Methods and compositions for modulating gene expression

Abstract

The present disclosure provides compositions with a modulating gene expression and methods for modulating transcription.

What is claimed is: 
     
         1 . A site-specific disrupting agent, comprising:
 a DNA-binding moiety that binds specifically to one or more target anchor sequences within a cell and not to non-targeted anchor sequences within the cell with sufficient affinity that it competes with binding of an endogenous nucleating polypeptide within the cell.   
     
     
         2 . The site-specific disrupting agent of  claim 1 , further comprising a negative effector moiety associated with the DNA-binding moiety so that, when the DNA-binding moiety is bound at the one or more target anchor sequences, the negative effector moiety is localized thereto, the negative effector moiety being characterized in that dimerization of the endogenous nucleating polypeptide is reduced when the negative effector moiety is present as compared with when it is absent. 
     
     
         3 . The site-specific disrupting agent of  claim 2 , wherein the negative effector moiety is or comprises a variant of the endogenous nucleating polypeptide's dimerization domain, or a dimerizing portion thereof. 
     
     
         4 . The site-specific disrupting agent of any one of the preceding claims, wherein the DNA binding moiety is or comprises a polymer. 
     
     
         5 . The site-specific disrupting agent of  claim 4 , wherein the polymer is or comprises a polyamide. 
     
     
         6 . The site-specific disrupting agent of  claim 4 , wherein the polymer is an oligonucleotide 
     
     
         7 . The site-specific disrupting agent of  claim 6 , wherein the oligonucleotide has a sequence that comprises a complement of the target anchor sequence. 
     
     
         8 . The site-specific disrupting agent of  claim 6  or  7 , wherein the oligonucleotide comprises a chemical modification. 
     
     
         9 . The site-specific disrupting agent of  claim 4 , wherein the polymer is a peptide nucleic acid. 
     
     
         10 . The site-specific disrupting agent of  claim 4 , wherein the DNA binding moiety is or comprises a peptide-nucleic acid mixmer. 
     
     
         11 . The site-specific disrupting agent of any one of  claim 4 , wherein the DNA binding moiety is or comprises a peptide or polypeptide. 
     
     
         12 . The site-specific disrupting agent of  claim 11 , wherein the polypeptide is a zinc finger polypeptide. 
     
     
         13 . The site-specific disrupting agent of  claim 13 , wherein the polypeptide is or comprises a Transcription activator-like effector nuclease (TALEN) polypeptide. 
     
     
         14 . The site-specific disrupting agent of any one of  claims 1 - 3 , wherein the DNA binding moiety is or comprises a small molecule. 
     
     
         15 . A method of modulating expression of a gene within an anchor sequence-mediated conjunction that comprises a first anchor sequence and a second anchor sequence, the method comprising a step of:
 contacting the first and/or second anchor sequence with the site-specific disrupting agent of any one of  claims 1 - 14 .   
     
     
         16 . The method of  claim 2 , wherein the anchor sequence-mediated conjunction comprises at least one internal transcriptional control sequence. 
     
     
         17 . The method of  claim 16 , wherein the transcriptional control sequence is an enhancing sequence. 
     
     
         18 . The method of  claim 16 , wherein the transcriptional control sequence is a silencing or repressive sequence. 
     
     
         19 . The method of any one of  claims 15 - 18 , wherein the gene is separated from the internal transcriptional control sequence by at least 300 base pairs. 
     
     
         20 . The method of any one of  claims 15 - 19 , wherein the first and/or the second anchor sequence is located within 500 kb of an external transcriptional control sequence. 
     
     
         21 . The method of  claim 20 , wherein the external transcriptional control sequence is an enhancing sequence. 
     
     
         22 . The method of  claim 20 , wherein the external transcriptional control sequence is a silencing or repressive sequence. 
     
     
         23 . A method of modulating expression of a gene within 10 kb of a first anchor sequence within an anchor sequence-mediated conjunction comprising a first anchor sequence and a second anchor sequence,
 the method comprising a step of:
 contacting the first and/or second anchor sequence with the site-specific disrupting agent of any one of  claims 1 - 14 . 
   
     
     
         24 . The method of  claim 23 , wherein the anchor sequence-mediated conjunction comprises at least one internal transcriptional control sequence. 
     
     
         25 . The method of  claim 24 , wherein the transcriptional control sequence is an enhancing sequence. 
     
     
         26 . The method of  claim 24 , wherein the transcriptional control sequence is a silencing or repressive sequence. 
     
     
         27 . The method of any one of  claims 24 - 26 , wherein the gene is separated from the internal transcriptional control sequence by at least 300 base pairs. 
     
     
         28 . The method of any one of  claims 23 - 27 , wherein the first and/or the second anchor sequence is located within 500 kb of an external transcriptional control sequence. 
     
     
         29 . The method of  claim 28 , wherein the external transcriptional control sequence is an enhancing sequence. 
     
     
         30 . The method of  claim 28 , wherein the external transcriptional control sequence is a silencing or repressive sequence. 
     
     
         31 . A method of decreasing expression of a gene within an anchor sequence-mediated conjunction that comprises a first anchor sequence, a second anchor sequence, and an internal enhancing sequence, the method comprising a step of:
 contacting the first and/or second anchor sequence with the site-specific disrupting agent of any one of  claims 1 - 14 .   
     
     
         32 . The method of  claim 31 , wherein the first and/or the second anchor sequence is located within 500 kb of an external silencing or repressive sequence. 
     
     
         33 . The method of  claim 31  or  32 , wherein the gene is separated from the internal enhancing sequence by at least 300 base pairs. 
     
