Targeted DNA demethylation and methylation
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.
1. A composition comprising:
a guide RNA (gRNA) comprising a PUF binding site (PBS) sequence;
a nuclease-deficient DNA endonuclease that binds to the gRNA; and
a PUF domain linked to a demethylation domain or a methylation domain, wherein the PUF domain binds to the PBS of the gRNA.
2. The composition of claim 1 , wherein the gRNA targets a sequence selected from gene sequences, transcriptional regulatory sequences, promoter sequences, enhancer sequences, and silencer sequences.
3. The composition of claim 1 , wherein the gRNA comprises 1-50 PBS sequences.
4. The composition of claim 1 , wherein the PBS sequence comprises the sequence of SEQ ID NO: 1.
5. The composition of claim 1 , wherein the nuclease-deficient DNA endonuclease enzyme is a nuclease-deficient Cas9 protein (dCas9).
6. The composition of claim 1 , wherein the nuclease-deficient DNA endonuclease enzyme further comprises a nuclear localization signal (NLS).
7. The composition of claim 1 , wherein the PUF domain comprises a PUFa domain, a PUFb domain, a PUFc domain, or a PUFw domain.
8. The composition of claim 7 , wherein the PUFa domain comprises the sequence of SEQ ID NO:2, the PUFb domain comprises the sequence of SEQ ID NO:3, the PUFc domain comprises the sequence of SEQ ID NO:4, and/or the PUFw domain comprises the sequence of SEQ ID NO:5.
9. The composition of claim 1 , wherein the PUF domain is linked to a demethylation domain.
10. The composition of claim 9 , wherein the demethylation domain comprises a ten eleven translocation (TET) domain.
11. The composition of claim 10 , wherein the TET domain is a TET1 domain.
12. The composition of claim 9 , wherein the gRNA targets a hypermethylated nucleic acid sequence.
13. The composition of claim 12 , wherein the hypermethylated nucleic acid sequence is a hypermethylated CpG sequence.
14. The composition of claim 9 , wherein the gRNA targets a human mutL homolog 1 (hMLH1) gene.
15. The composition of claim 1 , wherein the PUF domain is linked to a methylation domain.
16. The composition of claim 15 , wherein the methylation domain comprises a DNA methyltransferase (DNMT) domain.
17. The composition of claim 16 , wherein the DNMT domain is a DNMT3A domain or a DNMT3A-3L domain.
18. A mammalian cell comprising the composition of claim 1 .
19. A kit comprising: a guide RNA (gRNA) comprising a PUF binding site (PBS) sequence; a nuclease-deficient DNA endonuclease that binds to the gRNA; and a PUF domain linked to a demethylation domain or a methylation domain, wherein the PUF domain binds to the PBS of the gRNA.
20. A complex comprising: a guide RNA (gRNA) comprising a PUF binding site (PBS) sequence; and a protein comprising a PUF domain linked to a demethylation domain or a methylation domain, wherein the PUF domain is bound to the PBS of the gRNA.
21. The complex of claim 20 further comprising a nuclease-deficient DNA endonuclease that binds to the gRNA.
22. A method of modulating methylation state of a nucleic acid of a mammalian cell, the method comprising delivering to the mammalian cell the composition of claim 1 , wherein the gRNA targets the nucleic acid, and methylation state of the nucleic acid is modulated.