# US 12,215,343 B2 · Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription

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- Number: US 12,215,343 B2
- Document ID: US12215343B2
- Status: Granted
- Kind: B2
- Application number: 17/559,860
- Filed: 2021-12-22
- Published: 2022-08-25
- Granted: 2025-02-04
- 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/US12215343B2

## 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:
an activator-RNA comprising 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 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 2 (depends on 1)

The composition of claim 1, wherein the activator-RNA comprises said one or more non-natural internucleoside linkages and/or said one or more modified sugar moieties.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the activator-RNA comprises said one or more modified sugar moieties, wherein said one or more modified sugar moieties comprises one or more 2′—O-methyl modified sugar moieties and/or one or more 2′-fluoro modified sugar moieties.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the activator-RNA comprises:
(1) said one or more non-natural internucleoside linkages, wherein said one or more non-natural internucleoside linkages comprises a phosphorothioate, an inverted polarity linkage, an abasic nucleoside linkage, or any combination thereof;
(2) said one or more modified sugar moieties, wherein said one or more modified sugar moieties comprises a locked nucleic acid (LNA) sugar moiety, a 2′-substituted sugar moiety, 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, or any combination thereof,
(3) said one or more nucleic acid mimetics, wherein said one or more nucleic acid mimetics comprises a peptide nucleic acid (PNA), a morpholino nucleic acid, a cyclohexenyl nucleic acid (CeNA), or any combination thereof, or
(4) any combination thereof.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the activator-RNA comprises:
(1) said one or more non-natural internucleoside linkages, wherein said one or more non-natural internucleoside linkages comprises a phosphorothioate, a phosphoramidate, a non-phosphodiester, a heteroatom, a chiral phosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, a 3′-alkylene phosphonates, a 5′-alkylene phosphonate, a chiral phosphonate, a phosphinate, a, a 3′-amino phosphoramidate, an aminoalkylphosphoramidate, a phosphorodiamidate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, a selenophosphate, a boranophosphate, or any combination thereof, and/or
(2) said one or more modified nucleobases, wherein said one or more modified nucleobases comprises a 5-methylcytosine, a 5-hydroxymethyl cytosine, a xanthine, a hypoxanthine, a 2-aminoadenine, a 6-methyl derivative of adenine, a 6-methyl derivative of guanine, a 2-propyl derivative of adenine, a 2-propyl derivative of guanine, a 2-thiouracil, a 2-thiothymine, a 2-thiocytosine, a 5-propynyl uracil, a 5-propynyl cytosine, a 6-azo uracil, a 6-azo cytosine, a 6-azo thymine, a pseudouracil, a 4-thiouracil, an 8-haloadenin, an 8-aminoadenin, an 8-thioladenin, an 8-thioalkyladenin, an 8-hydroxyladenin, an 8-haloguanin, an 8-aminoguanin, an 8-thiolguanin, an 8-thioalkylguanin, an 8-hydroxylguanin, a 5-halouracil, a 5-bromouracil, a 5-trifluoromethyluracil, a 5-halocytosine, a 5-bromocytosine, a 5-trifluoromethylcytosine, a 5-substituted uracil, a 5-substituted cytosine, a 7-methylguanine, a 7-methyladenine, a 2-F-adenine, a 2-amino-adenine, an 8-azaguanine, an 8-azaadenine, a 7-deazaguanine, a 7-deazaadenine, a 3-deazaguanine, a 3-deazaadenine, a tricyclic pyrimidine, a phenoxazine cytidine, a phenothiazine cytidine, a substituted phenoxazine cytidine, a carbazole cytidine, a pyridoindole cytidine, a 7-deazaguanosine, a 2-aminopyridine, a 2-pyridone, a 5-substituted pyrimidine, a 6-azapyrimidine, an N-2, N-6 or O-6 substituted purine, a 2-aminopropyladenine, a 5-propynyluracil, a 5-propynylcytosine, or any combination thereof.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the activator-RNA further comprises a polyamine, a polyamide, a polyethylene glycol, a polyether, a cholesterol moiety, a cholic acid, a thioester, a thiocholesterol, a lipid, an aliphatic chain, a phospholipid, an adamantane acetic acid, a palmityl moiety, an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety, a biotin, a phenazine, a folate, a phenanthridine, an anthraquinone, an acridine, a fluorescein, a rhodamine, a coumarin, a dye, or any combination thereof.

