccessible to somewhere in the genome or a particular cell type or in between. Inducing transcriptional activation of the target locus before cleavage of that target locus can make the locus more accessible to the cleavage activity. Another way to increase the accessibility of a specific target locus is through the use of DNA binding regulatory enhancers.

[0179] One consideration regarding site-specific target locus cleavage activity is "off-target" effects. Off-target effects can be minimized by using DNA binding regulatory enhancers, gRNA with high target locus specificity, and high-fidelity gene editing reagents (e.g., high-fidelity Cas9), either alone or in combination.

[0180] Target locus changes: There are two main types of gene editing that are usually performed. These gene edits are to insert a nucleic acid molecule into the target locus and not to insert a nucleic acid molecule into the target locus, but to change the nucleotide sequence of the target locus. In addition, there are three possibilities when cleaving and "repairing" the target locus. The target locus can be (1) unchanged compared to the nucleotide sequence before cleavage; (2) modified by deletion or addition of one or more bases without donor nucleic acid insertion, or (3) donor DNA insertion can be introduced at or near the cleavage site. The first two of these possibilities are usually caused by NHEJ-based repair mechanisms. The third of these possibilities is usually based on HDR-based mechanisms. In many cases, particularly when it is necessary to insert donor DNA into the target locus, the third possibility is preferred. Therefore, provided herein are compositions and methods for enhancing HDR efficiency and / or being more conducive to HDR relative to NHEJ.

[0181] It has been found that many factors cause effective HDR and insert the donor nucleic acid molecule into the cleavage site. Some of these factors are related to the characteristics of the donor nucleic acid molecule. One of these factors is the length of the donor DNA homology arm. In many cases, the donor DNA molecule will have two homology arms, and its length range is independently about 20 to about 2,000 nucleotides or base pairs, depending on whether the donor DNA is single-stranded or double-stranded. In addition, the double-stranded donor DNA can have 3' protrusions at one or both ends, and the length range of these protrusions (and 5' protrusions) can be about 10 to about 40 nucleotides. Similarly, one or two chains of one or two homology arms of the donor DNA molecule can include one or more nuclease resistance groups (as discussed elsewhere herein) located at the end or other positions in the arm.

[0182] There are a number of ways to exploit HDR for NHEJ repair. One approach is to treat cells undergoing gene editing with one or more NHEJ inhibitors (see Figure 7B ). Another is to "reduce" intracellular NHEJ activity. This can be achieved by using, for example, antisense microRNA and / or RNAi agents designed to inhibit the expression of one or more NHEJ repair pathways (e.g., DNA-dependent protein kinase, catalytic subunit; Ku70 and / or Ku80).

[0183] definition

[0184] As used in accordance with this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0185] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded, double-stranded or multi-stranded forms, or their complements. The term "polynucleotide" refers to a linear sequence of nucleotides. The term "nucleotide" generally refers to a single unit, i.e., a monomer, of a polynucleotide. A nucleotide can be a ribonucleotide, a deoxyribonucleotide, or a modified form thereof. Examples of polynucleotides encompassed herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA (including siRNA), and hybrid molecules with a mixture of single-stranded and double-stranded DNA and RNA. Nucleic acids can be straight or branched. For example, a nucleic acid can be a straight chain of nucleotides, or a nucleic acid can be branched, for example, so that the nucleic acid comprises one or more nucleotide arms or branches. Optionally, branched nucleic acids are repeatedly branched to form higher-order structures, such as dendritic polymers, etc.

[0186] The term also includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, with binding properties similar to reference nucleic acids, and the mode of metabolism is similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphodiester derivatives, including, for example, phosphoramidates, diaminophosphoroesters, thiophosphates (phosphorothioate) (also referred to as thiophosphates (phosphothioate)), dithiophosphates, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methylphosphonates, borophosphonates, or O-methylphosphoramidate linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press, Oxford University Press); and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars and non-ribose backbones (e.g., diaminophosphoester morpholino oligonucleotides or locked nucleic acids (LNAs)), including those described in the following documents: U.S. Pat. Nos. 5,235,033 and 5,034,506 and ASC Symposium Series 580, Chapters 6 and 7, Carbohydrate Modifications in Antisense Research, Sanghui and Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included in a definition of nucleic acids. Modification of the ribose-phosphate backbone can be for a variety of reasons, such as increasing the stability and half-life of such molecules in a physiological environment or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be prepared; alternatively, mixtures of different nucleic acid analogs can be prepared, as well as mixtures of naturally occurring nucleic acids and analogs. In an embodiment, the internucleotide linkage in DNA is a phosphodiester, a phosphodiester derivative, or a combination of the two.

[0187] Nucleic acid can include non-specific sequence. As used herein, the term "non-specific sequence" refers to a nucleic acid sequence containing a series of residues that are not designed to be complementary to any other nucleic acid sequence or are only partially complementary. For example, a non-specific nucleic acid sequence is a sequence of nucleic acid residues that do not function as an inhibitory nucleic acid when contacted with a cell or an organism." Inhibitory nucleic acid" is a nucleic acid (e.g., a polymer of DNA, RNA, nucleotide analogs) that can bind to a target nucleic acid (e.g., an mRNA that can be translated into protein) and reduce the transcription of a target nucleic acid (e.g., mRNA from DNA) or reduce the translation of a target nucleic acid (e.g., mRNA) or change transcript splicing (e.g., single-stranded morpholino oligonucleotides).

[0188] As used herein, the term "nucleic acid molecule" refers to a covalently linked sequence of nucleotides or bases (e.g., ribonucleotides of RNA and deoxyribonucleotides of DNA, and includes DNA / RNA hybrids, wherein the DNA is in a separate chain or in the same chain), wherein the 3' position of the pentose of one nucleotide is joined to the 5' position of the pentose of the next nucleotide by a phosphodiester linkage. Nucleic acid molecules can be single-stranded or double-stranded or partially double-stranded. Nucleic acid molecules can appear in linear or cyclized forms with blunt or sticky ends when supercoiled or relaxed and can contain "nicks". Nucleic acid molecules can be composed of fully complementary single strands or partially complementary single strands that form at least one base mismatch. Nucleic acid molecules can additionally include two self-complementary sequences, which can form a double-stranded stem region, optionally separated by a loop sequence at one end. The two regions of the nucleic acid molecule comprising the double-stranded stem region are substantially complementary to each other, thereby producing self-hybridization. However, the stem may include one or more mismatches, insertions or deletions. As described above, nucleic acid molecules can include chemical, enzymatic or metabolic modified forms of nucleic acid molecules or combinations thereof. Chemically synthesized nucleic acid molecules may refer to nucleic acids that are generally less than or equal to 150 nucleotides long (e.g., 5 to 150, 10 to 100, 15 to 50 nucleotides long), while enzymatically synthesized nucleic acid molecules may cover smaller and larger nucleic acid molecules, as described elsewhere in this application. The enzymatic synthesis of nucleic acid molecules may include a stepwise method using an enzyme (such as a polymerase, a ligase, an exonuclease, an endonuclease, etc., or a combination thereof). As provided herein, the term "genome editing" or "gene editing" refers to a stepwise method involving an enzyme, such as a polymerase, a ligase, an exonuclease, an endonuclease, etc., or a combination thereof. For example, gene editing may include the following methods: cleaving a nucleic acid molecule, excising nucleotides at or near a cleavage site, newly synthesizing new nucleotides, and joining the cleaved chain.

[0189] The term nucleic acid molecule also refers to a short nucleic acid molecule, commonly referred to as, for example, a "primer" or "probe". Primers are commonly referred to as single-stranded starting nucleic acid molecules for enzymatic assembly reactions, and probes are commonly used to detect at least partially complementary nucleic acid molecules. Nucleic acid molecules have a "5' end" and a "3' end" because the nucleic acid molecule phosphodiester linkage is present between the 5' carbon and the 3' carbon of the pentose ring of the substituent mononucleotide. The end of the nucleic acid molecule newly linked to the 5' carbon is its 5' end nucleotide. The end of the nucleic acid molecule newly linked to the 3' carbon is its 3' end nucleotide. As used herein, a terminal nucleotide or base is a nucleotide at the terminal position of the 3' end or the 5' end. A nucleic acid molecule sequence may also be referred to as having a 5' end and a 3' end, even if it is inside a larger nucleic acid molecule (e.g., a sequence region within a nucleic acid molecule).

[0190] As used herein, "vector" is a nucleic acid molecule that can be used as a medium for transferring genetic material into a cell. A vector can be a plasmid, a virus or a phage, a cosmid or an artificial chromosome, such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or other sequences that can be replicated or replicated in vitro or in a host cell, or a desired nucleic acid segment is delivered to a desired position in a host cell. In an embodiment, a vector refers to a DNA molecule with at least one replication origin, a multiple cloning site (MCS) and one or more selection markers. A vector is generally composed of a backbone region and at least one insertion or transgenic region or a region (such as MCS) designed for inserting a DNA fragment or a transgenic. The backbone region generally contains a replication origin and one or more selection markers for breeding in at least one subject. A vector can have one or more restriction endonuclease recognition sites (such as two, three, four, five, seven, ten, etc.), and a sequence can be cut at the recognition site in a measurable manner without losing the basic biological function of the vector, and a nucleic acid fragment can be spliced ​​in the recognition site to achieve its replication and cloning. The vector may further provide primer sites (e.g., for PCR), transcription and / or translation initiation and / or regulatory sites, recombination signals, replicons, selectable markers, and the like. Obviously, methods for inserting the desired nucleic acid fragments can also be applied without the need to use recombination, transposition, or restriction enzymes (such as, but not limited to, uracil N-glycosylase (UDG) cloning PCR fragments (U.S. Pat. Nos. 5,334,575 and 5,888,795, both of which are fully incorporated herein by reference), T:A cloning, etc.) to clone the fragments into the cloning vector to be used according to the present invention. In an embodiment, the vector comprises additional features. Such additional features may include natural or synthetic promoters, genetic markers, antibiotic resistance cassettes or selection markers (e.g., toxins such as ccdB or tse2), antigenic determinants or tags for detection, manipulation, or purification (e.g., V5 antigenic determinants, c-myc, hemagglutinin (HA), FLAG TM, polyhistidine (His), glutathione-S-transferase (GST), maltose binding protein (MBP)), scaffold attachment region (SAR) or reporter gene (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP), luciferase, β-galactosidase, etc.). In an embodiment, a vector is used to separate, propagate or express the inserted DNA fragment in a target host. The vector can be, for example, a cloning vector, an expression vector, a functional vector, a capture vector, a co-expression vector (for expressing more than one open reading frame), a viral vector or an episome (i.e., a nucleic acid capable of extrachromosomal replication), etc.

[0191] As used herein, "cloning vector" includes any vector that can be used to delete, insert, replace or assemble one or more nucleic acid molecules. In an embodiment, the cloning vector may contain a counter-selection marker gene (e.g., ccdB or tse2) that can be removed or replaced by another transgene or DNA fragment. In an embodiment, the cloning vector may be referred to as a donor vector, entry vector, shuttle vector, destination vector, target vector, functional vector or capture vector. The cloning vector typically contains a series of unique restriction enzyme cleavage sites (e.g., type II or type IIS) for removing, inserting or replacing DNA fragments. Alternatively, the DNA fragment can be removed or replaced by another transgene or DNA fragment. Cloning or recombination to replace or insert, such as provided by Invitrogen / Life Technologies (Carlsbad, CA) Cloning or recombination employed in cloning systems and described in more detail elsewhere herein. A cloning vector that can be used to express a transgene in a target host may also be referred to as an expression vector. In an embodiment, a cloning vector is engineered to obtain a TAL effector conjugate.

[0192] An "expression vector" is designed to express a transgene and typically contains at least one promoter sequence that drives transgene expression. As used herein, expression refers to transcription of a transgene or transcription and translation of an open reading frame, and may occur in a cell-free environment (e.g., a cell-free expression system) or in a host cell. In an embodiment, expression of an open reading frame or gene causes the production of a polypeptide or protein. Expression vectors are typically designed to contain one or more regulatory sequences, such as enhancers, promoters, and terminator regions that control the expression of the inserted transgene. Suitable expression vectors include, but are not limited to, plasmids and viral vectors. Vectors and expression systems for various applications are available from commercial suppliers, such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Life Technologies Corp. (Carlsbad, CA). In an embodiment, the expression vector is engineered to express a TAL effector fusion.

[0193] "Viral vectors" generally refer to genetically engineered non-infectious viruses comprising modified viral nucleic acid sequences. In embodiments, viral vectors comprise at least one viral promoter and are designed to insert one or more transgenes or DNA fragments. In embodiments, viral vectors are delivered to a target host together with a helper virus that provides packaging or other functions. In embodiments, viral vectors are used to stably integrate transgenes into the genome of a host cell. Viral vectors can be used for delivery and / or expression of transgenes.

[0194] Viral vectors may be derived from bacteriophage, baculovirus, tobacco mosaic virus, vaccinia virus, retrovirus (avian leukosis sarcoma, mammalian C, B, D viruses, HTLV-BLV group, lentivirus, foamy virus), adenovirus, parvovirus (e.g., adeno-associated virus), coronavirus, negative-strand RNA virus (e.g., orthomyxovirus (e.g., influenza virus) or Sendai virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai virus), positive-strand RNA virus (e.g., picornavirus and alpha virus (e.g., Semliki Forest virus)) and double-stranded DNA virus, including adenovirus, herpes virus (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus) and pox virus (e.g., vaccinia, fowlpox and canarypox). Other viruses include, but are not limited to, Norwalk virus (Norwalk virus). Viruses, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. For example, a common viral vector for delivering genes is a lentiviral vector, based on its relatively large packaging capacity, reduced immunogenicity, and its ability to efficiently and stably transduce a variety of different cell types. Such lentiviral vectors can be "integrative" (i.e., capable of integrating into the genome of the target cell) or "non-integrative" (i.e., not integrated into the genome of the target cell). Expression vectors containing regulatory elements from eukaryotic viruses are often used. In eukaryotic expression vectors, such as SV40 vectors, papillomavirus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that allows protein expression under the guidance of the SV40 early promoter, the SV40 late promoter, the metallothionein promoter, the mouse mammary tumor virus promoter, the Rous sarcoma virus promoter, the polyhedrin promoter, or other promoters that have been shown to be effectively expressed in eukaryotic cells.

[0195] A "labeled nucleic acid or oligonucleotide" is a nucleic acid or oligonucleotide that is bound to a label covalently, via a linker or chemical bond, or non-covalently, via ionic, van der Waals, electrostatic or hydrogen bonding, so that the presence of the nucleic acid can be detected by detecting the presence of a detectable label bound to the nucleic acid. Alternatively, the same result can be obtained using a high affinity interaction method in which one of a pair of binding partners binds to the other, e.g., biotin, streptavidin. In an embodiment, the phosphorothioate nucleic acid or phosphorothioate polymer backbone includes a detectable label as disclosed herein and generally known in the art.

[0196] As used herein, the term "probe" or "primer" is defined as one or more nucleic acid fragments whose specific hybridization with a sample can be detected. The probe or primer can be of any length, depending on the specific technology for which it will be used. For example, the length of a PCR primer is usually between 10 and 40 nucleotides, and the length of a nucleic acid probe for, for example, Southern blotting can exceed one hundred nucleotides. The probe can be unlabeled or labeled as described below so that its binding to a target or sample can be detected. The probe can be produced by a nucleic acid source from one or more specific (preselected) parts of a chromosome, such as one or more clones, isolated full chromosomes or chromosome fragments, or a collection of polymerase chain reaction (PCR) amplification products. The length and complexity of the nucleic acid fixed on the target element are not critical for the present invention. The technician can adjust these factors to provide optimal hybridization and signal generation for a given hybridization program, and provide required resolution between different genes or genomic positions.

[0197] The probe can also be a separated nucleic acid fixed on a solid surface (e.g., nitrocellulose, glass, quartz, fused silica slide), such as in an array. In some embodiments, the probe can be a member of a nucleic acid array, such as described in WO 96 / 17958. Technologies capable of producing high-density arrays can also be used for this purpose (see, e.g., Fodor, Science, 251: 767-773 (1991); Johnston, Curr. Biol. 8: R171-R174 (1998); Schummer, Biotechniques, 23: 1087-1092 (1997); Kern, Biotechniques, 23: 120-124 (1997); U.S. Patent No. 5,143,854).

[0198] The words "complementary" or "complementarity" refer to the ability of a nucleic acid in a polynucleotide to form base pairs with another nucleic acid in a second polynucleotide. For example, the sequence AGT is complementary to the sequence TCA. Complementarity can be partial, in which only some of the nucleic acids match according to base pairing, or complete, in which all nucleic acids match according to base pairing.

[0199] The term "isolated" when applied to a nucleic acid or protein means that the nucleic acid or protein is substantially free of other cellular components with which it is naturally associated. It may, for example, be in a homogeneous state and may be present in anhydrous or aqueous solutions. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0200] The term "purified" means that the nucleic acid or protein produces essentially one band in an electrophoretic gel. In some embodiments, the nucleic acid or protein is at least 50% pure, optionally at least 65% pure, optionally at least 75% pure, optionally at least 85% pure, optionally at least 95% pure, and optionally at least 99% pure.

[0201] The term "separated" may also refer to a cell or sample cell. Separated cells or sample cells are single cell types that are substantially free of many components that normally accompany cells when the cell is in its native state or when the cell is initially removed from its native state. In certain embodiments, the separated cell sample retains those components required for maintaining the cell in the desired state from its native state. In certain embodiments, the separated (e.g., purified, separated) cell or separated cell is a cell that is substantially the only cell type in the sample. The purified cell sample may contain at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of a cell type. The separated cell sample may be obtained by using a cell marker or a cell marker combination, any of which is unique for a cell type in an unpurified cell sample. In certain embodiments, cells are separated by using a cell sorter. In certain embodiments, cells are separated using antibodies to cell proteins.

[0202] As used herein, "wild-type sequence" refers to any given sequence (e.g., an isolated sequence) that can be used as a template for subsequent reactions or modifications. As understood by the technician, a wild-type sequence can include a nucleic acid sequence (e.g., DNA or RNA or a combination thereof) or an amino acid sequence, or can be composed of different chemical entities. In some embodiments, a wild-type sequence can refer to a computer-simulated sequence, which can be such sequence information or sequence data that can be stored in a computer-readable medium in a form that is readable and / or editable by a mechanical device. A wild-type sequence (reflecting nucleotides or amino acid symbols in a given order) can be entered into, for example, a customer portal through a Web interface. In an embodiment, the sequence initially provided by a customer is considered to be a wild-type sequence in view of the downstream process based thereon, regardless of whether the sequence itself is a natural sequence or a modified sequence, that is, modified relative to another wild sequence, or completely artificial.

[0203] In an embodiment, wild-type sequence may also refer to entity molecules, such as nucleic acid molecules (such as RNA or DNA or a combination thereof) or proteins, polypeptides or peptides composed of amino acids. Methods for obtaining wild-type sequences by chemical, enzymatic or other means are known in the art. In one embodiment, entity nucleic acid wild-type sequences can be obtained by PCR amplification of the corresponding template region, or can be synthesized de novo based on the assembly of synthetic oligonucleotides. Wild-type sequences used herein may encompass naturally occurring and artificial (such as chemical or enzyme modified) parts or structural units. Wild-type sequences may be composed of two or more sequence portions. Wild-type sequences may be, for example, coding regions, open reading frames, expression cassettes, effector domains, repeat domains, promoter / enhancer or terminator regions, untranslated regions (UTRs), but may also be defined sequence motifs, such as binding, recognition or cleavage sites within a given sequence. Wild-type sequences may be DNA or RNA of any length, and may be linear, circular or branched, and may be single-stranded or double-stranded.

[0204] As used herein, the term "conjugate" refers to the combination between atoms or molecules. The combination can be direct or indirect. For example, the conjugate between the first part (e.g., nuclease part) and the second part (DNA binding part) provided herein can be direct, such as by covalent bonds, or indirect, such as by non-covalent bonds (e.g., electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (π effect), hydrophobic interactions, etc.). In an embodiment, the conjugate is formed using conjugate chemistry, including but not limited to nucleophilic substitution (e.g., reaction of amines and alcohols with acyl halides, active esters), electrophilic substitution (e.g., enamine reaction), and carbon-carbon and carbon-heteroatom multiple bond additions (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed, for example, in March, ADVANCED ORGANIC CHEMISTRY, 3rd ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, D.C., 1982. In embodiments, the first moiety (e.g., nuclease moiety) and the second moiety (peptide moiety) are non-covalently linked by a non-covalent chemical reaction between a component of the first moiety (e.g., nuclease moiety) and a component of the second moiety (DNA binding moiety). In other embodiments, the first moiety (e.g., polyamine moiety) includes one or more reactive moieties, e.g., a covalent reactive moiety as described herein (e.g., an alkyne, azide, maleimide, or thiol reactive moiety). In other embodiments, the first portion (e.g., the nuclease portion) comprises a linker having one or more reactive moieties, e.g., a covalent reactive moiety as described herein (e.g., an alkyne, azide, maleimide, or a thiol reactive moiety). In other embodiments, the second portion (the DNA binding portion) comprises one or more reactive moieties, e.g., a covalent reactive moiety as described herein (e.g., an alkyne, azide, maleimide, or a thiol reactive moiety). In other embodiments, the second portion (the DNA binding portion) comprises a linker having one or more reactive moieties, e.g., a covalent reactive moiety as described herein (e.g., an alkyne, azide, maleimide, or a thiol reactive moiety).

[0205] As used herein, the term "about" refers to a range of values ​​that includes the specified value and that one of ordinary skill in the art would consider to be reasonably similar to the specified value. In the embodiments, the term "about" refers to within the standard deviation of measurements generally accepted in the art. In the embodiments, about refers to a range extending to + / -10% of the specified value. In the embodiments, about refers to a specific value.

[0206] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may be bound to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of the corresponding amino acid occurring in nature, as well as to naturally occurring amino acid polymers and to non-naturally occurring amino acid polymers. The terms apply to macrocyclic peptides, peptides that have been modified with non-peptide functional groups, peptide mimetics, polyamides, and macrolactams. "Fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single part.

[0207] The terms "peptidyl," "peptide moiety," "protein moiety," and "peptidyl moiety" refer to a monovalent peptide or protein.

[0208] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphorylated serine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an alpha carbon bound to a hydrogen, carboxyl group, amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids. The terms "non-naturally occurring amino acids" and "non-natural amino acids" refer to amino acid analogs, synthetic amino acids, and amino acid mimetics that are not found in nature.

[0209] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides likewise may be referred to by their commonly accepted single-letter codes.

[0210] The amino acid or nucleotide base "position" is represented by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5' end). Due to deletions, insertions, truncations, fusions, etc. that must be considered when determining the optimal alignment, the number of amino acid residues in the test sequence, which is usually determined by simple counting from the N-terminus, is not necessarily the same as the number of its corresponding position in the reference sequence. For example, in the case where a variant has a deletion relative to the aligned reference sequence, there is no amino acid in the variant that corresponds to the position of the deletion site in the reference sequence. When there is an insertion in the aligned reference sequence, the insertion does not correspond to the amino acid position numbered in the reference sequence. In the case of truncations or fusions, an amino acid segment in the reference sequence or aligned sequence may not correspond to any amino acid in the corresponding sequence.