     
         34 . A method of increasing expression of a gene within an anchor sequence-mediated conjunction that comprises a first anchor sequence and a second anchor sequence, wherein the first and/or the second anchor sequence is located within 10 kb of an external enhancing sequence, the method comprising a step of:
 contacting the first and/or second anchor sequence with the site-specific disrupting agent of any one of  claims 1 - 14 .   
     
     
         35 . The method of  claim 34 , wherein the anchor sequence-mediated conjunction further comprises an internal enhancing sequence. 
     
     
         36 . The method of  claim 35 , wherein the gene is separated from the internal enhancing sequence by at least 300 base pairs. 
     
     
         37 . A method comprising a step of:
 (a) delivering the site-specific disrupting agent of any one of  claims 1 - 14  to a mammalian cell.   
     
     
         38 . The method of  claim 37 , wherein the mammalian cell is a somatic cell. 
     
     
         39 . The method of  claim 37  or  38 , wherein the mammalian cell is a primary cell. 
     
     
         40 . The method of any one of  claims 37 - 39 , wherein the step of delivering is performed ex vivo. 
     
     
         41 . The method of  claim 40 , further comprising, prior to the step of delivering, a step of removing the mammalian cell from a subject. 
     
     
         42 . The method of  claim 40 , further comprising, after the step of delivering, a step of (b) administering the mammalian cells to a subject. 
     
     
         43 . The method of any one of  claims 37 - 39 , wherein the step of delivering comprises administering a composition comprising the site-specific disrupting agent to a subject. 
     
     
         44 . The method of  claim 41  or  42 , wherein the subject has a disease or condition. 
     
     
         45 . The method of any one of  claims 37 - 43 , wherein the step of delivering comprises delivery across the cell membrane. 
     
     
         46 . A fusion molecule comprising:
 (i) a site-specific targeting moiety and   (ii) a deaminating agent,
 wherein the site-specific targeting moiety targets the fusion molecule to a target anchor sequence but not to at least one non-target anchor sequence. 
   
     
     
         47 . The fusion molecule of  claim 46 , wherein the target anchor sequence comprises a CTCF binding motif. 
     
     
         48 . The fusion molecule of  claim 47 , wherein the at least one non-target anchor sequence also comprises a CTCF binding motif. 
     
     
         49 . The fusion molecule of any one of  claims 46 - 48 , wherein the deaminating agent is a deaminase. 
     
     
         50 . The fusion molecule of any one of  claims 46 - 49 , wherein the site-specific targeting moiety comprises a Cas polypeptide and a site-specific guide RNA. 
     
     
         51 . The fusion molecule of  claim 50 , wherein the Cas polypeptide is enzymatically inactive. 
     
     
         52 . The fusion molecule of  claim 50  or  51 , wherein the Cas polypeptide is a Cas9 polypeptide. 
     
     
         53 . The fusion molecule of any one of  claims 46 - 48 , wherein the deaminating agent comprises an oligonucleotide. 
     
     
         54 . The fusion molecule of  claim 53 , wherein the oligonucleotide is conjugated to sodium bisulfite. 
     
     
         55 . The fusion molecule of any one of  claims 46 - 49 , wherein the site-specific targeting moiety is a polymer. 
     
     
         56 . The fusion molecule of any one of  claims 46 - 55 , wherein the DNA binding moiety is or comprises a polymer. 
     
     
         57 . The fusion molecule of  claim 56 , wherein the polymer is or comprises a polyamide. 
     
     
         58 . The fusion molecule of  claim 56 , wherein the polymer is an oligonucleotide 
     
     
         59 . The fusion molecule of  claim 58 , wherein the oligonucleotide has a sequence that comprises a complement of the target anchor sequence. 
     
     
         60 . The fusion molecule of  claim 58  or  59 , wherein the oligonucleotide comprises a chemical modification. 
     
     
         61 . The fusion molecule of  claim 56 , wherein the polymer is a peptide nucleic acid. 
     
     
         62 . The fusion molecule of  claim 46 , wherein the DNA binding moiety is or comprises a peptide-nucleic acid mixmer. 
     
     
         63 . The fusion molecule of  claim 56 , wherein the DNA binding moiety is or comprises a peptide or polypeptide. 
     
     
         64 . The fusion molecule of  claim 63 , wherein the polypeptide is a zinc finger polypeptide. 
     
     
         65 . The fusion molecule of  claim 63 , wherein the polypeptide is or comprises a Transcription activator-like effector nuclease (TALEN) polypeptide. 
     
     
         66 . The fusion molecule of any one of  claims 46 - 48 , wherein the DNA binding moiety is or comprises a small molecule. 
     
     
         67 . A composition comprising:
 (i) a fusion polypeptide comprising an enzymatically inactive Cas polypeptide and a deaminating agent, or a nucleic acid encoding the fusion polypeptide; and   (ii) a guide RNA, wherein the guide RNA targets the fusion polypeptide to a target anchor sequence but not to at least one non-target anchor sequence.   
     
     
         68 . A method of modulating expression of a gene within an anchor sequence-mediated conjunction that comprises a first anchor sequence and a second anchor sequence, the method comprising a step of:
 contacting the first and/or second anchor sequence with the fusion molecule of any one of  claims 46 - 66  or composition of  claim 67 .   
     
     
         69 . The method of  claim 68 , wherein the anchor sequence-mediated conjunction comprises at least one internal transcriptional control sequence. 
     
     
         70 . The method of  claim 69 , wherein the transcriptional control sequence is an enhancing sequence. 
     
     
         71 . The method of  claim 69 , wherein the transcriptional control sequence is a silencing or repressive sequence. 
     
