RNA-Guided Transcriptional Regulation

• US 20160237456A1
• Filed: 02/22/2016
• Published: 08/18/2016
• Est. Priority Date: 06/04/2013
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Esvelt, Kevin M., Church, George M., Mali, Prashant G.
• Granted Patent: US 10,640,789 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• 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 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/635 Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
• 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 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)

Abstract

Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

Claims

1-112. -112. (canceled)

113. A method of modulating expression of a target nucleic acid in a cell comprising providing to the cell a guide RNA complementary to the target nucleic acid sequence including a transcriptional activator or repressor domain connected thereto for modulating target nucleic acid expression in vivo,

providing to the cell a nuclease null Cas9 protein that interacts with the guide RNA and binds to the target nucleic acid sequence in a site specific manner,

wherein the guide RNA including the transcriptional activator or repressor domain connected thereto and the Cas9 protein co-localize to the target nucleic acid sequence and wherein the transcriptional activator or repressor domain modulates expression of the target nucleic acid.

122. A method of modulating expression of a target nucleic acid in a cell comprising

providing to the cell a guide RNA complementary to viral DNA including the target nucleic acid sequence, wherein the guide RNA includes a transcriptional activator or repressor domain connected thereto for modulating target nucleic acid expression in vivo,

providing to the cell a nuclease null Cas9 protein that interacts with the guide RNA and binds to the target nucleic acid sequence in a site specific manner,

wherein the guide RNA including the transcriptional activator or repressor domain connected thereto and the Cas9 protein co-localize to the target nucleic acid sequence and wherein the transcriptional activator or repressor domain modulates expression of the target nucleic acid.