# US 12,180,504 B2 · Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription

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- Number: US 12,180,504 B2
- Document ID: US12180504B2
- Status: Granted
- Kind: B2
- Application number: 17/348,619
- Filed: 2021-06-15
- Published: 2021-12-16
- Granted: 2024-12-31
- 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/US12180504B2

## 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 composition comprising
(a) a Cas9 protein or a nucleic acid encoding the Cas9 protein, wherein the Cas9 protein is a Streptococcus pyogenes Cas9; and
(b) a DNA-targeting RNA comprising:
(i) a targeter-RNA comprising: (1) a first nucleotide sequence that is complementary to a target sequence of a target DNA, and (2) a second nucleotide sequence that hybridizes with an activator-RNA; and
(ii) the activator-RNA, which hybridizes with the targeter-RNA to form a double-stranded RNA duplex, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs,
wherein (i) and (ii) are covalently linked,
wherein the DNA-targeting RNA is capable of forming a complex with the Cas9 protein and guiding the complex to the target sequence to cleave the target DNA.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the first nucleotide sequence is 18 to 25 nucleotides long.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the first and second nucleotide sequences are heterologous to one another.

### Claim 4 (depends on 1)

The composition of claim 1, further comprising a nuclease inhibitor and/or a buffering agent.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the composition is sterile.

### Claim 6 (depends on 1)

The composition of claim 1, further comprising a donor polynucleotide.

### Claim 7 (depends on 1)

The composition of claim 1, further comprising a reducing agent.

### Claim 8 (depends on 1)

The composition of claim 1, wherein the nucleic acid encoding the Cas9 protein is a recombinant expression vector.

### Claim 9 (depends on 1)

The composition of claim 1, wherein the Cas9 protein comprises one or more mutations in a RuvC and/or an HNH domain.

### Claim 10 (depends on 1)

The composition of claim 1, wherein the Cas9 protein comprises a heterologous polypeptide.

### Claim 11 (depends on 10)

The composition of claim 10, wherein the heterologous polypeptide comprises a 6×His tag, a hemagglutinin (HA) tag, or green fluorescent protein.

### Claim 12 (depends on 1)

The composition of claim 1, wherein the Cas9 protein is fused to a protein transduction domain (PTD).

### Claim 13 (depends on 12)

The composition of claim 12, wherein the PTD is covalently linked to the carboxyl terminus (C-terminus) or the amino terminus (N-terminus) of the Cas9 protein.

### Claim 14 (depends on 1)

The composition of claim 1, wherein the composition comprises a protein-RNA complex comprising the Cas9 protein and the DNA-targeting RNA.

### Claim 15 (depends on 1)

The composition of claim 1, comprising the DNA-targeting RNA and the nucleic acid encoding the Cas9 protein.

### Claim 16 (depends on 15)

The composition of claim 15, wherein the nucleic acid encoding the Cas9 protein is an RNA.

### Claim 17 (depends on 1)

The composition of claim 1, wherein:
(1) the activator-RNA comprises the 67 nt tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 432); or
(2) the activator RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 441); or
(3) the targeter-RNA comprises the 12 nucleotide (nt) crRNA sequence GUUUUAGAGCUA (SEQ ID NO: 679) and the activator-RNA comprises the 67 nt tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 432).

### Claim 18 (depends on 1)

The composition of claim 1, wherein the DNA-targeting RNA comprises one or more non-natural internucleoside linkages, one or more nucleic acid mimetics, one or more modified sugar moieties, one or more modified nucleobases, or any combination thereof.

### Claim 19 (depends on 18)

The composition of claim 18, wherein the DNA-targeting RNA comprises said one or more non-natural internucleoside linkages and/or said one or more modified sugar moieties.

### Claim 20 (depends on 1)

The composition of claim 1, wherein the DNA-targeting RNA comprises one or more phosphorothioates, one or more inverted polarity linkages, one or more abasic nucleoside linkages, one or more locked nucleic acids (LNAs), one or more 2′-O-methoxyethyl modified sugar moieties, one or more 2′-O-methyl modified sugar moieties, one or more 2′-O-(2-methoxyethyl) modified sugar moieties, one or more 2′-fluoro modified sugar moieties, one or more 2′-dimethylaminooxyethoxy modified sugar moieties, one or more 2′-dimethylaminoethoxyethoxy modified sugar moieties, one or more peptide nucleic acids (PNAs), one or more morpholino nucleic acids, one or more cyclohexenyl nucleic acids (CeNAs), or any combination thereof.

### Claim 21 (depends on 20)

The composition of claim 20, wherein the DNA-targeting RNA comprises said one or more 2′-O-methyl modified sugar moieties and/or said one or more 2′-fluoro modified sugar moieties.