     
         72 . The method of any one of  claims 69 - 71 , wherein the gene is separated from the internal transcriptional control sequence by at least 300 base pairs. 
     
     
         73 . The method of any one of  claims 69 - 72 , wherein the first and/or the second anchor sequence is located within 500 kb of an external transcriptional control sequence. 
     
     
         74 . The method of  claim 73 , wherein the external transcriptional control sequence is an enhancing sequence. 
     
     
         75 . The method of  claim 73 , wherein the external transcriptional control sequence is a silencing or repressive sequence. 
     
     
         76 . A method of modulating expression of a gene within 10 kb of a first anchor sequence within an anchor sequence-mediated conjunction comprising a first anchor sequence and a second anchor sequence, the method comprising a step of:
 contacting the first and/or second anchor sequence with the fusion molecule of any one of  claims 46 - 66  or composition of  claim 67 .   
     
     
         77 . The method of  claim 76 , wherein the anchor sequence-mediated conjunction comprises at least one internal transcriptional control sequence. 
     
     
         78 . The method of  claim 77 , wherein the transcriptional control sequence is an enhancing sequence. 
     
     
         79 . The method of  claim 77 , wherein the transcriptional control sequence is a silencing or repressive sequence. 
     
     
         80 . The method of any one of  claims 77 - 79 , wherein the gene is separated from the internal transcriptional control sequence by at least 300 base pairs. 
     
     
         81 . The method of any one of  claims 76 - 79 , wherein the first and/or the second anchor sequence is located within 500 kb of an external transcriptional control sequence. 
     
     
         82 . The method of  claim 81 , wherein the external transcriptional control sequence is an enhancing sequence. 
     
     
         83 . The method of  claim 81 , wherein the external transcriptional control sequence is a silencing or repressive sequence. 
     
     
         84 . A method of decreasing expression of a gene within an anchor sequence-mediated conjunction that comprises a first anchor sequence, a second anchor sequence, and an internal enhancing sequence, the method comprising a step of:
 contacting the first and/or second anchor sequence with the fusion molecule of any one of  claims 46 - 66  or composition of  claim 67 .   
     
     
         85 . The method of  claim 84 , wherein the first and/or the second anchor sequence is located within 500 kb of an external silencing or repressive sequence. 
     
     
         86 . The method of  claim 84  or  85 , wherein the gene is separated from the internal enhancing sequence by at least 300 base pairs. 
     
     
         87 . A method of increasing expression of a gene within an anchor sequence-mediated conjunction that comprises a first anchor sequence and a second anchor sequence, wherein the first and/or the second anchor sequence is located within 10 kb of an external enhancing sequence, the method comprising a step of:
 contacting the first and/or second anchor sequence with the fusion molecule of any one of  claims 46 - 66  or composition of  claim 67 .   
     
     
         88 . The method of  claim 87 , wherein the anchor sequence-mediated conjunction further comprises an internal enhancing sequence. 
     
     
         89 . The method of  claim 88 , wherein the gene is separated from the internal enhancing sequence by at least 300 base pairs. 
     
     
         90 . A method comprising a step of:
 (a) delivering the fusion molecule of any one of  claims 46 - 66  or the composition of  claim 67  to a mammalian cell.   
     
     
         91 . The method of  claim 90 , wherein the mammalian cell is a somatic cell. 
     
     
         92 . The method of  claim 90  or  91 , wherein the mammalian cell is a primary cell. 
     
     
         93 . The method of any one of  claims 90 - 92 , wherein the step of delivering is performed ex vivo. 
     
     
         94 . The method  claim 93 , further comprising, prior to the step of delivering, a step of removing the mammalian cell from a subject. 
     
     
         95 . The method of  claim 93  or  94 , further comprising a step of (b) administering the mammalian cell to a subject. 
     
     
         96 . The method of any one of  claims 90 - 92 , wherein the step of delivering comprises administering a composition comprising the fusion molecule of any one of  claims 46 - 66  or the composition of  claim 67  to a subject. 
     
     
         97 . The method of any one of  claims 90 - 93 , wherein the step of delivering comprises delivery across the cell membrane. 
     
     
         98 . A method comprising a step of:
 (a) substituting, adding, or deleting one or more nucleotides of an anchor sequence within a mammalian somatic cell.   
     
     
         99 . The method of  claim 98 , wherein the mammalian somatic cell is a primary cell. 
     
     
         100 . The method of  claim 98 , wherein the step of substituting, adding, or deleting is performed in vivo. 
     
     
         101 . The method of  claim 98 , wherein the step of substituting, adding, or deleting is performed ex vivo. 
     
     
         102 . The method of any one of  claims 98 - 101 , wherein the mammalian somatic cell is a non-embryonic cell. 
     
     
         103 . The method of any one of  claims 98 - 102 , wherein the anchor sequence is a genomic anchor sequence. 
     
     
         104 . A method comprising a step of delivering a mammalian somatic cell to a subject having a disease or condition, wherein one or more nucleotides of an anchor sequence within the mammalian somatic cell has been substituted, added, or deleted. 
     
     
         105 . A method comprising a step of:
 (a) administering somatic mammalian cells to a subject, wherein the somatic mammalian cells were obtained from the subject, and the fusion molecule of any one of  claims 46 - 66  or the composition of  claim 67  had been delivered ex vivo to the mammalian cells.   
     
     
         106 . The method of any one of  claims 94 - 96  or  105 , wherein the subject is a mammal. 
     
     
         107 . The method of  claim 106 , wherein the subject has a disease or condition. 
     
     
         108 . A fusion molecule comprising:
 (i) a site-specific targeting moiety and   (ii) an epigenetic modifying agent,
 wherein the site-specific targeting moiety targets the fusion molecule to a target anchor sequence but not to at least one non-target anchor sequence. 
   
