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US 2020/0071369 A1Published application
Targeted dna demethylation and methylation
AssigneeThe Jackson Laboratory
InventorAlbert Cheng
Life of this patent
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Abstract
Provided herein are, inter alia, compositions and methods for demethylating and methylating a 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.
Claims45
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;
- and
- (ii) a demethylation domain, said demethylation domain operably linked to the C-terminus of said PUF domain to form a protein conjugate,
- 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.
12 more elements
A methylation 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 methylation protein conjugate comprising:
- (i) a PUF domain;
- and
- (ii) a methylation domain, said methylation domain operably linked to the N-terminus of said PUF domain to form a protein conjugate,
- wherein said methylation protein conjugate binds to said ribonucleoprotein complex via said PUF domain binding to said one or more PBS sequences to form a methylation complex.
12 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;
- and
- (d) delivering to said mammalian cell a third polynucleotide encoding a demethylation protein conjugate comprising:
- (i) a PUF domain;
- and
- (ii) a demethylation domain, said demethylation domain operably linked to the C-terminus of said PUF domain,
- whereby said delivered demethylation protein conjugate demethylates said target nucleic acid sequence in said cell.
11 more elements
A method of methylating a target nucleic acid sequence in a mammalian cell, comprising:
- (a) providing a mammalian cell containing a target nucleic acid requiring methylation;
- (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;
- and
- (d) delivering to said mammalian cell a third polynucleotide encoding a methylation protein conjugate comprising:
- (i) a PUF domain;
- and
- (ii) a methylation domain, said methylation domain operably linked to the N-terminus of said PUF domain,
- whereby said delivered methylation protein conjugate methylates said target nucleic acid sequence in said cell.
11 more elements
Dependencies are inferred from the claim text.