# US 2019/0169648 A1 · Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription

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- Number: US 2019/0169648 A1
- Document ID: US20190169648A1
- Status: Published application
- Kind: A1
- Application number: 16/276,374
- 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/US20190169648A1

## 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 eukaryotic cell comprising a target DNA molecule and an engineered and/or non-naturally occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising
a nucleic acid comprising a nucleotide sequence encoding a Cas9 protein, and
a DNA-targeting RNA or one or more nucleic acids comprising one or more nucleotide sequences encoding the DNA-targeting RNA;
wherein the DNA-targeting RNA comprises
i) a targeter-RNA that is capable of hybridizing with a target sequence in a target DNA molecule, and
ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment,
wherein (I) the DNA-targeting RNA comprises a nucleotide sequence that is modified relative to naturally occurring DNA-targeting RNAs, and/or (11) the Cas9 protein comprises an amino acid sequence that is modified relative to naturally occurring Cas9 proteins,
whereby hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA molecule.

### Claim 4 (depends on 3)

The eukaryotic cell of claim 3, wherein the genome of said eukaryotic cell comprises the target DNA molecule.

### Claim 5 (depends on 3)

The eukaryotic cell of claim 3, wherein the eukaryotic cell is a mammalian cell or a human cell.

### Claim 6 (depends on 3)

The eukaryotic cell of claim 3, wherein the nucleic acid comprising the nucleotide sequence encoding the Cas9 protein is an RNA molecule.

### Claim 7 (depends on 3)

The eukaryotic cell of claim 3, comprising the DNA-targeting RNA as RNA.

### Claim 8 (depends on 3)

The eukaryotic cell of claim 3, wherein the nucleic acid comprising the nucleotide sequence encoding the Cas9 protein is an RNA molecule, and the eukaryotic cell comprises the DNA-targeting RNA as RNA.

### Claim 9 (depends on 8)

The eukaryotic cell of claim 8, wherein the eukaryotic cell is in vivo.

### Claim 10 (depends on 8)

The eukaryotic cell of claim 8, wherein the eukaryotic cell is a mammalian cell.

### Claim 11 (depends on 3)

The eukaryotic cell of claim 3, wherein the nucleic acid comprising the nucleotide sequence encoding the DNA-targeting RNA is a plasmid, and the eukaryotic cell comprises said plasmid.

### Claim 12 (depends on 3)

The eukaryotic cell of claim 3, wherein the nucleic acid comprising the nucleotide sequence encoding the Cas9 protein is a plasmid, and the eukaryotic cell comprises said plasmid.

### Claim 13 (depends on 3)

The eukaryotic cell of claim 3, comprising:
the nucleic acid comprising the nucleotide sequence encoding the DNA-targeting RNA, and
the nucleic acid comprising the nucleotide sequence encoding the Cas9 protein,
wherein both of said nucleic acids are plasmids.

### Claim 14 (depends on 13)

The eukaryotic cell of claim 13, wherein the eukaryotic cell is a mammalian cell.

### Claim 15 (depends on 14)

The eukaryotic cell of claim 14, wherein the Cas9 protein comprises the amino acid sequence set forth in SEQ ID NO: 2.

### Claim 16 (depends on 13)

The eukaryotic cell of claim 13, wherein the eukaryotic cell is a single-cell organism.

### Claim 17 (depends on 3)

The eukaryotic cell of claim 3, wherein the nucleic acid comprising the nucleotide sequence encoding the DNA-targeting RNA is a viral vector, and the eukaryotic cell comprises said viral vector.

### Claim 18 (depends on 3)

The eukaryotic cell of claim 3, wherein the nucleic acid comprising the nucleotide sequence encoding the Cas9 protein is a viral vector, and the eukaryotic cell comprises said viral vector.

### Claim 19 (depends on 3)

The eukaryotic cell of claim 3, comprising one or more viral vectors, wherein said one or more viral vectors comprises:
the nucleic acid comprising the nucleotide sequence encoding the DNA-targeting RNA, and
the nucleic acid comprising the nucleotide sequence encoding the Cas9 protein.

### Claim 20 (depends on 19)

The eukaryotic cell of claim 19, wherein the eukaryotic cell is a mammalian cell.

### Claim 21 (depends on 19)

The eukaryotic cell of claim 19, wherein the eukaryotic cell is a single-cell organism.

### Claim 22 (depends on 3)

The eukaryotic cell of claim 3, wherein the DNA-targeting RNA is a double-molecule DNA-targeting RNA such that said targeter-RNA and said activator-RNA are present on different RNA molecules.

### Claim 23 (depends on 3)

The eukaryotic cell of claim 3, wherein one or more of a) the nucleic acid comprising the nucleotide sequence encoding said Cas9 protein and b) the one or more nucleic acids comprising one or more nucleotide sequences encoding said DNA-targeting RNA, are one or more vectors, wherein the one or more vectors are selected from the group consisting of plasmids, cosmids, minicircles, phage, viral, retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors; and wherein the nucleotide sequence encoding said Cas9 protein and/or the one or more nucleotide sequences encoding said DNA-targeting RNA is operably linked to a control element operable in said eukaryotic cell.

### Claim 24 (depends on 3)

The eukaryotic cell of claim 3, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)) that comprise an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269.

### Claim 25 (depends on 3)

The eukaryotic cell of claim 3, wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.

### Claim 26 (depends on 3)

The eukaryotic cell of claim 3, wherein the system comprises a donor polynucleotide and the system is capable of editing the target DNA molecule by inserting a sequence of the donor polynucleotide into a cleaved strand of the target DNA molecule.

### Claim 27 (depends on 3)

The eukaryotic cell of claim 3, wherein the nucleotide sequence encoding said 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 28 (depends on 3)

The eukaryotic cell of claim 3, wherein the activator-RNA comprises the 88 nucleotide tracrRNA sequence set forth in SEQ ID NO:433.

### Claim 29 (depends on 3)

The eukaryotic cell of claim 3, wherein said Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain and is capable of cleaving only one strand of DNA.

### Claim 30

A eukaryotic cell comprising a target DNA and an engineered and/or non-naturally occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising
a) a nucleic acid comprising a nucleotide sequence encoding a Cas9 protein; and
b) a DNA-targeting RNA, or one or more nucleic acids comprising one or more nucleotide sequences encoding said DNA-targeting RNA;
wherein the DNA-targeting RNA comprises:
i) a targeter-RNA that is capable of hybridizing with a target sequence in the target DNA molecule, and
ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment;
wherein (I) the DNA-targeting RNA comprises a nucleotide sequence that is modified relative to naturally occurring DNA-targeting RNAs, and/or (II) the Cas9 protein comprises an amino acid sequence that is modified relative to naturally occurring Cas9 proteins,
wherein the DNA-targeting RNA is capable of forming a complex with the Cas9 protein, thereby targeting the Cas9 protein to the target DNA molecule,
whereby said system is capable of cleaving or editing the target DNA molecule or modulating transcription of at least one gene encoded by the target DNA molecule.

### Claim 31 (depends on 30)

The eukaryotic cell of claim 30, wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)) that comprise an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269.

### Claim 32 (depends on 3)

A non-human plant or animal comprising the eukaryotic cell of claim 3.