Compositions and Methods for Making Epigenetic Modifications
The disclosure provides, in various embodiments, fusion proteins comprising a DNA-binding domain, a DNMT3A-binding domain, and a H3K4me0; and polynucleotides and vectors encoding one or more of the fusion proteins. The disclosure also provides, in various embodiments, gene-delivery systems, cells, compositions (e.g., pharmaceutical compositions) and kits comprising one or more of the fusion proteins polynucleotides, or vectors; methods of epigenetically modifying a genomic locus in a cell; and methods of treating a subject (e.g., a human) in need thereof.
1 . A deoxyribonucleic acid (DNA) encoding a fusion protein comprising a H3K4me0, a DNMT3 methyltransferase-binding domain, and a DNA-binding domain.
2 . The DNA of claim 1 , wherein:
a) the fusion protein lacks nuclease activity;
b) the fusion protein lacks a DNA methyltransferase catalytic domain;
c) the DNMT3 methyltransferase-binding domain binds a catalytic domain of DNMT3A,
or any combination of the foregoing.
3 . The DNA of claim 1 , wherein the DNMT3 methyltransferase-binding domain comprises a DNA methyltransferase 3-like protein (Dnmt3L), or a C-terminal fragment of Dnmt3L.
4 . The DNA of claim 1 , wherein the DNA-binding domain comprises a DNA-binding domain of:
a) a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas);
b) a transcription activator-like effector nuclease (TALEN);
c) a zinc finger nuclease (ZFN);
d) a tetracycline-controlled repressor (tetR);
e) a meganuclease;
f) a homing (HO) endonuclease; or
g) a eukaryotic programmable RNA-guided endonuclease.
5 . The DNA of claim 1 , wherein the DNA-binding domain comprises:
a) Streptococcus pyogenes dCas9;
b) Staphylococcus aureus dCas9;
c) S. aureus dCas9;
d) dCas12a; or
e) dCas12f.
6 . The DNA of claim 1 , wherein the DNA-binding domain comprises a leucine zipper domain, a winged helix domain, a helix-turn-helix domain, a helix-loop-helix domain, a chromatin-associated high-mobility group (HMG)-box domain, a white-opaque regulator 3 (Wor3) domain, an oligonucleotide/oligosaccharide-binding (OB)-fold domain, an immunoglobulin domain, or a B3 DNA-binding domain.
7 . The DNA of claim 1 , wherein the fusion protein comprises, from N-terminus to C-terminus:
a) the H3K4me0, the DNMT3 methyltransferase-binding domain, and the DNA-binding domain; or
b) the H3K4me0, the DNA-binding domain, and the DNMT3 methyltransferase-binding domain.
8 . The DNA of claim 1 , wherein the fusion protein further comprises a Krüppel-associated box (KRAB) domain.
9 . The DNA of claim 8 , wherein the KRAB domain is a KOX1 KRAB domain or a ZIM3 KRAB domain.
10 . The DNA of claim 9 , wherein the fusion protein comprises, from N-terminus to C-terminus:
a) the H3K4me0, the DNMT3 methyltransferase-binding domain, the DNA-binding domain, and the KRAB domain;
b) the H3K4me0, the KRAB domain, the DNMT3 methyltransferase-binding domain, and the DNA-binding domain;
c) the H3K4me0, the DNA-binding domain, and the DNMT3 methyltransferase-binding domain, and the KRAB domain;
d) the H3K4me0, the KRAB domain, the DNA-binding domain, and the DNMT3 methyltransferase-binding domain; or
e) the H3K4me0, the DNA-binding domain, the KRAB domain, and the DNMT3 methyltransferase-binding domain.
11 . The DNA of claim 1 , wherein:
a) the H3K4me0 has at least 80% sequence identity to at least one sequence set forth in SEQ ID NO:87, SEQ ID NO:81, and SEQ ID NOs:393-396;
b) the DNMT3 methyltransferase-binding domain has at least 80% sequence identity to at least one sequence set forth in SEQ ID NOs:31-50 and 71-75;
c) the DNA-binding domain has at least 80% sequence identity to at least one sequence set forth in SEQ ID NO:1, SEQ ID NO:489, SEQ ID NOs:3-15, and SEQ ID NOs:454-469;
or any combination of the foregoing.
12 . The DNA of claim 1 , wherein:
a) the H3K4me0 has 100% sequence identity to a sequence set forth in SEQ ID NO:87, SEQ ID NO:81, and SEQ ID NOs:393-396;
b) the DNMT3 methyltransferase-binding domain has 100% sequence identity to a sequence set forth in SEQ ID NOs:31-50 and 71-75;
c) the DNA-binding domain has 100% sequence identity to a sequence set forth in SEQ ID NO:1, SEQ ID NO:489, SEQ ID NOs:3-15, and SEQ ID NOs:454-469;
or any combination of the foregoing.
13 . The DNA of claim 1 , wherein:
a) the H3K4me0 has at least 80% sequence identity to SEQ ID NO:87, SEQ ID NO:81, or both;
b) the DNMT3 methyltransferase-binding domain has at least 80% sequence identity to SEQ ID NO:31, SEQ ID NO:32, or both;
c) the DNA-binding domain has at least 80% sequence identity to at least one sequence set forth in SEQ ID NO:1, SEQ ID NO:489, SEQ ID NOs:3-9, and SEQ ID NOs:454-461,
or any combination of the foregoing.
14 . The DNA of claim 1 , wherein:
a) the H3K4me0 has 100% sequence identity to SEQ ID NO:87 or SEQ ID NO:81;
b) the DNMT3 methyltransferase-binding domain has 100% sequence identity to SEQ ID NO:31 or SEQ ID NO:32;
c) the DNA-binding domain has 100% sequence identity to a sequence set forth in SEQ ID NO:1, SEQ ID NO:489, SEQ ID NOs:3-9, and SEQ ID NOs:454-461,
or any combination of the foregoing.
15 . The DNA of claim 8 , wherein the KRAB domain has at least 80% sequence identity to SEQ ID NO:155.
16 . The DNA of claim 8 , wherein the KRAB domain has 100% sequence identity to SEQ ID NO:155.
17 . The DNA of claim 1 , wherein the H3K4me0 is connected to the DNMT3 methyltransferase-binding domain with a linker of about 30-50 amino acids in length.
18 . The DNA of claim 17 , wherein the linker has 100% sequence identity to a sequence set forth in SEQ ID NOs:89-100, SEQ ID NOs:113-128, SEQ ID NO:145, SEQ ID NO:147, and SEQ ID NO:506.
19 . The DNA of claim 17 , wherein the linker has 100% sequence identity to SEQ ID NO:96 or SEQ ID NO:506.
20 . The DNA of claim 8 , wherein the H3K4me0 is connected to the KRAB domain with a linker of about 30-50 amino acids in length.
21 . The DNA of claim 20 , wherein the linker has 100% sequence identity to a sequence set forth in SEQ ID NOs:89-100, SEQ ID NOs:113-128, SEQ ID NO:145, SEQ ID NO:147, and SEQ ID NO:506.
22 . The DNA of claim 20 , wherein the linker has 100% sequence identity to SEQ ID NO:96 or SEQ ID NO:506.