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

• US 20160010076A1
• Filed: 03/14/2014
• Published: 01/14/2016
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Joung, J. Keith, Maeder, Morgan
• Granted Patent: US 10,378,027 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/01 containing a localisation/targetting motif
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2710/00033 Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
• CPC Class Codes: C12N 2770/00033 Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1007 Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/96 : Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Y 114/11 : with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
• CPC Class Codes: C12Y 201/01 : Methyltransferases (2.1.1)
• CPC Class Codes: C12Y 301/00 : Hydrolases acting on ester bonds (3.1)
• CPC Class Codes: C12Y 301/21004 : Type II site-specific deoxyribonuclease (3.1.21.4)

Abstract

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

Claims

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-κ

B 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).

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 claim 1.

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 claim 2 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 claim 4 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 claim 7 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 claim 10 in the cell, and one or more guide RNAs directed to the relevant target gene sequence.