### Claim 22 (depends on 1)

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

### Claim 23

A composition comprising
(a) a Cas9 protein or a nucleic acid encoding the Cas9 protein, wherein the Cas9 protein is a Streptococcus pyogenes Cas9; and
(b) a DNA-targeting RNA comprising:
(i) a targeter-RNA comprising: (1) a first nucleotide sequence that is complementary to a target sequence of a target DNA, and (2) a second nucleotide sequence that hybridizes with an activator-RNA; and
(ii) the activator-RNA, which hybridizes with the targeter-RNA to form a double-stranded RNA duplex, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 15-18 base pairs,
wherein (i) and (ii) are covalently linked,
wherein the DNA-targeting RNA is capable of forming a complex with the Cas9 protein and guiding the complex to the target sequence to cleave the target DNA.

### Claim 24 (depends on 23)

The composition of claim 23, wherein the first nucleotide sequence is 18 to 25 nucleotides long.

### Claim 25 (depends on 23)

The composition of claim 23, wherein the first and second nucleotide sequences are heterologous to one another.

### Claim 26 (depends on 23)

The composition of claim 23, further comprising a nuclease inhibitor and/or a buffering agent.

### Claim 27 (depends on 23)

The composition of claim 23, wherein the composition is sterile.

### Claim 28 (depends on 23)

The composition of claim 23, further comprising a donor polynucleotide.

### Claim 29 (depends on 23)

The composition of claim 23, further comprising a reducing agent.

### Claim 30 (depends on 23)

The composition of claim 23, wherein the nucleic acid encoding the Cas9 protein is a recombinant expression vector.

### Claim 31 (depends on 23)

The composition of claim 23, wherein the Cas9 protein comprises one or more mutations in a RuvC and/or an HNH domain.

### Claim 32 (depends on 23)

The composition of claim 23, wherein the Cas9 protein comprises a heterologous polypeptide.

### Claim 33 (depends on 32)

The composition of claim 32, wherein the heterologous polypeptide comprises a 6×His tag, a hemagglutinin (HA) tag, or green fluorescent protein.

### Claim 34 (depends on 23)

The composition of claim 23, wherein the Cas9 protein is fused to a protein transduction domain (PTD).

### Claim 35 (depends on 34)

The composition of claim 34, wherein the PTD is covalently linked to the carboxyl terminus (C-terminus) or the amino terminus (N-terminus) of the Cas9 protein.

### Claim 36 (depends on 23)

The composition of claim 23, wherein the composition comprises a protein-RNA complex comprising the Cas9 protein and the DNA-targeting RNA.

### Claim 37 (depends on 23)

The composition of claim 23, comprising the DNA-targeting RNA and the nucleic acid encoding the Cas9 protein.

### Claim 38 (depends on 37)

The composition of claim 37, wherein the nucleic acid encoding the Cas9 protein is an RNA.

### Claim 39 (depends on 23)

The composition of claim 23, wherein:
(1) the activator-RNA comprises the 67 nt tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 432); or
(2) the activator RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 441); or
(3) the targeter-RNA comprises the 12 nucleotide (nt) crRNA sequence GUUUUAGAGCUA (SEQ ID NO: 679) and the activator-RNA comprises the 67 nt tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 432).

### Claim 40 (depends on 23)

The composition of claim 23, wherein the DNA-targeting RNA comprises one or more non-natural internucleoside linkages, one or more nucleic acid mimetics, one or more modified sugar moieties, one or more modified nucleobases, or any combination thereof.

### Claim 41 (depends on 40)

The composition of claim 40, wherein the DNA-targeting RNA comprises said one or more non-natural internucleoside linkages and/or said one or more modified sugar moieties.

### Claim 42 (depends on 23)

The composition of claim 23, wherein the DNA-targeting RNA comprises one or more phosphorothioates, one or more inverted polarity linkages, one or more abasic nucleoside linkages, one or more locked nucleic acids (LNAs), one or more 2′-O-methoxyethyl modified sugar moieties, one or more 2′-O-methyl modified sugar moieties, one or more 2′-O-(2-methoxyethyl) modified sugar moieties, one or more 2′-fluoro modified sugar moieties, one or more 2′-dimethylaminooxyethoxy modified sugar moieties, one or more 2′-dimethylaminoethoxyethoxy modified sugar moieties, one or more peptide nucleic acids (PNAs), one or more morpholino nucleic acids, one or more cyclohexenyl nucleic acids (CeNAs), or any combination thereof.

### Claim 43 (depends on 42)

The composition of claim 42, wherein the DNA-targeting RNA comprises said one or more 2′-O-methyl modified sugar moieties and/or said one or more 2′-fluoro modified sugar moieties.

### Claim 44 (depends on 23)

The composition of claim 23, wherein the activator-RNA and/or the targeter-RNA are conjugated to a heterologous moiety.