# US 10,308,961 B2 · Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription

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- Number: US 10,308,961 B2
- Document ID: US10308961B2
- Status: Granted
- Kind: B2
- Application number: 16/033,016
- Filed: 2018-07-11
- Published: 2018-11-01
- Granted: 2019-06-04
- Earliest claimed: 2012-05-25
- Assignees: The Regents of the University of California; University of Vienna; Emmanuelle Charpentier
- Applicants: The Regents of the University of California; University of Vienna; Emmanuelle Charpentier
- Inventors: Jennifer A. Doudna; Martin Jinek; Krzysztof Chylinski; Emmanuelle Charpentier
- Web page: https://patentgrep.com/patent/US10308961B2

## 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 method of targeting and binding a target DNA, modifying a target DNA, or modulating transcription from a target DNA, the method comprising:
contacting a target DNA inside of a cell with a complex that comprises:
(a) a Cas9 protein; and
(b) a nonnaturally occurring DNA-targeting RNA comprising:
(i) a targeter-RNA comprising: a first nucleotide sequence that hybridizes with a target sequence of the target DNA, and a second nucleotide sequence that hybridizes with an activator-RNA, wherein the first and second nucleotide sequences are heterologous to one another; and
(ii) the activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs,
wherein: the complex binds to the target DNA, the target DNA is modified, the target DNA is cleaved, the target DNA is edited, and/or transcription from the target DNA is modulated.

### Claim 2 (depends on 1)

The method of claim 1, wherein the target sequence of the target DNA is 15 nucleotides (nt) to 18 nt long.

### Claim 3 (depends on 1)

The method of claim 1, wherein the target sequence of the target DNA is 18 nucleotides (nt) to 25 nt long.

### Claim 4 (depends on 1)

The method of claim 1, wherein said contacting comprises inducing expression of and/or (b).

### Claim 5 (depends on 1)

The method of claim 1, wherein said contacting comprises introducing into the cell one or more of:
(1) the Cas9 protein, or a nucleic acid encoding the Cas9 protein;
(2) the targeter-RNA, or a nucleic acid encoding the targeter-RNA; and
(3) the activator-RNA, or a nucleic acid encoding the activator-RNA.

### Claim 6 (depends on 5)

The method of claim 5, wherein one or more of:
(A) the nucleic acid encoding the Cas9 protein;
(B) the nucleic acid encoding the targeter-RNA; and
(C) the nucleic acid encoding the activator-RNA,
is a plasmid, a cosmid, a minicircle, a phage, or a viral vector.

### Claim 7 (depends on 5)

The method of claim 5, comprising introducing into the cell (1), (2), and (3).

### Claim 8 (depends on 5)

The method of claim 5, wherein the nucleic acid encoding the Cas9 protein comprises a nucleotide sequence modification that replaces one or more codons of a wild-type Cas9-encoding nucleotide sequence with one or more different codons encoding the same amino acid.

### Claim 9 (depends on 5)

The method of claim 5, wherein said introducing comprises: introducing the Cas9 protein into the cell as protein, and introducing the targeter-RNA and the activator-RNA into the cell as RNA.

### Claim 10 (depends on 9)

The method of claim 9, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.

### Claim 11 (depends on 5)

The method of claim 5, wherein said introducing comprises: introducing into the cell the nucleic acid encoding the Cas9 protein, wherein the nucleic acid encoding the Cas9 protein is RNA.

### Claim 12 (depends on 5)

The method of claim 5, wherein said introducing comprises: introducing into the cell, the nucleic acid encoding the Cas9 protein, the nucleic acid encoding the targeter-RNA, and the nucleic acid encoding the activator-RNA.

### Claim 13 (depends on 12)

The method of claim 12, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.

### Claim 14 (depends on 1)

The method of claim 1, wherein the method comprises introducing a donor polynucleotide into the cell.

### Claim 15 (depends on 1)

The method of claim 1, wherein the method comprises introducing into the cell a DNA molecule that encodes both the non-naturally occurring DNA-targeting RNA and the Cas9 protein.

### Claim 16 (depends on 1)

The method of claim 1, wherein the targeter-RNA and the activator-RNA are covalently linked via the 3′ end of the targeter-RNA and the 5′ end of the activator-RNA.

### Claim 17 (depends on 1)

The method of claim 1, wherein the method comprises introducing two or more of the non-naturally occurring DNA-targeting RNAs into the cell, wherein the two or more non-naturally occurring DNA-targeting RNAs are complementary to different target sequences within the same or different target DNAs.

### Claim 18 (depends on 1)

The method of claim 1, wherein the Cas9 protein comprises a mutation in a RuvC domain and/or an HNH domain.

### Claim 19 (depends on 1)

The method of claim 1, wherein the Cas9 protein is fused to a heterologous polypeptide.

### Claim 20 (depends on 1)

The method of claim 1, wherein the targeter-RNA and the activator-RNA are not covalently linked by intervening nucleotides.

### Claim 21 (depends on 1)

The method of claim 1, wherein the targeter-RNA and/or the activator-RNA comprises one or more of: a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, and a modified nucleobase.

### Claim 22 (depends on 1)

The method of claim 1, wherein the targeter-RNA and/or the activator-RNA comprises one or more of: a phosphorothioate, an inverted polarity linkage, an abasic nucleoside linkage, a locked nucleic acid (LNA), a 2′-O-methoxyethyl modified sugar moiety, a 2′-O-methyl modified sugar moiety, a 2′-O-(2-methoxyethyl) modified sugar moiety, a 2′-fluoro modified sugar moiety, a 2′-dimethylaminooxyethoxy modified sugar moiety, a 2′-dimethylaminoethoxyethoxy modified sugar moiety, a peptide nucleic acid (PNA), a morpholino nucleic acid, and a cyclohexenyl nucleic acid (CeNA).

### Claim 23 (depends on 1)

The method of claim 1, wherein the targeter-RNA and the activator-RNA are conjugated to a heterologous moiety.

### Claim 24 (depends on 1)

The method of claim 1, wherein the targeter-RNA and/or the activator-RNA is conjugated to one or more of: a polyamine; a polyimide; a polyethylene glycol; a polyether; a cholesterol moiety; a cholic acid; a thioether; a thiocholesterol; an aliphatic chain; a phospholipid; an adamantane acetic acid; a palmityl moiety; an octadecylamine moiety; a hexylamino-carbonyl-oxycholesterol moiety; a biotin; a phenazine; a folate; a phenanthridine; an anthraquinone; an acridine; a fluorescein; a rhodamine; a dye; and a coumarin.

### Claim 25 (depends on 1)

The method of claim 1, wherein the target DNA is chromosomal DNA.

### Claim 26 (depends on 1)

The method of claim 1, wherein the activator-RNA comprises the 67 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG CUUUUUUU (SEQ ID NO: 432).

### Claim 27 (depends on 1)

The method of claim 1, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 441).

### Claim 28 (depends on 1)

The method of claim 1, wherein said contacting results in cleavage of the target DNA.

### Claim 29 (depends on 1)

The method of claim 1, wherein the Cas9 protein is capable of cleaving only one strand of DNA and comprises one or more mutations in a RuvC domain or an HNH domain.

### Claim 30 (depends on 1)

The method of claim 1, wherein the cell is a bacterial cell.