[0211] The terms "numbering reference" or "corresponding to" when used in the context of numbering a given amino acid or polynucleotide sequence refers to the numbering of the residues of the reference sequence to which the given amino acid or polynucleotide is compared.

[0212] "Conservatively modified variants" apply to both amino acid sequences and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to those nucleic acids that encode identical or substantially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence; substantially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variations. Each nucleic acid sequence encoding a polypeptide herein also describes each possible silent variation of the nucleic acid. The skilled person will recognize that each codon in the nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce functionally identical molecules. Therefore, each silent variation of a nucleic acid encoding a polypeptide is implicit in each of the described sequences for the expression product, but does not involve the actual probe sequence.

[0213] With respect to amino acid sequences, the skilled artisan will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or protein sequence that alter, add or delete a single amino acid or a small portion of amino acids in the encoded sequence are "conservatively modified variants," wherein the alteration results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs and alleles of the present invention.

[0214] The following eight groups each contain amino acids that are conservative substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M).

[0215] The "sequence identity percentage" is determined by comparing two optimally aligned sequences in a comparison window, wherein the polynucleotide or polypeptide sequence portion in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence (not including additions or deletions) to achieve optimal alignment of the two sequences. The percentage is calculated as follows: the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences is determined to produce the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window and the result is multiplied by 100 to obtain the sequence identity percentage.

[0216] The term "identical" or "identity" percentage in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that have the same amino acid residues or nucleotides or have a specified percentage of the same amino acid residues or nucleotides (i.e., 60% identical, as appropriate 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical over, for example, a specified region of the entire polypeptide sequence of the invention or an individual domain of a polypeptide of the invention) when compared and aligned according to maximum consistency over a comparison window or a specified region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then referred to as "substantially identical". The definition also refers to the complement of the test sequence. Optionally, the identity exists over a region of at least about 50 nucleotides in length, or more preferably over a region of 100 to 500 or 1000 or more nucleotides in length.

[0217] When comparing sequences, usually one sequence acts as a reference sequence for comparison with a test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters.

[0218] As used herein, "comparison window" includes a segment referring to any of a plurality of consecutive positions, selected from the group consisting of, for example, a full-length sequence or 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides, wherein after the two sequences are optimally aligned, a sequence can be compared to a reference sequence having the same number of consecutive positions. Methods for comparing sequences are well known in the art. Sequences can be optimally aligned for comparison, for example, according to the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, according to the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, according to the similarity search method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, 1996). Biology) (1995 supplement)).

[0219] Examples of algorithms suitable for determining percentages of sequence identity and sequence similarity are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25: 3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215: 403-410, respectively. Software for BLAST analysis can be publicly obtained through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which match or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., as above). These initial neighborhood word hits serve as seeds for starting searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of the word hits in each direction is interrupted when the cumulative alignment score decreases by an amount X from its maximum achieved value; when the cumulative score becomes zero or below zero due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation (E) or 10, M=5, N=-4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3 and expectation (E) of 10 and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)), alignments (B) 50, expectation (E) 10, M=5, N=-4, and a comparison of both strands.

[0220] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. 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 that 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 when the test nucleic acid is compared to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0221] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only in conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules, or their complements, hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[0222] "Contacting" is used according to its clear ordinary meaning and refers to a method of allowing at least two different species to become close enough to react, interact or physically contact. However, it should be understood that the resulting reaction product can be prepared directly from the reaction between the added reagents or from intermediates that can be produced in the reaction mixture from one or more of the added reagents. In an embodiment, contacting includes, for example, allowing a ribonucleic acid as described herein to interact with an endonuclease and an enhancer element.

[0223] "Control" sample or value refers to a sample used as a reference (usually a known reference for comparison with a test sample). For example, a test sample can be taken from a test condition, such as in the presence of a test compound (such as a first or second DNA binding modulator), and compared with a sample from a known condition, such as in the absence of a test compound (negative control) or in the presence of a known compound (positive control). Control can also represent a mean value collected from a large number of tests or results. Those skilled in the art will recognize that controls for evaluating any number of parameters can be designed. Those skilled in the art will understand which standard controls are most appropriate in a given situation and can analyze data based on comparisons with standard control values. Standard controls are also valuable for determining the significance (such as statistical significance) of data. For example, if the value of a given parameter varies greatly in a standard control, the change in the test sample will not be considered significant.

[0224] A "label" or "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., enzymes commonly used in ELISA), biotin, digoxigenin, or haptens and proteins or other entities that can be detected, for example, by incorporating a radioactive label into a peptide or antibody that specifically reacts with the target peptide. Any suitable method known in the art for conjugating an antibody to a label can be used, for example, using the method described in Hermanson, Bioconjugate Techniques, 1996, Academic Press, Inc., San Diego.

[0225] A "labeled protein or polypeptide" is a protein or polypeptide that is covalently bound to a label by a linker or chemical bond or non-covalently bound by ionic, van der Waals, electrostatic or hydrogen bonding, such that the presence of the labeled protein or polypeptide can be detected by detecting the presence of the label bound to the labeled protein or polypeptide. Alternatively, the same result can be obtained using a high affinity interaction method, in which one of a pair of binding partners binds to the other, e.g., biotin, streptavidin.

[0226] "Biological sample" or "sample" refers to a substance obtained from or derived from a subject or patient. Biological samples include tissue sections, such as biopsy samples and autopsy samples, and frozen sections taken for histological purposes. Such samples include body fluids, such as blood and blood parts or products (such as serum, plasma, platelets, red blood cells, etc.), sputum, tissue, cultured cells (such as primary cultures, explants, and transformed cells), feces, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. Biological samples are typically obtained from eukaryotic organisms, such as mammals, such as primates, such as chimpanzees or humans; cattle; dogs; cats; rodents, such as guinea pigs, rats, mice; rabbits; or birds; reptiles; or fish.

[0227] As used herein, "cell" refers to a cell that performs a metabolic or other function sufficient to preserve or replicate its genomic DNA. Cells can be identified by methods well known in the art, including, for example, the presence of an intact membrane, staining with a specific dye, the ability to produce offspring, or, in the case of gametes, the ability to combine with a second gamete to produce viable offspring. Cells can include prokaryotic cells and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, such as mammals, insects (e.g., Spodoptera) and human cells.

[0228] The term "gene" refers to a DNA segment involved in producing a protein; it includes regions before and after the coding region (enhancers, promoters, leader sequences and tail sequences) and intermediate sequences (introns) between individual coding segments (exons). Enhancers, promoters, leader sequences, tail sequences and introns include regulatory elements necessary during gene transcription and translation. In addition, a "protein gene product" is a protein expressed from a specific gene.

[0229] As used herein, the words "expression" or "expressed" with respect to a gene refer to the transcription and / or translation product of the gene. The expression level of a DNA molecule in a cell can be determined based on the amount of the corresponding mRNA present in the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88).

[0230] The expression of the transfected gene can occur transiently or stably in the cell. During "transient expression", the transfected gene is not transferred to daughter cells during cell division. Because its expression is confined to the transfected cells, the expression of the gene is lost over time. In contrast, when the gene is co-transfected with another gene that confers a selective advantage to the transfected cells, stable expression of the transfected gene can occur. Such a selective advantage may be resistance to a certain toxin presented to the cell.

[0231] The term "plasmid" refers to a nucleic acid molecule that encodes genes and / or regulatory elements necessary for gene expression. Expression of genes from a plasmid can occur in cis or in trans. If a gene is expressed in cis, the gene and regulatory elements are encoded by the same plasmid. Trans expression refers to the situation where the gene and regulatory elements are encoded by separate plasmids.

[0232] The term "episome" refers to the extrachromosomal state of a plasmid in a cell. An episomal plasmid is a nucleic acid molecule that is not part of the chromosomal DNA and replicates independently of it.

[0233] The term "exogenous" refers to molecules or substances (eg, nucleic acids or proteins) that originate from outside a given cell or organism. In contrast, the term "endogenous" refers to molecules or substances that are native to or originate from within a given cell or organism.

[0234] "Cell culture" is an in vitro cell group present outside the organism.Cell culture can be established by primary cells separated from cell banks or animals, or by immortalizing cells derived from one of these sources and carrying out long-term in vitro culture.Cell culture as provided herein further refers to an environment including suitable cell nutrients and capable of maintaining cells in vitro.The environment can be a liquid environment, a solid environment and / or a semi-solid environment (e.g., agar, gel, etc.) in a suitable container (e.g., a cell culture dish).Cell culture medium can be used.As used herein, "cell culture medium" is used according to its generally accepted meaning in the art.Cell culture medium (also referred to as "culture medium" in the art and herein) includes liquids (e.g., growth factors, minerals, vitamins, etc.) or gels designed to support cell growth (e.g., division, differentiation, maintenance, etc.).In an embodiment, the composition provided herein (including embodiments) further includes a physiologically acceptable solution.As provided herein, "physiologically acceptable solution" refers to any acceptable aqueous solution (e.g., buffer), wherein the composition provided herein can be included without losing its biological properties.In an embodiment, a physiologically acceptable solution is a cell culture medium.

[0235] The terms "transfection", "transduction", "transfecting" or "transducing" are used interchangeably and are defined as the process of introducing nucleic acid molecules and / or proteins into cells. Nucleic acids can be introduced into cells using non-viral or viral-based methods. Nucleic acid molecules can be sequences encoding complete proteins or functional portions thereof. Typically, nucleic acid vectors contain elements necessary for protein expression (e.g., promoters, transcription start sites, etc.). Non-viral transfection methods include any suitable method for introducing nucleic acid molecules into cells without using viral DNA or viral particles as a delivery system. Exemplary non-viral transfection methods include calcium phosphate transfection, liposome transfection, nucleofection, sonoporation, transfection by heat shock, magnetization transfection, and electroporation. For viral-based methods, any available viral vector can be used in the methods described herein. Examples of viral vectors include, but are not limited to, retroviral, adenoviral, lentiviral, and adeno-associated viral vectors. In some aspects, retroviral vectors are used to introduce nucleic acid molecules into cells according to standard procedures known in the art. The term "transfection" or "transduction" also refers to the introduction of proteins from the external environment into cells. Typically, protein transduction or transfection relies on the attachment of a peptide or protein that can cross the cell membrane to the protein of interest. See, for example, Ford et al., Gene Therapy 8:1-4 (2001) and Prochiantz, Nat. Methods 4:119-120 (2007).

[0236] As used herein, the term "specifically binds" or "specifically binds" refers to the formation of a complex between two molecules (eg, a DNA binding enhancer and an enhancer binding sequence) that is relatively stable under physiological conditions.

[0237] "Ribonucleoprotein complexes", "ribonucleoprotein particles", "deoxyribonucleoprotein complexes" or "deoxyribonucleoprotein particles" provided herein refer to complexes or particles comprising nucleoproteins and ribonucleic acids or deoxyribonucleic acids. "Nucleoproteins" as provided herein refer to proteins that can bind nucleic acids (e.g., RNA, DNA). In the case where nucleoproteins bind to ribonucleic acids, they are referred to as "ribonucleoproteins". In the case where nucleoproteins bind to deoxyribonucleic acids, they are referred to as "deoxyribonucleoproteins". The interaction between a ribonucleoprotein and a ribonucleic acid or the interaction between a deoxyribonucleoprotein and a ribonucleic acid can be direct, such as through a covalent bond, or indirect, such as through a non-covalent bond (e.g., electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (π effect), hydrophobic interactions, etc.). In an embodiment, a ribonucleoprotein comprises an RNA binding motif that is non-covalently bound to a ribonucleic acid. In an embodiment, the ribonucleoprotein comprises an RNA binding motif that is non-covalently bound to deoxyribonucleic acid. For example, positively charged aromatic amino acid residues (e.g., lysine residues) in RNA binding motifs or DNA binding motifs can form electrostatic interactions with the negative nucleic acid phosphate backbone of RNA or DNA, thereby forming a ribonucleoprotein complex or a deoxyribonucleoprotein complex (e.g., the Ager protein complex mentioned herein). Non-limiting examples of ribonucleoproteins include ribosomes, telomerase, RNAseP, hnRNP, CRISPR-associated protein 9 (Cas9) and small nuclear RNP (snRNP). An example of a deoxyribonucleoprotein is Ager protein. Ribonucleoprotein or deoxyribonucleoprotein can be an enzyme. In an embodiment, a ribonucleoprotein or a deoxyribonucleoprotein is an endonuclease. Therefore, in an embodiment, the ribonucleoprotein complex includes an endonuclease and ribonucleic acid. In an embodiment, the endonuclease is CRISPR-associated protein 9. Therefore, in an embodiment, the deoxyribonucleoprotein complex includes an endonuclease and deoxyribonucleic acid. In embodiments, the endonuclease is an arginase.

[0238] As provided herein, "guide RNA" or "gRNA" refers to a ribonucleotide sequence that can bind to a nucleoprotein to form a ribonucleoprotein complex. Similarly, as provided herein, "guide DNA" or "gDNA" refers to a deoxyribonucleotide sequence that can bind to a nucleoprotein to form a deoxyribonucleoprotein complex. In an embodiment, the guide RNA includes one or more RNA molecules. In an embodiment, the guide DNA includes one or more DNA molecules. In an embodiment, the gRNA includes a nucleotide sequence complementary to the target site (e.g., a regulator binding sequence). In an embodiment, the gDNA includes a nucleotide sequence complementary to the target site (e.g., a regulator binding sequence). The complementary nucleotide sequence can mediate the binding of a ribonucleoprotein complex or a deoxyribonucleoprotein complex to the target site, thereby providing the sequence specificity of a ribonucleoprotein complex or a deoxyribonucleoprotein complex. Therefore, in an embodiment, the guide RNA or guide DNA is complementary to a target nucleic acid (e.g., a regulator binding sequence). In an embodiment, the guide RNA binds to a target nucleic acid sequence (e.g., a regulator binding sequence). In an embodiment, the guide DNA binds to a target nucleic acid sequence (e.g., a regulator binding sequence). In an embodiment, the guide RNA is complementary to a CRISPR nucleic acid sequence. In an embodiment, the complement of the guide RNA or guide DNA has about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the target nucleic acid (e.g., a regulator binding sequence). The target nucleic acid sequence as provided herein is a nucleic acid sequence expressed by a cell. In an embodiment, the target nucleic acid sequence is an exogenous nucleic acid sequence. In an embodiment, the target nucleic acid sequence is an endogenous nucleic acid sequence. In an embodiment, the target nucleic acid sequence (e.g., a regulator binding sequence) forms a part of a cell gene. Therefore, in an embodiment, the guide RNA or guide DNA is complementary to a cell gene or a fragment thereof. In an embodiment, the guide RNA or guide DNA is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% complementary to the target nucleic acid sequence (e.g., a regulator binding sequence). In embodiments, the guide RNA or guide DNA is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% complementary to a cellular gene sequence. In embodiments, the guide RNA or guide DNA binds to a cellular gene sequence. The term "target nucleic acid sequence" refers to a regulator binding sequence as provided herein.

[0239] In an embodiment, the guide RNA or guide DNA is a single-stranded ribonucleic acid. In an embodiment, the guide RNA or guide DNA is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleic acid residues in length. In an embodiment, the guide RNA or guide DNA is about 10 to about 30 nucleic acid residues in length. In an embodiment, the guide RNA or guide DNA is about 20 nucleic acid residues in length. In an embodiment, the guide RNA or guide DNA may be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 94, 95, 96, 97, 98, 99, 100, 100 or more nucleic acid residues or sugar residues in length. In embodiments, the length of the guide RNA or guide DNA is 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 5 to 75, 10 to 75, 15 to 75, 20 to 75, 25 to 75, 30 to 75, 35 to 75, 40 to 75, 45 to 75, 50 to 75, 55 to 75, 60 to 75, 65 to 75. 100, 10-100, 15-100, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100, 55-100, 60-100, 65-100, 70-100, 75-100, 80-100, 85-100, 90-100, 95-100 or more residues in length. In embodiments, the guide RNA or guide DNA is 10-15, 10-20, 10-30, 10-40, or 10-50 residues in length.

[0240] PAM refers to "protospacer adjacent motif". These sites are typically 2-6 base pair DNA sequences adjacent to the DNA sequence to which Cas9 binds. Therefore, in some cases, DNA binding regulatory enhancers other than Cas9 can be used, while in other cases, a single Cas9 / RNA complex can be used as a DNA binding regulatory enhancer (either alone or in combination with a different DNA binding regulatory enhancer).

[0241] For the specific proteins described herein (e.g., Cas9, Ag protein), the protein includes any naturally occurring form of the protein, or a variant or homolog that maintains protein transcription factor activity (e.g., activity is at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 150 or 200 consecutive amino acid portions) compared to the naturally occurring form. In other embodiments, the protein is a protein identified according to its NCBI sequence reference. In other embodiments, the protein is a protein identified according to its NCBI sequence reference or its functional fragment or homolog.

[0242] Therefore, "CRISPR-associated protein 9", "Cas9" or "Cas9 protein" referred to herein includes any recombinant or naturally occurring form of Cas9 endonuclease or a variant or homolog thereof that maintains Cas9 endonuclease activity (e.g., activity within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% compared to Cas9). In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 150 or 200 consecutive amino acid portions) compared to the naturally occurring Cas9 protein. In an embodiment, the Cas9 protein is substantially identical to a protein identified according to UniProt reference number Q99ZW2 or a variant or homolog having substantial identity thereto. Cas9 refers to a protein also known in the art as a "nickase". In an embodiment, Cas9 binds to a CRISPR (clustered regularly spaced short palindromic repeats) nucleic acid sequence. In an embodiment, a CRISPR nucleic acid sequence is a prokaryotic nucleic acid sequence. Examples of Cas9 proteins suitable for use in the present invention provided herein include, but are not limited to, cas9 mutant proteins, such as HiFi Cas9 described by Kleinstiver, Benjamin P. et al. ("High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects." Nature (2016). PubMed PMID: 26735016); Cas9 proteins bind to modified PAMs and orthologous Cas9 proteins, such as CRISPR (Cpf1) of Prevotella and Francisella 1. Any mutant Cas9 forms generally known and described in the art can be used in the methods and compositions provided herein.Non-limiting examples of mutant Cas9 proteins contemplated for use in the methods and compositions provided herein are described in Slaymaker, Ian M. et al. ("Rationally engineered Cas9 nucleases with improved specificity" Science (2015): aad5227. PubMed PMID: 26628643) and Kleinstiver, Benjamin P. et al. ("High-fidelity CRISPR-Cas9 nuclease with undetectable off-target effects throughout the genome" Nature (2016). PubMed PMID: 26735016).

[0243] As referred to herein, the term "Ago protein", "NgAgo" or "Natronobacterium gregoryi Ago protein", "Natronobacterium gregoryi SP2 Ago protein" includes recombinant or naturally occurring forms of NgAgo or its variants or homologs that maintain NgAgo endonuclease activity (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to wild-type NgAgo). In an embodiment, the variant or homolog has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 150 or 200 consecutive amino acid portions) compared to the naturally occurring NgAgo protein. In an embodiment, the NgAgo protein is substantially identical to a protein identified by the National Center for Biotechnology Information (NCBI) protein identifier AFZ73749.1 or a variant or homolog thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity. In an embodiment, the Ago protein may also include a nuclease domain (i.e., a deoxyribonuclease or ribonuclease domain), an additional DNA binding domain, a helicase domain, a protein-protein interaction domain, a dimerization domain, and other domains.

[0244] Ag protein also refers to a protein that forms a complex that binds a nucleic acid molecule. Thus, one Ag protein can bind to, for example, a guide RNA and another protein can have endonuclease activity. These proteins are all considered Ag proteins because they act as part of a complex that performs the same function as a single protein (such as NgAgo).

[0245] As used herein, the term "Arg protein system" refers to a set of Arg proteins and nucleic acids that, when combined, produce at least an activity associated with an Arg protein (eg, target locus-specific double-stranded cleavage of double-stranded DNA).

[0246] As used herein, the term "Ago complex" refers to an Ago protein and a nucleic acid (e.g., RNA) that bind to each other to form a functionally active aggregate. An example of an Ago complex is the NgAgo protein bound to a guide DNA specific for a target locus.

[0247] In an embodiment, the "Ago protein", "NgAgo" or "Halomimus gambiae Ago protein", "Halomimus gambiae SP2 Ago protein" mentioned herein includes any recombinant or naturally occurring form of NgAgo or its variant or homolog that maintains NgAgo endonuclease activity (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to wild-type NgAgo). In an embodiment, the variant or homolog has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 150 or 200 consecutive amino acid portions) compared to the naturally occurring NgAgo protein. In an embodiment, the NgAgo protein is substantially identical to a protein identified by the National Center for Biotechnology Information (NCBI) protein identifier AFZ73749.1 or a variant or homolog thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity. In an embodiment, the Ago protein may also include a nuclease domain (i.e., a deoxyribonuclease or ribonuclease domain), an additional DNA binding domain, a helicase domain, a protein-protein interaction domain, a dimerization domain, and other domains.

[0248] Ag protein also refers to a protein that forms a complex that binds a nucleic acid molecule. Thus, one Ag protein can bind to, for example, a guide RNA and another protein can have endonuclease activity. These proteins are all considered Ag proteins because they act as part of a complex that performs the same function as a single protein (such as NgAgo).

[0249] As used herein, the term "transcriptional regulatory sequence" refers to a functional nucleotide fragment in any configuration or geometry contained in a nucleic acid molecule, which is used to regulate (1) the transcription of one or more structural genes (e.g., two, three, four, five, seven, ten, etc.) into messenger RNA or (2) the transcription of one or more genes into non-translated RNA. Examples of transcriptional regulatory sequences include, but are not limited to, promoters, enhancers, repressors, etc.

[0250] As used herein, the term "nucleic acid targeting ability" refers to the ability of a molecule or molecular complex to recognize and / or bind to a nucleic acid based on sequence specificity. For example, the binding of a regulatory protein or regulatory complex to a regulator binding sequence or hybridization region on a guide DNA (gDNA) molecule confers the ability of a nucleic acid to target an Ag protein complex.

[0251] As used herein, the "TAL effector" or "TAL effector protein" provided herein refers to a protein that includes more than one TAL repeat sequence and is capable of binding to a nucleic acid in a sequence-specific manner. In an embodiment, the TAL effector protein includes at least six (e.g., at least 8, at least 10, at least 12, at least 15, at least 17, about 6 to about 25, about 6 to about 35, about 8 to about 25, about 10 to about 25, about 12 to about 25, about 8 to about 22, about 10 to about 22, about 12 to about 22, about 6 to about 20, about 8 to about 20, about 10 to about 22, about 12 to about 20, about 6 to about 18, about 10 to about 18, about 12 to about 18, etc.) TAL repeats. In an embodiment, the TAL effector protein includes 18 or 24 or 17.5 or 23.5 TAL nucleic acid binding boxes. In an embodiment, the TAL effector protein comprises a 15.5, 16.5, 18.5, 19.5, 20.5, 21.5, 22.5 or 24.5 TAL nucleic acid binding cassette. The TAL effector protein comprises at least one polypeptide region flanked by a region containing a TAL repeat sequence. In an embodiment, the flanking region is present at the amino terminus and / or carboxyl terminus of the TAL repeat sequence.

