TARGETED ENHANCED DNA DEMETHYLATION
Provided herein are, inter alia, compositions and methods for the delivery of enhanced demethylation activity to target DNA sequences in a mammalian cell. The compositions and methods are, useful for activity modulation of a targeted gene, or to create a gene regulatory network.
1 . A demethylation complex, comprising:
(a) a ribonucleoprotein complex comprising:
(i) a nuclease-deficient RNA-guided DNA endonuclease enzyme; and
(ii) a polynucleotide comprising:
(1) a DNA-targeting sequence that is complementary to a target polynucleotide sequence;
(2) a binding sequence for said nuclease-deficient RNA-guided DNA endonuclease enzyme; and
(3) one or more PUF binding site (PBS) sequences,
wherein said nuclease-deficient RNA-guided DNA endonuclease enzyme is bound to said polynucleotide via said binding sequence; and
(b) a demethylation protein conjugate comprising:
(i) a PUF domain having a C-terminus and a N-terminus;
(ii) a TET demethylation domain operably linked to the C-terminus of said PUF domain; and
(iii) a demethylation enhancer domain operably linked to the N-terminus of said PUF domain, to form a protein conjugate, and
wherein said demethylation protein conjugate binds to said ribonucleoprotein complex via said PUF domain binding to said one or more PBS sequences to form a demethylation complex.
2 . The complex of claim 1 , wherein said TET demethylation domain is a TET1 domain, a TET2 domain or a TET3 domain.
3 . The complex of claim 2 , wherein said TET 1 domain has the sequence of SEQ ID NO:51.
4 . The complex of one of claims 1 - 3 , wherein said demethylation enhancer domain is a Growth Arrest and DNA-Damage-inducible Alpha (GADD45A) domain.
5 . The complex of claim 4 , wherein said GADD45 domain has the amino acid sequence of SEQ ID NO:85.
6 . The complex of one of claims 1 - 3 , wherein said demethylation enhancer domain is a NEIL2 domain.
7 . The complex of claim 5 , wherein said NEIL2 domain has the amino acid sequence of SEQ ID NO:86.
8 . The complex of one of claims 1 - 7 , wherein said nuclease-deficient RNA-guided DNA endonuclease enzyme comprises a nuclear localization signal (NLS).
9 . The complex of one of claims 1 - 8 , wherein said nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9.
10 . The complex of one of claims 1 - 9 , wherein said target polynucleotide sequence is part of a gene.
11 . The complex of one of claims 1 - 9 , wherein said target polynucleotide sequence is part of a transcriptional regulatory sequence.
12 . The complex of one of claims 1 - 9 or 11 , wherein said target polynucleotide sequence is part of a promoter, enhancer or silencer.
13 . The complex of one of claims 1 - 12 , wherein said target polynucleotide sequence is a hypermethylated nucleic acid sequence.
14 . The complex of one of claims 1 - 13 , wherein said target polynucleotide sequence is a hypermethylated CpG sequence.
15 . The complex of one of claims 1 - 9 or 11 - 14 , wherein said target polynucleotide sequence is part of an hMLH1 promoter.
16 . The complex of one of claims 1 - 10 or 13 - 14 , wherein said target polynucleotide sequence is part of a Sox gene.
17 . The complex of one of claims 1 - 16 , wherein said one or more PBS sequences contain 8 nucleotides in length.
18 . The complex of one of claims 1 - 17 , wherein said one or more PBS sequences are identical.
19 . The complex of one of claims 1 - 18 , wherein said polynucleotide comprises 1 to 50 PBS sequences.
20 . The complex of one of claims 1 - 19 , wherein one or more PBS sequences comprise the nucleotide sequence of SEQ ID NO: 1.
21 . The complex of one of claims 1 - 20 , wherein said PUF domain comprises a PUFa domain, a PUFb domain, a PUFc domain, or a PUFw domain.
22 . The complex of claim 21 , wherein said PUFa domain has the amino acid sequence of SEQ ID NO:2.
23 . The complex of claim 21 , wherein said PUFb domain has the amino acid sequence of SEQ ID NO:3.
24 . The complex of claim 21 , wherein said PUFc domain has the amino acid sequence of SEQ ID NO:4.
