TARGETED DNA DEMETHYLATION AND METHYLATION

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; 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.

2 .- 28 . (canceled)

29 . 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.

30 . The method of claim 29 , wherein said demethylation protein conjugate is bound to said second polynucleotide via binding of said PUF domain to said one or more PBS sequences.

31 . The method of claim 29 , wherein said first polynucleotide is contained within a first vector.

32 . The method of claim 29 , wherein said second polynucleotide is contained within a second vector.

33 . The method of claim 29 , wherein said third polynucleotide is contained within a third vector.

34 . The method of claim 29 , wherein either said first, second or third vector is the same.

35 . The method of claim 29 , wherein said delivering is performed by transfection.

36 . 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.

37 . The method of claim 36 , wherein said methylation protein conjugate is bound to said second polynucleotide via binding of said PUF domain to said one or more PBS sequences.

38 . The method of claim 36 , wherein said first polynucleotide is contained within a first vector.

39 . The method of claim 36 , wherein said second polynucleotide is contained within a second vector.

40 . The method of claim 36 , wherein said third polynucleotide is contained within a third vector.

41 . The method of claim 36 , wherein either said first, second or third vector is the same.

42 . The method of claim 36 , wherein said delivering is performed by transfection.

43 . A kit comprising:

(i) a ribonucleoprotein complex of claim 1 or a nucleic acid encoding the same; and

(ii) a methylation modifying protein conjugate of claim 1 or a nucleic acid encoding the same.

44 .- 45 . (canceled)