[0252] As used herein, "regulatory sequence" refers to a nucleic acid sequence that affects the rate, stability and / or mobility of transcription and / or translation initiation and transcripts or polypeptide products.Regulatory sequences include, but are not limited to, promoter sequences or control elements, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, transcription start sites, termination sequences, polyadenylation sequences, introns, 5' and 3' untranslated regions (UTRs) and other regulatory sequences that may be present in coding sequences, such as splice sites, inhibitory sequence elements (commonly referred to as CNS or INS, as known from certain viruses), secretion signals, nuclear localization signal (NLS) sequences, introns, translation coupling sequences, protease cleavage sites, as described in more detail elsewhere herein.5' untranslated regions (UTRs) are transcribed but not translated, and are located between the start site and the translation start codon of the transcript, and may include +1 nucleotides.3'UTRs may be located between the translation stop codon and the end of the transcript.UTRs may have specific functions, such as increasing mRNA message stability or translation attenuation. Examples of 3'UTRs include, but are not limited to, polyadenylation signals and transcription termination sequences.Regulatory sequences may be universal or host- or tissue-specific.

[0253] As used herein, "promoter" is a transcriptional regulatory sequence that can guide the transcription of a nucleic acid fragment when it is operably linked to a nucleic acid fragment (e.g., a transgenic comprising an open reading frame). A promoter is a nucleotide sequence located upstream of a transcription initiation site (usually near the initiation site of RNA polymerase II). A promoter generally comprises at least one core or basic motif, and may include at least one or more control elements or synergize with at least one or more control elements, such as an upstream element (e.g., an upstream activation region (UAR)) or other regulatory sequences or synthetic elements. The basic motif constitutes the minimum sequence required for the assembly of the transcription complex required for transcription initiation. In an embodiment, such a minimum sequence includes a "TATA box" element, which may be located between about 15 to about 35 nucleotides upstream of the transcription initiation site. A basic promoter may also include a "CCAAT box" element (usually a sequence CCAAT) and / or a GGGGG sequence, which may be located between about 40 to about 200 nucleotides upstream of the transcription initiation site, typically about 60 to about 120 nucleotides. Transcription of adjacent nucleic acid fragments initiates at the promoter region. The transcription rate of an inhibitory promoter decreases in response to an inhibitor. The transcription rate of an inducible promoter increases in response to an inducing agent. The transcription rate of a constitutive promoter is not specifically regulated, however it can vary under the influence of general metabolic conditions.

[0254] The selection of the promoter contained in the expression vector depends on several factors, including but not limited to efficiency, selectivity, inducibility, desired expression level, and cell or tissue specificity. For example, tissue, organ, and cell specific promoters that only or mainly confer transcription in specific tissues, organs, and cell types, respectively, can be used. In an embodiment, a promoter that is substantially specific to seeds ("seed preferred promoter") can be useful. In an embodiment, a constitutive promoter is used, which can promote transcription in most or all tissues of a particular species. Other types of promoters include but are not limited to inducible promoters, such as promoters that confer transcription in response to external stimuli (such as chemical reagents, developmental stimuli, or environmental stimuli). Inducible promoters can be induced by pathogens or cold or stress (such as cold, heat, ultraviolet light, or high ion concentrations), or can be induced by chemicals. Examples of inducible promoters are eukaryotic metallothionein promoters, which are induced by elevated heavy metal content; prokaryotic lacL promoters, which are induced in response to isopropyl-β-D-thiogalactopyranoside (IPTG); and eukaryotic heat shock promoters induced by elevated temperature. Many other bacterial and eukaryotic promoters suitable for use in the present invention are known in the art and are described in, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed., 1989; 3rd ed., 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel et al., Current Protocols in Molecular Biology. Bacterial expression systems for expressing ZFPs are available, for example, from Escherichia coli, Bacillus, and Salmonella (Palva et al., Secretion of interferon by Bacillus subtilis. Gene 22:229-235 (1983)). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known to those skilled in the art and are also commercially available.

[0255] Common promoters for prokaryotic protein expression are, for example, the lac promoter or the trc and tac promoters (IPTG inducible), the tetA promoter / operator (anhydrotetracycline inducible), the PPBAD promoter (L-arabinose inducible), the r / zaPBAD promoter (L-rhamnose inducible) or bacteriophage promoters, for example the phage promoters pL (sensitive to temperature changes), T7, T3, SP6 or T5.

[0256] Common promoters used for mammalian protein expression are, for example, the cytomegalovirus (CMV) promoter, the SV40 promoter / enhancer, the vaccinia virus promoter, viral LTRs (MMTV, RSV, HIV, etc.), the E1B promoter, promoters of constitutively expressed genes (actin, GAPDH), promoters of genes expressed in a tissue-specific manner (albumin, NSE), promoters of inducible genes (metallothioneins, steroid hormones).

[0257] Many promoters for expressing nucleic acid in plant are known and can be used in the implementation of the present invention. Such promoters can be constitutive, regulatable and / or tissue-specific (e.g. seed-specific, stem-specific, leaf-specific, root-specific, fruit-specific, etc.). Exemplary promoters that can be used for plant expression include cauliflower mosaic virus 35S promoter and promoters of following genes: ACT 11 and CAT 3 genes from Arabidopsis thaliana, genes (GenBank numbering X74782) encoding stearoyl-acyl carrier protein desaturase from Brassica napus, and genes encoding GPC1 (GenBank numbering X15596) and GPC2 (GenBank numbering U45855) from corn. Other promoters include tobacco mosaic virus subgenomic promoters, cassava vein mosaic virus (CVMV) promoters (showing high transcriptional activity in vascular element, mesophyll cell and root tip), drought-inducible promoters of corn, and cold, drought and high-salt inducible promoters of potato. Many other promoters suitable for plant expression are found in US Pat. No. 8,067,222, the disclosure of which is incorporated herein by reference.

[0258] Heterologous expression in chloroplasts of microalgae (e.g., Chlamydomonas reinhardtii) can be achieved in a genetic background lacking psbA using, for example, a psbA promoter / 5' untranslated region (UTR) (due to psbA / Dl-dependent autoattenuation) or by fusing a strong 16S rRNA promoter to the 5'UTR of the psbA and atpA genes in an expression cassette, as disclosed, for example, in Rasala et al., "Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5' untranslated region optimization," Plant Biotechnology Journal, Vol. 9, No. 6, pp. 674-683 (2011). The promoter used to direct expression of a TAL effector encoding nucleic acid depends on the specific application. For example, strong constitutive promoters are often used for expression and purification of TAL effector fusion proteins. In contrast, when TAL effector nuclease fusion proteins are administered in vivo for gene regulation, it may be desirable to use a constitutive or inducible promoter, depending on the specific use of the TAL effector nuclease fusion protein and other factors. In addition, a promoter suitable for administration of TAL effector nuclease fusion proteins may be a weak promoter, such as HSV thymidine kinase or a promoter with similar activity.Promoters can also typically include elements that respond to transactivation, such as hypoxia response elements, Gal4 response elements, lac repressor response elements, and small molecule control systems, such as the tet regulatory system and the RU-486 system (see, e.g., Gossen and Bujard. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89:5547 ​​(1992); Oligino et al., Drug inducible transgene expression in brain using a herpes simplex virus vector. Gene Ther. 5:491-496 (1998); Wang et al., Positive and negative regulation of gene expression in eukaryotic cells with an inducible transcriptional regulator. transcriptional regulator). Gene Therapy 4:432-441 (1997); Neering et al., Transduction of primitive human hematopoietic cells with recombinant adenovirus vectors. Blood 88:1147-1155 (1996); and Rendahl et al., Regulation of gene expression in vivo following transduction by two separate rAAV vectors. Nat. Biotechnol. 16:151-161 (1998). The MNDU3 promoter may also be used, and is preferably active in CD34+ hematopoietic stem cells.

[0259] "Host" refers to a cell or organism that supports vector replication or expression of proteins or polypeptides encoded by vector sequences. Host cells can be prokaryotic cells, such as E. coli, or eukaryotic cells, such as yeast, fungi, protozoa, higher plants, insects or amphibian cells, or mammalian cells, such as CHO, HeLa, 293, COS-1, etc., such as cultured cells (in vitro), explants and primary cultures (in vitro and ex vivo), and in vivo cells.

[0260] As used herein, the phrase "recombinant protein" includes excision-type or integration-type proteins, enzymes, cofactors or related proteins that participate in the recombination reaction involving one or more recombination sites (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which can be wild-type proteins (see Landy, Current Opinion in Biotechnology 3: 699-707 (1993)) or their mutants, derivatives (e.g., fusion proteins containing recombinant protein sequences or fragments thereof), fragments and variants. Examples of recombinant proteins include Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, Phi-C31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, SpCCEl and Par A.

[0261] As used herein, the phrase "recombination site" refers to a recognition sequence on a nucleic acid molecule that participates in integration / recombination reactions through recombinant proteins. A recombination site is a discontinuous portion or segment of a nucleic acid on a participating nucleic acid molecule that is recognized and combined by site-specific recombinant proteins in the initial stages of integration or reorganization. For example, the recombination site of the Cre recombinase is loxV, which is a 34 base pair sequence comprising two 13 base pair inverted repeats (used as recombinase binding sites), with a core sequence of 8 base pairs flanking it (see Figure 1 in the following document: Sauer, B., Site-specific recombination: developments and applications. Curr. Opin. Biotech. 5: 521-527 (1994)). Other examples of recognition sequences include the attB, attP, attL and attR sequences described herein, as well as mutants, fragments, variants and derivatives thereof, which are recognized by the recombinant protein λ phage integrase and the auxiliary proteins integration host factor (IHF), Fis and excision enzyme (λ phage is).

[0262] As used herein, the phrase "recognition sequence" refers to a specific sequence that a protein, compound, DNA or RNA molecule (e.g., restriction endonuclease, modified methylase or recombinase) recognizes and binds. In the present invention, a recognition sequence generally refers to a recombination site. For example, the recognition sequence of the Cre recombinase is loxP, which is a sequence of 34 base pairs, comprising two 13 base pairs of inverted repeats of a core sequence of 8 base pairs flanking the core sequence (serving as a recombinase binding site) (see Sauer, B., "New Views in Biotechnology" 5: Figure 1 of 521-527 (1994)). Other examples of recognition sequences are attB, attP, attL and attR sequences recognized by the recombinase lambda phage integrase. AttB is a sequence of about 25 base pairs, containing two 9 base pairs of core-type Int binding sites and an overlapping region of 7 base pairs. attP is a sequence of about 240 base pairs that contains the core-type Int binding site and the arm-type Int binding site, as well as sites for the accessory proteins integration host factor (IHF), FIS, and excision enzyme (λ phage is). (See Landy, Current Opinion in Biotechnology 3: 699-707 (1993)).

[0263] Throughout this document, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising,” or “contain,” “contains,” or “containing,” will be understood to imply the inclusion of a stated step or element or a group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0264] As used herein, the term "homologous recombination" refers to the mechanism of genetic recombination, wherein two DNA chains containing similar nucleotide sequences exchange genetic material. Cells utilize homologous recombination during meiosis, wherein homologous recombination is not only used to rearrange DNA to produce a set of completely unique haploid chromosomes, but also to repair damaged DNA, especially for repairing double-strand breaks. The mechanism of homologous recombination is well known to technicians and has been described in, for example, Paques and Haber (Paques F, Haber J E.; Microbial. Mal. Biol. Rev. 63: 349-404 (1999)). In the method of the present invention, homologous recombination is achieved by placing the first side joint element and the second side joint element respectively upstream (5') and downstream (3') of the donor DNA sequence, each of which is homologous to the continuous DNA sequence in the target sequence.

[0265] As used herein, the term "non-homologous end joining" (NHEJ) refers to a cellular process that joins the two ends of a double-strand break (DSB) by a process that is largely independent of homology. Naturally occurring DSBs are spontaneously generated during DNA synthesis (when the replication fork encounters a damaged template) and during certain special cellular processes (including V(D)J recombination, class switch recombination occurring at the immunoglobulin heavy chain (IgH) locus, and meiosis). In addition, DSBs are generated by cell exposure to ionizing radiation (X-rays and gamma rays), ultraviolet light, topoisomerase poisons, or radiomimetic drugs. The NHEJ (non-homologous end joining) pathway joins the two ends of a DSB by a process that is largely independent of homology. NHEJ can be precise or mutation-induced, depending on the specific sequence and chemical modifications generated at the DSB (Lieber MR, The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu. Rev. Biochem. 79: 181-211).

[0266] As used herein, the term "donor DNA" or "donor nucleic acid" refers to a nucleic acid designed to be introduced into a locus by homologous recombination. The donor nucleic acid has at least one region homologous to the locus sequence. In an embodiment, the donor nucleic acid has two regions homologous to the locus sequence. These homologous regions can be located at one of the two ends or can be located inside the donor nucleic acid. In an embodiment, the nucleic acid "insertion" region in the nucleic acid molecule present in the cell that is desired to be introduced will be located between the two homologous regions.

[0267] The donor nucleic acid molecule (e.g., donor DNA molecule) can be double-stranded, single-stranded, or partially double-stranded and single-stranded, and thus, can have overhangs at one or both ends (e.g., two 5' overhangs, two 3' overhangs, one 5' and one 3' overhang, a single 3' overhang, or a single 5' overhang). In addition, the nucleic acid molecule can be a linear nucleic acid molecule (a closed circular or nicked nucleic acid molecule).

[0268] As used herein, the term "homologous recombination system" or "HR system" refers to components of the systems described herein that can be used to alter cells by homologous recombination. Specifically, zinc finger nucleases, TAL effector nucleases, CRISPR endonucleases, homing endonucleases, and Argo editing systems.

[0269] As used herein, the term "nucleic acid cleavage entity" refers to a single molecule or molecular complex having nucleic acid cleavage activity (e.g., double-stranded nucleic acid cleavage activity). Exemplary nucleic acid cleavage entities include Agg protein complexes, zinc finger proteins, transcription activator-like effectors (TALEs), CRISPR complexes, and homing megabase large-range nucleases. In an embodiment, nucleic acid cleavage entities will have an activity that allows them to be nuclear localized (e.g., will contain a nuclear localization signal (NLS)).

[0270] As used herein, the term "double-strand break site" refers to a position in a nucleic acid molecule where a double-strand break occurs. In an embodiment, this will be produced by cutting the nucleic acid molecule at two close positions (e.g., within a range of about 3 to about 50 base pairs, about 5 to about 50 base pairs, about 10 to about 50 base pairs, about 15 to about 50 base pairs, about 20 to about 50 base pairs, about 3 to about 40 base pairs, about 5 to about 40 base pairs, about 10 to about 40 base pairs, about 15 to about 40 base pairs, about 20 to about 40 base pairs, etc.). Generally, the spacing of the cuts in a nucleic acid region containing a higher AT content can be further increased compared to a nucleic acid region containing a higher GC content.

[0271] As used herein, the term "matched end" refers to the end of a nucleic acid molecule having a sequence identity greater than 90%. The matched end of the DS break of the target locus can be double-stranded or single-stranded. The matched end of the donor nucleic acid molecule is usually single-stranded.

[0272] As used herein, "homologous directed repair" or "HDR" is a mechanism in cells to repair DNA double-strand breaks (DSBs). In some embodiments, HDR is greater than or equal to 10%, 25%, 50%, 75%, 90%, 95%, 98%, 99% or 100%.

[0273] A common form of HDR is "homologous recombination", which refers to a genetic recombination mechanism in which two DNA chains containing similar nucleotide sequences exchange genetic material. Cells utilize homologous recombination during meiosis, where homologous recombination is used not only to rearrange DNA to produce a set of completely unique haploid chromosomes, but also to repair damaged DNA, especially for repairing double-strand breaks. The mechanism of homologous recombination is well known to technicians and has been described in, for example, Paques F., Haber J.E., Microbiology and Molecular Biology Review (Microbiol.Mol.Biol.Rev.) 63:349-404 (1999). In some embodiments, homologous recombination is achieved by the presence of matching ends located upstream (5') and downstream (3') of the donor nucleic acid molecule, each of which is homologous to a continuous DNA sequence in the cracked nucleic acid molecule.

[0274] Some embodiments include compositions and methods designed to improve homologous recombination efficiency in cells (e.g., eukaryotic cells, such as plant cells and animal cells, such as insect cells and mammalian cells, including mice, rats, hamsters, rabbits, and human cells). In some embodiments, the homologous recombination efficiency should be such that greater than 20% of the cells in the population will have undergone homologous recombination at the desired target locus or loci. In some embodiments, homologous recombination can occur in a range of 10% to 65%, 15% to 65%, 20% to 65%, 30% to 65%, 35% to 65%, 10% to 55%, 20% to 55%, 30% to 55%, 35% to 55%, 40% to 55%, 10% to 45%, 20% to 45%, 30% to 45%, 40% to 45%, 30% to 50% of the cells in the population, etc.

[0275] In addition, some embodiments include compositions and methods for increasing the efficiency of homologous recombination in cells. For example, if homologous recombination occurs in 10% of a cell population under one set of conditions and in 40% of a cell population under another set of conditions, the efficiency of homologous recombination is increased by 300%. In some embodiments, the efficiency of homologous recombination can be increased by 100% to 500% (e.g., 100% to 450%, 100% to 400%, 100% to 350%, 100% to 300%, 200% to 500%, 200% to 400%, 250% to 500%, 250% to 400%, 250% to 350%, 300% to 500%, etc.).

[0276] As used herein, "double-strand break" or "DSB" refers to a double-strand break in a nucleic acid molecule. In various embodiments, a DSB will be generated by cutting a nucleic acid molecule at two proximal positions (e.g., within a range of 3 to 50 base pairs, 5 to 50 base pairs, 10 to 50 base pairs, 15 to 50 base pairs, 20 to 50 base pairs, 3 to 40 base pairs, 5 to 40 base pairs, 10 to 40 base pairs, 15 to 40 base pairs, 20 to 40 base pairs, etc.). The spacing of the cuts may be further increased in nucleic acid regions containing higher AT content compared to nucleic acid regions containing higher GC content. In some embodiments, the double-strand break is less than or equal to 250 bp from the ATG start codon for N-terminal tags of nucleic acid molecules, or less than or equal to 250 bp from the stop codon for C-terminal tags of nucleic acid molecules.

[0277] As used herein, "donor nucleic acid molecule" or "donor DNA" refers to a nucleic acid designed for introduction into a cleaved nucleic acid molecule by homologous recombination. The donor nucleic acid molecule will have at least one region homologous to the cleaved nucleic acid molecule sequence. In many embodiments, the donor nucleic acid molecule will have two regions homologous to the locus sequence. These homologous regions can be located at one or both ends, or can be located inside the donor nucleic acid molecule.

[0278] As used herein, "integration efficiency" refers to the frequency with which the foreign DNA segment of interest is incorporated into the initial nucleic acid molecule. In some embodiments, the integration efficiency of the donor nucleic acid molecule is greater than or equal to 50%, 75%, 90%, 95%, 98%, 99% or 100%.

[0279] Table 1 shows that nearly 100% integration efficiency and up to 100% accurate HDR were found at four different genomic loci in three different mammalian cell lines. At some loci, deletions and insertions at the junction or at the Cas9 cleavage site were observed.

[0280]

[0281] a: determined by flow cytometry; b: determined by ELISA analysis; c: due to the low expression level of the chimeric protein, it could not be determined by flow cytometry.

[0282] In some embodiments, end modification of the donor DNA with phosphorothioate or amino groups and / or treatment with a non-homologous end joining (NHEJ) inhibitor can further improve the efficiency of HDR.

[0283] As used herein, "matching end" refers to the end of a nucleic acid molecule having a sequence identity greater than or equal to 90%. In some embodiments, the matching end on the 5' and 3' ends has a length of 12bp to 250bp, 12bp to 200bp, 12bp to 150bp, 12bp to 100bp, 12bp to 50bp, or 12bp to 40bp. In some embodiments, the matching end has a length of 35bp. In some embodiments, the matching end will share a sequence identity greater than or equal to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99% or equal to 100%. The matching end of the double-strand break at the target locus can be double-stranded or single-stranded DNA. In some embodiments, the matching end of the donor nucleic acid molecule will be single-stranded.

[0284] The larger the amount of the shared sequence identity of the matching end and the nucleic acid at the target locus, the higher the homologous recombination efficiency is usually. When the homologous region is quite short (e.g., 50 bases), a high level of sequence identity is particularly desired. In certain embodiments, the amount of the sequence identity between the target locus and the matching end is greater than 90% (e.g., 90% to 100%, 90% to 99%, 90% to 98%, 95% to 100%, 95% to 99%, 95% to 98%, 97% to 100%, etc.).

[0285] As used herein, "sequence identity percentage" means a value determined by comparing two optimally aligned nucleotide sequences over a comparison window, wherein the nucleotide sequence portion in the comparison window may include additions or deletions (i.e., sequence alignment gaps) relative to a reference sequence (which does not include additions or deletions) so that the two sequences are optimally aligned. In other words, for quantitative purposes, sequence alignment gaps are removed. The sequence identity percentage is calculated as follows: the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences is determined to produce the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window and the result is multiplied by 100 to obtain the sequence identity percentage.

[0286] One method for determining sequence identity values ​​is to use the BLAST 2.0 suite of programs using default parameters (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). Software for performing BLAST analyses is publicly available, for example, through the National Center for Biotechnology Information.

[0287] In some embodiments, the ends may differ in one or more features associated with homologous recombination. For example, the length of the end "matching" region complementary to the target locus sequence may be different. Therefore, one end may have 40 nucleotides with sequence complementarity, while the other end may only have 15 nucleotides with sequence complementarity. In some embodiments, one or both ends of the donor nucleic acid molecule may be partially or completely single-stranded.

[0288] As used herein, "promoterless selection marker" refers to a foreign gene of interest that does not have a promoter, such that it is expressed only after insertion into a genomic locus containing a promoter. In some embodiments, the promoterless selection marker is a protein, an antibiotic resistance selection marker, a cell surface marker, a cell surface protein, a metabolite, or an active fragment thereof. In some embodiments, the promoterless selection marker is a marker (e.g., EmGFP or OFP). In one embodiment, the promoterless selection marker is puromycin, dihydrofolate reductase, or glutamine synthetase.

[0289] The promoter-free selection marker can be directly connected to the reporter gene, or alternatively, the donor nucleic acid molecule can include another amino acid sequence that serves as a "joint" between the promoter-free selection marker and the reporter gene. The joint can be a polypeptide or any other suitable joint known in the art. In some embodiments, the joint comprises more than or equal to 2, 3, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80 or 90 amino acids. In some embodiments, the joint comprises 100 amino acids. In some embodiments, the joint comprises more than or equal to two amino acids selected from the group consisting of glycine, serine, alanine and threonine. In some embodiments, the joint is a polyglycine joint. In some embodiments, the polyglycine joint comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 glycine residues. In one embodiment, the joint is a polyglycine of 6 residues. In some embodiments, the distance between the promoterless selection marker and the reporter gene is less than or equal to 300 nt, 240 nt, 180 nt, 150 nt, 120 nt, 90 nt, 60 nt, 30 nt, 15 nt, 12 nt, or 9 nt.