25 . The complex of claim 21 , wherein said PUFw domain has the amino acid sequence of SEQ ID NO:5.
26 . A method of demethylating a target nucleic acid sequence in a mammalian cell, comprising:
(a) providing a mammalian cell containing a target nucleic acid requiring demethylation;
(b) delivering to said mammalian cell a first polynucleotide encoding a nuclease-deficient RNA-guided DNA endonuclease enzyme;
(c) delivering to said mammalian cell a second polynucleotide comprising:
(i) a DNA-targeting sequence that is complementary to a target polynucleotide sequence;
(ii) a binding sequence for said nuclease-deficient RNA-guide DNA endonuclease enzyme, and
(iii) one or more PUF binding site (PBS) sequences,
wherein said nuclease-deficient RNA-guided DNA endonuclease enzyme is bound to said second polynucleotide via said binding sequence;
(d) delivering to said mammalian cell a third polynucleotide encoding a demethylation protein conjugate comprising:
(i) a PUF domain having a C-terminus and a N-terminus;
(ii) a TET demethylation domain operably linked to the C-terminus of said PUF domain; and
(iii) a demethylation enhancer domain operably linked to the N-terminus of said PUF domain, to form a protein conjugate, and
whereby said delivered demethylation protein conjugate demethylates said target nucleic acid sequence in said cell.
27 . The method of claim 26 , wherein said demethylation protein conjugate binds to said ribonucleoprotein complex via said PUF domain binding to said one or more PBS sequences to form a demethylation complex.
28 . The method of claim 26 or 27 , wherein said first polynucleotide is contained within a first vector.
29 . The method of one of claims 26 - 28 , wherein said second polynucleotide is contained within a second vector.
30 . The method of one of claims 26 - 29 , wherein said third polynucleotide is contained within a third vector.
31 . The method of one of claims 26 - 30 , wherein either said first, second or third vector is the same.
32 . The method of one of claims 26 - 31 , wherein said delivering is performed by transfection.
33 . A kit comprising:
(i) a ribonucleoprotein complex of one of claims 1 - 25 or a nucleic acid encoding the same; and
(ii) a demethylation protein conjugate of one of claims 1 - 25 or a nucleic acid encoding the same.
34 . The kit of claim 33 , further comprising a transfection agent.
35 . The kit of claim 33 or 34 , further comprising a sample collection device for collecting a sample from a cancer patient.
36 . A demethylation complex, comprising:
(a) a ribonucleoprotein complex comprising:
(i) a nuclease-deficient RNA-guided DNA endonuclease enzyme; and
(ii) a polynucleotide comprising:
(1) a DNA-targeting sequence that is complementary to a target polynucleotide sequence;
(2) a binding sequence for said nuclease-deficient RNA-guided DNA endonuclease enzyme; and
(3) one or more PUF binding site (PBS) sequences,
wherein said nuclease-deficient RNA-guided DNA endonuclease enzyme is bound to said polynucleotide via said binding sequence; and
(b) a demethylation protein conjugate comprising:
(i) a PUF domain having a C-terminus;
(ii) a demethylation enhancer domain, having a N-terminus and a C-terminus, wherein said N-terminus of said demethylation enhancer domain is operably linked to the C-terminus of said PUF domain; and
(iii) a TET demethylation domain operably linked to the C-terminus of said demethylation enhancer domain; and
wherein said demethylation protein conjugate binds to said ribonucleoprotein complex via said PUF domain binding to said one or more PBS sequences to form a demethylation complex.
37 . The complex of claim 36 , wherein said TET demethylation domain is a TET1 domain, a TET2 domain or a TET3 domain.
38 . The complex of claim 37 , wherein said TET1 domain has the sequence of SEQ ID NO:51.
39 . The complex of one of claims 36 - 38 , wherein said demethylation enhancer domain is a Growth Arrest and DNA-Damage-inducible Alpha (GADD45A) domain.
40 . The complex of claim 39 , wherein said GADD45 domain has the amino acid sequence of SEQ ID NO:85.
41 . The complex of one of claims 36 - 38 , wherein said demethylation enhancer domain is a NEIL2 domain.
