# US 2021/0340575 A1 · Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription

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- Number: US 2021/0340575 A1
- Document ID: US20210340575A1
- Status: Published application
- Kind: A1
- Application number: 17/326,805
- Filed: 2021-05-21
- Published: 2021-11-04
- 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/US20210340575A1

## 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 at position 1

1-2. (canceled)

### Claim 3

A composition comprising: an activator-RNA, or a nucleic acid encoding the activator-RNA, wherein the activator-RNA comprises a duplex-forming segment comprising a nucleotide sequence capable of hybridizing with a targeter-RNA to form a DNA-targeting RNA that forms a complex with a Cas9 protein and guides the complex to a target sequence of a target DNA,
wherein the activator-RNA is truncated at the 5′ and/or 3′ end relative to wildtype mature tracrRNA, and
wherein the activator-RNA comprises one or more non-natural internucleoside linkages, one or more nucleic acid mimetics, one or more modified sugar moieties, and/or one or more modified nucleobases.

### Claim 4 (depends on 3)

The composition of claim 3, wherein the activator-RNA is capable of forming a total of 8-15 base pairs with the targeter-RNA when the targeter-RNA is of wildtype length.

### Claim 5 (depends on 3)

The composition of claim 3, wherein said one or more non-natural internucleoside linkages are one or more phosphorothioates, one or more inverted polarity linkages, one or more a basic nucleoside linkages, or any combination thereof;
wherein said one or more modified sugar moieties are one or more locked nucleic acid (LNA) sugar moieties, one or more 2′-substituted sugar moieties, 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, or any combination thereof;
wherein said one or more nucleic acid mimetics are one or more peptide nucleic acids (PNAs), one or more morpholino nucleic acids, one or more cyclohexenyl nucleic acids (CeNAs), or any combination thereof; and
wherein said one or more modified nucleobases is one or more 5-methylcytosines, one or more 5-hydroxymethyl cytosines, one or more xanthines, one or more hypoxanthines, one or more 2-aminoadenines, one or more 6-methyl derivatives of adenine, one or more 6-methyl derivatives of guanine, one or more 2-propyl derivatives of adenine, one or more 2-propyl derivatives of guanine, one or more 2-thiouracils, one or more 2-thiothymines, one or more 2-thiocytosines, one or more 5-propynyl uracils, one or more 5-propynyl cytosines, one or more 6-azo uracils, one or more 6-azo cytosines, one or more 6-azo thymines, one or more pseudouracils, one or more 4-thiouracils, an 8-haloadenins, one or more 8-aminoadenins, one or more 8-thioladenins, one or more 8-thioalkyladenins, one or more 8-hydroxyladenins, one or more 8-haloguanins, one or more 8-aminoguanins, one or more 8-thiolguanins, one or more 8-thioalkylguanins, one or more 8-hydroxylguanins, one or more 5-halouracils, one or more 5-bromouracils, one or more 5-trifluoromethyluracils, one or more 5-halocytosines, one or more 5-bromocytosines, one or more 5-trifluoromethylcytosines, one or more 5-substituted uracils, one or more 5-substituted cytosines, one or more 7-methylguanines, one or more 7-methyladenines, one or more 2-F-adenines, one or more 2-amino-adenines, one or more 8-azaguanines, one or more 8-azaadenines, one or more 7-deazaguanines, one or more 7-deazaadenines, one or more 3-deazaguanines, one or more 3-deazaadenines, one or more tricyclic pyrimidines, one or more phenoxazine cytidines, one or more phenothiazine cytidines, one or more substituted phenoxazine cytidines, one or more carbazole cytidines, one or more pyridoindole cytidines, one or more 7-deazaguanosines, one or more 2-aminopyridines, one or more 2-pyridones, one or more 5-substituted pyrimidines, one or more 6-azapyrimidines, one or more N-2, N-6 or 0-6 substituted purines, one or more 2-aminopropyladenines, one or more 5-propynyluracils, one or more 5-propynylcytosines, or any combination thereof.

### Claim 6 (depends on 3)

The composition of claim 3, wherein the composition is lyophilized.

### Claim 7 (depends on 3)

The composition of claim 3, wherein the nucleotide sequence of the duplex-forming segment of the activator-RNA comprises a sequence having greater than 80% identity to the tracrRNA sequence UAGCAAGUUAAAAU (SEQ ID NO: 562).

### Claim 8 (depends on 3)

The composition of claim 3, wherein said nucleotide sequence of the duplex-forming segment of the activator-RNA is a sequence having greater than 80% identity to the tracrRNA sequence UAGCAAGUUAAAAU (SEQ ID NO: 562).

### Claim 9 (depends on 3)

The composition of claim 3, wherein the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 432)
UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC
GAGUCGGUGCUUUUUUU,
(SEQ ID NO: 396)
UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC,
and
(SEQ ID NO: 397)
UAGCAAGUUAAAAUAAGGCUAGUCCG.

### Claim 10

A composition comprising: an activator-RNA, or a nucleic acid encoding the activator-RNA, wherein the activator-RNA comprises a duplex-forming segment comprising a nucleotide sequence capable of hybridizing with a targeter-RNA to form a DNA-targeting RNA that forms a complex with a Cas9 protein and guides the complex to a target sequence of a target DNA,
wherein the activator-RNA is truncated at the 5′ and/or 3′ end relative to wildtype mature tracrRNA, and
wherein the activator-RNA comprises one or more heterologous moieties.

### Claim 11 (depends on 10)

The composition of claim 10, wherein the activator-RNA is capable of forming a total of 8-15 base pairs with the targeter-RNA when the targeter-RNA is of wildtype length.