[0290] As used herein, "reporter gene" refers to a gene whose product is easily analyzed, and can be used as a marker for screening successfully modified cells, studying the regulation of gene expression, or as a control for standardized recombination efficiency. In some embodiments, the reporter gene is a selectable marker. In some embodiments, the reporter gene is a fluorescent reporter, such as an emerald green fluorescent protein (EmGFP) reporter or an orange fluorescent protein (OFP) reporter. In some embodiments, the reporter gene is a luminescent reporter, such as a luciferase (e.g., North American firefly (P.pyralisluciferase)). Other commonly used reporter genes are β-glucuronidase and β-galactosidase. Ideally, the reporter gene should not be present in the cells used for the study, or be easily distinguished from the native form of the gene, easy to analyze, with a wide linear detection range, and does not affect the normal physiology and overall health of the cell.

[0291] As used herein, "self-cleaving peptide" refers to a peptide that dissociates into component proteins during translation. In some embodiments, the self-cleaving peptide is linked to a promoterless selection marker and a reporter gene, and after recombining into the initial nucleic acid molecule, the promoterless selection marker and the reporter gene are able to dissociate during translation. In some embodiments, the self-cleaving peptide is a self-cleaving 2A peptide or other self-cleaving peptide known to the skilled person.

[0292] In some embodiments, "loxP" or "X-over P1 locus" is located on either side of the promoterless selection marker as an alternative or supplement to the self-cleaving peptide. LoxP can be used as part of the Cre-lox recombination strategy to promote the replication of the promoterless selection marker. The Cre-lox strategy requires at least two components: 1) Cre recombinase, an enzyme that catalyzes recombination between two loxP sites; and 2) loxP sites (e.g., a specific 34 base pair sequence consisting of an 8bp core sequence and two flanking 13bp inverted repeats where recombination occurs) or mutant lox sites. (See, e.g., Araki et al., PNAS 92:160-4 (1995); Nagy, A. et al., Genesis 26:99-109 (2000); Araki et al., Nuc Acids Res 30(19):e103 (2002); and US20100291626A1, all of which are incorporated herein by reference). Exemplary loxP sites include, but are not limited to, wild-type lox511, lox5171, lox2272, M2, M3, M7, M11, lox71, and lox66. loxP allows for the removal of the promoterless selection marker at a later time. In this way, the edited population can be selected and then the promoterless selection marker removed. This allows the promoterless selection marker to be reused if further editing is desired.

[0293] As used herein, "non-homologous end joining" (NHEJ) refers to a cellular process in which the two ends of a double-strand break (DSB) are connected by a process that is largely independent of homology. Naturally occurring DSBs are spontaneously generated during DNA synthesis (when the replication fork encounters a damaged template) and during certain special cellular processes (including V (D) J recombination, class switching recombination occurring at the immunoglobulin heavy chain (IgH) locus, and meiosis). In addition, cells are exposed to ionizing radiation (X-rays and gamma rays), ultraviolet light, topoisomerase poisons, or radiomimetic drugs to produce DSBs. Depending on the specific sequence and chemical modification produced at the DSB, NHEJ can be precise or mutation-induced (Lieber, MR, Annals of Biochemistry 79: 181-211 (2010)).

[0294] As used herein, "non-homologous end joining inhibitor" or "NHEJ inhibitor" refers to a molecule that inhibits the non-homologous end joining process. In some embodiments, the donor nucleic acid molecule is treated with at least one NHEJ inhibitor. Examples of NHEJ inhibitors include, but are not limited to, DNA-dependent protein kinase (DNA-PK), DNA ligase IV, DNA polymerase 1 or 2 (PARP-1 or PARP-2), or a combination thereof. Exemplary DNA-PK inhibitors include Nu7206 (2-(4-morpholinyl)-4H-naphtho[1,2-b]pyran-4-one), Nu7441 (8-(4-dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one), Ku-0060648 (4-ethyl-N-[4-[2-(4-morpholinyl)-4-oxo-4H-1-benzopyran-8-yl]-1-dibenzothienyl]-1-piperazineacetamide), Compound 401 (2-(4-morpholinyl)-4H-pyrimido[2,1-a]isoquinolin-4-one), DMNB (4,5-dimethoxy-2-nitrobenzaldehyde), ETP 45658 (3-[1-methyl-4-(4-morpholinyl)-1H-pyrazolo[3,4-d]pyrimidin-6-ylphenol), LTURM 34 (8-(4-dibenzothienyl)-2-(4-morpholinyl)-4H-1,3-benzoxazin-4-one), and P1103 hydrochloride (3-[4-(4-morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol hydrochloride).

[0295] As used herein, "target locus" refers to a site within a nucleic acid molecule that is recognized and cleaved by a nucleic acid cleavage entity. When, for example, a single CRISPR complex is designed to cleave a double-stranded nucleic acid, the target locus is the cleavage site and surrounding area recognized by the CRISPR complex. When, for example, two CRISPR complexes are designed to cleave double-stranded nucleic acids in close proximity to produce double-strand breaks, the surrounding area including the breakpoints recognized by the two CRISPR complexes is called the target locus.

[0296] As used herein, "nuclease resistance group" refers to a chemical group that can be incorporated into a nucleic acid molecule and can inhibit the degradation of nucleic acid molecules containing the group by enzymes (exonucleases and / or endonucleases). Examples of such groups are thiophosphate groups, amino groups, 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxy nucleotides and 5-C-methyl nucleotides. Nuclease resistance groups can be located at many positions in donor nucleic acid molecules. In certain embodiments, cellular nucleases will digest this part of the donor nucleic acid molecule. These nucleases will be terminated or slowed down by nuclease resistance groups, thereby stabilizing the structure of the donor nucleic acid molecule.

[0297] Embodiments of the invention include compositions comprising nucleic acid molecules containing one or more (e.g., one, two, three, four, five, six, seven, etc.) nuclease resistance groups; and methods of making and using such donor nucleic acid molecules. In many embodiments, the nuclease resistance group will be located at one or both ends of the donor nucleic acid molecule. The donor nucleic acid molecule may contain internal groups relative to one or both ends. In many embodiments, some or all of such donor nucleic acid molecules will be processed within the cell to produce ends that match the DS break site.

[0298] As used herein, the term "intracellular targeting moiety" refers to a chemical entity (eg, a polypeptide) that facilitates localization to an intracellular location. Examples of intracellular targeting moieties include nuclear localization signals, chloroplast targeting signals, and mitochondrial targeting signals.

[0299] As used herein, "subject" refers to a human or non-human animal (eg, a mammal) or a plant.

[0300] As used herein, "treating" refers to alleviating at least one symptom of a disease, disorder or condition in a subject by administering an effective amount of a promoterless selectable marker to the subject.

[0301] As used herein, "nucleic acid cleavage entity" refers to one or more molecules, enzymes or molecular complexes with nucleic acid cleavage activity (e.g., double-stranded nucleic acid cleavage activity). In most embodiments, nucleic acid cleavage entity components will be proteins or nucleic acids or a combination of the two, but they can be combined with cofactors and / or other molecules. Nucleic acid cleavage entities will generally be selected based on many factors, such as the efficiency of producing DS fractures at the target locus, the ability to produce DS fractures at or near the target locus, the low potential, low toxicity and cost issues of producing DS fractures at the target locus. Many of these factors will vary with the cells used and the target locus. Many nucleic acid cleavage entities are known in the art. For example, in some embodiments, nucleic acid cleavage entities include one or more zinc finger proteins, transcription activator-like effectors (TALEs), CRISPR complexes (e.g., Cas9 or CPF1), homing endonucleases or meganucleases, Ager protein-nucleic acid complexes, or meganucleases. In some embodiments, nucleic acid cleavage entities will have an activity that allows them to be nuclear localized (e.g., will include a nuclear localization signal (NLS)). In some embodiments, a single-stranded DNA donor may function in conjunction with nicking or a combination of nicking.

[0302] Zinc Finger Protein (ZFP)

[0303] As used herein, "zinc finger protein" (ZFP) refers to a chimeric protein comprising a nuclease domain and a zinc-stabilized nucleic acid (e.g., DNA) binding domain. Individual DNA binding domains are often referred to as "fingers," such that zinc finger proteins or polypeptides have at least one finger, more typically two fingers or three fingers, or even four or five fingers, to at least six or more fingers. In some embodiments, the ZFP will comprise three or four zinc fingers. Each finger typically binds two to four base pairs of DNA. Each finger may comprise a zinc-chelating DNA binding region of about 30 amino acids (see, e.g., U.S. Patent Publication No. 2012 / 0329067A1, the disclosure of which is incorporated herein by reference).

[0304] An example of a nuclease domain is the nonspecific cleavage domain from the type IIs restriction endonuclease FokI (Kim, YG et al., Proc. Natl. Acad. Sci. USA 93: 1156-60 (1996)), which is typically separated by a linker sequence of 5-7 base pairs. A pair of FokI cleavage domains is typically required to allow domain dimerization and non-palindromic target sequence cleavage from opposite strands. The DNA binding domain of an individual Cys2His2ZFN typically contains 3 to 6 individual zinc finger repeats and each is capable of recognizing 9 to 18 base pairs.

[0305] One issue associated with ZFPs is the possibility of off-target cleavage, which may cause random integration of the donor DNA or cause chromosomal rearrangements or even cell death, which still raises concerns about applicability to higher organisms (Radecke, S. et al., Mol. Ther. 18:743-753 (2010)).

[0306] Transcription activator-like effector (TALE)

[0307] As used herein, "transcription activator-like effector" (TALE) refers to a protein composed of more than one TAL repeat sequence and is capable of binding nucleic acids in a sequence-specific manner. TALE represents a class of DNA-binding proteins secreted by plant pathogenic bacterial species (e.g., Xanthomonas and Ralstonia) via their type III secretion system after infecting plant cells. Natural TALEs have been shown to bind to plant promoter sequences, thereby regulating gene expression and activating effector-specific host genes, thereby promoting bacterial reproduction. P. et al., Science, 318:645-648 (2007); Boch, J. et al., Annu. Rev. Phytopathol. 48:419-436 (2010); Kay, S. et al., Science, 318:648-651 (2007); Kay, S. et al., Curr. Opin. Microbiol. 12:37-43 (2009).

[0308] Natural TALEs are typically characterized by a central repeat domain and a carboxyl-terminal nuclear localization signal sequence (NLS) and a transcription activation domain (AD). In addition to the usually shorter carboxyl-terminal repeat sequence (called a half-repeat sequence), the central repeat domain typically consists of: 1.5 to 33.5 different amounts of amino acid repeat sequences, which are usually 33-35 residues in length. Most of the repeat sequences are consistent, but differ in certain hypervariable residues. The DNA recognition specificity of TALE is mediated by hypervariable residues (so-called repeat variable diresidues (RVDs), each of which targets a specific nucleotide in a given DNA sequence) that are usually located at positions 12 and 13 of each repeat sequence. Therefore, the order of repeat sequences in TAL proteins tends to be related to the defined linear order of nucleotides in a given DNA sequence. Potential RVD codes for some naturally occurring TALEs have been identified, allowing prediction of the order of repeat sequences necessary to bind to a given DNA sequence (Boch, J. et al., Science 326: 1509-1512 (2009); Moscou, MJ et al., Science 326: 1501 (2009)). In addition, it has been shown that TAL effectors generated with novel repeat sequence combinations bind to target sequences predicted from this code. It has been shown that target DNA sequences typically begin with a 5' thymine base that is recognized by TAL proteins.

[0309] The TAL module structure allows the combination of DNA binding domains with effector molecules such as nucleases. Specifically, TALE nucleases allow the development of new genome engineering tools.

[0310] The TALE used in some embodiments can generate DS breaks, or can have a combined effect for generating DS breaks. For example, a TAL-FokI nuclease fusion can be designed to bind at or near the target locus and form double-stranded nucleic acid cleavage activity through the binding of two FokI domains.

[0311] In some embodiments, the TALE will comprise greater than or equal to 6 (e.g., greater than or equal to 8, 10, 12, 15, or 17, or 6 to 25, 6 to 35, 8 to 25, 10 to 25, 12 to 25, 8 to 22, 10 to 22, 12 to 22, 6 to 20, 8 to 20, 10 to 22, 12 to 20, 6 to 18, 10 to 18, 12 to 18, etc.) TAL repeat sequences. In some embodiments, the TALE may comprise 18 or 24 or 17.5 or 23.5 TAL nucleic acid binding boxes. In further embodiments, the TALE may comprise 15.5, 16.5, 18.5, 19.5, 20.5, 21.5, 22.5, or 24.5 TAL nucleic acid binding boxes. The TALE will typically have at least one polypeptide region flanking a region comprising a TAL repeat sequence. In many embodiments, the flanking regions will be present at the amino terminus and carboxyl terminus of the TAL repeat sequence. Exemplary TALEs are described in U.S. Patent Publication No. 2013 / 0274129A1, the disclosure of which is incorporated herein by reference, and can be modified forms of naturally occurring proteins found in Burkholderia, Xanthamonas, and Ralstonia bacteria.

[0312] In some embodiments, the TALE protein will contain a nuclear localization signal (NLS) that allows it to be transported to the nucleus.

[0313] CRISPR-based systems

[0314] The term "CRISPR" or "Clustered Regularly Interspaced Short Palindromic Repeats" is a general term that applies to three types of systems and subtypes of systems. In general, the term CRISPR refers to the repetitive regions that encode the components of the CRISPR system (e.g., the encoded crRNAs). Three types of CRISPR systems have been identified, each with different characteristics (see Table 2).

[0315]

[0316] As used herein, the term "CRISPR complex" refers to a CRISPR protein and a nucleic acid (e.g., RNA) that bind to each other to form a functionally active aggregate. An example of a CRISPR complex is a wild-type Cas9 (sometimes referred to as Csn1) protein bound to a guide RNA specific for a target locus.

[0317] As used herein, the term "CRISPR protein" refers to a protein comprising a nucleic acid (e.g., RNA) binding domain nucleic acid and an effector domain (e.g., Cas9, such as Streptococcus pyogenes Cas9 or CPF1 (cleavage and polyadenylation factor 1)). The nucleic acid binding domain interacts with a first nucleic acid molecule having a region capable of hybridizing to a desired target nucleic acid (e.g., a guide RNA) or allows binding to a second nucleic acid having a region capable of hybridizing to a desired target nucleic acid (e.g., crRNA). CRISPR proteins may also include nuclease domains (i.e., deoxyribonuclease or ribonuclease domains), additional DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains.

[0318] CRISPR protein also refers to a protein that forms a complex that binds to the first nucleic acid molecule described above. Thus, one CRISPR protein may bind to, for example, a guide RNA and another protein may have endonuclease activity. These are all considered CRISPR proteins because they act as part of a complex that performs the same function as a single protein (such as Cas9 or CPF1).

[0319] In some embodiments, the CRISPR protein will contain a nuclear localization signal (NLS) that allows it to translocate to the nucleus.

[0320] The CRISPR used in some embodiments can produce DS breaks or can have a combined action for producing DS breaks. For example, mutations can be introduced into CRISPR components to prevent CRISPR complexes from causing DS breaks, but still allow these complexes to cut DNA. Mutations have been identified in Cas9 proteins that allow the preparation of Cas9 proteins that can cut DNA instead of performing double-stranded cleavage. Therefore, some embodiments include the use of Cas9 proteins with mutations in the RuvC and / or HNH domains that limit the nuclease activity of this protein to nicking activity.

[0321] The term "dCas9" as provided herein refers to a nuclease-inactivated Cas9. In an embodiment, the DNA binding regulatory enhancer can be a guide RNA bound to a dCas9 domain. In other embodiments, the regulatory complex is a Cas9 domain bound to a gRNA, wherein the regulatory complex further includes a VP16 transcriptional activation domain operably linked to the Cas9 domain. Such systems can be used to induce expression of, for example, endogenous genes in mammalian cells. One of ordinary skill in the art will immediately recognize that the type of DNA binding regulatory enhancer used will vary depending on the cell type and specific application.

[0322] In many cases, the dCas9 protein has at least one mutation in each of the RuvC and HNH domains that inactivates the nuclease activity of the protein.

[0323] The CRISPR systems that can be used vary widely. These systems generally have functional activity that can form a complex comprising a protein and a first nucleic acid, wherein the complex recognizes a second nucleic acid. The CRISPR system can be a Type I, Type II, or Type III system. Non-limiting examples of suitable CRISPR proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas1 Od, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4 and Cu1966.

[0324] In some embodiments, the CRISPR protein (e.g., Cas9) is derived from a type II CRISPR system. In specific embodiments, the CRISPR system is designed to act as an endonuclease derived from a Cas9 protein guided by an oligonucleotide (e.g., DNA or RNA). Cas9 proteins useful for this and other functions described herein can be derived from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius. salivarius), Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.), Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus halophilus, Nitrosococcus watsoni, Pseudoalteromonas shaloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngby asp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina).

[0325] Arg protein gene editing system

[0326] The Arg protein family is a family of endonucleases that use 5' phosphorylated single-stranded nucleic acids as guides to cleave nucleic acid targets. These proteins, such as Cas9, are believed to have roles in gene expression repression and defense against foreign nucleic acids.

[0327] Ag protein is different from Cas9 in many aspects. Unlike Cas9, which only exists in prokaryotes, Ag protein is evolutionarily conservative and exists in almost all organisms. Some Ag proteins have been found to bind single-stranded DNA and cleave target DNA molecules. In addition, Ag protein binding does not require a specific common secondary guide structure, and does not require a sequence such as the PAM site of the CRISPR system. It has been shown that the Ag protein of Halobacterium glaucoma can be programmed with a single-stranded DNA guide and used as a genome editor in mammalian cells (Gao, F. et al., "Nature Biotechnology (Nat.Biotechnol.)" 34:768-73 (2016)).

[0328] The Ag protein requires 5' phosphorylation of a single-stranded guide DNA molecule, which is about 24 nucleotides in length. See, for example, the amino acid sequence of the Ag protein in SEQ ID NO: 6 in Table 12.

[0329] Introducing materials into cells

[0330] Introduction of various molecules into cells can be performed by a variety of methods, including those described in many standard laboratory manuals, such as Davis, L. et al., Basic Methods in Molecular Biology (1986) and Sambrook, J. et al., Molecular Cloning: A laboratory manual, Vol. 1, 2nd ed., Cold Spring Harbour Lab. Press, NY (1989). Examples include, but are not limited to, calcium phosphate transfection, DEAE-polydextrose-mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scratch loading, ballistic introduction, nuclear poration, hydrodynamic impact, and infection.

[0331] Different components of nucleic acid cleavage entities and / or donor nucleic acid molecules can be introduced into cells in different ways. In some embodiments, a single type of nucleic acid cleavage entity molecule can be introduced into a cell, but some nucleic acid cleavage entity molecules can be expressed in the cell. An example is the use of two zinc finger-Fok I fusions to generate double-strand breaks in intracellular nucleic acids. In some cases, only one zinc finger-Fok I fusion can be introduced into a cell, while another zinc finger-Fok I fusion can be produced in the cell.

[0332] Suitable transfection agents include those that facilitate the introduction of RNA, DNA, and proteins into cells. Exemplary transfection agents include, but are not limited to, TurboFect transfection reagent (Thermo Fisher Scientific), Pro-Ject reagent (Thermo Fisher Scientific), TRANSPASS TM P protein transfection reagent (New England Biolabs), CHARIOT TM Protein delivery reagents (Active Motif), PROTEOJUICE TM Protein transfection reagent (EMD Millipore), 293fectin, LIPOFECTAMINE TM 2000、LIPOFECTAMINE TM 3000 (Thermo Fisher Scientific), LIPOFECTAMINE TM (Thermo Fisher Scientific), LIPOFECTIN TM (Thermo Fisher Scientific), DMRIE-C, CELLFECTIN TM (Thermo Fisher Scientific), Oligofectamine TM (Thermo Fisher Scientific), LIPOFECTACE TM 、Fugene TM (Roche, Basel, Switzerland), Fugene TM HD (Roche), Transfectam TM (Transfectam, Promega, Madison, Wis.), Tfx-10 TM (Promega), Tfx-20 TM (Promega), Tfx-50 TM (Promega), Transfectin TM (BioRad, Hercules, Calif.), SilentFect TM (Bio-Rad), Effectene TM (Qiagen, Valencia, Calif.), DC-chol (Avanti Polar Lipids), GENEPORTER TM (Gene Therapy Systems, San Diego, Calif.), DHARMAFECT1 TM(Dharmacon, Lafayette, Colo.), DHARMAFECT 2 TM (Dharmacon), DHARMAFECT 3 TM (Dharmacon), DHARMAFECT 4 TM (Dharmacon), ESCORT TM III(Sigma,St.Louis,Mo.) and Escort TM IV (Sigma Chemical Company).

[0333] The compositions and methods of the present invention include methods that can be used for high-throughput screening. An example of such methods is reverse transfection. For the purpose of illustration, it is assumed that a library of gRNA molecules and corresponding NLS-binding donor DNA molecules has been produced. It is further assumed that each library composition comprises (1) a gRNA molecule having sequence homology with a specific locus in the cell genome, and (2) an NLS-binding donor DNA molecule having a region homologous to the expected genomic cleavage site flanking the cell. It is also assumed that three hundred such library compositions have been produced, and each of these compositions is added to a separate position on a glass slide. Finally, under conditions that allow (1) absorption of the library composition and (2) gene editing at the gRNA-specified target locus, a 293FT cell line expressing Cas9 protein is covered on a glass slide. Of course, many variations of such methods are possible, including variations in which the gene editing reagents used are different (e.g., TAL-FokI mRNA instead of gRNA) and array formats are different (e.g., the wells of a 96-well plate instead of the surface of a glass slide).

[0334] Thus, the present invention includes libraries of gene editing reagents (e.g., gRNAs, TAL mRNAs, donor nucleic acid molecules, etc.) and high-throughput methods for modifying various target loci in cells.

[0335] Nucleic acid localization and gene editing efficiency

[0336] The present invention also includes compositions and methods for improving gene editing efficiency. In some embodiments, such compositions and methods relate to nucleic acid molecules connected to one or more intracellular targeting moieties, and the intracellular targeting moieties locate nucleic acid molecules in intracellular locations (e.g., nuclei, mitochondria, chloroplasts, etc.) where gene editing is required. Some embodiments will use intracellular targeting moieties to promote the increase in local concentrations of nucleic acid molecules at one or more intracellular locations. Although it is not desired to be bound by theory, it is believed that the increased gene editing efficiency is caused by an increase in the concentration of donor nucleic acids at the location where gene editing is desired.

[0337] exist Fig. 9An example is shown in this figure showing a nuclear localization signal (NLS) (an example of an intracellular targeting moiety) connected to a single-stranded donor DNA molecule via two different linkers. This type of construct can be used to facilitate nucleic acid delivery to the nucleus. Many variations of this construct are possible.