42 . The complex of claim 41 , wherein said NEIL2 domain has the amino acid sequence of SEQ ID NO:86.
43 . The complex of one of claims 36 - 42 , wherein said nuclease-deficient RNA-guided DNA endonuclease enzyme comprises a nuclear localization signal (NLS).
44 . The complex of one of claims 36 - 43 , wherein said nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9.
45 . The complex of one of claims 36 - 44 , wherein said target polynucleotide sequence is part of a gene.
46 . The complex of one of claims 36 - 44 , wherein said target polynucleotide sequence is part of a transcriptional regulatory sequence.
47 . The complex of one of claims 36 - 44 or 46 , wherein said target polynucleotide sequence is part of a promoter, enhancer or silencer.
48 . The complex of one of claims 36 - 47 , wherein said target polynucleotide sequence is a hypermethylated nucleic acid sequence.
49 . The complex of one of claims 36 - 48 , wherein said target polynucleotide sequence is a hypermethylated CpG sequence.
50 . The complex of one of claims 36 - 44 or 46 - 49 , wherein said target polynucleotide sequence is part of an hMLH1 promoter.
51 . The complex of one of claims 36 - 45 or 48 - 49 , wherein said target polynucleotide sequence is part of a Sox gene.
52 . The complex of one of claims 36 - 51 , wherein said one or more PBS sequences contain 8 nucleotides in length.
53 . The complex of one of claims 36 - 52 , wherein said one or more PBS sequences are identical.
54 . The complex of one of claims 36 - 53 , wherein said polynucleotide comprises 1 to 50 PBS sequences.
55 . The complex of one of claims 36 - 54 , wherein one or more PBS sequences comprise the nucleotide sequence of SEQ ID NO: 1.
56 . The complex of one of claims 36 - 55 , wherein said PUF domain comprises a PUFa domain, a PUFb domain, a PUFc domain, or a PUFw domain.
57 . The complex of claim 56 , wherein said PUFa domain has the amino acid sequence of SEQ ID NO:2.
58 . The complex of claim 56 , wherein said PUFb domain has the amino acid sequence of SEQ ID NO:3.
59 . The complex of claim 56 , wherein said PUFc domain has the amino acid sequence of SEQ ID NO:4.
60 . The complex of claim 56 , wherein said PUFw domain has the amino acid sequence of SEQ ID NO:5.
61 . A method of demethylating a target nucleic acid sequence in a mammalian cell, comprising:
(a) providing a mammalian cell containing a target nucleic acid requiring demethylation;
(b) delivering to said mammalian cell a first polynucleotide encoding a nuclease-deficient RNA-guided DNA endonuclease enzyme;
(c) delivering to said mammalian cell a second polynucleotide comprising:
(i) a DNA-targeting sequence that is complementary to a target polynucleotide sequence;
(ii) a binding sequence for said nuclease-deficient RNA-guide DNA endonuclease enzyme; and
(iii) one or more PUF binding site (PBS) sequences,
wherein said nuclease-deficient RNA-guided DNA endonuclease enzyme is bound to said second polynucleotide via said binding sequence;
(d) delivering to said mammalian cell a third polynucleotide encoding a demethylation protein conjugate comprising:
(i) a PUF domain having a C-terminus;
(ii) a demethylation enhancer domain, having a N-terminus and a C-terminus,
wherein said N-terminus of said demethylation enhancer domain is operably linked to the C-terminus of said PUF domain; and
(iii) a TET demethylation domain operably linked to the C-terminus of said demethylation enhancer domain, whereby said delivered demethylation protein conjugate demethylates said target nucleic acid sequence in said cell.
62 . The method of claim 61 , wherein said demethylation protein conjugate binds to said ribonucleoprotein complex via said PUF domain binding to said one or more PBS sequences to form a demethylation complex.
63 . The method of claim 61 or 62 , wherein said first polynucleotide is contained within a first vector.
64 . The method of one of claims 61 - 63 , wherein said second polynucleotide is contained within a second vector.
65 . The method of one of claims 61 - 64 , wherein said third polynucleotide is contained within a third vector.
66 . The method of one of claims 61 - 65 , wherein either said first, second or third vector is the same.