### Claim 7 (depends on 6)

The composition of claim 6, wherein the activator-RNA comprises said polyamine, said polyamide, said polyethylene glycol, said cholesterol moiety, or any combination thereof.

### Claim 8 (depends on 1)

The composition of claim 1, wherein said one or more non-natural internucleoside linkages comprises one or more phosphorothioates, one or more inverted polarity linkages, one or more abasic nucleoside linkages, or any combination thereof.

### Claim 9 (depends on 1)

The composition of claim 1, wherein said one or more modified sugar moieties comprises 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.

### Claim 10 (depends on 1)

The composition of claim 1, wherein said one or more nucleic acid mimetics comprises 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 11 (depends on 1)

The composition of claim 1, wherein said one or more modified nucleobases comprises 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 O-6 substituted purines, one or more 2-aminopropyladenines, one or more 5-propynyluracils, one or more 5-propynylcytosines, or any combination thereof.

### Claim 12 (depends on 1)

The composition of claim 1, wherein:
said one or more non-natural internucleoside linkages comprises one or more phosphorothioates, one or more inverted polarity linkages, one or more abasic nucleoside linkages, or any combination thereof;
said one or more modified sugar moieties comprises 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,
said one or more nucleic acid mimetics comprises 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
said one or more modified nucleobases comprises 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 O-6 substituted purines, one or more 2-aminopropyladenines, one or more 5-propynyluracils, one or more 5-propynylcytosines, or any combination thereof.

### Claim 13 (depends on 12)

The composition of claim 12, wherein the activator-RNA is lyophilized.

### Claim 14 (depends on 1)

The composition of claim 1, wherein said 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 15 (depends on 1)

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

### Claim 16 (depends on 1)

The composition of claim 1, wherein the activator-RNA comprises the 31 nucleotide tracrRNA sequence: UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC (SEQ ID NO: 396).

### Claim 17 (depends on 1)

The composition of claim 1, wherein the activator-RNA comprises the 67 nucleotide tracrRNA sequence
(SEQ ID NO: 432)
UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA
AAGUGGCACCGAGUCGGUGCUUUUUUU.

### Claim 18 (depends on 1)

The composition of claim 1, wherein the activator-RNA comprises a nucleotide sequence having greater than 80% nucleotide sequence identity with any one of the following tracrRNA sequences:
(SEQ ID NO: 478)
AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC
AACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU;
(SEQ ID NO: 1425)
AAAUAACAUAGCAAGUUAAAAUAAGGCUUUGUCCGUUAU
CAACUUUUAAUUAAGUAGCGCCGUUUCGGCGCUUUUUU;
(SEQ ID NO: 1426)
AAACAACACAGCAAGUUAAAUAAGGCAGUGAUUUUUAAU
CCAGUCCGUACACAACUUGAAAAAGUGCGCACCGAUUCG
GUGCUUUUUU;
(SEQ ID NO: 1427)
AAACAACACAGCGAGUUAAAUAAGGCUUAGUCCGUACUC
AACUUGAAAAGGUGGCACCGAUUCGGUGUUUUUU;
(SEQ ID NO: 1428)
AAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGA
AAUCAACACCCUGUCAUUUUAUGGCAGGGUGUUUUCGUU
AUUU;
(SEQ ID NO: 1429)
AAAUGAGAACCGUUGCUACAAUAAGGCCGUCUGAAAAGA
UGUGCCGCAACGCUCUGCCCCUUAAAGCUUCUGCUUUAA
GGGGCAUCGUUUAUUUC;
and
(SEQ ID NO: 1430)
AAAUGAGAGAGUUGCUACAAUAAGGCUUCUGAAAAGAAU
GACCGUAACGCUCUGCCCCUUGUGAUUCUUAAUUGCAAG
GGGCAUCGUUUU.