     
     
         109 . The fusion molecule of  claim 108 , wherein the target anchor sequence comprises a CTCF binding motif. 
     
     
         110 . The fusion molecule of  claim 109 , wherein the at least one non-target anchor sequence also comprises a CTCF binding motif. 
     
     
         111 . The fusion molecule of any one of  claims 108 - 110 , wherein the epigenetic modifying agent is selected from the group consisting of a DNA methylase, DNA demethylase, histone methyltransferase, a histone deacetylase, and combinations thereof. 
     
     
         112 . The fusion molecule of any one of  claims 108 - 111 , wherein the site-specific targeting moiety comprises a Cas polypeptide and a site-specific guide RNA. 
     
     
         113 . The fusion molecule of  claim 112 , wherein the Cas polypeptide is enzymatically inactive. 
     
     
         114 . The fusion molecule of  claim 112  or  113 , wherein the Cas polypeptide is a Cas9 polypeptide. 
     
     
         115 . The fusion molecule of any one of  claims 108 - 111 , wherein the site-specific targeting moiety is a polymer. 
     
     
         116 . The fusion molecule of  claim 115 , wherein the polymer is or comprises a polyamide. 
     
     
         117 . The fusion molecule of  claim 115 , wherein the polymer is an oligonucleotide 
     
     
         118 . The fusion molecule of  claim 116 , wherein the oligonucleotide has a sequence that comprises a complement of the target anchor sequence. 
     
     
         119 . The fusion molecule of  claim 116 , wherein the oligonucleotide comprises a chemical modification. 
     
     
         120 . The fusion molecule of  claim 115 , wherein the polymer is a peptide nucleic acid. 
     
     
         121 . The fusion molecule of  claim 108 , wherein the site-specific targeting moiety is or comprises a peptide-nucleic acid mixmer. 
     
     
         122 . The fusion molecule of  claim 115 , wherein the site-specific targeting binding moiety is or comprises a peptide or polypeptide. 
     
     
         123 . The fusion molecule of  claim 122 , wherein the polypeptide is a zinc finger polypeptide. 
     
     
         124 . The fusion molecule of  claim 122 , wherein the polypeptide is or comprises a Transcription activator-like effector nuclease (TALEN) polypeptide. 
     
     
         125 . The fusion molecule of any one of  claims 108 - 110 , wherein the site-specific binding moiety is or comprises a small molecule. 
     
     
         126 . A site-specific guide RNA that comprises a targeting domain complementary to a target nucleic acid comprising an anchor sequence. 
     
     
         127 . The site-specific guide RNA of  claim 126 , wherein the targeting domain is not complementary to at least one non-target nucleic acid comprising the anchor sequence. 
     
     
         128 . The site-specific guide RNA of  claim 126  or  127 , wherein the anchor sequence comprises a CTCF binding motif. 
     
     
         129 . A composition comprising:
 (i) a fusion polypeptide comprising an enzymatically inactive Cas polypeptide and an epigenetic modifying agent, or a nucleic acid encoding the fusion polypeptide; and   (ii) a guide RNA, wherein the guide RNA targets the fusion polypeptide to a target anchor sequence but not to at least one non-target anchor sequence.   
     
     
         130 . A method of modulating expression of a gene within an anchor sequence-mediated conjunction that comprises a first anchor sequence and a second anchor sequence, the method comprising a step of:
 contacting the first and/or second anchor sequence with the fusion molecule of any one of  claims 108 - 125 , the site-specific guide RNA of any one of  claims 126 - 128 , or the composition of  claim 129 .   
     
     
         131 . The method of  claim 130 , wherein the anchor sequence-mediated conjunction comprises at least one internal transcriptional control sequence. 
     
     
         132 . The method of  claim 131 , wherein the transcriptional control sequence is an enhancing sequence. 
     
     
         133 . The method of  claim 131 , wherein the transcriptional control sequence is a silencing or repressive sequence. 
     
     
         134 . The method of any one of  claims 130 - 133 , wherein the gene is separated from the internal transcriptional control sequence by at least 300 base pairs. 
     
     
         135 . The method of any one of  claims 130 - 134 , wherein the first and/or the second anchor sequence is located within 500 kb of an external transcriptional control sequence. 
     
     
         136 . The method of  claim 135 , wherein the external transcriptional control sequence is an enhancing sequence. 
     
     
         137 . The method of  claim 135 , wherein the external transcriptional control sequence is a silencing or repressive sequence. 
     
     
         138 . A method of modulating expression of a gene within 10 kb of a first anchor sequence within an anchor sequence-mediated conjunction comprising a first anchor sequence and a second anchor sequence,
 the method comprising a step of:
 contacting the first and/or second anchor sequence with the fusion molecule of any one of  claims 108 - 125 , the site-specific guide RNA of any one of  claims 126 - 128 , or the composition of  claim 129 . 
   
     
     
         139 . The method of  claim 138  wherein the anchor sequence-mediated conjunction comprises at least one internal transcriptional control sequence. 
     
     
         140 . The method of  claim 139 , wherein the internal transcriptional control sequence is an enhancing sequence. 
     
     
         141 . The method of  claim 139 , wherein the internal transcriptional control sequence is a silencing or repressive sequence. 
     
     
         142 . The method of any one of  claims 139 - 141 , wherein the gene is separated from the internal transcriptional control sequence by at least 300 base pairs. 
     
     
         143 . The method of any one of  claims 138 - 142 , wherein the first and/or the second anchor sequence is located within 500 kb of an external transcriptional control sequence. 
     
