RNA-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci

Patent metadata

Field	Value
Publication number	US-10378027-B2
Application number	US-201414775869-A
Country	US
Kind code	B2
Filing date	Mar 14, 2014
Priority date	Mar 15, 2013
Publication date	Aug 13, 2019
Grant date	Aug 13, 2019

Abstract

Methods and constructs for RNA-guided targeting of heterologous functional domains such as transcriptional activators to specific genomic loci.

First claim

What is claimed is:

1. A fusion protein comprising a catalytically inactive CRISPR associated 9 (dCas9) protein linked to a heterologous functional domain, wherein the heterologous functional domain is a Ten-Eleven Translocation (TET) protein.

2. The fusion protein of claim 1 , wherein the TET protein is Tet Methylcytosine Dioxygenase 1 (TET1).

3. The fusion protein of claim 1 , wherein the catalytically inactive Cas9 protein is from S. pyogenes.

4. The fusion protein of claim 3 , wherein the catalytically inactive Cas9 protein comprises mutations corresponding to positions D10, E762, H983, or D986 of a wild-type or a codon-optimized Cas 9; and corresponding to positions-H840 or N863 of a wild-type or a codon-optimized Cas 9.

5. The fusion protein of claim 4 , wherein the mutations corresponding to positions D10 and H840 of a wild-type or a codon-optimized Cas 9 are:
(i) D10A or D10N, and
(ii) H840A, H840N, or H840Y.

6. The fusion protein of claim 1 , wherein the heterologous functional domain is linked to the N terminus or C terminus of the catalytically inactive Cas9 protein, with an optional intervening linker, wherein the linker does not interfere with activity of the fusion protein.

7. The fusion protein of claim 1 , further comprising one or both of a nuclear localization sequence and one or more epitope tags on the N-terminus, C-terminus, and/or in between the catalytically inactive CRISPR associated 9 (Cas9) protein and the heterologous functional domain, optionally with one or more intervening linkers.

8. The fusion protein of claim 7 , wherein the epitope tag is c-myc, 6His, or FLAG.

9. A nucleic acid encoding the fusion protein of claim 1 .

10. An expression vector comprising the nucleic acid of claim 9 .

11. A method of reducing DNA methylation of a target gene or its promoter or enhancer(s) in a cell, the method comprising expressing the fusion protein of claim 1 in the cell, and one or more guide RNAs directed to the relevant target gene sequence, wherein the expressed fusion protein is directed to the target gene by the guideRNA, and wherein the heterologous functional domain mediates the reduction in methylation.

Assignees

• Massachusetts Gen Hospital

Inventors

• Joung J Keith
• Maeder Morgan

Classifications

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N9/1007

  Methyltransferases (general) (2.1.1.) · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12Y301/21004

  Type II site-specific deoxyribonuclease (3.1.21.4) · CPC title

• C07K14/005

  from viruses · CPC title