67 . The method of one of claims 61 - 66 , wherein said delivering is performed by transfection.
68 . A kit comprising:
(i) a ribonucleoprotein complex of one of claims 36 - 60 or a nucleic acid encoding the same; and
(ii) a demethylation protein conjugate of one of claims 36 - 60 or a nucleic acid encoding the same.
69 . The kit of claim 68 , further comprising a transfection agent.
70 . The kit of claim 68 or 69 , further comprising a sample collection device for collecting a sample from a cancer patient.
71 . A demethylation complex, comprising:
(a) a ribonucleoprotein complex comprising:
(i) a nuclease-deficient RNA-guided DNA endonuclease enzyme; and
(ii) a polynucleotide comprising:
(1) a DNA-targeting sequence that is complementary to a target polynucleotide sequence;
(2) a binding sequence for said nuclease-deficient RNA-guided DNA endonuclease enzyme;
(3) a first PUF binding site (PBS) sequence; and
(4) a second PUF binding site (PBS) sequence,
wherein said nuclease-deficient RNA-guided DNA endonuclease enzyme is bound to said polynucleotide via said binding sequence;
(b) a demethylation protein conjugate comprising:
(i) a first PUF domain having a C-terminus, and
(ii) a TET demethylation domain operably linked to the C-terminus of said first PUF domain,
wherein said demethylation protein conjugate binds to said ribonucleoprotein complex via said first PUF domain binding to said first PBS sequence; and
(c) a demethylation enhancer conjugate comprising:
(i) a second PUF domain; and
(ii) a demethylation enhancer domain operably linked to said second PUF domain,
wherein said demethylation enhancer conjugate binds to said ribonucleoprotein complex via said second PUF domain binding to said second PBS sequence to form a demethylation complex.
72 . The complex of claim 71 , wherein said TET demethylation domain is a TET1 domain, a TET2 domain or a TET3 domain.
73 . The complex of claim 72 , wherein said TET1 domain has the sequence of SEQ ID NO:51.
74 . The complex of one of claims 71 - 73 , wherein said demethylation enhancer domain is a Growth Arrest and DNA-Damage-inducible Alpha (GADD45A) domain.
75 . The complex of claim 74 , wherein said GADD45 domain has the amino acid sequence of SEQ ID NO:85.
76 . The complex of one of claims 71 - 73 , wherein said demethylation enhancer domain is a NEIL2 domain.
77 . The complex of claim 76 , wherein said NEIL2 domain has the sequence of SEQ ID NO:86.
78 . The complex of one of claims 71 - 77 , wherein said first PUF domain is a PUFa domain.
79 . The complex of claim 78 , wherein said PUFa domain has the sequence of SEQ ID NO:2.
80 . The complex of one of claims 71 - 79 , wherein said second PUF domain is a PUFc domain.
81 . The complex of claim 80 , wherein said PUFc domain has the sequence of SEQ ID NO:4.
82 . The complex of one of claims 71 - 81 , wherein said demethylation enhancer domain is operably linked to the N-terminus of said second PUF domain.
83 . The complex of one of claims 71 - 81 , wherein said demethylation enhancer domain is operably linked to the C-terminus of said second PUF domain.
84 . The complex of one of claims 71 - 83 , wherein said nuclease-deficient RNA-guided DNA endonuclease enzyme comprises a nuclear localization signal (NLS).
85 . The complex of one of claims 71 - 43 , wherein said nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9.
86 . The complex of one of claims 71 - 85 , wherein said target polynucleotide sequence is part of a gene.
87 . The complex of one of claims 71 - 85 , wherein said target polynucleotide sequence is part of a transcriptional regulatory sequence.
88 . The complex of one of claims 71 - 85 or 87 , wherein said target polynucleotide sequence is part of a promoter, enhancer or silencer.
89 . The complex of one of claims 71 - 88 , wherein said target polynucleotide sequence is a hypermethylated nucleic acid sequence.
90 . The complex of one of claims 71 - 89 , wherein said target polynucleotide sequence is a hypermethylated CpG sequence.
91 . The complex of one of claims 71 - 85 or 87 - 90 , wherein said target polynucleotide sequence is part of an hMLH1 promoter.