### Claim 19 (depends on 1)

The composition of claim 1, wherein the activator-RNA comprises a nucleotide sequence having greater than 90% nucleotide sequence identity with any one of the following tracrRNA sequences:
(SEQ ID NO: 397)
UAGCAAGUUAAAAUAAGGCUAGUCCG;
(SEQ ID NO: 1431)
UAGCAAGUUAAAAUAAGGCUUUGUCCG;
(SEQ ID NO: 1432)
CAGCGAGUUAAAUAAGGCUUAGUCCG;
(SEQ ID NO: 1433)
AGAAGCUACAAAGAUAAGGCUUCAUGCCG;
(SEQ ID NO: 1434)
GUUGCUACAAUAAGGCCGUCUGAAAAGAUGUGCCG;
and
(SEQ ID NO: 1435)
AGAGUUGCUACAAUAAGGCUUCUGAAAAGAAUGACCG.

### Claim 20 (depends on 1)

The composition of claim 1, wherein the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 478)
AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAU
CAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU;
(SEQ ID NO: 1425)
AAAUAACAUAGCAAGUUAAAAUAAGGCUUUGUCCGUU
AUCAACUUUUAAUUAAGUAGCGCCGUUUCGGCGCUUU
UUU;
(SEQ ID NO: 1426)
AAACAACACAGCAAGUUAAAUAAGGCAGUGAUUUUUA
AUCCAGUCCGUACACAACUUGAAAAAGUGCGCACCGA
UUCGGUGCUUUUUU;
(SEQ ID NO: 1427)
AAACAACACAGCGAGUUAAAUAAGGCUUAGUCCGUAC
UCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUUU;
(SEQ ID NO: 1428)
AAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCC
GAAAUCAACACCCUGUCAUUUUAUGGCAGGGUGUUUU
CGUUAUUU;
(SEQ ID NO: 1429)
AAAUGAGAACCGUUGCUACAAUAAGGCCGUCUGAAAA
GAUGUGCCGCAACGCUCUGCCCCUUAAAGCUUCUGCU
UUAAGGGGCAUCGUUUAUUUC;
and
(SEQ ID NO: 1430)
AAAUGAGAGAGUUGCUACAAUAAGGCUUCUGAAAAGA
AUGACCGUAACGCUCUGCCCCUUGUGAUUCUUAAUUG
CAAGGGGCAUCGUUUU.

### Claim 21 (depends on 1)

The composition of claim 1, wherein the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 397)
UAGCAAGUUAAAAUAAGGCUAGUCCG;
(SEQ ID NO: 1431)
UAGCAAGUUAAAAUAAGGCUUUGUCCG;
(SEQ ID NO: 1432)
CAGCGAGUUAAAUAAGGCUUAGUCCG;
(SEQ ID NO: 1433)
AGAAGCUACAAAGAUAAGGCUUCAUGCCG;
(SEQ ID NO: 1434)
GUUGCUACAAUAAGGCCGUCUGAAAAGAUGUGCCG;
and
(SEQ ID NO: 1435)
AGAGUUGCUACAAUAAGGCUUCUGAAAAGAAUGACCG.

