Cas9-based transcription modulation systems

Patent metadata

Field	Value
Publication number	US-11629342-B2
Application number	US-201816756995-A
Country	US
Kind code	B2
Filing date	Oct 17, 2018
Priority date	Oct 17, 2017
Publication date	Apr 18, 2023
Grant date	Apr 18, 2023

Abstract

The present disclosure provides methods and compositions of modulating expression of a target nucleic acid in a eukaryotic cell. The methods include providing to the cell a guide RNA complementary to the target nucleic acid sequence, providing to the cell a fusion protein, wherein the fusion protein comprises a nuclease null Cas9 protein and a transcriptional effector domain, wherein the nuclease null Cas9 protein interacts with the guide RNA and binds to the target nucleic acid sequence in a site specific manner and wherein the transcriptional effector domain modulates expression of the target nucleic acid.

First claim

What is claimed is:

1. A method of modulating expression of a target nucleic acid in a eukaryotic cell comprising providing to the cell a guide RNA complementary to the target nucleic acid sequence, providing to the cell a fusion protein, wherein the fusion protein comprises a nuclease null Cas9 protein (dCas9) and an effector domain, wherein the effector domain comprises a fusion of two or more transcriptional repressor domains selected from the group consisting of Kruppel-associated box (KRAB), MeCP2, SIN3A, HDT1, MBD2B, NIPP1, and HP1A, and wherein the nuclease null Cas9 protein interacts with the guide RNA and binds to the target nucleic acid sequence in a site specific manner and wherein the effector domain modulates expression of the target nucleic acid.

2. The method of claim 1
wherein the guide RNA is provided to the cell by introducing to the cell a nucleic acid encoding the guide RNA,
wherein the fusion protein is provided to the cell by introducing to the cell a nucleic acid encoding the fusion protein, and
wherein the cell expresses the guide RNA and the fusion protein.

3. The method of claim 1 wherein the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell.

4. The method or claim 3 wherein the eukaryotic cell is a human cell.

5. The method of claim 1 wherein the effector domain comprises a fusion of two transcriptional repressor domains selected from the group consisting of Kruppel-associated box (KRAB), MeCP2, SIN3A, HDT1, MBD2B, NIPP1, and HP1A.

6. The method of claim 1 wherein the effector domain comprises a fusion of three or more transcriptional repressor domains selected from the group consisting of Krüppel-associated box (KRAB), MeCP2, SIN3A, HDT1, MBD2B, NIPP1, and HP1A.

7. The method of claim 1 wherein the effector domain comprises a bipartite fusion of KRAB-MeCP2.

8. The method of claim 1 wherein the fusion protein comprises a fusion of dCas9-KRAB-MeCP2.

9. The method of claim 8 wherein the dCas9-KRAB-MeCP2 fusion exhibited at least two fold stronger repression of target gene expression compared to nuclease null Cas9 or nuclease null Cas9 fused to KRAB.

Assignees

• Harvard College

Inventors

• Chavez Alejandro
• Yeo Nan Cher

Classifications

• C07K14/4703

  Inhibitors; Suppressors · CPC title

• C12N2740/15043

  viral genome or elements thereof as genetic vector · CPC title

• C12N15/11

  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 title

• C12N15/8509

  for producing genetically modified animals, e.g. transgenic · CPC title

• C07K2319/70

  containing domain for protein-protein interaction · CPC title