     
         144 . The method of  claim 143 , wherein the external transcriptional control sequence is an enhancing sequence. 
     
     
         145 . The method of  claim 143 , wherein the external transcriptional control sequence is a silencing or repressive sequence. 
     
     
         146 . A method of decreasing expression of a gene within an anchor sequence-mediated conjunction that comprises a first anchor sequence, a second anchor sequence, and an internal enhancing sequence, the method comprising a step of:
 contacting the first and/or second anchor sequence with the fusion molecule of any one of  claims 108 - 125 , the site-specific guide RNA of any one of  claims 126 - 128 , or the composition of  claim 129 .   
     
     
         147 . The method of  claim 146 , wherein the first and/or the second anchor sequence is located within 500 kb of an external silencing or repressive sequence. 
     
     
         148 . The method of  claim 146  or  147 , wherein the gene is separated from the internal enhancing sequence by at least 300 base pairs. 
     
     
         149 . A method of increasing expression of a gene within an anchor sequence-mediated conjunction that comprises a first anchor sequence and a second anchor sequence, wherein the first and/or the second anchor sequence is located within 10 kb of an external enhancing sequence, the method comprising a step of:
 contacting the first and/or second anchor sequence with the fusion molecule of any one of  claims 108 - 125 , the site-specific guide RNA of any one of  claims 126 - 128 , or the composition of  claim 129 .   
     
     
         150 . The method of  claim 149 , wherein the anchor sequence-mediated conjunction further comprises an internal enhancing sequence. 
     
     
         151 . The method of  claim 150 , wherein the gene is separated from the internal enhancing sequence by at least 300 base pairs. 
     
     
         152 . A method comprising a step of:
 (a) delivering the fusion molecule of any one of  claims 108 - 125 , the site-specific guide RNA of any one of  claims 126 - 128 , or the composition of  claim 129  to a mammalian cell.   
     
     
         153 . The method of  claim 152 , wherein the mammalian cell is a somatic cell. 
     
     
         154 . The method of  claim 152  or  153 , wherein the mammalian cell is a primary cell. 
     
     
         155 . The method of any one of  claims 152 - 154 , wherein the step of delivering is performed ex vivo. 
     
     
         156 . The method of  claim 155 , further comprising, prior to the step of delivering, a step of removing the mammalian cell from a subject. 
     
     
         157 . The method of  claim 155  or  156 , further comprising, after the step of delivering, a step of (b) administering the mammalian cells to a subject. 
     
     
         158 . The method of any one of  claims 152 - 155 , wherein the step of delivering comprises administering a composition comprising the fusion molecule of any one of  claims 108 - 125 , the site-specific guide RNA of any one of  claims 126 - 128 , or the composition of  claim 129  to a subject. 
     
     
         159 . The method of any one of  claims 156 ,  157 , or  158 , wherein the subject has a disease or condition. 
     
     
         160 . The method of any one of  claims 152 - 159 , wherein the step of delivering comprises delivery across the cell membrane. 
     
     
         161 . An engineered site-specific nucleating agent, comprising:
 an engineered DNA-binding moiety that binds specifically to one or more target sequences within a cell and not to non-targeted sequences within the cell with sufficient affinity that it competes binding of an endogenous nucleating polypeptide within the cell; and   a nucleating polypeptide dimerization domain associated with the engineered DNA-binding moiety so that, so that, when the engineered DNA-binding moiety is bound at the at least one target sequences, the nucleating polypeptide dimerization domain is localized thereto, and each at least one targeted sequence is a target anchor sequence   wherein the at least one or more target anchor sequences is positioned relative to an anchor sequence to which a nucleating polypeptide binds so that, when the nucleating polypeptide dimerization domain is localized to the target anchor sequence, interaction between the nucleating polypeptide dimerization domain and the nucleating polypeptide generates an anchor-sequence-mediated conjunction.   
     
     
         162 . The engineered site-specific nucleating agent of  claim 1 , wherein the target anchor sequence does not comprise a CTCF binding motif. 
     
     
         163 . A method of modulating expression of a gene within an anchor sequence-mediated conjunction that comprises a first anchor sequence and a second anchor sequence, the method comprising a step of:
 contacting the first and/or second anchor sequence with the engineered site-specific nucleating agent of  claim 161  or  162 .   
     
     
         164 . The method of  claim 163 , wherein the anchor sequence-mediated conjunction comprises at least one internal transcriptional control sequence. 
     
     
         165 . The method of  claim 163 , wherein the internal transcriptional control sequence is an enhancing sequence. 
     
     
         166 . The method of  claim 163 , wherein the internal transcriptional control sequence is a silencing or repressive sequence. 
     
     
         167 . The method of any one of  claims 163 - 166 , wherein the gene is separated from the internal transcriptional control sequence by at least 300 base pairs. 
     
     
         168 . The method of any one of  claims 163 - 167 , wherein the first and/or the second anchor sequence is located within 500 kb of an external transcriptional control sequence. 
     
     
         169 . The method of  claim 168 , wherein the external transcriptional control sequence is an enhancing sequence. 
     
     
         170 . The method of  claim 168 , wherein the external transcriptional control sequence is a silencing or repressive sequence. 
     
     
         171 . A method of modulating expression of a gene within 10 kb of a first anchor sequence within an anchor sequence-mediated conjunction comprising a first anchor sequence and a second anchor sequence,
 the method comprising a step of:
 contacting the first and/or second anchor sequence with the engineered site-specific nucleating agent of  claim 161  or  162 . 
   
     
     
         172 . The method of  claim 171  wherein the anchor sequence-mediated conjunction comprises at least one internal transcriptional control sequence. 
     