### Claim 22 (depends on 1)

The composition of claim 1, wherein said nucleotide sequence of the duplex-forming segment of the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 395)
AAACAGCAUAGCAAGUUAAAAU;
(SEQ ID NO: 1436)
AAAUAACAUAGCAAGUUAAAAU;
(SEQ ID NO: 1437)
AAACAACACAGCAAGUUAAA;
(SEQ ID NO: 1438)
AAACAACACAGCGAGUUAAA;
(SEQ ID NO: 1439)
AAAUCUUGCAGAAGCUACAAAGAU;
(SEQ ID NO: 1440)
AAAUGAGAACCGUUGCUACAAU;
and
(SEQ ID NO: 1441)
AAAUGAGAGAGUUGCUACAAU.

### Claim 23 (depends on 1)

The composition of claim 1, wherein said nucleotide sequence of the duplex-forming segment of the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 562)
UAGCAAGUUAAAAU;
(SEQ ID NO: 1442)
CAGCGAGUUAAA;
(SEQ ID NO: 1443)
AGAAGCUACAAAGAU;
(SEQ ID NO: 1444)
GUUGCUACAAU;
and
(SEQ ID NO: 1445)
AGAGUUGCUACAAU.

### Claim 24 (depends on 1)

The composition of claim 1, wherein the activator-RNA comprises a nucleotide sequence having 90% nucleotide sequence identity with the tracrRNA sequence set forth in any one of SEQ ID NOs.: 478 and 1425-1430 over a stretch of at least 25 nucleotides.

### Claim 25 (depends on 1)

The composition of claim 1, wherein the activator-RNA comprises a nucleotide sequence having 90% nucleotide sequence identity with the tracrRNA sequence set forth in any one of SEQ ID NOs.: 397 and 1431-1435 over a stretch of at least 25 nucleotides.

### Claim 26 (depends on 1)

The composition of claim 1, wherein the activator-RNA comprises a nucleotide sequence having 100% nucleotide sequence identity with the tracrRNA sequence set forth in any one of SEQ ID NOs.: 478 and 1425-1430 over a stretch of at least 25 nucleotides.

### Claim 27 (depends on 1)

The composition of claim 1, wherein the activator-RNA comprises a nucleotide sequence having 100% nucleotide sequence identity with the tracrRNA sequence set forth in any one of SEQ ID NOs.: 397 and 1431-1435 over a stretch of at least 25 nucleotides.

### Claim 28 (depends on 1)

The composition of claim 1, wherein the activator-RNA is lyophilized.

### Claim 29 (depends on 1)

The composition claim 1, further comprising a buffering agent.

### Claim 30 (depends on 29)

The composition of claim 29, wherein the buffering agent stabilizes nucleic acids.

### Claim 31

A composition comprising:
an activator-RNA comprising 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 comprises a polyamine; a polyamide; a polyethylene glycol; a polyether; a cholesterol moiety; a cholic acid; a thioester; a thiocholesterol; a lipid; an aliphatic chain; a phospholipid; an adamantane acetic acid; a palmityl moiety; an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety; a biotin; a phenazine; a folate; a phenanthridine; an anthraquinone; an acridine; a fluorescein; a rhodamine; a coumarin; a dye; or any combination thereof.

### Claim 32 (depends on 31)

The composition of claim 31, wherein the activator-RNA comprises said polyamine; said polyamide; said polyethylene glycol; said polyether; said cholesterol moiety; said cholic acid; said thioester; said thiocholesterol; said lipid; said aliphatic chain; said phospholipid; said adamantane acetic acid; said palmityl moiety; said octadecylamine or hexylamino-carbonyl-oxycholesterol moiety, or any combination thereof.

### Claim 33 (depends on 31)

The composition of claim 31, wherein said 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 34 (depends on 31)

The composition of claim 31, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 441).

### Claim 35 (depends on 31)

The composition of claim 31, wherein the activator-RNA comprises the 31 nucleotide tracrRNA sequence: UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC (SEQ ID NO: 396).

### Claim 36 (depends on 31)

The composition of claim 31, wherein the activator-RNA comprises the 67 nucleotide tracrRNA sequence
(SEQ ID NO: 432)
UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
UGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU.