### Claim 12 (depends on 10)

The composition of claim 10, comprising a buffering agent or a nuclease inhibitor.

### Claim 13 (depends on 10)

The composition of claim 10, wherein the composition is lyophilized.

### Claim 14 (depends on 10)

The composition of claim 10, wherein said one or more heterologous moieties is one or more polyamines, one or more polyamides, one or more polyethylene glycols, one or more polyethers, one or more cholesterol moieties, one or more cholic acids, one or more thioesters, one or more thiocholesterols, one or more lipids, one or more aliphatic chains, one or more phospholipids, one or more adamantane acetic acids, one or more palmityl moieties, one or more octadecylamine or hexylamino-carbonyl-oxycholesterol moieties, one or more biotins, one or more phenazines, one or more folates, one or more phenanthridines, one or more anthraquinones, one or more acridines, one or more fluoresceins, one or more rhodamines, one or more coumarins, one or more dyes, or any combination thereof.

### Claim 15 (depends on 10)

The composition of claim 10, wherein the nucleotide sequence of the duplex-forming segment of the activator-RNA comprises a sequence having greater than 80% identity to the tracrRNA sequence UAGCAAGUUAAAAU (SEQ ID NO: 562).

### Claim 16 (depends on 10)

The composition of claim 10, wherein said nucleotide sequence of the duplex-forming segment of the activator-RNA is a sequence having greater than 80% identity to the tracrRNA sequence UAGCAAGUUAAAAU (SEQ ID NO: 562).

### Claim 17 (depends on 10)

The composition of claim 10, wherein the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 432)
UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC
GAGUCGGUGCUUUUUUU,
(SEQ ID NO: 396)
UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC,
and
(SEQ ID NO: 397)
UAGCAAGUUAAAAUAAGGCUAGUCCG.

### Claim 18

A composition comprising: an activator-RNA, or a nucleic acid encoding the activator-RNA, wherein the activator-RNA comprises a duplex-forming segment comprising a nucleotide sequence capable of hybridizing with a targeter-RNA to form a DNA-targeting RNA that forms a complex with a Cas9 protein and guides the complex to a target sequence of a target DNA,
wherein the activator-RNA is truncated at the 5′ and/or 3′ end relative to wildtype mature tracrRNA, and
wherein the composition comprises one or more of: a nuclease inhibitor, a buffering agent, a detergent, a polyamine, an adjuvant, a wetting agent, a stabilizing agent, an antioxidant, and a complexing agent.

### Claim 19 (depends on 18)

The composition of claim 18, wherein the activator-RNA is capable of forming a total of 8-15 base pairs with the targeter-RNA when the targeter-RNA is of wildtype length.

### Claim 20 (depends on 18)

The composition of claim 18, comprising the buffering agent.

### Claim 21 (depends on 18)

The composition of claim 18, comprising the nuclease inhibitor.

### Claim 22 (depends on 18)

The composition of claim 18, wherein the composition is lyophilized.

### Claim 23 (depends on 18)

The composition of claim 18, wherein the nucleotide sequence of the duplex-forming segment of the activator-RNA comprises a sequence having greater than 80% identity to the tracrRNA sequence UAGCAAGUUAAAAU (SEQ ID NO: 562).

### Claim 24 (depends on 18)

The composition of claim 18, wherein said nucleotide sequence of the duplex-forming segment of the activator-RNA is a sequence having greater than 80% identity to the tracrRNA sequence UAGCAAGUUAAAAU (SEQ ID NO: 562).

### Claim 25 (depends on 18)

The composition of claim 18, wherein the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 432)
UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC
GAGUCGGUGCUUUUUUU,
(SEQ ID NO: 396)
UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC,
and
(SEQ ID NO: 397)
UAGCAAGUUAAAAUAAGGCUAGUCCG.

### Claim 26

A composition comprising: a nucleic acid that comprises a nucleotide sequence that encodes an activator-RNA, wherein the activator-RNA comprises a duplex-forming segment comprising a nucleotide sequence capable of hybridizing with a targeter-RNA to form a DNA-targeting RNA that forms a complex with a Cas9 protein and guides the complex to a target sequence of a target DNA,
wherein the activator-RNA is truncated at the 5′ and/or 3′ end relative to wildtype mature tracrRNA, and
wherein the nucleotide sequence that encodes the activator-RNA is operably linked to a heterologous DNA regulatory sequence.

### Claim 27 (depends on 26)

The composition of claim 26, wherein the activator-RNA is capable of forming a total of 8-15 base pairs with the targeter-RNA when the targeter-RNA is of wildtype length.

### Claim 28 (depends on 26)

The composition of claim 26, wherein the DNA regulatory sequence is a promoter.

### Claim 29 (depends on 26)

The composition of claim 26, wherein said nucleic acid is a plasmid, a cosmid, a minicircle, a phage, or a viral vector.

### Claim 30 (depends on 26)

The composition of claim 26, wherein said nucleic acid is a vaccinia, polio, retro, lenti, adeno, adeno-associated, or herpes simplex viral vector.

### Claim 31 (depends on 26)

The composition of claim 26, wherein the nucleotide sequence of the duplex-forming segment of the activator-RNA comprises a sequence having greater than 80% identity to the tracrRNA sequence UAGCAAGUUAAAAU (SEQ ID NO: 562).

### Claim 32 (depends on 26)

The composition of claim 26, wherein said nucleotide sequence of the duplex-forming segment of the activator-RNA is a sequence having greater than 80% identity to the tracrRNA sequence UAGCAAGUUAAAAU (SEQ ID NO: 562).