# US 2019/0376090 A1 · RNA-Guided Targeting of Genetic and Epigenomic Regulatory Proteins to Specific Genomic Loci

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- Number: US 2019/0376090 A1
- Document ID: US20190376090A1
- Status: Published application
- Kind: A1
- Application number: 16/535,199
- Filed: 2019-08-08
- Published: 2019-12-12
- Earliest claimed: 2013-03-15
- Assignees: The General Hospital Corporation
- Applicants: The General Hospital Corporation
- Inventors: J. Keith Joung; Morgan Maeder
- Web page: https://patentgrep.com/patent/US20190376090A1

## CPC

- C12N 15/907: {in mammalian cells}
- C07K 14/005: from viruses
- C07K 14/195: from bacteria
- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12N 9/1007: {Methyltransferases (general) (2.1.1.)}
- C12N 9/16: acting on ester bonds (3.1)
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/224: {Class 1 CAS enzyme complex, e.g. multi-enzyme}
- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/1031: {mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR}
- 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}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/85: for animal cells
- C12Y 301/21004: Type II site-specific deoxyribonuclease (3.1.21.4)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/01: containing a localisation/targetting motif
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2710/00033: Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
- C12N 2770/00033: Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12Y 114/11: with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
- C12Y 201/01: Methyltransferases (2.1.1)
- C12Y 301/00: Hydrolases acting on ester bonds (3.1)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES

## Abstract

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

## Claims

### Claim 1

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

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 2)

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

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 4)

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).

### Claim 6 (depends on 4)

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

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 7)

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.

### Claim 9 (depends on 8)

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

### Claim 10 (depends on 1)

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

### Claim 11 (depends on 1)

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.

### Claim 12 (depends on 1)

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

### Claim 13 (depends on 12)

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

### Claim 14 (depends on 1)

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

### Claim 15 (depends on 1)

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.

### Claim 16 (depends on 15)

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

### Claim 17 (depends on 1)

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.

### Claim 18 (depends on 1)

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.

### Claim 19 (depends on 18)

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

### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)

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

### Claim 21 (depends on 20)

An expression vector comprising the nucleic acid of claim 20.

### Claim 22 (depends on 2, 3)

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.

### Claim 23 (depends on 4, 5, 6)

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.

### Claim 24 (depends on 7, 8, 9)

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.

### Claim 25 (depends on 10, 11)

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.