[0338] like Fig.11 and 13 As shown and discussed in the nuclear localization example below, it has been found that Fig. 9 Those constructs shown can significantly improve the efficiency of gene editing in cells and allow the use of less donor nucleic acid. Specifically, the above-mentioned data prove that the use of NLS-modified donor DNA can improve the efficiency of genome engineering at nucleic acid cleavage sites (e.g., chromosomal loci cut with gRNA / Cas9).

[0339] Fig.11 and 13 The data in the study showed a gene editing efficiency of nearly 80%. In addition, it was found that every approximately 2x 10 5 As little as 0.03 pmoles of NLS-donor DNA conjugates per cell. Thus, embodiments include compositions and methods for genetic engineering of cells wherein at least 75% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, 50% to 75%, 50% to 80%, 50% to 85%, 50% to 95%, 60% to 95%, 70% to 95%, 70% to 90%, 75% to 90%, 80% to 99%, 80% to 97%, 80% to 99%, 80% to 96%, 88% to 98%, etc.) of the cells are modified at a particular locus of interest. Additionally, some embodiments include compositions and methods wherein when cells are treated with 2×10 5 When the donor DNA is contacted with 0.3 pmoles or less (e.g., 0.001 to 0.3, 0.005 to 0.3, 0.01 to 0.3, 0.05 to 0.3, 0.001 to 0.2, 0.005 to 0.2, 0.001 to 0.15, 0.001 to 0.1 pmoles, etc.) per cell, at least 50% of the transfected cells in the reaction mixture are modified at the target locus. This assumes 100% transfection. For example, in the case of 50% transfection, about half of the total number of cells and at least 75% of the transfected cells will be obtained.

[0340] Some embodiments also relate to compositions and methods for increasing the local concentration of donor nucleic acid at a location (e.g., nucleus) where there is an intracellular nucleic acid molecule that needs to be changed. Some embodiments include compositions and methods for increasing the concentration at an intracellular location using an intracellular targeting moiety, wherein the increase in local nucleic acid concentration is at least 10 times (10 to 1,000, 10 to 800, 10 to 600, 10 to 1,000, 10 to 1,000, 10 to 1,000, 10 to 400, 50 to 1,000, 50 to 600, 100 to 1,000, 100 to 700 times, etc.) when the intracellular targeting moiety is not used. For example, the increase in the intracellular localization of the nucleic acid molecule can be measured using, for example, fluorescently labeled nucleic acid molecules. For illustration, both NLS-bound and unbound nucleic acid molecules can be used for comparison in such analyses.

[0341] Fig. 9 A variation of the construct shown in is that the NLS is positioned at the 3' end (rather than the 5' end) of the donor DNA molecule, at both ends, in the middle of the donor DNA molecule, etc. In addition, more than one NLS may be present at one or both ends. In addition, the nucleic acid molecule may be: (1) DNA or RNA; (2) single-stranded or double-stranded; (3) linear, circular, or having a stem or hairpin loop; and / or (4) chemically modified (e.g., containing thiophosphate linkages, 2'-O-methyl bases, etc.). In addition, as described below, the NLS may be replaced with an intracellular targeting moiety that directs the positioning to a cellular space other than the nucleus (e.g., mitochondria, chloroplasts, etc.). Therefore, some embodiments include nucleic acid molecules that are operably linked to one or more intracellular targeting moieties positioned at an intracellular location where gene editing is desired, and methods using such nucleic acid molecules (e.g., for genome engineering). Table 3 lists the amino acid sequences of some exemplary intracellular targeting moieties that may be used in some embodiments.

[0342] Table 3: Exemplary subcellular / organelle localization sequences

[0343]

[0344] In addition, in many cases where intracellular targeting moieties (e.g., polypeptides) are used, these targeting moieties can be designed to localize nucleic acid molecules to locations where intracellular nucleic acids are expected to be present (e.g., nuclei, the matrix of chloroplasts, the matrix of mitochondria, etc.). In other words, in many cases, it may be desirable to localize nucleic acids to specific subspaces within cells. Some embodiments include compositions and methods for localizing donor nucleic acid molecules to locations within cells, and for enhancing the efficiency of genome engineering reactions at locations within cells where donor nucleic acid molecules are localized.

[0345] In the practice of the present invention, a variety of methods can be used to link the intracellular targeting moiety to the nucleic acid molecule. Two methods described in the examples are succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker and System (Thermo Fisher Scientific). In any case, the linker used to connect the intracellular targeting moiety to the nucleic acid molecule generally has certain characteristics, some of which are (1) low cytotoxicity, (2) promote cellular uptake or at least low interference with cellular uptake; and (3) low molecular weight (mwt) (e.g., less than 500 mwt). The connection of the intracellular targeting moiety to the nucleic acid molecule can be performed by PCR amplification using NLS-bound DNA oligonucleotides as primers. In addition, the NLS-bound DNA oligonucleotides can be used as universal primers, where the nucleic acid moiety is connected to a specific region of the gene for PCR amplification. In addition, the NLS-bound DNA oligonucleotide can be bonded to a single-stranded DNA donor or a double-stranded DNA donor with single-stranded protrusions instead of covalently binding the intracellular targeting moiety, and then the donor is brought into the intracellular compartment.

[0346] The size, type and other characteristics of the nucleic acid molecule components in the conjugate often change with application, and wherein the SNP changes are usually shorter than the coding region insertion. In addition, the length of the region (when present) homologous to the endogenous nucleic acid will also change with application. The length of the nucleic acid molecule components in the conjugate (for example donor DNA) can be 1 to 2000 or larger (for example less than or equal to 1500, 1000, 750, 500, 300, 250, 200, 150, 100, 75, 50, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2 or 1) nucleotide or base pair (this depends on whether it is single-stranded or double-stranded). In certain embodiments, the length of the nucleic acid molecule components is 1 to 500, 10 to 400, 20 to 300, 30 to 250, 30 to 200 or 30 to 100 etc. nucleotide or base pair.

[0347] Also included are compositions and methods comprising gene editing proteins (e.g., Cas9 proteins, TAL proteins, etc.), wherein the gene editing proteins are operably linked to one or more intracellular targeting moieties, which are capable of locating donor nucleic acid molecules to the location of endogenous nucleic acid molecules in the cell, and the one or more intracellular targeting moieties are associated with the gene editing protein.

[0348] Nuclear localization signals that can be used in the practice of the present invention can have a variety of structures and can be, for example, monopartite or bipartite. Monopartite NLSs are generally composed of a single cluster of basic residues. Bipartite NLSs are generally composed of two clusters of basic residues separated by 10-12 residues. Exemplary NLS amino acid sequences are shown below:

[0349] Listed in Tables 4, 5 and 6.

[0350]

[0351]

[0352] Fig.39 A series of schematic diagrams of Cas9 proteins operably connected to NLS are shown. Any number and type of NLS can be used, and their position in the protein or nucleic acid molecule will vary with the specificity and intended purpose of the molecule to which the NLS is connected. For proteins such as Cas9 or TAL effectors, it is generally necessary to introduce a large amount of protein into the cell, then keep the protein in the cytoplasm for a relatively short period of time, and localize most of the protein to the nucleus. This is so because it is believed that the longer the protein is retained in the cytoplasm, the higher the degree of protein degradation. It is also believed that the higher the concentration of Cas9 in the nucleus, the higher the cutting efficiency (of course assuming that all Cas9 have cutting activity (such as related to gRNA)). Therefore, the number of proteins (and other molecules) associated with NLS "collected" in the nucleus is based on: (1) the number of proteins introduced into the cell and (2) the rate at which the protein is carried to the nucleus.

[0353] Fig.45 Schematic diagram showing the common TALE structural form. In many cases, TALE works by stimulating DNA present in specific locations within the cell (e.g., nucleus, mitochondria, chloroplasts, etc.). In many cases, interruption of the TALE protein region involved in DNA recognition and binding will result in reduced or eliminated DNA recognition and / or binding activity. Sites 1, 2, and 3 are located outside the TALE region that is believed to be related to DNA recognition and binding. Therefore, when high levels of target DNA binding are required, these are suitable sites for NLS positioning.

[0354] use Fig.45 As a reference point, NLS localization at site 1 may occur anywhere to the left (N-terminally) of amino acid 25. NLS localization at sites 2 and 3 may occur anywhere to the right (N-terminally) of amino acid 814. This includes situations where there are naturally occurring longer TALE protein regions that extend beyond Fig.45 Amino acid 25 on the left and amino acid 814 on the right. In addition, site 3 is located at the C-terminus of the effector domain.

[0355] One or more NLSs may be located at one or more of sites 1, 2 and / or 3. Furthermore, when multiple NLSs are present (at one or more of these sites), they may be of the same type or of different types.

[0356] In many cases, the location of the NLS (e.g. Fig.45 Sites 1, 2 and / or 3) and types in the nucleus will be selected in a manner that causes: (1) high levels of gene editing agents localized to the nucleus and / or (2) high levels of functional activity in the nucleus. These two effects are generally associated with the fact that nuclear functional activity is generally lower than the amount of nuclear localization. This is so because in many cases, not all gene editing agents that enter the nucleus will bind to the target loci for which they are specific, and those gene editing agents that do bind may not always act on the target locus nucleic acid in the manner for which they are designed (e.g., nucleic acid cleavage, transcriptional activation, etc.). In this regard, an exemplary reason is that the nucleic acids of the nucleus in one cell type are more accessible than those of another cell type. In addition, even within cells of the same cell type, there are variations that can make the target loci of one cell in the population more or less accessible than those of another cell in the same population.

[0357] Nuclear localization assays and functional assays (eg, genomic cleavage assays) are described elsewhere herein. To correct for differences in target loci and cell types, the same target loci and cell types will generally be used in comparative analyses.

[0358] In addition, gene editing efficiency will often vary with the locus and cell type being edited. This is due to a variety of factors, including the accessibility of gene editing reagents to the target locus and the efficiency of the cell type in homology-directed repair (HDR). In terms of HDR, cells with higher HDR efficiency (such as 293FT and U2OS cells) generally exhibit higher gene editing rates than cells with lower HDR efficiency levels (such as A549 cells).

[0359] Regarding the location and type of NLS, various forms of TALE proteins are shown in Table 7 below.

[0360]

[0361] The exemplary TALE / NLS formats listed in Table 7 differ in the type of NLS and the position of the NLS within the TALE protein. In some cases, the TALE protein will contain about 1 to about 15 (e.g., about 2 to about 14, about 3 to about 14, about 4 to about 14, about 2 to about 10, about 2 to about 8, about 2 to about 6, about 3 to about 5, about 3 to about 4, etc.) NLSs. In addition, when multiple NLSs are present in a TALE protein, these NLSs can be monopartite or bipartite.

[0362] In addition, two or more NLSs can be located in the same region of the TALE protein (e.g., N-terminal region, etc.). When more than one NLS is located in the TALE protein (e.g., in the same region of the TALE protein), two or more of these NLSs can be located within about 1 to about 50 (e.g., about 2 to about 50, about 3 to about 50, about 5 to about 50, about 10 to about 50, about 15 to about 50, about 2 to about 30, about 5 to about 50, about 5 to about 25, etc.) amino acids from each other. In some cases, two NLSs can be separated by two amino acids from each other. In addition, these amino acids can be of a type intended to form a flexible linker (e.g., Gly-Gly, Gly-Ser, etc.).

[0363] Table 7 lists six specific TALE / NLS forms with respect to the regional position and amino acid sequence of the NLS. Two more general TALE / NLS forms are further listed. Any number of such forms are possible. For example, each region of the TALE protein can independently contain about 1 to about 5 NLSs.

[0364] Fig.46 and 47 The amino acid sequences of two different TALEN proteins with NLS in different positions are shown. Cas9 and TALEN proteins may differ in the number, type, and position of NLS present in the molecule. Fig.46 In the amino acid sequence shown, when the NLS is located at the N-terminus relative to the repeat region, the position of the NLS is farther from the N-terminus than from the R-3 region. In addition, when the NLS is located at the C-terminus relative to the repeat region, the position of the NLS is usually farther from the amino acid HRVA ( Fig.46 amino acids 811-814 in ("HRVA" is disclosed as residues 811-814 of SEQ ID NO:95) are generally closer to the C-terminus, between the repeat region and the effector domain ( Fig.46 Therefore, using Fig.46 Based on the amino acid sequence in , the NLS can be located in three general positions: (1) N-terminal to the repeat region; (2) between the repeat region and the effector domain, and (3) after the effector domain.

[0365] In some cases, use Fig.46 With reference to the amino acid sequence shown in , one or more NLSs may be located in the region of amino acids 768 to 814. For example, in Fig.45 In the sequence of the present invention, one or more NLSs may be immediately following one or more of the following amino acids: 768, 777, 779, 788 and / or 789.

[0366] Specifically, Fig.46In particular, the TAL protein region located to the left of the N-terminal end of the repeat region is shown ( Fig.46 The TAL protein region is generally conserved among Xanthomonas species, with over 90% identity at the amino acid level. In addition, this region contains four regions (R0, R-1, R-2, and R-3) that have some sequence homology to TAL repeat sequences. The NLS is typically located outside of this region and will typically be located closer to the N-terminus of the TAL protein.

[0367] in addition, Fig.46 The amino acid sequence shown in contains, for example, only 153 amino acids at the N-terminus of the repeat region. This N-terminal region can have different lengths and the length can be, for example, about 140 to about 400 (e.g., about 150 to about 350, about 150 to about 300, about 150 to about 250, about 150 to about 200, about 180 to about 350, about 185 to about 300, about 200 to about 350, about 200 to about 300, about 200 to about 300, etc.) amino acids.

[0368] In addition, use Fig.46 The amino acid sequence shown in , the region C-terminal to the amino acid repeat region is also generally conserved among Xanthomonas species. Likewise, the NLS will generally be located outside of this region and will generally be located closer to the C-terminus of the TAL protein.

[0369] Depending on the desired intracellular level of the gene editing molecule (e.g., TAL protein, CRISPR protein, gRNA, etc.) and the desired duration of gene editing activity, the gene editing molecule can be introduced into the cell as RNA / mRNA or by DNA encoding RNA or protein gene editing reagents. In addition, when the nucleic acid encoding the gene editing molecule is located in the cell, the coding region will generally be operably linked to an expression control sequence, such as a promoter (e.g., a constitutive promoter, an inducible promoter, a repressible promoter, etc.).

[0370] Provided herein are various forms of TAL proteins (and other gene editing molecules), nucleic acid molecules encoding these proteins, and methods of using these proteins to modify the genome of a cell.

[0371] The analysis of nuclear absorption for measuring proteins and other molecules is known. Such analysis can be based on the measurement results of functional activity in the core (e.g., GCD analysis described in Example 1). Other analyses directly measure molecular absorption and include fluorescence-based analysis. Such analysis usually requires that the measured molecule shows fluorescence. Fluorescence can be naturally present in the molecule, or fluorescence can be produced by combining with fluorescent molecules (e.g., GFP, OFP, chemical labels (e.g., dyes), etc.).

[0372] Wu et al., Biophysical Journal, 96:3840-3849 (2009) proposed a suitable analysis in which two-photon fluorescence correspondence microscopy was used to measure nuclear import. These methods are based on measuring the average fluorescence intensity of multiple points inside the cytoplasm and the nucleus by microscopy and then determining the ratio. Although some gene editing reagents can be trapped in membranes and endosomes, the cytoplasmic fluorescence level can be compared with the nuclease fluorescence level to determine the rate at which the gene editing reagent enters the nucleus and the amount of gene editing reagent present in the nucleus at one or more time points.

[0373] The method as described by Wu et al. can be used to measure the concentration of fluorescently labeled gene editing reagents based on absorption and position. An exemplary method is to use a two-photon fluorescence corresponding microscope to measure the nuclear localization of Cas9 protein. In this illustration of the method, a series of different Cas9-NLS-GFP fusion protein / gRNA complexes are introduced into the cell line, and 50 fluorescence measurements are performed on the cells, half in the cytoplasm and half in the nucleus. The steady-state nucleus to cytoplasm ratio of each Cas9-NLS-GFP fusion protein / gRNA complex is then determined. Provided herein are compositions and methods for producing cells wherein the nuclear to cytoplasmic ratio of gene editing agents within the cells is, on average, about 5 to about 120 (e.g., about 5 to about 100, about 15 to about 100, about 20 to about 100, about 25 to about 100, about 30 to about 100, about 35 to about 100, about 40 to about 100, about 50 to about 100, about 60 to about 100, about 70 to about 100, about 40 to about 120, about 50 to about 120, etc.).

[0374] Also provided herein are compositions and methods that allow for the generation of cell populations, wherein for diploid cells, at least 90% cleavage occurs in at least one of the two target loci of the population members (e.g., about 90% to about 100%, about 90% to about 98%, about 90% to about 96%, about 93% to about 100%, about 95% to about 100%, about 92% to about 96%, etc.). In some cases, the above percentages of cleavage will apply when the conditions are adjusted so that 50,000 (+ / - 10%) are contacted with about 0.5 to about 200 ng (e.g., about 0.5 to about 150, about 0.5 to about 100, about 0.5 to about 90, about 0.5 to about 75, about 1 to about 200, about 1.5 to about 200, about 3 to about 200, about 1 to about 50 ng, about 10 to about 45, about 12 to about 60 ng, etc.) Cas9 / gRNA complexes under the conditions described in Example 7.

[0375] Compositions and methods using intracellular targeting moieties can be used to alter endogenous nucleic acid molecules by many methods. For example, these compositions and methods can be used to promote homologous recombination at the position where the endogenous nucleic acid is "intact". This means that the endogenous nucleic acid has not been cut by editing gene reagents (e.g., CRISPR, TAL, zinc finger-FokI fusions, etc.). However, in some cases, the genetic alteration site will be cut or have a double-strand break.

[0376] method

[0377] The methods and compositions provided herein are particularly useful for regulating a target locus (e.g., a gene, a genomic region, or a transcriptional regulatory sequence (e.g., a promoter, an enhancer)), including chromatin (a histone bound to DNA), DNA, a protein bound to DNA, or a combination thereof. As used herein, the term "target locus" refers to a region within a cell genome. The target locus includes one or more binding sequences that bind proteins or nucleic acids, the binding of which causes the target locus to be structurally and or chemically modified. Using the methods and compositions provided herein, the target locus can be structurally or chemically modified by binding one or more DNA binding agents (e.g., a first or second DNA binding regulatory enhancer) to a specific site that forms a portion of the target locus. The binding of the DNA binding agent (e.g., a first or second DNA binding regulatory enhancer) can cause, for example, chromatin displacement or reconstruction at the target locus, and / or it can enhance the accessibility of other endogenous or exogenous regulators to further modification of the target locus. For example, by enhancing the accessibility of the cleavage site and surrounding sequences of the DNA at the locus, the methods provided herein are useful for improving the efficiency and specificity of nucleases (TALEN, Cas9) at genomic loci. Thus, the methods and compositions provided herein are particularly suitable for genome editing and enhancing the enzymatic processes involved therein.

[0378] Thus, in one aspect, a method of enhancing accessibility of a target locus in a cell is provided. The method comprises: (1) introducing a first DNA binding regulatory enhancer into a cell comprising a nucleic acid encoding the target locus, wherein the first DNA binding regulatory enhancer is not endogenous to the cell; and (2) allowing the first DNA binding regulatory enhancer to bind to a first enhancer binding sequence of the target locus, thereby enhancing accessibility of the target locus relative to the absence of the first DNA binding regulatory enhancer.

[0379] The accessibility of the target locus can be enhanced upstream or downstream of the enhancer binding sequence provided herein. Thus, after the DNA binding regulatory enhancer binds to the enhancer binding site, the chromatin located 5' and 3' of the enhancer binding sequence can be more accessible relative to the absence of the DNA binding regulatory enhancer.

[0380] In an embodiment, the target locus includes a plurality of DNA binding regulating enhancers of a plurality of enhancer binding sequences (such as 2,4,6,8,10 enhancer binding sequences) that are bonded to the target locus. Each of a plurality of enhancer binding sequences can be separated from each other by a sequence that is 20-60 nucleotides in length. In an embodiment, the target locus includes the first, second, third, fourth, fifth and sixth enhancer binding sequences, wherein the first enhancer binding sequence is connected with the third enhancer binding sequence by the second enhancer binding sequence, the third enhancer binding sequence is connected with the fifth enhancer binding sequence by the fourth enhancer binding sequence, and the fourth enhancer binding sequence is connected with the sixth enhancer binding sequence by the fifth enhancer binding sequence. The first and second enhancer binding sequences, the second and third enhancer binding sequences, the third and fourth enhancer binding sequences, the fourth and fifth enhancer binding sequences and the fifth and sixth enhancer binding sequences can be separated by 20-50 nucleotides respectively. In embodiments, the first and second enhancer binding sequences, the second and third enhancer binding sequences, the third and fourth enhancer binding sequences, the fourth and fifth enhancer binding sequences, and the fifth and sixth enhancer binding sequences are each separated by 50 nucleotides.

[0381] In another aspect, a method for replacing chromatin of a target locus in a cell is provided. The method comprises: (1) introducing a first DNA binding regulatory enhancer into a cell comprising a nucleic acid encoding the target locus, wherein the first DNA binding regulatory enhancer is not endogenous to the cell; and (2) allowing the first DNA binding regulatory enhancer to bind to a first enhancer binding sequence of the target locus, thereby replacing chromatin of the target locus.

[0382] In another aspect, a method for reconstructing chromatin of a target locus in a cell is provided. The method comprises: (1) introducing a first DNA binding regulatory enhancer into a cell comprising a nucleic acid encoding the target locus, wherein the first DNA binding regulatory enhancer is not endogenous to the cell; and (2) allowing the first DNA binding regulatory enhancer to bind to a first enhancer binding sequence of the target locus, thereby reconstructing the chromatin of the target locus.

[0383] As described above, the methods and compositions provided herein may include the binding of one or more DNA binding agents (e.g., a first or second DNA binding regulatory enhancer) to complete the regulation of the target locus. Therefore, in another aspect, a method for enhancing the accessibility of a target locus in a cell is provided. The method comprises (1) introducing into a cell comprising a nucleic acid encoding a target locus: (i) a first DNA binding regulatory enhancer, wherein the first DNA binding regulatory enhancer is not endogenous to the cell; and (ii) a second DNA binding regulatory enhancer, wherein the second DNA binding regulatory enhancer is not endogenous to the cell. (2) allowing the first DNA binding regulatory enhancer to bind to a first enhancer binding sequence of the target locus; and (3) allowing the second DNA binding regulatory enhancer to bind to a second enhancer binding sequence of the target locus, thereby enhancing the accessibility of the target locus relative to the lack of the first DNA binding regulatory enhancer or the second DNA binding regulatory enhancer. As provided herein, enhancing (increasing) the accessibility of the target locus refers to structural regulation of the target locus, which causes the functional activity of a regulatory protein or complex (e.g., an enzyme (e.g., a nuclease)) on the target locus to be enhanced. The target locus is removed from the chromatin and / or the DNA at the target locus is reconstructed so that the regulatory protein can better bind and / or enhance activity. Therefore, the term enhances (increases) the accessibility of the target locus and includes regulating the structure of the target locus so that the activity of the regulatory protein is increased, wherein the activity includes, for example, enzymatic activity, DNA binding activity, transcriptional activity.

