# US 2019/0169641 A1 · Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription

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- Number: US 2019/0169641 A1
- Document ID: US20190169641A1
- Status: Published application
- Kind: A1
- Application number: 16/276,343
- Filed: 2019-02-14
- Published: 2019-06-06
- Earliest claimed: 2012-05-25
- Assignees: The Regents of the University of California; University of Vienna
- Applicants: The Regents of the University of California; University of Vienna
- Inventors: Jennifer A. Doudna; Martin Jinek; Krzysztof Chylinski; Emmanuelle Charpentier
- Web page: https://patentgrep.com/patent/US20190169641A1

## CPC

- C12N 15/907: {in mammalian cells}
- A01H 6/4684: {Zea mays [maize]}
- A01K 67/027: New or modified breeds of vertebrates
- A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases}
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/04: Antibacterial agents
- A61P 31/12: Antivirals
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- 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)}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 15/746: {for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)}
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- C12N 15/902: {using homologous recombination}
- C12Q 1/686: Polymerase chain reaction [PCR]
- H10H 20/0137: {the light-emitting regions comprising nitride materials}
- H10P 14/20: of semiconductor materials
- H10P 14/6512: {by exposure to a gas or vapour}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 2310/11: Antisense
- C12N 2310/13: Decoys
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/31: of the backbone
- C12N 2310/32: of the sugar
- C12N 2310/33: of the base
- C12N 2310/3519: Fusion with another nucleic acid
- C12N 2310/531: Stem-loop; Hairpin
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12Y 301/04: Phosphoric diester hydrolases (3.1.4)
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- H10H: INORGANIC LIGHT-EMITTING SEMICONDUCTOR DEVICES HAVING POTENTIAL BARRIERS
- H10P: GENERIC PROCESSES OR APPARATUS FOR THE MANUFACTURE OR TREATMENT OF DEVICES COVERED BY CLASS H10
- C12Y: ENZYMES
- Y02A: TECHNOLOGIES FOR ADAPTATION TO 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

### Claim 1

A single-molecule DNA-targeting RNA having the structure:
wherein the linker is GAAA and the nucleotide sequence of the single-molecule DNA-targeting RNA is GAUUUCUUCUUGCGCUUUUUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCG (SEQ ID NO: 321).

### Claim 2 (depends on 1)

A method of cleaving a target DNA, the method comprising:
contacting a target DNA with a complex comprising:
(a) the single molecule DNA-targeting RNA of claim 1; and
(b) a Cas9 protein comprising the S. pyogenes Cas9 amino acid sequence set forth as SEQ ID NO.: 2,
wherein prior to said contacting the target DNA is double stranded and comprises a nucleotide sequence taatgaattccccaatacccaaaaagcgcaagaagaaatcaaccagcgca (SEQ ID NO: 302) hybridized to a nucleotide sequence tgcgctggttgatttcttettgcgctttttgggtattggggaattcatta (SEQ ID NO: 301),
wherein said contacting is in vitro and does not take place inside of a cell, and
wherein the method results in cleavage of the target DNA.