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US 2019/0218261 A1Published application
Targeted enhanced dna demethylation
AssigneeThe Jackson Laboratory
InventorsAlbert Cheng, Aziz Taghbalout, Nathaniel Jillette
Life of this patent
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Abstract
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.
Claims113
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.
13 more elements
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.
10 more elements
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.
14 more elements
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.
11 more elements
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.
17 more elements
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.
13 more elements
Dependencies are inferred from the claim text.