[0384] In one aspect, a method for replacing chromatin of a target locus in a cell is provided. The method comprises: (1) introducing into a cell comprising a nucleic acid encoding the target locus: (i) a first DNA binding regulatory enhancer, wherein the first DNA binding regulatory enhancer is not endogenous to the cell; and (ii) a second DNA binding regulatory enhancer, wherein the second DNA binding regulatory enhancer is not endogenous to the cell. (2) allowing the first DNA binding regulatory enhancer to bind to a first enhancer binding sequence of the target locus; and (3) allowing the second DNA binding regulatory enhancer to bind to a second enhancer binding sequence of the target locus, thereby replacing chromatin of the target locus.

[0385] In one aspect, a method for reconstructing chromatin of a target locus in a cell is provided. The method comprises: (1) introducing into a cell comprising a nucleic acid encoding the target locus: (i) a first DNA binding regulatory enhancer, wherein the first DNA binding regulatory enhancer is not endogenous to the cell; and (ii) a second DNA binding regulatory enhancer, wherein the second DNA binding regulatory enhancer is not endogenous to the cell. (2) allowing the first DNA binding regulatory enhancer to bind to a first enhancer binding sequence of the target locus; and (3) allowing the second DNA binding regulatory enhancer to bind to a second enhancer binding sequence of the target locus, thereby reconstructing the chromatin of the target locus.

[0386] As described above, the methods and compositions provided herein can enhance the accessibility of the target locus and thereby allow the regulatory activity of the target locus to be raised. Therefore, a method for regulating the target locus in a cell is provided. The method includes (1) introducing into a cell comprising a nucleic acid encoding the target locus: (i) a first regulatory protein or a first regulatory complex capable of binding to a regulator binding sequence of the target locus, wherein the regulator binding sequence includes a regulatory site; and (ii) a first DNA binding regulatory enhancer capable of binding to a first enhancer binding sequence of the target locus. And (2) allowing the first regulatory protein or the first regulatory complex to regulate the regulatory site, thereby regulating the target locus in the cell.

[0387] Since one or more DNA binders (e.g., the first or second DNA binding regulatory enhancer) are bound to the target locus, the target locus becomes more accessible, thereby allowing the efficiency and / or specificity of the regulatory protein or regulatory complex to be enhanced at the target locus. For example, the efficiency of gene editing reactions can be enhanced by, for example, homologous recombination using the methods and compositions provided herein. In an embodiment, due to the presence of one or more DNA binders (e.g., the first or second DNA binding regulatory enhancer), the nuclease activity of the nuclease at the target locus is enhanced.

[0388] Therefore, in one aspect, a method for enhancing the activity of a regulatory protein or a regulatory complex at a target locus in a cell is provided. The method comprises (1) introducing into a cell comprising a nucleic acid encoding a target locus: (i) a first regulatory protein or a first regulatory complex capable of binding to a regulator binding sequence of the target locus, wherein the regulator binding sequence comprises a regulatory site; and (ii) a first DNA binding regulatory enhancer capable of binding to a first enhancer binding sequence of the target locus. And (2) binding the first DNA binding regulatory enhancer to the first enhancer binding sequence, thereby enhancing the activity of the first regulatory protein or the first regulatory complex at the target locus in the cell.

[0389] Also provided herein are compositions and methods for using DNA binding protein-transcription activator fusion proteins to generate regions of chromatin structure accessible to gene editing agents. In some aspects, provided herein are uses of DNA binding protein-transcription activator fusion proteins and methods of using such fusion proteins to reshape chromatin to allow for enhanced site-directed nucleic acid cleavage. Variations of some aspects of this method are shown in Fig.51 middle.

[0390] It is known that transcriptional activation remodels chromatin and disrupts the normally defined pattern of nucleosomes at a particular locus. (See, e.g., Gilbert and Ramsahoye, "The relationship between chromatin structure and transcriptional activity in mammalian genomes," BRIEFINGS IN FUNCTIONAL GENOMICS AND PROTEOMICS 4: 129-142 (2005)).

[0391] Fig.51 The upper part of the diagram shows a schematic diagram of the intracellular nucleic acid region, where the nucleic acids are in the form of 10 nm chromatin fibers. Fig.51 The upper portion shows the promoter, nucleosomes, desired editing sites, and potential Buddy-TAL binding sites. The position of the nucleosome may vary depending on the nucleic acid region and / or the specific cell in which the specific nucleic acid region is located. For example, in any specific cell, the promoter nucleic acid may be completely or partially located in the nucleosome or entirely located outside the nucleosome. In addition, the position of a specific nucleic acid region (e.g., an editing site) relative to the nucleosome in a specific cell may vary depending on factors such as a specific time point, transcriptional state, cell cycle stage, etc.

[0392] For illustration purposes, use Fig.51 Schematic diagram of a TAL-transcription activator fusion protein binding to a TAL binding site ("TBS"), causing transcriptional activation. This results in chromatin remodeling in the transcribed nucleic acid region and surrounding local regions. This chromatin remodeling enhances the accessibility of gene editing agents with nucleic acid cleavage activity (e.g., TAL-FokI fusion protein) to nucleic acids. The net result is increased nucleic acid cleavage activity by gene editing agents.

[0393] Fig.51Also shown is an "editing site". As used herein, an "editing site" refers to a nucleic acid site where one or more gene editing reagents are designed to cleave a locus that changes the nucleotide sequence level (e.g., deletion, insertion, and / or substitution). In this schematic, transcription is used to increase the accessibility of gene editing reagents to the editing site.

[0394] Buddy-TAL, also known as DNA binding regulatory enhancers, can also be used in combination with DNA binding protein transcription activator fusion proteins. These Buddy-TALs can be used to enhance the binding of DNA binding protein-transcription activator fusion proteins to TAL binding sites ("TBS") and / or enhance the accessibility of gene editing agents with nucleic acid cleavage activity to editing sites. Therefore, the present invention provides compositions and methods for enhancing nucleic acid cleavage using DNA binding protein-transcription activator fusion proteins alone and in combination with Buddy-TAL.

[0395] In some aspects, provided herein is a method for editing a first nucleic acid locus in a cell, the method comprising: (A) contacting a second nucleic acid locus with a DNA binding protein-transcription activator fusion protein under conditions that allow nucleic acid transcription; and (B) contacting the first nucleic acid locus with one or more gene editing agents having nucleic acid cleavage activity under conditions that allow nucleic acid cleavage at the first nucleic acid locus, wherein nucleic acid transcription causes the chromatin structure of the first nucleic acid locus to change. In some cases, one or more DNA binding regulatory enhancers can also be used, the DNA binding regulatory enhancers being designed to bind to one or more nucleic acid positions within two hundred (e.g., about 30 to about 200, about 50 to about 200, about 60 to about 200, about 30 to about 180, about 30 to about 130, about 45 to about 150, etc.) base pairs of (a) the first nucleic acid locus and / or (b) the second nucleic acid locus. In some cases, one or more DNA binding regulatory enhancers form a first nucleic acid locus, and the DNA binding regulatory enhancer is designed to bind to a nucleic acid position within two hundred base pairs upstream of the second nucleic acid locus and / or downstream of the first nucleic acid locus. In some cases, the DNA binding protein-transcription activator fusion protein can be a TAL-transcription activation domain (e.g., p53, NFAT, NF-κB, VP16, VP32, VP64, etc.) fusion protein. In some cases, at least one of the one or more DNA binding regulatory enhancers can be a TAL-nuclease fusion protein (e.g., TAL-FokI fusion protein).

[0396] DNA binding regulator enhancer

[0397] As provided herein, "DNA binding regulatory enhancer" is an agent that can bind to the corresponding sequence (enhancer binding sequence) of the target locus in the cell and thereby chemically or structurally regulate the target locus. After binding to the target locus, the DNA binding regulatory enhancer provided herein (including its embodiments) can regulate the chromatin at the locus. After binding, the DNA binding regulatory enhancer can convert the densely packed heterochromatin region upstream (5') or downstream (3') of the enhancer binding sequence into a less densely packed euchromatin region. The conversion can be achieved by dissociating histones from the DNA bound to them on the target site (chromatin replacement). Alternatively, histones can be rearranged in the chromatin at the target locus (chromatin reconstruction). Once the chromatin structure on the target locus is changed, DNA becomes more accessible so as to subsequently modify the target locus. This effect can be achieved by the combination of one or more DNA binding regulatory enhancers (e.g., the first or second DNA binding regulatory enhancer). Therefore, in an embodiment, the method described herein includes introducing a second DNA binding regulatory enhancer that can bind to the second enhancer binding sequence of the target locus.

[0398] For the method provided herein, enhancers and regulatory proteins or complexes can be introduced into cells in a variety of ways. Enhancers and regulatory proteins or complexes can be introduced by transfection of nucleic acids (vectors) encoding enhancers and regulatory proteins or complexes. Alternatively, enhancers and regulatory proteins or complexes can be introduced by transfection of mRNA encoding enhancers and regulatory proteins or complexes. Enhancers and regulatory proteins or complexes can be further introduced by direct transfection of actual medicaments, regulatory proteins or regulatory complexes. Those of ordinary skill in the art will immediately recognize that the half-life of medicaments, regulatory proteins or complexes in cells (the time when medicaments have activity and / or expression in cells) is determined by the physical form of the cells delivered to them. Not bound by any particular scientific theory, compared to enhancers and regulatory proteins or complexes transfected as actual proteins or complexes, the delivery of nucleic acids encoding enhancers, regulatory proteins or complexes will extend the enhancers, regulatory proteins or complexes expressed / present in the cells.

[0399] In embodiments, the introduction of the first DNA binding modulator enhancer comprises the introduction of a vector encoding the first DNA binding modulator enhancer. In embodiments, the introduction of the first DNA binding modulator enhancer comprises the introduction of an mRNA encoding the first DNA binding modulator enhancer. In embodiments, the introduction of the first DNA binding modulator enhancer comprises the introduction of a first DNA binding protein or a first DNA binding nucleic acid.

[0400] In an embodiment, the introduction of a second DNA binding modulator enhancer comprises the introduction of a vector encoding the second DNA binding modulator enhancer. In an embodiment, the introduction of a second DNA binding modulator enhancer comprises the introduction of an mRNA encoding the second DNA binding modulator enhancer. In an embodiment, the introduction of a second DNA binding modulator enhancer comprises the introduction of a second DNA binding protein or a second DNA binding nucleic acid.

[0401] In embodiments, introducing a first regulatory protein comprises introducing a vector encoding a first regulatory protein. In embodiments, introducing a first regulatory protein comprises introducing an mRNA encoding a first regulatory protein. In embodiments, introducing a first regulatory protein comprises introducing a first regulatory protein. In embodiments, introducing a first regulatory complex comprises introducing a vector encoding a first regulatory complex. In embodiments, introducing a first regulatory complex comprises introducing an mRNA encoding a first regulatory complex. In embodiments, introducing a first regulatory complex comprises introducing a first regulatory complex.

[0402] In an embodiment, the introduction of a second regulatory protein comprises introducing a vector encoding a second regulatory protein. In an embodiment, the introduction of a second regulatory protein comprises introducing an mRNA encoding a second regulatory protein. In an embodiment, the introduction of a second regulatory protein comprises introducing a second regulatory protein. In an embodiment, the introduction of a second regulatory complex comprises introducing a vector encoding a second regulatory complex. In an embodiment, the introduction of a second regulatory complex comprises introducing an mRNA encoding a second regulatory complex. In an embodiment, the introduction of a second regulatory complex comprises introducing a second regulatory complex.

[0403] Exemplary DNA binding regulatory enhancers that can be used for the methods and compositions provided herein include DNA binding proteins or DNA binding nucleic acids. The first DNA binding regulatory enhancer and the second DNA binding regulatory enhancer can be the same or chemically different. In an embodiment, the first DNA binding regulatory enhancer is not endogenous to the cell. In an embodiment, the second DNA binding regulatory enhancer is not endogenous to the cell. In an embodiment, the first DNA binding regulatory enhancer is a first DNA binding protein or a first DNA binding nucleic acid. In an embodiment, the first DNA binding regulatory enhancer is a first transcription activator-like (TAL) effector protein or a truncated first guide RNA (gRNA). In an embodiment, the first DNA binding regulatory enhancer is a first zinc finger DNA binding protein. In an embodiment, the second DNA binding regulatory enhancer is a second DNA binding protein or a second DNA binding nucleic acid. In an embodiment, the second DNA binding regulatory enhancer is a TAL effector protein or a truncated gRNA.

[0404] "Truncate gRNA" or "truncated guide RNA" is a ribonucleic acid corresponding to a wild-type guide RNA, but contains fewer nucleotides than the wild-type guide RNA. As provided herein, a truncated gRNA can bind to a Cas9 protein. Therefore, a truncated guide RNA as provided herein can be an RNA that binds to a Cas9 protein and is capable of binding to a regulator binding sequence. The Cas9 protein bound to the truncated gRNA cannot cleave the regulator binding sequence. Therefore, in an embodiment, a DNA binding regulatory enhancer is a truncated gRNA bound to a Cas9 protein. In an embodiment, the Cas9 protein bound to the truncated gRNA is a Streptococcus pyogenes Cas9 protein. The Streptococcus pyogenes Cas9 protein as provided herein is a Cass9 protein derived from the bacterium Streptococcus pyogenes.

[0405] The length of the truncated gRNA provided herein may be less than 16 nucleotides. In an embodiment, the length of the truncated gRNA is no more than 15 nucleotides. In an embodiment, the length of the truncated gRNA is 10 to 15 nucleotides. In an embodiment, the length of the truncated gRNA is 11 to 15 nucleotides. In an embodiment, the length of the truncated gRNA is 12 to 15 nucleotides. In an embodiment, the length of the truncated gRNA is 13 to 15 nucleotides. In an embodiment, the length of the truncated gRNA is 10 to 14 nucleotides. In an embodiment, the length of the truncated gRNA is 10 to 13 nucleotides. In an embodiment, the length of the truncated gRNA is 10 to 12 nucleotides. In an embodiment, the length of the truncated gRNA is 16 nucleotides. In an embodiment, the length of the truncated gRNA is less than 15 nucleotides. In an embodiment, the length of the truncated gRNA is 15 nucleotides. In an embodiment, the length of the truncated gRNA is less than 14 nucleotides. In an embodiment, the length of the truncated gRNA is 14 nucleotides. In an embodiment, the length of the truncated gRNA is less than 13 nucleotides. In an embodiment, the length of the truncated gRNA is 13 nucleotides. In an embodiment, the length of the truncated gRNA is less than 12 nucleotides. In an embodiment, the length of the truncated gRNA is 12 nucleotides. In an embodiment, the length of the truncated gRNA is less than 11 nucleotides. In an embodiment, the length of the truncated gRNA is 11 nucleotides. In an embodiment, the length of the truncated gRNA is less than 10 nucleotides. In an embodiment, the length of the truncated gRNA is 10 nucleotides. In an embodiment, the length of the truncated gRNA is less than 9 nucleotides. In an embodiment, the length of the truncated gRNA is 9 nucleotides. In an embodiment, the length of the truncated gRNA is less than 8 nucleotides. In an embodiment, the length of the truncated gRNA is 8 nucleotides. In an embodiment, the length of the truncated gRNA is less than 7 nucleotides. In an embodiment, the length of the truncated gRNA is 7 nucleotides. In an embodiment, the length of the truncated gRNA is less than 6 nucleotides. In an embodiment, the length of the truncated gRNA is 6 nucleotides. In an embodiment, the length of the truncated gRNA is less than 5 nucleotides. In an embodiment, the length of the truncated gRNA is 5 nucleotides. In an embodiment, the length of the truncated gRNA is less than 4 nucleotides. In an embodiment, the length of the truncated gRNA is 4 nucleotides.

[0406] enhancer binding sequence

[0407] As provided herein, an "enhancer binding sequence" is a nucleic acid sequence that forms part of a target locus and is bound by a DNA binding regulatory enhancer. In an embodiment, the enhancer binding sequence is a TAL nucleic acid binding box. As used herein, a "TAL nucleic acid binding box" (also referred to as a "TAL box") refers to a nucleic acid encoding a polypeptide that allows a protein including the polypeptide to bind to a single base pair (e.g., A, T, C, or G). In an embodiment, the protein will contain more than one polypeptide encoded by the TAL nucleic acid binding box. The individual amino acid sequences of the encoded polymer are referred to as "TAL repeats." In an embodiment, the TAL repeat sequence will be between twenty-eight and forty amino acids in length and will share at least 60% (e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, about 60% to about 95%, about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 60% to about 90%, about 60% to about 85%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, etc.) identity with the following thirty-four amino acid sequence: LTPDQVVAIA SXXGGKQALE TVQRLLPVLC QAHG (SEQ ID NO: 118).

[0408] In an embodiment, the two Xs at positions twelve and thirteen in the above sequence represent amino acids in the TAL nucleic acid binding box that also recognize specific bases in nucleic acid molecules.

[0409] In embodiments, the last TAL repeat sequence present at the carboxyl terminus of a series of repeat sequences is typically a partial TAL repeat sequence, wherein the carboxyl terminus may be deleted (eg, approximately the amino-terminal 15 to 20 amino acids of the last TAL repeat sequence).

[0410] In an embodiment, the enhancer binding sequence is a nucleic acid sequence that can bind (hybridize) to a guide RNA binding sequence or a guide DNA binding sequence. In an embodiment, the first enhancer binding sequence has a sequence of SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40. In an embodiment, the second enhancer binding sequence has a sequence of SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, or SEQ ID NO: 41.

[0411] Regulatory proteins and regulatory complexes

[0412] The regulatory protein and regulatory complex provided herein can be cell endogenous, or may not be cell endogenous.Terms " regulatory protein " and " regulatory complex " as provided herein refer to molecules (such as protein or protein conjugate) or molecular complexes (such as ribonucleoprotein complex) that can structurally and / or chemically change the target locus.The change of the target locus structure or chemical composition can include the change of the whole target locus or its part.The example of regulatory protein includes but is not limited to double-stranded nuclease, nickase, transcription activator, transcription repressor, nucleic acid methylase, nucleic acid demethylase, topoisomerase, rotating screw, ligase, methyltransferase, transposase, glycosylase, integrase, kinase, phosphatase, thiourease, polymerase, fluorescence activity and recombinase.The non-limiting examples of the regulatory complex provided herein include ribonucleoprotein complex and deoxyribonucleoprotein complex.

[0413] In an embodiment, the first regulatory protein or the second regulatory protein comprises a DNA binding protein or a DNA regulatory enzyme. The DNA binding protein can be a transcriptional repressor or a transcriptional activator. In an embodiment, the DNA regulatory enzyme is a nuclease, a deaminase, a methylase or a demethylase. In an embodiment, the first regulatory protein or the second regulatory protein comprises a histone regulatory enzyme. In an embodiment, the histone regulatory enzyme is a deacetylase or an acetylase.

[0414] In an embodiment, the first regulatory protein or the second regulatory protein includes a first DNA binding domain operably connected to a first DNA modification domain. In an embodiment, the first DNA binding domain is a TAL effector domain, and the first DNA modification domain is a transcriptional activation domain or a transcriptional repression domain. In an embodiment, the first DNA modification domain is a VP16 domain. In an embodiment, the first DNA modification domain is a VP64 domain. In an embodiment, the first DNA modification domain is a VP16 domain, a VP32 domain, or a VP64 transcriptional activation domain, or a KRAB transcriptional repression domain.

[0415] In an embodiment, the first regulatory protein is a first DNA binding nuclease conjugate. In an embodiment, the second regulatory protein is a second DNA binding nuclease conjugate. As used herein, "DNA binding nuclease conjugate" refers to one or more molecules, enzymes or molecular complexes with nucleic acid cleavage activity (e.g., double-stranded nucleic acid cleavage activity). In most embodiments, the DNA binding nuclease conjugate component will be a protein or nucleic acid or a combination of the two, but they can be combined with cofactors and / or other molecules. DNA binding nuclease conjugates will usually be selected based on a variety of factors, such as the DS fracture production efficiency at the target locus, the ability to produce DS fractures at or near the target locus, the potential low, low toxicity and cost issues of producing DS fractures at non-desired loci. Many of these factors will vary with used cells and target loci. Many DNA binding nuclease conjugates are known in the art. For example, in some embodiments, the DNA binding nuclease conjugate includes one or more zinc finger proteins, transcription activator-like effectors (TALEs), CRISPR complexes (e.g., Cas9 or CPF1), homing endonucleases or megabase meganucleases, Ager protein-nucleic acid complexes, or meganucleases. In some embodiments, the DNA binding nuclease conjugate will have an activity that allows them to be nuclear localized (e.g., will include a nuclear localization signal (NLS)). In some embodiments, a single-stranded DNA donor can work with a nick or a combination of nicks.

[0416] In an embodiment, the DNA-binding nuclease conjugate is a TAL effector fusion. As provided herein, "TAL effector fusion" refers to a TAL effector connected to another polypeptide or protein (e.g., Ag protein) that is not naturally associated with it in nature. In an embodiment, the non-TAL component in the TAL effector fusion will confer functional activity (e.g., enzymatic activity) to the fusion protein. In an embodiment, the TAL effector fusion may have binding activity or may have activity that directly or indirectly triggers nucleic acid modification, such as nuclease activity.

[0417] In an embodiment, the first DNA binding nuclease conjugate comprises a first nuclease, and the second DNA binding nuclease conjugate comprises a second nuclease. In an embodiment, the first nuclease and the second nuclease form a dimer. In an embodiment, the first nuclease and the second nuclease are independently transcription activator-like effector nucleases (TALENs). In an embodiment, the first nuclease and the second nuclease are independently FokI nuclease cleavage domain mutant KKR Sharkey. In an embodiment, the first nuclease and the second nuclease are independently FokI nuclease cleavage domain mutant ELD Sharkey.

[0418] In an embodiment, the first DNA-binding nuclease conjugate comprises a first transcription activator-like (TAL) effector domain (e.g., a DNA-binding portion of a TAL protein) operably linked to a first nuclease (TALEN). In an embodiment, the first DNA-binding nuclease conjugate comprises a first TAL effector domain operably linked to a first FokI nuclease. In an embodiment, the second DNA-binding nuclease conjugate comprises a second TAL effector domain operably linked to a second nuclease (TALEN). In an embodiment, the second DNA-binding nuclease conjugate comprises a second TAL effector domain operably linked to a second FokI nuclease. In an embodiment, the first DNA-binding nuclease conjugate comprises a first zinc finger nuclease. In an embodiment, the second DNA-binding nuclease conjugate comprises a first zinc finger nuclease.