     
         173 . The method of  claim 172 , wherein the internal transcriptional control sequence is an enhancing sequence. 
     
     
         174 . The method of  claim 172 , wherein the internal transcriptional control sequence is a silencing or repressive sequence. 
     
     
         175 . The method of any one of  claims 171 - 174 , wherein the gene is separated from the internal transcriptional control sequence by at least 300 base pairs. 
     
     
         176 . The method of any one of  claims 171 - 175 , wherein the first and/or the second anchor sequence is located within 500 kb of an external transcriptional control sequence. 
     
     
         177 . The method of  claim 176 , wherein the external transcriptional control sequence is an enhancing sequence. 
     
     
         178 . The method of  claim 176 , wherein the external transcriptional control sequence is a silencing or repressive sequence. 
     
     
         179 . A method of decreasing expression of a gene within an anchor sequence-mediated conjunction that comprises a first anchor sequence, a second anchor sequence, and an internal enhancing sequence, the method comprising a step of:
 contacting the first and/or second anchor sequence with engineered site-specific nucleating agent of  claim 161  or  162 .   
     
     
         180 . The method of  claim 179 , wherein the first and/or the second anchor sequence is located within 500 kb of an external silencing or repressive sequence. 
     
     
         181 . The method of  claim 179  or  180 , wherein the gene is separated from the internal enhancing sequence by at least 300 base pairs. 
     
     
         182 . A method of increasing expression of a gene within an anchor sequence-mediated conjunction that comprises a first anchor sequence and a second anchor sequence, wherein the first and/or the second anchor sequence is located within 10 kb of an external enhancing sequence, the method comprising a step of:
 contacting the first and/or second anchor sequence with the engineered site-specific nucleating agent of  claim 161  or  162 .   
     
     
         183 . The method of  claim 182 , wherein the anchor sequence-mediated conjunction further comprises an internal enhancing sequence. 
     
     
         184 . The method of  claim 183 , wherein the gene is separated from the internal enhancing sequence by at least 300 base pairs. 
     
     
         185 . A method comprising a step of:
 (a) delivering the engineered site-specific nucleating agent of  claim 161  or  162  to a mammalian cell.   
     
     
         186 . The method of  claim 185 , wherein the mammalian cell is a somatic cell. 
     
     
         187 . The method of  claim 186 , wherein the mammalian cell is a primary cell. 
     
     
         188 . The method of any one of  claims 185 - 187  wherein the step of delivering is performed ex vivo. 
     
     
         189 . The method of  claim 188 , further comprising, prior to the step of delivering, a step of removing the mammalian cell from a subject. 
     
     
         190 . The method of  claim 188  or  189 , further comprising, after the step of delivering, a step of
 (b) administering the mammalian cells to a subject. 
 
     
     
         191 . The method of any one of  claims 185 - 187 , wherein the step of delivering comprises administering a composition comprising the engineered site-specific nucleating agent of  claim 161  or  162  to a mammalian cell to a subject. 
     
     
         192 . The method of any one of  claims 189 ,  190 , or  191 , wherein the subject has a disease or condition. 
     
     
         193 . The method of any one of  claims 185 - 192 , wherein the step of delivering comprises delivery across the cell membrane. 
     
     
         194 . A method of modulating expression of a target gene in an expression unit comprising:
 altering formation of an anchor sequence-mediated conjunction with a targeting moiety that targets a sequence outside of or that is not part of the target gene or its associated transcriptional control sequences that influence transcription of the gene, e.g., an anchor sequence, thereby modulating the target gene's expression.   
     
     
         195 . A method of modulating transcription of a nucleic acid sequence, e.g., a target gene in an expression unit, comprising
 altering formation of an anchor sequence-mediated conjunction with a targeting moiety that targets a sequence non-contiguous with the target gene or its associated transcriptional control sequences that influence transcription of the target gene to alter formation of the anchor sequence-mediated conjunction.   
     
     
         196 . A pharmaceutical preparation comprising:
 a composition comprising a targeting moiety that binds an anchor sequence of an anchor sequence-mediated conjunction and alters formation of the anchor sequence-mediated conjunction, wherein the composition modulates transcription, e.g., in a human cell, of a target gene associated with the anchor sequence-mediated conjunction.   
     
     
         197 . A composition comprising:
 a targeting moiety that binds an anchor sequence of an anchor sequence-mediated conjunction and alters formation of the anchor sequence-mediated conjunction (e.g., alters affinity of the anchor sequence to a conjunction nucleating molecule, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.   
     
     
         198 . The method or composition of any one of  claims 194 - 197 , wherein the targeting moiety comprises an effector moiety that:
 (i) is a chemical, e.g., a chemical that modulates a cytosine (C) or an adenine (A) (e.g., Na bisulfite, ammonium bisulfite)   (ii) has enzymatic activity (methyl transferase, demethylase, nuclease (e.g., Cas9), deaminase)   (iii) sterically hinders formation of the anchor sequence-mediated conjunction, e.g., ssDNA oligos, locked nucleic acids (LNAs), peptide oligonucleotide conjugates (e.g., membrane translocating polypeptides with nucleic acid side chains), bridged nucleic acids (BNAs), polyamides, and antisense oligonucleotide-conjugates comprising a DNA binding molecule.   
     
     
         199 . The method or composition of any one of  claims 194 - 198 , wherein one or more transcriptional control sequences are inside the anchor sequence-mediated conjunction, e.g., a type 1 anchor sequence-mediated conjunction. 
     
     
         200 . The method or composition of any one of  claims 194 - 199 , wherein one or more one or more transcriptional control sequences are outside the anchor sequence-mediated conjunction comprises, e.g., a type 2 anchor sequence-mediated conjunction. 
     
