RNA-Guided Targeting of Genetic and Epigenomic Regulatory Proteins to Specific Genomic Loci

Patent metadata

Field	Value
Publication number	US-2019376090-A1
Application number	US-201916535199-A
Country	US
Kind code	A1
Filing date	Aug 8, 2019
Priority date	Mar 15, 2013
Publication date	Dec 12, 2019
Grant date	—

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 catalytically inactive CRISPR associated 9 (dCas9) protein linked to a heterologous functional domain.

2 . The fusion protein of claim 1 , wherein the heterologous functional domain is a transcriptional activation domain.

3 . The fusion protein of claim 2 , wherein the transcriptional activation domain is from VP64 or NF-κB p65.

4 . The fusion protein of claim 1 , wherein the heterologous functional domain is a transcriptional silencer or transcriptional repression domain.

5 . The fusion protein of claim 4 , wherein the transcriptional repression domain is a Krueppel-associated box (KRAB) domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID).

6 . The fusion protein of claim 4 , wherein the transcriptional silencer is Heterochromatin Protein 1 (HP1), e.g., HP1α or HP1β.

7 . The fusion protein of claim 1 , wherein the heterologous functional domain is an enzyme that modifies the methylation state of DNA.

8 . The fusion protein of claim 7 , wherein the enzyme that modifies the methylation state of DNA is a DNA methyltransferase (DNMT) or a TET protein.

9 . The fusion protein of claim 8 , wherein the TET protein is TET1.

10 . The fusion protein of claim 1 , wherein the heterologous functional domain is an enzyme that modifies a histone subunit.

11 . The fusion protein of claim 1 , wherein the enzyme that modifies a histone subunit is a histone acetyltransferase (HAT), histone deacetylase (HDAC), histone methyltransferase (HMT), or histone demethylase.

12 . The fusion protein of claim 1 , wherein the heterologous functional domain is a biological tether.

13 . The fusion protein of claim 12 , wherein the biological tether is MS2, Csy4 or lambda N protein.

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

15 . The fusion protein of claim 1 , wherein the catalytically inactive Cas9 protein comprises mutations at D10, E762, H983, or D986; and at H840 or N863.

16 . The fusion protein of claim 15 , wherein the mutations are:
(i) D10A or D10N, and (ii) H840A, H840N, or H840Y.

17 . 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.

18 . 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.

19 . The fusion protein of claim 18 , wherein the epitope tag is c-myc, 6His, or FLAG.

20 . A nucleic acid encoding the fusion protein of any of claims 1 - 19 .

21 . An expression vector comprising the nucleic acid of claim 20 .

22 . A method of increasing expression of a target gene in a cell, the method comprising expressing the fusion protein of claims 2 - 3 in the cell, and one or more guide RNAs directed to the target gene.

23 . A method of decreasing of a target gene in a cell, the method comprising expressing the fusion protein of claims 4 - 6 in the cell, and one or more guide RNAs directed to the target gene.

24 . 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 claims 7 - 9 in the cell, and one or more guide RNAs directed to the relevant target gene sequence.

25 . A method of modifying histones associated with a target gene or its promoter or enhancer(s) in a cell, the method comprising expressing the fusion protein of claims 10 - 11 in the cell, and one or more guide RNAs directed to the relevant target gene sequence.

Assignees

• Massachusetts Gen Hospital

Inventors

• Joung J Keith
• Maeder Morgan

Classifications

• C07K2319/01

  containing a localisation/targetting motif · CPC title

• C07K14/195

  from bacteria · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• C12N9/1007

  Methyltransferases (general) (2.1.1.) · CPC title