92 . The complex of one of claims 71 - 86 or 89 - 90 , wherein said target polynucleotide sequence is part of a Sox gene.
93 . The complex of one of claims 71 - 92 , wherein said first or said second PBS sequence contains 8 nucleotides in length.
94 . The complex of one of claims 71 - 93 , wherein said first or said second PBS sequences comprise the nucleotide sequence of SEQ ID NO:1.
95 . The complex of one of claims 71 - 94 , wherein said first or said second PUF domain comprises a PUFa domain, a PUFb domain, a PUFc domain, or a PUFw domain.
96 . The complex of claim 95 , wherein said first or said second PUFa domain has the amino acid sequence of SEQ ID NO:2.
97 . The complex of claim 95 , wherein said first or said second PUFb domain has the amino acid sequence of SEQ ID NO:3.
98 . The complex of claim 95 , wherein said first or said second PUFc domain has the amino acid sequence of SEQ ID NO:4.
99 . The complex of claim 95 , wherein said first or said second PUFw domain has the amino acid sequence of SEQ ID NO:5.
100 . A method of demethylating a target nucleic acid sequence in a mammalian cell, comprising:
(a) providing a mammalian cell containing a target nucleic acid requiring demethylation;
(b) delivering to said mammalian cell a first polynucleotide encoding a nuclease-deficient RNA-guided DNA endonuclease enzyme;
(c) delivering to said mammalian cell a second polynucleotide comprising:
(i) a DNA-targeting sequence that is complementary to a target polynucleotide sequence;
(ii) a binding sequence for said nuclease-deficient RNA-guide DNA endonuclease enzyme;
(iii) a first PUF binding site (PBS) sequence, and
(iv) a second PUF binding site (PBS) sequence,
wherein said nuclease-deficient RNA-guided DNA endonuclease enzyme is bound to said second polynucleotide via said binding sequence;
(d) delivering to said mammalian cell a third polynucleotide encoding a demethylation protein conjugate comprising:
(i) a first PUF domain; and
(ii) a demethylation domain, said demethylation domain operably linked to the C-terminus of said first PUF domain, and
(e) delivering to said mammalian cell a fourth polynucleotide encoding a demethylation enhancer conjugate comprising:
(i) a second PUF domain; and
(ii) a demethylation enhancer domain operably linked to said second PUF domain, whereby said delivered demethylation protein conjugate demethylates said target nucleic acid sequence in said cell.
101 . The method of claim 100 , wherein said demethylation protein conjugate binds to said ribonucleoprotein complex via said first PUF domain binding to said first PBS sequence.
102 . The method of claim 100 or 101 , wherein said demethylation enhancer conjugate binds to said ribonucleoprotein complex via said second PUF domain binding to said second PBS sequence.
103 . The method of one of claims 100 - 102 , wherein said demethylation enhancer domain is operably linked to the N-terminus of said second PUF domain.
104 . The method of one of claims 100 - 102 , wherein said demethylation enhancer domain is operably linked to the C-terminus of said second PUF domain.
105 . The method of one of claims 100 - 104 , wherein said first polynucleotide is contained within a first vector.
106 . The method of one of claims 100 - 105 , wherein said second polynucleotide is contained within a second vector.
107 . The method of one of claims 100 - 106 , wherein said third polynucleotide is contained within a third vector.
108 . The method of one of claims 100 - 107 , wherein said fourth polynucleotide is contained within a fourth vector.
109 . The method of one of claims 100 - 108 , wherein either said first, second, third or fourth vector is the same.
110 . The method of one of claims 100 - 109 , wherein said delivering is performed by transfection.
111 . A kit comprising:
(i) a ribonucleoprotein complex of one of claims 71 - 99 or a nucleic acid encoding the same;
(ii) a demethylation protein conjugate of one of claims 71 - 99 or a nucleic acid encoding the same, and
(iii) a demethylation enhancer conjugate of one of claims 71 - 99 or a nucleic acid encoding the same.
112 . The kit of claim 111 , further comprising a transfection agent.
113 . The kit of claim 111 or 112 , further comprising a sample collection device for collecting a sample from a cancer patient.