     
         201 . The method or composition of any one of  claims 194 - 200 , wherein one or more one or more transcriptional control sequences are inside, e.g., enhancing sequences, and outside, at least partially, e.g., silencing sequences, the anchor sequence-mediated conjunction, e.g., a type 3 anchor sequence-mediated conjunction. 
     
     
         202 . The method or composition of any one of  claims 194 - 201 , wherein one or more one or more transcriptional control sequences are inside, e.g., enhancing sequences, and outside, at least partially, e.g., enhancing sequences, the anchor sequence-mediated conjunction, e.g., a type 4 anchor sequence-mediated conjunction. 
     
     
         203 . A pharmaceutical composition comprising (a) a targeting moiety and (b) a DNA sequence, e.g., comprising an anchor sequence. 
     
     
         204 . A composition comprising:
 a protein comprising a domain, e.g., an enzyme domain, that acts on DNA (e.g., a nuclease domain, e.g., a Cas9 domain, e.g., a dCas9 domain; a DNA methyltransferase, a demethylase, a deaminase),   in combination with at least one guide RNA (gRNA) or antisense DNA oligonucleotide that targets the protein to an anchor sequence of a target anchor sequence mediated conjunction, wherein the composition is effective to alter, in a human cell, the target anchor sequence mediated conjunction.   
     
     
         205 . A composition comprising:
 a targeting moiety that binds an anchor sequence in an anchor sequence-mediated conjunction to alter a topology of the anchor sequence-mediated conjunction.   
     
     
         206 . A composition comprising:
 a protein comprising a first polypeptide comprising a Cas or modified Cas protein domain and a second polypeptide comprising a polypeptide having DNA methyltransferase activity [or associated with demethylation or deaminase activity],   in combination with at least one guide RNA (gRNA) or antisense DNA oligonucleotide that targets the protein to an anchor sequence of a target anchor sequence mediated conjunction, wherein the system is effective to alter, in a human cell, the target anchor sequence mediated conjunction.   
     
     
         207 . A pharmaceutical composition comprising:
 a Cas protein and at least one guide RNA (gRNA) that targets the Cas protein to an anchor sequence of a target anchor sequence mediated conjunction, wherein the Cas protein is effective to cause a mutation of the target anchor sequence that decreases the formation of an anchor sequence mediated conjunction associated with the target anchor sequence.   
     
     
         208 . A kit comprising:
 (a) a nucleic acid encoding a protein comprising a first polypeptide domain that comprises a Cas or modified Cas protein and a second polypeptide domain that comprises a polypeptide having DNA methyltransferase activity [or associated with demethylation or deaminase activity]; and   (b) at least one guide RNA (gRNA) or antisense DNA oligonucleotide for targeting the protein to an anchor sequence of a target anchor sequence mediated conjunction in a target cell.   
     
     
         209 . A method of [altering gene expression/altering an anchor sequence mediated conjunction] in a mammalian subject comprising administering to the subject (separately or in the same pharmaceutical composition):
 a) (i) a protein comprising a first polypeptide domain that comprises a Cas or modified Cas protein and a second polypeptide domain that comprises a polypeptide having DNA methyltransferase activity [or associated with demethylation or deaminase activity] or (ii) a nucleic acid encoding a protein comprising a first polypeptide domain that comprises a Cas protein and a second polypeptide domain that comprises a polypeptide having DNA methyltransferase activity [or associated with demethylation or deaminase activity], and   b) at least one guide RNA (gRNA) or antisense DNA oligonucleotide that targets an anchor sequence of an anchor sequence mediated conjunction.   
     
     
         210 . A method of modifying a chromatin structure, e.g., a two-dimensional structure, comprising:
 altering a topology of an anchor sequence-mediated conjunction, e.g., a loop, to modulate transcription of a nucleic acid sequence,   
       wherein the altered topology of the anchor sequence-mediated conjunction modulates transcription of the nucleic acid sequence. 
     
     
         211 . A method of modifying a chromatin structure, e.g., a two-dimensional structure, comprising:
 altering a topology of a plurality of anchor sequence-mediated conjunctions, e.g., multiple loops, to modulate transcription of a nucleic acid sequence,   
       wherein the altered topology modulates transcription of the nucleic acid sequence. 
     
     
         212 . A method of modulating transcription of a nucleic acid sequence comprising:
 altering an anchor sequence-mediated conjunction, e.g., a loop, that influences transcription of a nucleic acid sequence,   
       wherein altering the anchor sequence-mediated conjunction modulates transcription of the nucleic acid sequence. 
     
     
         213 . An engineered cell comprising a targeted alteration in an anchor sequence-mediated conjunction. 
     
     
         214 . An engineered nucleic acid sequence comprising an anchor sequence-mediated conjunction with a targeted alteration. 
     
     
         215 . A pharmaceutical composition comprising the engineered cell of any one of the previous claims, or the engineered nucleic acid sequence of any one of the previous claims. 
     
     
         216 . A plurality of cells comprising the engineered cell of any one of the previous claims. 
     
     
         217 . A vector comprising the engineered nucleic acid sequence of any one of the previous claims. 
     
     
         218 . A composition for introducing a targeted alteration to an anchor sequence-mediated conjunction to modulate transcription of a nucleic acid sequence, the composition comprising a targeting moiety that binds the anchor sequence. 
     
     
         219 . A composition comprising a synthetic conjunction nucleating molecule with a selected binding affinity for an anchor sequence within the anchor sequence-mediated conjunction. 
     
