# US 12,065,668 B2 · RNA-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci

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- Number: US 12,065,668 B2
- Document ID: US12065668B2
- Status: Granted
- Kind: B2
- Application number: 17/450,135
- Filed: 2021-10-06
- Published: 2022-03-24
- Granted: 2024-08-20
- 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/US12065668B2

## 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 method of silencing or repressing expression of a target gene in a cell, the method comprising expressing
(i) a fusion protein comprising catalytically inactive CRISPR associated 9 (dCas9) protein linked to a heterologous functional domain, wherein the heterologous functional domain is a Heterochromatin Protein 1 (HP1), and
(ii) one or more guide RNAs directed to the target gene.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 2)

The method of claim 2, wherein the catalytically inactive Cas9 protein comprises mutations at D10, E762, H983, or D986; and at H840 or N863.

### Claim 4 (depends on 3)

The method of claim 3, wherein the mutations are:
(i) D10A or D10N, and
(ii) H840A, H840N, or H840Y.

### Claim 5 (depends on 1)

The method 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 6 (depends on 1)

The method 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 7 (depends on 6)

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