[0419] As used herein, the term "zinc finger nuclease" refers to a protein comprising a polypeptide having a zinc-stabilized nucleic acid (e.g., DNA) binding domain. Individual DNA binding domains are often referred to as "fingers," such that zinc finger proteins or polypeptides have at least one finger, more often two fingers or three fingers, or even four or five fingers, to at least six or more fingers. In certain aspects, a zinc finger nuclease will comprise three or four zinc fingers. Each finger typically binds two to four base pairs of DNA. Each finger typically comprises a zinc-chelating DNA binding region of about 30 amino acids (see, e.g., U.S. Patent Publication No. 2012 / 0329067A1, the disclosure of which is incorporated herein by reference).

[0420] An example of a nuclease protein that forms part of a conjugate provided herein is the nonspecific cleavage domain from the type IIS restriction endonuclease FokI (Kim, YG et al., Proc. Natl. Acad. Sci. USA 93: 1156-60 (1996)), which is typically separated by a linker sequence of 5-7 base pairs. A pair of FokI cleavage domains is typically required to allow domain dimerization and non-palindromic target sequence cleavage from opposite strands. The DNA binding domain of an individual Cys2His2ZFN typically contains 3 to 6 individual zinc finger repeats and each is capable of recognizing 9 to 18 base pairs.

[0421] As used herein, "transcription activator-like effector" (TALE) refers to a protein composed of more than one TAL repeat sequence and is capable of binding nucleic acids in a sequence-specific manner. TALE represents a class of DNA-binding proteins secreted by plant pathogenic bacterial species (e.g., Xanthomonas and Ralstonia) via their type III secretion system after infecting plant cells. Natural TALEs have been shown to bind to plant promoter sequences, thereby regulating gene expression and activating effector-specific host genes, thereby promoting bacterial reproduction. P. et al., Science, 318:645-648 (2007); Boch, J. et al., Annu. Rev. Phytopathol. 48:419-436 (2010); Kay, S. et al., Science, 318:648-651 (2007); Kay, S. et al., Curr. Opin. Microbiol. 12:37-43 (2009).

[0422] Natural TALEs are typically characterized by a central repeat domain and a carboxyl-terminal nuclear localization signal sequence (NLS) and a transcription activation domain (AD). In addition to the usually shorter carboxyl-terminal repeat sequence (called a half-repeat sequence), the central repeat domain typically consists of: 1.5 to 33.5 different amounts of amino acid repeat sequences, which are usually 33-35 residues in length. Most of the repeat sequences are consistent, but differ in certain hypervariable residues. The DNA recognition specificity of TALE is mediated by hypervariable residues (so-called repeat variable diresidues (RVDs), each of which targets a specific nucleotide in a given DNA sequence) that are usually located at positions 12 and 13 of each repeat sequence. Therefore, the order of repeat sequences in TAL proteins tends to be related to the defined linear order of nucleotides in a given DNA sequence. Potential RVD codes for some naturally occurring TALEs have been identified, allowing prediction of the order of repeat sequences necessary to bind to a given DNA sequence (Boch, J. et al., Science 326: 1509-1512 (2009); Moscou, MJ et al., Science 326: 1501 (2009)). In addition, it has been shown that TAL effectors generated with novel repeat sequence combinations bind to target sequences predicted from this code. It has been shown that target DNA sequences typically begin with a 5' thymine base that is recognized by TAL proteins.

[0423] The TAL module structure allows the DNA binding domain to be combined with an effector molecule (such as a nuclease). Specifically, TALE nucleases allow the development of new genome engineering tools. The TALE used in some embodiments can produce a DS break, or can have a combined effect for producing a DS break. For example, a TAL-FokI nuclease fusion can be designed to bind at or near the target locus and form a double-stranded nucleic acid cleavage activity through the binding of two FokI domains.

[0424] In some embodiments, the TALE will comprise greater than or equal to 6 (e.g., greater than or equal to 8, 10, 12, 15, or 17, or 6 to 25, 6 to 35, 8 to 25, 10 to 25, 12 to 25, 8 to 22, 10 to 22, 12 to 22, 6 to 20, 8 to 20, 10 to 22, 12 to 20, 6 to 18, 10 to 18, 12 to 18, etc.) TAL repeat sequences. In some embodiments, the TALE may comprise 18 or 24 or 17.5 or 23.5 TAL nucleic acid binding boxes. In further embodiments, the TALE may comprise 15.5, 16.5, 18.5, 19.5, 20.5, 21.5, 22.5, or 24.5 TAL nucleic acid binding boxes. The TALE will typically have at least one polypeptide region flanking a region comprising a TAL repeat sequence. In many embodiments, the flanking regions will be present at the amino terminus and carboxyl terminus of the TAL repeat sequence. Exemplary TALEs are described in U.S. Patent Publication No. 2013 / 0274129A1, the disclosure of which is incorporated herein by reference, and may be modified forms of naturally occurring proteins found in Burkholderia, Xanthamonas, and Ralstonia bacteria. In some embodiments, the TALE protein will contain a nuclear localization signal (NLS) that allows it to be transported to the nucleus.

[0425] For the methods and compositions provided herein, the nucleic acid targeting ability of the regulatory protein or regulatory complex is enhanced relative to the absence of a DNA binding regulatory enhancer. In embodiments, the homologous recombination rate at the target locus is increased relative to the absence of a DNA binding regulatory enhancer.

[0426] In an embodiment, the first regulatory complex is a first ribonucleoprotein complex. In an embodiment, the second regulatory complex is a second ribonucleoprotein complex. In an embodiment, the first ribonucleoprotein complex includes a CRISPR-associated protein 9 (Cas9) domain bound to a gRNA or an Agg protein domain bound to a guide DNA (gDNA). In an embodiment, the second ribonucleoprotein complex includes a CRISPR-associated protein 9 (Cas9) domain bound to a gRNA or an Agg protein domain bound to a guide DNA (gDNA).

[0427] In embodiments, the first DNA binding regulatory enhancer is a first TAL effector protein and the second DNA binding regulatory enhancer is a second TAL effector protein. In embodiments, the first DNA binding regulatory enhancer is a TAL effector protein and the second DNA binding regulatory enhancer is a truncated gRNA. In embodiments, the first DNA binding regulatory enhancer is a truncated first gRNA and the second DNA binding regulatory enhancer is a truncated second gRNA. In embodiments, the first DNA binding regulatory enhancer is a truncated gRNA and the second DNA binding regulatory enhancer is a TAL effector protein.

[0428] In an embodiment, the first regulatory protein is a first DNA-binding nuclease conjugate and the second regulatory protein is a second DNA-binding nuclease conjugate. In an embodiment, the first regulatory protein is a DNA-binding nuclease conjugate and the second regulatory complex is a ribonucleoprotein complex. In an embodiment, the first regulatory complex is a first ribonucleoprotein complex and the second regulatory complex is a second ribonucleoprotein complex. In an embodiment, the first regulatory complex is a ribonucleoprotein complex and the second regulatory protein is a DNA-binding nuclease conjugate.

[0429] The agents provided herein may be endogenous or non-endogenous to the cells expressing them. Therefore, in an embodiment, the first regulatory protein or the first regulatory complex is not endogenous to the cell. In an embodiment, the first regulatory protein, the first regulatory complex, the second regulatory protein, or the second regulatory complex is not endogenous to the cell. In an embodiment, the first regulatory protein and the second regulatory protein are not endogenous to the cell. In an embodiment, the first regulatory complex and the second regulatory complex are not endogenous to the cell. In an embodiment, the first DNA binding regulatory enhancer or the second DNA binding regulatory enhancer is not endogenous to the cell. In an embodiment, the first DNA binding regulatory enhancer and the second DNA binding regulatory enhancer are not endogenous to the cell.

[0430] The applicant is surprised to find that the distance of the first and / or second enhancer binding site relative to the regulator binding sequence affects the DNA binding regulation enhancer on the activity of the regulator protein or the regulation complex. The distance between the first enhancer binding site and the regulator binding sequence is the number of nucleotides that make the most of 3' nucleotides of the first DNA binding regulation enhancer be connected to the most of 5' nucleotides of the regulator binding sequence. Similarly, the distance between the second enhancer binding site and the regulator binding sequence is the number of nucleotides that make the most of 3' nucleotides of the regulator binding sequence and the most of 5' nucleotides of the first DNA binding regulation enhancer be connected. The regulator binding sequence can be combined by protein (such as DNA binding protein) or nucleic acid (such as gRNA or gDNA). The regulation site included in the regulator binding sequence is the position of the nucleotides in the regulator binding sequence, which is recognized by the regulator protein or the regulation complex, and the nucleotides corresponding to the key hydrolyzed with the remainder of the regulator binding sequence.

[0431] In an embodiment, the first enhancer binding sequence or the second enhancer binding sequence is less than 200 nucleotides away from the regulator binding sequence. In an embodiment, the first enhancer binding sequence or the second enhancer binding sequence is less than 150 nucleotides away from the regulator binding sequence. In an embodiment, the first enhancer binding sequence or the second enhancer binding sequence is less than 100 nucleotides away from the regulator binding sequence. In an embodiment, the first enhancer binding sequence or the second enhancer binding sequence is less than 50 nucleotides away from the regulator binding sequence. In an embodiment, the first enhancer binding sequence or the second enhancer binding sequence is 4 to 30 nucleotides away from the regulator binding sequence. In an embodiment, the first enhancer binding sequence or the second enhancer binding sequence is 7 to 30 nucleotides away from the regulator binding sequence. In an embodiment, the first enhancer binding sequence or the second enhancer binding sequence is 4 nucleotides away from the regulator binding sequence. In an embodiment, the first enhancer binding sequence or the second enhancer binding sequence is 7 nucleotides away from the regulator binding sequence. In an embodiment, the first enhancer binding sequence or the second enhancer binding sequence is 12 nucleotides away from the regulator binding sequence. In an embodiment, the first enhancer binding sequence or the second enhancer binding sequence is separated from the regulator binding sequence by 20 nucleotides. In an embodiment, the first enhancer binding sequence or the second enhancer binding sequence is separated from the regulator binding sequence by 30 nucleotides.

[0432] In embodiments, the first enhancer binding sequence or the second enhancer binding sequence is separated from the regulatory site by 10 to 40 nucleotides. In embodiments, the first enhancer binding sequence or the second enhancer binding sequence is separated from the regulatory site by 33 nucleotides.

[0433] In an embodiment, the first enhancer binding sequence is separated from the regulator binding sequence by 30 nucleotides, and the second enhancer binding sequence is separated from the regulator binding sequence by 19 nucleotides. In other embodiments, the length of the first enhancer binding sequence and the second enhancer binding sequence is independently 18 nucleotides. In another other embodiment, the regulator binding sequence includes a first binding sequence and a second binding sequence, wherein the length of the first binding sequence and the second binding sequence is independently 18 nucleotides and separated by 16 nucleotide sequences.

[0434] In embodiments, the first DNA binding regulatory enhancer or the second DNA binding regulatory enhancer enhances the activity of the first regulatory protein, the first regulatory complex, the second regulatory protein, or the second regulatory complex at the regulatory site.

[0435] Provided herein are various forms for enhancing the accessibility of other components present in the cell (e.g., donor DNA molecules, regulatory proteins, regulatory complexes, etc.) to the target locus. The binding of a regulatory enhancer (which binds to a specific DNA sequence (enhancer binding sequence) in the target locus) enhances accessibility. The DNA binding regulatory enhancer can be a truncated gRNA or TAL effector domain. In an embodiment, two DNA binding regulatory enhancers (e.g., a first and a second DNA binding regulatory enhancer) bind to the target locus. In the case where two DNA binding regulatory enhancers (e.g., two TAL effector domains or two truncated gRNAs) bind to the target locus, they can be flanked by regulatory sequences, including, for example, nuclease cleavage sites. Relative to the lack of DNA binding regulatory enhancers, the regulatory sequences of the target locus can be more accessible through the binding of DNA binding regulatory enhancers to their corresponding enhancer binding sequences.

[0436] The present invention particularly provides a target locus comprising two TAL effector domains each bound to its corresponding binding sequence (enhancer binding sequence), wherein the enhancer binding sequence is flanked by a regulator binding sequence having a regulatory site (e.g., a nuclease cleavage site). In the case where the enhancer binding sequence is flanked by the regulator binding sequence, the first enhancer binding sequence is connected to the second enhancer binding sequence through the regulator binding sequence. Therefore, in the 5' to 3' direction, the target locus can encode a first enhancer binding sequence connected to the regulator binding sequence, and the regulator binding sequence is connected to the second enhancer binding sequence. The binding of the two TAL effector domains to their corresponding binding sequences (enhancer binding sequences) allows accessibility to the target locus, especially when the regulator binding sequence is bound and / or modified by two TALEN binders. Each of the two enhancer binding sequences can be separated from the regulator binding sequence by, for example, 7 nucleotides. In the case where each of the two enhancer binding sequences is separated from the regulator binding sequence by 7 nucleotides, most of the 3' nucleotides (i.e., the last nucleotide) of the first enhancer binding sequence are connected to most of the 5' nucleotides (i.e., the first nucleotide) of the regulator binding sequence through a sequence of 7 consecutive nucleotides. Similarly, most of 5' nucleotides (i.e., first nucleotides) of the second enhancer binding sequence are connected to most of 3' nucleotides (i.e., last nucleotides) of the regulator binding sequence via a sequence of 7 continuous nucleotides. In the case of two kinds of regulatory proteins or regulatory complexes or their combination in conjunction with regulator binding sequences, they can be combined by combining independent binding sequences (first binding sequence and second binding sequence) respectively. The first binding sequence can be included in the 5' part of the regulator binding sequence, and the second binding sequence can form a part of the 3' part of the regulator binding sequence. Therefore, in the 5' to 3' direction, the regulator binding sequence can include the first binding sequence that is connected to the second binding sequence by at least one nucleotide. In an embodiment, most of 5' nucleotides (i.e., first nucleotides) of the regulator binding sequence are most of 5' nucleotides of the first binding site, and most of 3' nucleotides (i.e., last nucleotides) of the regulator binding sequence are most of 3' nucleotides of the first binding site.

[0437] In addition, each of the two enhancer binding sequences can be separated by 33 nucleotides from the cleavage site (regulatory site). In the case where each of the two enhancer binding sequences is separated by 33 nucleotides from the regulatory site, most of the 3' nucleotides of the first enhancer binding sequence are connected to the 5' nucleotides of the regulatory site by a sequence of 33 continuous nucleotides. Similarly, most of the 5' nucleotides of the second enhancer binding sequence are connected to the 3' nucleotides of the regulatory site by a sequence of 33 continuous nucleotides.

[0438] Thus, in one embodiment, the target locus comprises a first enhancer binding sequence that binds to a first TAL effector protein; a second enhancer binding sequence that binds to a second TAL effector protein; a first DNA-binding nuclease conjugate consisting of a first TAL effector domain operably linked to a first TALEN, wherein the first binder binds to a regulator binding sequence at a first binding site; and a second DNA-binding nuclease conjugate consisting of a second TAL effector domain operably linked to a second TALEN, wherein the second binder binds to a regulator binding sequence at a second binding site. In another embodiment, the first enhancer binding sequence is 7 nucleotides away from the regulator binding sequence, and the second enhancer binding sequence is 7 nucleotides away from the regulator binding sequence. In another embodiment, the first enhancer binding sequence is 7 nucleotides away from the first binding sequence of the regulator binding sequence, and the second enhancer binding sequence is 7 nucleotides away from the second binding sequence of the regulator binding sequence. In another embodiment, the first enhancer binding sequence is 33 nucleotides away from the regulatory site, and the second enhancer binding sequence is 33 nucleotides away from the regulatory site. In another embodiment, the first enhancer binding sequence is separated from the regulator binding sequence by 12 nucleotides, and the second enhancer binding sequence is separated from the regulator binding sequence by 12 nucleotides. In another embodiment, the first enhancer binding sequence is separated from the regulator binding sequence by 4 nucleotides, and the second enhancer binding sequence is separated from the regulator binding sequence by 4 nucleotides.

[0439] In one embodiment, the target locus includes a first enhancer binding sequence that binds to a first TAL effector protein; a second enhancer binding sequence that binds to a second TAL effector protein; a first DNA-binding nuclease conjugate consisting of a first TAL effector domain operably linked to a first TALEN, wherein the first binder binds to a regulator binding sequence at a first binding site; and a second DNA-binding nuclease conjugate consisting of a second TAL effector domain operably linked to a second TALEN, wherein the second binder binds to a regulator binding sequence at a second binding site. In another embodiment, the first enhancer binding sequence is 30 nucleotides away from the regulator binding sequence, and the second enhancer binding sequence is 19 nucleotides away from the regulator binding sequence. In another embodiment, the first enhancer binding sequence is 30 nucleotides away from the first binding sequence of the regulator binding sequence, and the second enhancer binding sequence is 19 nucleotides away from the second binding sequence of the regulator binding sequence. In another embodiment, the length of the first enhancer binding sequence is 18 nucleotides, and the length of the second enhancer binding sequence is 18 nucleotides. In another embodiment, the first binding sequence of the regulator binding sequence is 16 nucleotides away from the second binding sequence of the regulator binding sequence.

[0440] In one embodiment, the target locus includes a first enhancer binding sequence that is bound to a first TAL effector protein; a second enhancer binding sequence that is bound to a second TAL effector protein; and a ribonucleoprotein complex consisting of a Cas9 domain bound to a guide RNA, wherein the ribonucleoprotein complex is bound to a regulator binding sequence. In another embodiment, the first enhancer binding sequence is separated from the regulator binding sequence by 7 nucleotides, and the second enhancer binding sequence is separated from the regulator binding sequence by 7 nucleotides. In another embodiment, the first enhancer binding sequence is separated from the regulator binding sequence by 20 nucleotides, and the second enhancer binding sequence is separated from the regulator binding sequence by 20 nucleotides.

[0441] In one embodiment, the target locus includes a first enhancer binding sequence that binds to a first TAL effector protein; a second enhancer binding sequence that binds to a second TAL effector protein; and a DNA binding conjugate consisting of a TAL effector domain operably linked to a transcriptional activation domain, wherein the DNA binding conjugate binds to a regulator binding sequence. In another embodiment, the first enhancer binding sequence is 30 nucleotides apart from the regulator binding sequence, and the second enhancer binding sequence is 30 nucleotides apart from the regulator binding sequence. In another embodiment, the length of the first enhancer binding sequence is 18 nucleotides, and the length of the second enhancer binding sequence is 18 nucleotides. In another embodiment, the length of the regulator binding sequence is 18 nucleotides.

[0442] In another embodiment, the target locus includes a first enhancer binding sequence bound to a first truncated guide RNA bound to a Cas9 protein; a second enhancer binding sequence bound to a second truncated guide RNA bound to a Cas9 protein; and a ribonucleoprotein complex consisting of a Cas9 domain bound to the guide RNA, wherein the ribonucleoprotein complex is bound to a regulator binding sequence. In another embodiment, the first enhancer binding sequence is 30 nucleotides away from the regulator binding sequence, and the second enhancer binding sequence is 15 nucleotides away from the regulator binding sequence.

[0443] In one embodiment, the length of the regulator binding sequence is 52 nucleotides. In another embodiment, the length of the first binding sequence is 18 nucleotides. In another embodiment, the length of the second binding sequence is 18 nucleotides.

[0444] In one embodiment, the first DNA binding modulator enhancer is a first TAL effector protein and the second DNA binding modulator enhancer is a second TAL effector protein.

[0445] In one embodiment, the first DNA binding regulatory enhancer is a first TAL effector protein and the second DNA binding regulatory enhancer is a truncated gRNA. In another embodiment, the truncated gRNA is bound to the Cas9 protein.

[0446] In one embodiment, the first DNA binding regulatory enhancer is a truncated first gRNA, and the second DNA binding regulatory enhancer is a truncated second gRNA. In another embodiment, the truncated first gRNA binds to the first Cas9 protein, and the truncated second gRNA binds to the second Cas9 protein.

[0447] In one embodiment, the first DNA-binding regulatory enhancer is a first TAL effector protein; the second DNA-binding regulatory enhancer is a second TAL effector protein; the first regulatory protein is a first DNA-binding nuclease conjugate consisting of a first TAL effector domain operably linked to a first TALEN; and the second regulatory protein is a second DNA-binding nuclease conjugate consisting of a second TAL effector domain operably linked to a second TALEN.

[0448] In one embodiment, the first DNA binding regulatory enhancer is a first TAL effector protein; the second DNA binding regulatory enhancer is a second TAL effector protein; and the regulatory protein complex is a Cas9 domain bound to a guide RNA.

[0449] In one embodiment, the first DNA binding regulatory enhancer is a truncated first gRNA that binds to the Cas9 protein; the second DNA binding regulatory enhancer is a truncated second gRNA that binds to the Cas9 protein; and the regulatory protein complex is a Cas9 domain that binds to the guide RNA.

[0450] In one embodiment, the first DNA binding regulatory enhancer is a truncated first gRNA that binds to the Cas9 protein; the second DNA binding regulatory enhancer is a truncated second gRNA that binds to the Cas9 protein; the first regulatory protein is a first DNA binding nuclease conjugate, which is composed of a first TAL effector domain operably linked to a first TALEN; and the second regulatory protein is a second DNA binding nuclease conjugate, which is composed of a second TAL effector domain operably linked to a second TALEN.

[0451] Nucleic acid molecules for intracellular changes

[0452] Donor nucleic acid molecules (e.g., donor DNA molecules) typically contain at least one region of homology corresponding to a nucleic acid at or near the target locus and an inert region designed to modify the target locus. Donor nucleic acid molecules designed for homologous recombination will generally have at least three regions in the following order: (1) a first region of homology corresponding to a nucleic acid at or near the target locus; (2) an insertion region; and (3) a second region of homology corresponding to a nucleic acid at or near the target locus (see Fig.38 ). In addition, the donor nucleic acid molecules can be single-stranded (SS) or double-stranded (DS), and one or both of their ends can be blunt ends, or can have overhangs on one or both ends. Overhangs, when present, can be 5', 3', or 3' and 5'. In addition, the length of the overhangs can vary. The donor nucleic acid molecules will also typically include an "insert" region, which can be from about one nucleotide to about several thousand nucleotides.

[0453] As mentioned above, the overhangs, when present, may be of different sizes. The overhang can have about 1 to about 1,000 nucleotides (e.g., about 1 to about 1,000, about 5 to about 1,000, about 10 to about 1,000, about 25 to about 1,000, about 30 to about 1,000, about 40 to about 1,000, about 50 to about 1,000, about 60 to about 1,000, about 70 to about 1,000, about 80 to about 1,000, about 100 to about 1,000, about 1 to about 800, about 1 to about 700, about 1 to about 500, about 1 to about 400, about 1 to about 300, about 10 to about 600, about 10 to about 400, about 10 to about 250, about 30 to about 700, about 50 to about 600, about 50 to about 250, about 75 to about 800, about 80 to about 500, about 100 to about 800, about 100 to about 600 nucleotides, etc.).

[0454] The efficiency of homologous recombination is increased when one or both ends of the donor nucleic acid molecule "match" the ends of the double-strand break designed to be introduced. In addition, the donor nucleic acid molecule can be exposed to nucleases (e.g., endonucleases, endonucleases, etc.) after entering the cell (as well as before entering the cell). In order to limit the effect of the endonuclease in changing the donor nucleic acid molecule, one or more nuclease resistance groups can be present.