     
         220 . A synthetic nucleic acid comprising a plurality of anchor sequences, a gene sequence, and a transcriptional modifier sequence. 
     
     
         221 . A vector comprising the nucleic acid of any one of the previous claims. 
     
     
         222 . A cell comprising the nucleic acid of any one of the previous claims. 
     
     
         223 . A pharmaceutical composition comprising the nucleic acid of any one of the previous claims. 
     
     
         224 . A method of modulating expression of a gene by administering a composition comprising the nucleic acid of any one of the previous claims. 
     
     
         225 . A method of preparing a conjunction nucleating molecule with selected binding affinity. 
     
     
         226 . A method of treating a disease or condition comprising administering a targeting moiety selected from an exogenous conjunction nucleating molecule, a nucleic acid encoding the conjunction nucleating molecule, or a fusion of a sequence targeting polypeptide and a conjunction nucleating molecule, wherein the targeting moiety alters an anchor sequence-mediated conjunction to a subject. 
     
     
         227 . The composition and method of any one of the previous claims further comprising:
 at least one polypeptide with each comprising at least one sequence of ABX n C, where A is selected from a hydrophobic amino acid or an amide containing backbone, e.g., aminoethyl-glycine, with a nucleic acid side chain; B and C may be the same or different, and are each independently selected from arginine, asparagine, glutamine, lysine, and analogs thereof; X is each independently a hydrophobic amino acid or X is each independently an amide containing backbone, e.g., aminoethyl-glycine, with a nucleic acid side chain; and n is an integer from 1 to 4,   
       wherein the polypeptide hybridizes a nucleic acid sequence within an anchor sequence-mediated conjunction (e.g., anchor sequence of an anchor sequence-mediated conjunction, e.g., CTCF binding motif, BORIS binding motif, cohesin binding motif, USF1 binding motif, YY1 binding motif, TATA-box, ZNF143 binding motif, etc). 
     
     
         228 . A method of modifying expression of a target gene, comprising:
 altering an anchor sequence-mediated conjunction associated with the target gene, wherein the alteration modulates transcription of the target gene.   
     
     
         229 . A method of modifying expression of a target gene, comprising:
 administering the composition of any one of the prior claims to a cell, tissue or subject.   
     
     
         230 . A method of modulating transcription of a nucleic acid sequence comprising:
 administering the composition of any one of the prior claims to alter an anchor sequence-mediated conjunction, e.g., a loop, that modulates transcription of a nucleic acid sequence, wherein the altered anchor sequence-mediated conjunction modulates transcription of the nucleic acid sequence.   
     
     
         231 . A method of altering expression of a target gene comprising: administering to the genome a pharmaceutical composition comprising (a) a targeting moiety and (b) a DNA sequence comprising an anchor sequence, wherein the anchor sequence promotes the formation of a conjunction that brings a gene expression factor (an enhancing sequence, a silencing/repressive sequences) into operable linkage with the target gene. 
     
     
         232 . A method of modulating gene expression comprising providing the composition of any one of the prior claims, e.g., the targeting moiety comprises an effector moiety that inhibits CpG binding, is an endogenous effector, is an exogenous effector, or agonist or antagonist thereof. 
     
     
         233 . A method of delivering a therapeutic comprising:
 administering the composition of any one of the prior claims to a subject, wherein the targeting moiety comprises an effector moiety that is the therapeutic, and wherein the composition increases intracellular delivery of the therapeutic as compared to the therapeutic alone, composition modulates transcription of a gene.   
     
     
         234 . A method of modulating a membrane protein on a cell comprising:
 contacting the cell with the composition of any one of the prior claims, wherein the composition targets the cell and modulates the membrane protein.   
     
     
         235 . A method of inducing cell death comprising contacting a cell with the composition of any one of the prior claims, wherein the composition targets the cell and induces apoptosis. 
     
     
         236 . A method of increasing bioavailability of a therapeutic comprising:
 administering the composition of any one of the prior claims, wherein the therapeutic is the heterologous moiety.   
     
     
         237 . A method of treating a disease/disorder/condition in a subject comprising:
 administering the composition of any of the prior claims, wherein the composition modulates transcription to treat the disease/disorder/condition.   
     
     
         238 . A method of treating an acute or chronic infection comprising:
 administering the composition of any one of the prior claims.   
     
     
         239 . A method of treating a cancer comprising:
 administering the composition of any one of the prior claims.   
     
     
         240 . A method of treating a neurological disease or disorder comprising administering the composition of any one of the prior claims. 
     
     
         241 . A method of inducing immune tolerance comprising providing the composition of any one of the prior claims, e.g., the heterologous moiety is an antigen. 
     
     
         242 . A system for pharmaceutical use comprising:
 a protein comprising a first polypeptide domain that comprises a Cas or modified Cas protein and a second polypeptide domain that comprises a polypeptide having DNA methyltransferase activity [or associated with demethylation or deaminase activity],   in combination with at least one guide RNA (gRNA) or antisense DNA oligonucleotide that targets the protein to an anchor sequence of a target anchor sequence mediated conjunction, wherein the system is effective to alter, in a human cell, the target anchor sequence mediated conjunction.   
     
     
         243 . A system for altering, in a human cell, expression of a target gene, comprising:
 a targeting moiety (e.g., a gRNA, an LDB) that associates with an anchor sequence associated with the target gene,   
       optionally, a heterologous moiety (e.g., an enzyme, e.g., a nuclease or deactivated nuclease (e.g., a Cas9, dCas9), a methylase, a de-methylase, a deaminase) operably linked to the targeting moiety, wherein the system is effective to modulate a conjunction mediated by the anchor sequence and alter expression of the target gene.