# US 11,028,412 B2 · Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription

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- Number: US 11,028,412 B2
- Document ID: US11028412B2
- Status: Granted
- Kind: B2
- Application number: 17/084,023
- Filed: 2020-10-29
- Published: 2021-03-04
- Granted: 2021-06-08
- 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/US11028412B2

## 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 nucleic acid encoding a single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises, in 5′ to 3′ order:
(i) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of a target DNA; and (ii) an activator-RNA that 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, and
wherein the activator-RNA and the targeter-RNA are covalently linked by intervening nucleotides,
wherein the single molecule DNA-targeting RNA is capable of forming a complex with a Cas9 protein and hybridization of the targeter-RNA to the target sequence is capable of targeting the complex to the target DNA.

### Claim 2 (depends on 1)

The composition of claim 1, further comprising the Cas9 protein or a nucleic acid encoding the Cas9 protein, wherein the activator-RNA and the Cas9 protein do not naturally occur together.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the nucleic acid encoding the single molecule DNA-targeting RNA is a plasmid, a cosmid, a minicircle, a phage, or a viral vector.

### Claim 4 (depends on 2)

The composition of claim 2, wherein the activator-RNA comprises the 67 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGCUUUUUUU (SEQ ID NO: 432) or the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 441).

### Claim 5 (depends on 1)

The composition of claim 1, wherein the single molecule DNA-targeting RNA comprises a 103 nucleotide (nt) sequence that comprises, in 5′ to 3′ order:
a 20 nucleotide (nt) targeting sequence that is said nucleotide sequence that is complementary to the target sequence of the target DNA;
a 12 nt crRNA sequence GUUUUAGAGCUA (SEQ ID NO: 679),
a 4 nt linker sequence GAAA, and
a 67 nt tracrRNA sequence
(SEQ ID NO: 432)
UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCAC
CGAGUCGGUGCUUUUUUU.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the activator-RNA and the targeter-RNA are covalently linked by 3-5 intervening nucleotides.

### Claim 7 (depends on 1)

The composition of claim 1, wherein the activator-RNA and the targeter-RNA are covalently linked by 4 intervening nucleotides.

### Claim 8 (depends on 1)

The composition of claim 1, wherein the nucleotide sequence that is complementary to the target sequence in the target DNA molecule is 18 to 25 nucleotides long.

### Claim 9

A composition comprising:
a nucleic acid encoding a single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises, in 5′ to 3′ order:
(i) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of a target DNA; and (ii) an activator-RNA that 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 to 18 base pairs, and wherein the activator-RNA and the targeter-RNA are covalently linked by intervening nucleotides,
wherein the single molecule DNA-targeting RNA is capable of forming a complex with a Cas9 protein and hybridization of the targeter-RNA to the target sequence is capable of targeting the complex to the target DNA.

### Claim 10 (depends on 9)

The composition of claim 9, further comprising the Cas9 protein or a nucleic acid encoding the Cas9 protein, wherein the activator-RNA and the Cas9 protein do not naturally occur together.

### Claim 11 (depends on 10)

The composition of claim 10, wherein the nucleic acid encoding the single molecule DNA-targeting RNA is a plasmid, a cosmid, a minicircle, a phage, or a viral vector.

### Claim 12 (depends on 10)

The composition of claim 10, wherein the activator-RNA comprises the 67 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGCUUUUUUU (SEQ ID NO: 432) or the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 441).

### Claim 13 (depends on 9)

The composition of claim 9, wherein the activator-RNA and the targeter-RNA are covalently linked by 3-5 intervening nucleotides.

### Claim 14 (depends on 9)

The composition of claim 9, wherein the activator-RNA and the targeter-RNA are covalently linked by 4 intervening nucleotides.

### Claim 15 (depends on 9)

The composition of claim 9, wherein the nucleotide sequence that is complementary to the target sequence in the target DNA molecule is 15 to 25 nucleotides long.

### Claim 16

A composition comprising:
(1) Cas9 protein or a nucleic acid encoding the Cas9 protein; and
(2) a single molecule DNA-targeting RNA or a nucleic acid encoding the single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises, in 5′ to 3′ order:
(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 that 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 the activator-RNA and the targeter-RNA are covalently linked by intervening nucleotides,
wherein the single molecule DNA-targeting RNA is capable of forming a complex with a Cas9 protein and hybridization of the targeter-RNA to the target sequence is capable of targeting the complex to the target DNA.

### Claim 17 (depends on 16)

The composition of claim 16, wherein the first and second nucleotide sequences are heterologous to one another.

### Claim 18 (depends on 17)

The composition of claim 17, wherein the Cas9 protein is an S. pyogenes Cas9.

### Claim 19 (depends on 17)

The composition of claim 17, comprising the nucleic acid encoding the Cas9 protein and the nucleic acid encoding the single molecule DNA-targeting RNA.

### Claim 20 (depends on 17)

The composition of claim 17, comprising a protein-RNA complex that comprises the Cas9 protein and the single molecule DNA-targeting RNA.

### Claim 21 (depends on 17)

The composition of claim 17, wherein the activator-RNA comprises the 67 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGCUUUUUUU (SEQ ID NO: 432) or the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 441).

### Claim 22 (depends on 16)

The composition of claim 16, wherein the Cas9 protein is fused to a heterologous polypeptide.

### Claim 23 (depends on 16)

The composition of claim 16, wherein the activator-RNA and the targeter-RNA are covalently linked by 3-5 intervening nucleotides.

### Claim 24 (depends on 16)

The composition of claim 16, comprising the single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises one or more of: a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, and a modified nucleobase.

### Claim 25 (depends on 16)

The composition of claim 16, comprising the single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting 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′-dirnethylaminooxyethoxy 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 26 (depends on 16)

The composition of claim 16, wherein the single molecule DNA-targeting RNA, or the nucleic acid encoding the single molecule DNA-targeting RNA, is conjugated to one or more of: a polyamine; a polyamide; 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 27

A composition comprising:
(1) Cas9 protein or a nucleic acid encoding the Cas9 protein; and
(2) a single molecule DNA-targeting RNA or a nucleic acid encoding the single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises, in 5′ to 3′ order:
(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 that 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 to 18 base pairs,
wherein the activator-RNA and the targeter-RNA are covalently linked by intervening nucleotides, wherein the single molecule DNA-targeting RNA is capable of forming a complex with a Cas9 protein and hybridization of the targeter-RNA to the target sequence is capable of targeting the complex to the target DNA.

### Claim 28 (depends on 27)

The composition of claim 27, wherein the first and second nucleotide sequences are heterologous to one another.

### Claim 29 (depends on 27)

The composition of claim 27, wherein the Cas9 protein is fused to a heterologous polypeptide.

### Claim 30 (depends on 28)

The composition of claim 28, wherein the Cas9 protein is an S. pyogenes Cas9.