Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription

• US 10,385,360 B2
• Filed: 03/19/2018
• Issued: 08/20/2019
• Est. Priority Date: 05/25/2012
• Status: Active Grant

• Current Assignee: The University Of Vienna, Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Doudna, Jennifer A., Jinek, Martin, Chylinski, Krzysztof, Charpentier, Emmanuelle
• Primary Examiner(s): Hammell, Neil P
• Application Number:
  US15/925,544
• Publication Number:
  US 20180208931A1
• Time in Patent Office: 519 Days
• Field of Search: None
• CPC Class Codes: A01H 6/4684 Zea mays [maize]
• CPC Class Codes: A01K 67/027 New or modified breeds of vertebrates
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/746 for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/13 Decoys
• CPC Class Codes: C12N 2310/14 : interfering N.A.
• CPC Class Codes: C12N 2310/20 : involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2310/31 : of the backbone
• CPC Class Codes: C12N 2310/32 : of the sugar
• CPC Class Codes: C12N 2310/33 : of the base
• CPC Class Codes: C12N 2310/3519 : Fusion with another nucleic acid
• CPC Class Codes: C12N 2310/531 : Stem-loop; Hairpin
• CPC Class Codes: C12N 2800/80 : Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C12Y 301/04 : Phosphoric diester hydrolases (3.1.4)
• CPC Class Codes: H01L 21/02312 : treatment by exposure to a gas or vapour
• CPC Class Codes: H01L 21/02365 : Forming inorganic semiconducting materials on a substrate for light-sensitive devices H01L31/00
• CPC Class Codes: H01L 33/0075 : comprising nitride compounds
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present disclosure provides a DNA-targeting RNA that comprises a targeting sequence and, together with a modifying polypeptide, provides for site-specific modification of a target DNA and/or a polypeptide associated with the target DNA. The present disclosure further provides site-specific modifying polypeptides. The present disclosure further provides methods of site-specific modification of a target DNA and/or a polypeptide associated with the target DNA The present disclosure provides methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA. Kits and compositions for carrying out the methods are also provided. The present disclosure provides genetically modified cells that produce Cas9; and Cas9 transgenic non-human multicellular organisms.

Claims

1. A nucleic acid molecule encoding a single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises:

(a) a DNA-targeting segment comprising a nucleotide sequence that is complementary to a target sequence in a target DNA molecule, and

(b) a protein-binding segment comprising two complementary stretches of nucleotides that hybridize to form a double stranded RNA (dsRNA) duplex, wherein said two complementary stretches of nucleotides are covalently linked to one another with intervening nucleotides,

wherein the DNA-targeting segment is positioned 5′

of both of said two complementary stretches of nucleotides, and wherein the single molecule DNA-targeting RNA is capable of forming a complex with a Cas9 protein and targeting the complex to the target sequence of the target DNA molecule.

2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is a recombinant expression vector.

3. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is a vector selected from the group consisting of plasmids, cosmids, minicircles, phage, and viral vectors.

4. A composition comprising:

(1) a single molecule DNA-targeting RNA, or a nucleic acid encoding said single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises;

(a) a DNA-targeting segment comprising a nucleotide sequence that is complementary to a target sequence in a target DNA molecule, and

(b) a protein-binding segment comprising two complementary stretches of nucleotides that hybridize to form a double stranded RNA (dsRNA) duplex, wherein said two complementary stretches of nucleotides are covalently linked to one another with intervening nucleotides,

wherein the DNA-targeting segment is positioned 5′

of both of said two complementary stretches of nucleotides, and wherein the single molecule DNA-targeting RNA is capable of forming a complex with a Cas9 protein and targeting the complex to the target sequence of the target DNA molecule; and

(2) one or more of;

(i) a nuclease inhibitor, (ii) a buffering agent, and (iii) a pharmaceutically-acceptable, non-toxic carrier or diluent.

5. The composition of claim 4, comprising the buffering agent.

6. The composition of claim 5, wherein the buffering agent is a buffer for stabilizing nucleic acids.

7. The composition of claim 4, further comprising one or more of:

a detergent, a carbohydrate, a polyamine, an amino acid, a peptide, sodium, potassium, calcium, magnesium, manganese, and a lipid.

8. The composition of claim 4, wherein the composition is sterile.