Compositions and methods for modulating expression of methyl-cpg binding protein 2 (mecp2)

A DNA-targeting system comprising:

• (a) a DNA-targeting domain that binds to a target site in a regulatory DNA element of a methyl-CpG-binding protein 2 (MeCP2) locus;
• and
• (b) at least one effector domain that increases transcription of the MeCP2 locus.

A DNA-targeting system comprising a DNA-targeting domain that is a Cas-guide RNA (gRNA) combination comprising:

• (a) a deactivated Cas (dCas) protein;
• and
• (b) at least one gRNA comprising a gRNA spacer sequence that is capable of hybridizing to a target site in a regulatory DNA element of a methyl-CpG-binding protein 2 (MeCP2) locus or is complementary to the target site.

A DNA-targeting system comprising a DNA-targeting domain that is a Cas-guide RNA (gRNA) combination comprising:

• (a) a Streptococcus pyogenes dCas9 (dSpCas9) protein;
• and
• (b) at least one gRNA comprising a gRNA spacer sequence that is capable of hybridizing to a target site in a regulatory DNA element of a MeCP2 locus or is complementary to the target site.

A DNA-targeting system comprising a DNA-targeting domain that is a Cas-guide RNA (gRNA) combination comprising:

• (a) a Streptococcus pyogenes deactivated Cas9 protein (dSpCas9) protein set forth in SEQ ID NO:95 fused to at least one effector domain that induces transcription de-repression;
• and
• (b) at least one gRNA that is a gRNA comprising a gRNA spacer sequence set forth in SEQ ID NO:39.

A DNA-targeting system comprising a DNA-targeting domain that is a Cas-guide RNA (gRNA) combination comprising:

• (a) a Streptococcus pyogenes deactivated Cas9 protein (dSpCas9) protein set forth in SEQ ID NO:95 fused to at least one effector domain that induces transcription de-repression;
• and
• (b) at least one gRNA that is a gRNA comprising a gRNA spacer sequence set forth in SEQ ID NO:57.

A DNA-targeting system comprising a DNA-targeting domain that is a Cas-guide RNA (gRNA) combination comprising:

• (a) a first polypeptide of a split variant Cas9 protein comprising an N-terminal fragment of a Streptococcus pyogenes deactivated Cas9 protein (dSpCas9) protein fused to an N-terminal intein and at least one effector domain that induces transcription de-repression;
• and
• (b) at least one gRNA that is a gRNA comprising a gRNA spacer sequence set forth in SEQ ID NO:39.

A DNA-targeting system comprising a DNA-targeting domain that is a Cas-guide RNA (gRNA) combination comprising:

• (a) a first polypeptide of a split variant Cas9 protein comprising an N-terminal fragment of a Streptococcus pyogenes deactivated Cas9 protein (dSpCas9) protein fused to an N-terminal intein and at least one effector domain that induces transcription de-repression;
• and
• (b) at least one gRNA that is a gRNA comprising a gRNA spacer sequence set forth in SEQ ID NO:57.

A DNA-targeting system comprising a DNA-targeting domain that is a Cas-guide RNA (gRNA) combination comprising:

• (a) a second polypeptide of a split variant Cas9 protein comprising a C-terminal fragment of a Streptococcus pyogenes deactivated Cas9 protein (dSpCas9) protein fused to an C-terminal intein and at least one effector domain that induces transcription de-repression;
• and
• (b) at least one gRNA that is a gRNA comprising a gRNA spacer sequence set forth in SEQ ID NO:39.

A DNA-targeting system comprising a DNA-targeting domain that is a Cas-guide RNA (gRNA) combination comprising:

• (a) a second polypeptide of a split variant Cas9 protein comprising a C-terminal fragment of a Streptococcus pyogenes deactivated Cas9 protein (dSpCas9) protein fused to a C-terminal intein and at least one effector domain that induces transcription de-repression;
• and
• (b) at least one gRNA that is a gRNA comprising a gRNA spacer sequence set forth in SEQ ID NO:57.

A guide RNA (gRNA) that binds a target site located within the genomic coordinates human genome assembly GRCh38 (hg38) chrX:154,097,151-154,098,158.

A guide RNA (gRNA) that binds a target site comprising the sequence set forth in any one of SEQ ID NOs:

• 1-29, a contiguous portion thereof of at least 14 nt, or a complementary sequence of any of the foregoing.

A combination, comprising:

• a first gRNA that binds a first target site in a regulatory DNA element of a methyl-CpG-binding protein 2 (MeCP2) locus, wherein the first target site is located within the genomic coordinates human genome assembly GRCh38 (hg38) chrX:154,097,151-154,098,158;
• and
• a second gRNA that binds a second target site in a regulatory DNA element of a MeCP2 locus, wherein the second target site is located within the genomic coordinates hg38 chrX:154,097,151-154,098,158.

A fusion protein comprising (1) a DNA-targeting domain or a component thereof and (2) at least one effector domain, wherein:

• the DNA-targeting domain or a component thereof binds to a target site in a regulatory DNA element of a methyl-CpG-binding protein 2 (MeCP2) locus;
• and
• the effector domain increases transcription of the MeCP2 locus.

A fusion protein comprising (1) a DNA-targeting domain or a component thereof and (2) at least one effector domain, wherein:

• the DNA-targeting domain or a component thereof binds to a target site in a regulatory DNA element of a methyl-CpG-binding protein 2 (MeCP2) locus;
• and
• the effector domain induces transcription activation, transcription co-activation, transcription elongation, transcription de-repression, histone modification, nucleosome remodeling, chromatin remodeling, reversal of heterochromatin formation, DNA demethylation, or DNA base oxidation.

A fusion protein comprising (1) a Cas protein or a variant thereof and (2) at least one effector domain, wherein the effector domain increases transcription of the MeCP2 locus.

A fusion protein comprising (1) a first polypeptide of a split variant Cas protein comprising an N-terminal fragment of a Cas protein and an N-terminal Intein, and (2) at least one effector domain, wherein the effector domain induces transcription activation, transcription co-activation, transcription elongation, transcription de-repression, histone modification, nucleosome remodeling, chromatin remodeling, reversal of heterochromatin formation, DNA demethylation, or DNA base oxidation.

A fusion protein comprising (1) a first polypeptide of a split variant Cas protein comprising an N-terminal fragment of a Cas protein and an N-terminal Intein, and (2) at least one effector domain, wherein the effector domain increases transcription of the MeCP2 locus.

A fusion protein comprising (1) a second polypeptide of a split variant Cas protein comprising a C-terminal fragment of a Cas protein and a C-terminal Intein and (2) at least one effector domain, wherein the effector domain induces transcription activation, transcription co-activation, transcription elongation, transcription de-repression, histone modification, nucleosome remodeling, chromatin remodeling, reversal of heterochromatin formation, DNA demethylation, or DNA base oxidation.

A fusion protein comprising (1) a second polypeptide of a split variant Cas protein comprising a C-terminal fragment of a Cas protein and a C-terminal Intein and (2) at least one effector domain, wherein the effector domain increases transcription of the MeCP2 locus.