[0455] The intracellular nucleic acid molecule predetermined to be modified can be any intracellular nucleic acid molecule, including chromosome, nuclear plasmid, chloroplast genome and mitochondrial genome. In addition, the intracellular nucleic acid molecule predetermined to be modified can be located at any position in the cell.

[0456] Fig.38Many variants of donor nucleic acid molecules that can be used in the methods described herein are shown. The open circles at the ends represent nuclease resistance groups. Such groups can be located at many positions in the donor nucleic acid molecules. Donor nucleic acid molecule number 6 shows the 3' terminal region of the lower chain after the nuclease resistance group. In some cases, cellular nucleases will digest this part of the donor nucleic acid molecule. These nucleases will be terminated or slowed by the nuclease resistance group, thereby stabilizing the structure of the 3' region end of the lower chain.

[0457] Compositions comprising nucleic acid molecules containing one or more (e.g., one, two, three, four, five, six, seven, etc.) nuclease resistance groups can be used to implement the methods described herein. In many cases, the nuclease resistance group will be located at one or both ends of the donor nucleic acid molecule. Relative to one or both ends, the donor nucleic acid molecule may include internal groups. In many cases, some or all of such donor nucleic acid molecules will be processed in the cell to produce ends that match the double-strand break site.

[0458] The homologous regions can have different lengths and can have different amounts of sequence identity with the nucleic acid at the target locus. Generally, the efficiency of homologous recombination increases with the length and sequence identity of the homologous regions. The length of the homologous regions used is generally determined by factors such as the fragility of large nucleic acid molecules, transfection efficiency, and ease of generating nucleic acid molecules containing the homologous regions.

[0459] The total length of the homologous region can be from about 20 bases to about 10,000 bases (e.g., from about 20 bases to about 100 bases, from about 30 bases to about 100 bases, from about 40 bases to about 100 bases, from about 50 bases to about 8,000 bases, from about 50 bases to about 7,000 bases, from about 50 bases to about 6,000 bases, from about 50 bases to about 5,000 bases, from about 50 bases to about 5,000 bases, bases to about 3,000 bases, about 50 bases to about 2,000 bases, about 50 bases to about 1,000 bases, about 50 bases to about 800 bases, about 50 bases to about 600 bases, about 50 bases to about 500 bases, about 50 bases to about 400 bases, about 50 bases to about 300 bases, about 50 bases to about 200 bases, about 100 bases to about 8, bases, about 100 bases to about 2,000 bases, about 100 bases to about 1,000 bases, about 100 bases to about 700 bases, about 100 bases to about 600 bases, about 100 bases to about 400 bases, about 100 bases to about 300 bases, about 150 bases to about 1,000 bases, about 150 bases to about 500 bases, about 150 bases to about to about 400 bases, about 200 bases to about 1,000 bases, about 200 bases to about 600 bases, about 200 bases to about 400 bases, about 200 bases to about 300 bases, about 250 bases to about 2,000 bases, about 250 bases to about 1,000 bases, about 350 bases to about 2,000 bases, about 350 bases to about 1,000 bases, etc.).

[0460] In some cases, it may be desirable to use a region of sequence homology that is less than 200 bases in length. This is typically the case when the donor nucleic acid molecule contains a small insert (e.g., less than about 300 bases) and / or when the donor nucleic acid molecule has one or two overhangs that match the double-strand break site.

[0461] The overhangs can be of various lengths, and can be of different lengths at each end of the same donor nucleic acid molecule. In many cases, these overhangs will form regions of sequence homology. Fig.38 For example, a series of donor nucleic acid molecules with 30 nucleotide single-stranded overhangs are shown. These donor nucleic acid molecules are shown as single-stranded and double-stranded. Fig.38 The donor nucleic acid molecule No. 1 in is a single-stranded molecule having a 30-nucleotide sequence with a predetermined double-strand break site, a 30-nucleotide insertion, and two nuclease resistance groups located at each end.

[0462] The larger the amount of shared sequence identity between the homologous region and the target locus nucleic acid, the higher the homologous recombination efficiency is usually. When the homologous region is quite short (e.g., 50 bases), a high level of sequence identity is particularly desired. Typically, the amount of sequence identity between the target locus and the homologous region will be greater than 90% (e.g., about 90% to about 100%, about 90% to about 99%, about 90% to about 98%, about 95% to about 100%, about 95% to about 99%, about 95% to about 98%, about 97% to about 100%, etc.).

[0463] The insertion region of the donor nucleic acid molecule can have a variety of lengths, depending on its intended application. In many cases, the length of the donor nucleic acid molecule is about 1 to about 4,000 bases (e.g., about 1 to 3,000, about 1 to 2,000, about 1 to 1,500, about 1 to 1,000, about 2 to 1,000, about 3 to 1,000, about 5 to 1,000, about 10 to 1,000, about 10 to 400, about 10 to 50, about 15 to 65, about 2 to 15 bases, etc.).

[0464] Also provided herein is a composition and method for introducing a small amount of bases (e.g., about 1 to about 10, about 1 to about 6, about 1 to about 5, about 1 to about 2, about 2 to about 10, about 2 to about 6, about 3 to about 8, etc.) into intracellular nucleic acid. For illustrative purposes, a donor nucleic acid molecule of 51 base pairs in length can be prepared. This donor nucleic acid molecule can have two homology regions of 25 base pairs in length, wherein the insertion region is a single base pair. When the nucleic acid around the target locus is substantially matched with the homology region without an intermediate base pair, homologous recombination will introduce a single base pair at the target locus. Such homologous recombination reactions can be used, for example, to destroy a protein coding reading frame, thereby introducing a frameshift in intracellular nucleic acid. Therefore, the invention provides a composition and method for introducing one or a small amount of bases into an intracellular nucleic acid molecule.

[0465] The present invention further provides the composition and method for changing the short nucleotide sequence in the intracellular nucleic acid molecule.One example is the change of the single nucleotide position, and one example is the correction or change of single nucleotide polymorphism (SNP).For the purpose of illustration, using SNP to change, it is possible to design a donor nucleic acid molecule with two homology regions of 25 base pairs in length.Single base pair is positioned between these homology regions, and the base pair is basically " mispairing " of the corresponding base pair in the intracellular nucleic acid molecule.Therefore, homologous recombination can be used to change SNP by changing the base pair into a base pair or another base (such as different SNP) that is considered to be wild type.The cell that has correctly carried out homologous recombination can be identified by sequencing the target locus subsequently.

[0466] The present invention also includes compositions and methods for altering genomes for therapeutic applications, including SNP alterations. For illustrative purposes, two genetic diseases caused by SNP alterations are listed below.

[0467] The most common SNP associated with sickle cell anemia is rs334, which causes a change in a single codon from GAG to GTG. This change results in the replacement of a glutamic acid residue by a valine residue. The compositions and methods described herein are suitable for changing this SNP from GTG to GAG, particularly individual homozygotes of SNP rs334. One of these reasons relates to the fact that the introduction of nucleic acid molecules into cells can induce toxicity-related effects. In addition, the magnitude of these effects is that they increase with the amount of nucleic acid introduced into the cell. As shown in the examples below, the genome insertion efficiency is such that a relatively small amount of donor DNA needs to be introduced into the cell (see, e.g., Fig.11 and 13 (donor DNA-NLS conjugate data in ).

[0468] An exemplary ex vivo workflow for altering SNP rs334 in a patient would include removing bone marrow tissue from the patient, altering SNP rs334, and then reintroducing the edited cells into the patient.

[0469] One of the most common genomic alterations associated with cystic fibrosis is based on a three-base pair deletion (SNP rs199826652) in the cystic fibrosis transmembrane conductance regulator (CFTR), which results in the loss of the amino acid phenylalanine at position 508.

[0470] The in vivo workflow for altering SNP rs199826652 in a patient involves delivering a donor DNA molecule to the patient's airway cells under conditions where a three base pair insertion occurs to correct SNP rs199826652.

[0471] The low doses of donor nucleic acids required for effective gene editing can also be used for systemic delivery. This is so because low doses are associated with reduced toxicity. Low donor DNA molecule levels are particularly important when using modified nucleic acid molecules (e.g., nucleic acid molecules with phosphorothioate bonds).

[0472] Donor nucleic acid molecules can be combined with extracellular targeting moieties and intracellular targeting moieties. "Extracellular targeting moieties" are molecules that guide donor nucleic acid molecules to one or more cell types. Such moieties include cell surface receptor ligands and antibodies. Domain II of Pseudomonas has been shown to participate in transcellular transport. (Jinno et al., "Journal of Biological Chemistry (J.Biol.Chem.)" 263:13203-13207 (1988)). Therefore, an exemplary system for delivering nucleic acid molecules to subcellular locations in an organism may involve the following components: (1) donor DNA molecules, (2) nuclear binding signals (NLS), and (3) fusion proteins, which include antibodies that are bound to domain II of cell surface receptors and Pseudomonas exotoxins, wherein NLS and fusion proteins are covalently bound to donor DNA molecules. This type of donor DNA molecule allows systemic delivery of donor DNA molecules, wherein the donor DNA molecule will be delivered to the subcellular location within the cell containing the cell surface receptor.

[0473] In each of the two examples above, only one copy of an allele needs to be altered in order for the patient to achieve a significant benefit. However, in many cells, both copies of the SNP will be altered. Therefore, the present invention includes treatments for ailments caused by both homozygous and ...

Claims

1. A method for altering an endogenous nucleic acid molecule present in a cell, the method comprising introducing a donor DNA molecule into the cell, The donor DNA molecule is operably linked to one or more intracellular targeting moieties, which are capable of localizing the donor DNA molecule to the location of the endogenous nucleic acid molecule in the cell.

2. The method of claim 1, wherein the location of the endogenous nucleic acid molecule in the cell is in the nucleus, mitochondria or chloroplasts.

3. The method of claim 1, wherein the one or more intracellular targeting moieties are nuclear localization signals.

4. The method of claim 1, wherein the donor DNA molecule is about 25 to about 8,000 nucleotides in length.

5. The method of claim 1, wherein the donor DNA molecule is single-stranded.

6. The method of claim 1, wherein the donor DNA molecule has one or more nuclease resistance groups within 50 nucleotides of one or more termini.

7. The method of claim 6, wherein the nuclease resistance group is selected from the group consisting of a phosphorothioate group, an amino group, a 2'-O-methyl nucleotide, a 2'-deoxy-2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 5-C-methyl nucleotide, or a combination thereof.

8. The method of claim 1, wherein the donor DNA is double-stranded or partially double-stranded.

9. The method of claim 1, wherein the donor DNA molecule contains a positive selectable marker and a negative selectable marker.

10. The method of claim 9, wherein the negative selectable marker is a nucleic acid encoding herpes simplex virus thymidine kinase.

11. The method of claim 1, wherein the donor DNA molecule has two regions whose sequence is complementary to a target locus present in the cell.

12. The method of claim 11, wherein the selectable marker is located between two regions of sequence complementarity in the donor DNA molecule.

13. The method of claim 1, wherein the cell is contacted with one or more of: (1) one or more nucleic acid cleavage entities, (2) one or more nucleic acid molecules encoding one or more components of a nucleic acid cleavage entity, (3) one or more DNA binding modulators, (4) one or more nucleic acid molecules encoding one or more components of a DNA binding modulator enhancer, or (5) one or more non-homologous end joining (NHEJ) inhibitors.

14. The method of claim 13, wherein the one or more non-homologous end joining (NHEJ) inhibitors are DNA-dependent protein kinase inhibitors.

15. The method of claim 13, wherein one or more of the one or more nucleic acid cleavage entities are selected from the group consisting of: (1) Zinc finger nuclease (2) TAL effector nucleases, and (3) CRISPR complex.

16. The method of claim 13, wherein one or more of the one or more DNA binding modulators are selected from the group consisting of: (1) Zinc finger proteins, (2) TALE proteins, and (3) CRISPR complex.

17. The method of claim 16, wherein the TALE protein does not have a heterologous nuclease domain.

18. The method of claim 16, wherein the CRISPR complex comprises a dCas9 protein.

19. The method of claim 13, wherein one or more of the one or more DNA binding modulator enhancers are designed to bind within 50 nucleotides of the target locus.

20. A TALE protein comprising amino acids: amino acids 811-830 of Figure 46, wherein the amino acids at positions 815-816 and 824-825 can be Gly-Ser or Gly-Gly.

21. The TALE protein of claim 20, comprising amino acids: amino acids 810-1029 of Figure 46, wherein the amino acids at positions 1022-1023 may be Gly-Ser or Gly-Gly.

22. The TALE protein of claim 20, comprising the amino acids of Figure 46: amino acids 752-1021.

23. A TALE protein comprising amino acids: amino acids 20-27, 30-107 and 111-165 of Figure 47, wherein the amino acids at positions 28-29 are Gly-Ser or Gly-Gly and wherein the amino acids at positions 108-110 are Arg-Gly-Ala or Gln-Trp-Ser.

24. A TALE protein comprising amino acids: amino acids 821-826 and 829-840 of Figure 47, wherein the amino acids at positions 827-828 are Gly-Ser or Gly-Gly.

25. The TALE protein of claim 24, comprising amino acids corresponding to Figure 47.

26. The TALE protein of claim 24, comprising a repeat region comprising 4 to 25 repeat units.

27. A method for engineering an intracellular nucleic acid in a cell, the method comprising introducing a TALE protein according to claim 23 or a nucleic acid encoding a TALE protein according to claim 23 into the cell.

28. The method of claim 27, further comprising introducing a donor nucleic acid molecule into the cell, wherein the donor nucleic acid molecule has one or more regions of sequence homology to a nucleic acid located within 50 nucleotides of the target locus.

29. A Cas9 protein comprising two or more bipartite nuclear localization signals.

30. The Cas9 protein of claim 29, wherein the two or more bipartite nuclear localization signals are located within twenty amino acids of one or more termini.

31. The Cas9 protein of claim 29, wherein the two or more bi-type nuclear localization signals are individually located within twenty amino acids of the N-terminus and C-terminus of the protein.

32. The Cas9 protein of claim 29, wherein the two or more bi-typed nuclear localization signals comprise different amino acid sequences.

33. The Cas9 protein of claim 29, further comprising one or more monotypic nuclear localization signals.

34. The Cas9 protein of claim 29, further comprising an affinity tag.

35. A method for engineering intracellular nucleic acids in a cell, the method comprising introducing the Cas9 protein according to claim 29 or a nucleic acid encoding the Cas9 protein according to claim 29 into the cell, wherein the Cas9 protein is designed to bind to a target locus within the cell.

36. The method of claim 35, wherein the Cas9 protein is introduced into the cell in the form of a Cas9 / gRNA complex.

37. The method of claim 35, further comprising introducing a donor nucleic acid molecule into the cell, wherein the donor nucleic acid molecule has one or more regions of sequence homology to a nucleic acid located within 50 nucleotides of the target locus.

38. A method for causing homologous recombination of intracellular nucleic acid molecules in a cell population at a cleavage site, the method comprising: (a) causing a double-strand break in the nucleic acid molecule in the cell at the cleavage site to produce a cleaved nucleic acid molecule, and (b) contacting the cleaved nucleic acid molecule with a donor nucleic acid molecule, wherein the donor nucleic acid molecule has at least ten nucleotides or base pairs homologous to a nucleic acid within 100 base pairs on each side of the cleavage site, wherein at least 95% of the cells within the cell population undergo homology-directed repair of the donor nucleic acid molecule at the cleavage site.

39. The method of claim 38, wherein the donor nucleic acid molecule contains a selection marker or a reporter gene, which is operably linked to a promoter present in the nucleic acid molecule in the cell after homology-directed repair.

40. The method of claim 38, wherein the donor nucleic acid molecules are linked to one or more nuclear localization signals that allow the donor nucleic acid molecules to localize to the nuclei of the cell population.

41. The method of claim 38, wherein the cell population is contacted with one or more of: (1) one or more nucleic acid cleavage entities, (2) one or more nucleic acid molecules encoding one or more components of a nucleic acid cleavage entity, (3) one or more DNA binding modulators, (4) one or more nucleic acid molecules encoding one or more components of a DNA binding modulator enhancer, or (5) one or more non-homologous end joining (NHEJ) inhibitors.

42. The method of claim 38, wherein the donor nucleic acid molecule is single-stranded.

43. The method of claim 38, wherein the donor DNA molecule is operably linked to one or more nuclear localization signals.

44. The method of claim 38, wherein the cell population is contacted with the one or more nucleic acid cleavage entities or one of a plurality of nucleic acid molecules encoding one or more nucleic acid cleavage entities, and subsequently the cell population is contacted with one or more donor nucleic acid molecules.

45. The method of claim 44, wherein the cell population is contacted with one or more donor nucleic acid molecules for 5 to 60 minutes after contacting the cell population with the one or more nucleic acid cleavage entities or one of a plurality of nucleic acid molecules encoding one or more nucleic acid cleavage entities.

46. ​​A method for performing homologous recombination in an initial nucleic acid molecule, comprising: (a) causing double-strand breaks in the initial nucleic acid molecule to produce cleaved nucleic acid molecules, and (b) contacting the cleaved nucleic acid molecule with a donor nucleic acid molecule, The initial nucleic acid molecule comprises a promoter and a gene, and The donor nucleic acid molecule comprises: (i) a 5' end with a length of 12 bp to 250 bp and a matching end at the 3' end; (ii) a promoterless selection marker; (iii) a reporter gene; (iv) a self-cleaving peptide that connects the promoterless selection marker to the reporter gene or loxP located on either side of the promoterless selection marker; and (iv) optionally a linker between the promoterless selection marker and the reporter gene.

47. The method of claim 46, wherein the double-strand break in the nucleic acid molecule is: (i) less than or equal to 250 bp from the ATG start codon, so that the cleaved nucleic acid molecule is N-terminally labeled; or (ii) less than or equal to 250 bp from the stop codon, so that the cleaved nucleic acid molecule is C-terminally labeled.

48. The method of claim 46, wherein the double-strand break is induced by one or more nucleic acid cleaving entities.

49. The method of claim 48, wherein the one or more nucleic acid cleavage entities comprise a nuclease containing one or more zinc finger proteins, one or more transcription activator-like effectors (TALEs), one or more CRISPR complexes, one or more Agg protein-nucleic acid complexes, or one or more meganucleases.

50. The method of claim 48, wherein the one or more nucleic acid cleavage entities are administered using an expression vector, a plasmid, a ribonucleoprotein complex (RNC), or mRNA.

51. The method of claim 46, wherein the promoterless selectable marker comprises a protein, an antibiotic resistance selectable marker, a cell surface marker, a cell surface protein, a metabolite, or an active fragment thereof.

52. The method of claim 46, wherein the self-cleaving peptide is a self-cleaving 2A peptide.

53. The method of claim 46, wherein the matching ends comprise single-stranded DNA or double-stranded DNA.

54. The method of claim 46, wherein the matching ends of the 5' and 3' ends of the donor nucleic acid molecule have a length of 12 bp to 200 bp, 12 bp to 150 bp, 12 bp to 100 bp, 12 bp to 50 bp, or 12 bp to 40 bp.

55. The method of claim 46, wherein the donor nucleic acid molecule is integrated into the cleaved nucleic acid molecule by homology directed repair (HDR).

56. The method of claim 46, further comprising modifying the donor nucleic acid molecule at the 5' end, the 3' end, or both the 5' and 3' ends.

57. The method of claim 56, wherein the donor nucleic acid molecule is modified at the 5' and 3' ends.

58. The method of claim 56, wherein the donor nucleic acid molecule is modified to have one or more nuclease resistance groups in one or more strands at one or more ends.

59. The method of claim 58, wherein the one or more nuclease resistance groups comprise one or more phosphorothioate groups, one or more amino groups, 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxy nucleotides, 5-C-methyl nucleotides, or a combination thereof.

60. The method of claim 46, further comprising treating the donor nucleic acid molecule with one or more non-homologous end joining (NHEJ) inhibitors.

61. A nucleic acid molecule prepared according to the method of claim 46.

62. A recombinant antibody expression cassette comprising: (i) matching ends located at the 5' and 3' ends of the expression cassette, wherein the length of the matching ends is less than or equal to 250 bp; (ii) without a promoter selection marker; (iii) reporter gene; (iv) a self-cleaving peptide linking the promoterless selectable marker to the reporter gene; and (v) optionally, a linker between the promoterless selection marker and the reporter gene, The promoterless selection marker is connected to the 5' end of the reporter gene to carry out N-terminal labeling of the cleaved nucleic acid molecule, or is connected to the 3' end of the reporter gene to carry out C-terminal labeling of the cleaved nucleic acid molecule.

63. A method of enhancing the activity of a regulatory protein or regulatory complex at a target locus in a cell, the method comprising: (1) introducing into a cell comprising a nucleic acid encoding the target locus: (i) a first regulatory protein or a first regulatory complex capable of binding to a first regulator binding sequence of the target locus, wherein the first regulator binding sequence comprises a regulatory site; and (ii) a first DNA binding regulatory enhancer capable of binding to a first enhancer binding sequence of the target locus; as well as (2) allowing the first DNA binding regulatory enhancer to bind to the first enhancer binding sequence, thereby enhancing the activity of the first regulatory protein or the first regulatory complex at the target locus in the cell.

64. The method of claim 63, wherein said introducing a first DNA-binding modulator enhancer comprises introducing a vector encoding said first DNA-binding modulator enhancer.

65. The method of claim 63, wherein said introducing a first DNA binding modulator enhancer comprises introducing mRNA encoding said first DNA binding modulator enhancer.

66. The method of claim 63, wherein the first DNA binding regulatory enhancer is a first transcription activator-like (TAL) effector.

67. The method of claim 63, further comprising: (1) introducing a second DNA binding modulator enhancer into the cell; and (2) allowing the second DNA binding regulatory enhancer to bind to the second enhancer binding sequence of the target locus.

68. The method of claim 64, wherein the first enhancer binding sequence and the second enhancer binding sequence are located within 180 base pairs of each other.

69. The method of claim 64, wherein the first enhancer binding sequence and the second enhancer binding sequence are located on opposite sides of the regulator binding sequence.

70. The method of claim 63, wherein the first regulatory protein is a DNA binding nuclease fusion protein.

71. The method of claim 70, wherein the DNA binding nuclease fusion protein is a TALE-FokI fusion protein.

72. The method of claim 63, wherein the first regulatory complex is a CRISPR / gRNA complex with nuclease activity.

73. The method of claim 72, wherein the first regulatory complex is a Cas9 / gRNA complex with nuclease activity.

74. The method of claim 63, further comprising introducing into the cell a second regulatory protein or a second regulatory complex capable of binding to a second regulator binding sequence of the target locus, wherein the second regulator binding sequence comprises the regulatory site.

75. The method of claim 74, wherein the first regulatory protein is a DNA binding nuclease fusion protein.

76. The method of claim 75, wherein the DNA binding nuclease fusion protein is a TALE-FokI fusion protein.

77. The method of claim 76, wherein the second regulatory complex is a CRISPR / gRNA complex with nuclease activity.

78. The method of claim 77, wherein the second regulatory complex is a Cas9 / gRNA complex with nuclease activity.

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