### Claim 37 (depends on 31)

The composition of claim 31, wherein the activator-RNA comprises a nucleotide sequence having at least 90% nucleotide sequence identity with any one of the following tracrRNA sequences:
(SEQ ID NO: 478)
AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCC
GUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU
GCUUUUUUU;
(SEQ ID NO: 1425)
AAAUAACAUAGCAAGUUAAAAUAAGGCUUUGUC
CGUUAUCAACUUUUAAUUAAGUAGCGCCGUUUC
GGCGCUUUUUU;
(SEQ ID NO: 1426)
AAACAACACAGCAAGUUAAAUAAGGCAGUGAUU
UUUAAUCCAGUCCGUACACAACUUGAAAAAGUG
CGCACCGAUUCGGUGCUUUUUU;
(SEQ ID NO: 1427)
AAACAACACAGCGAGUUAAAUAAGGCUUAGUCC
GUACUCAACUUGAAAAGGUGGCACCGAUUCGGU
GUUUUUU;
(SEQ ID NO: 1428)
AAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCA
UGCCGAAAUCAACACCCUGUCAUUUUAUGGCAG
GGUGUUUUCGUUAUUU;
(SEQ ID NO: 1429)
AAAUGAGAACCGUUGCUACAAUAAGGCCGUCUG
AAAAGAUGUGCCGCAACGCUCUGCCCCUUAAAG
CUUCUGCUUUAAGGGGCAUCGUUUAUUUC;
and
(SEQ ID NO: 1430)
AAAUGAGAGAGUUGCUACAAUAAGGCUUCUGAA
AAGAAUGACCGUAACGCUCUGCCCCUUGUGAUU
CUUAAUUGCAAGGGGCAUCGUUUU.

### Claim 38 (depends on 31)

The composition of claim 31, wherein the activator-RNA comprises a nucleotide sequence having at least 90% nucleotide sequence identity with any one of the following tracrRNA sequences:
(SEQ ID NO: 397)
UAGCAAGUUAAAAUAAGGCUAGUCCG;
(SEQ ID NO: 1431)
UAGCAAGUUAAAAUAAGGCUUUGUCCG;
(SEQ ID NO: 1432)
CAGCGAGUUAAAUAAGGCUUAGUCCG;
(SEQ ID NO: 1433)
AGAAGCUACAAAGAUAAGGCUUCAUGCCG;
(SEQ ID NO: 1434)
GUUGCUACAAUAAGGCCGUCUGAAAAGAUGUGCCG;
and
(SEQ ID NO: 1435)
AGAGUUGCUACAAUAAGGCUUCUGAAAAGAAUGACCG.

### Claim 39 (depends on 31)

The composition of claim 31, wherein the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 478)
AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGU
UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU
UUUUU;
(SEQ ID NO: 1425)
AAAUAACAUAGCAAGUUAAAAUAAGGCUUUGUCCG
UUAUCAACUUUUAAUUAAGUAGCGCCGUUUCGGCG
CUUUUUU;
(SEQ ID NO: 1426)
AAACAACACAGCAAGUUAAAUAAGGCAGUGAUUUU
UAAUCCAGUCCGUACACAACUUGAAAAAGUGCGCA
CCGAUUCGGUGCUUUUUU;
(SEQ ID NO: 1427)
AAACAACACAGCGAGUUAAAUAAGGCUUAGUCCGU
ACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUU
UUU;
(SEQ ID NO: 1428)
AAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUG
CCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG
UUUUCGUUAUUU;
(SEQ ID NO: 1429)
AAAUGAGAACCGUUGCUACAAUAAGGCCGUCUGAA
AAGAUGUGCCGCAACGCUCUGCCCCUUAAAGCUUC
UGCUUUAAGGGGCAUCGUUUAUUUC;
and
(SEQ ID NO: 1430)
AAAUGAGAGAGUUGCUACAAUAAGGCUUCUGAAAA
GAAUGACCGUAACGCUCUGCCCCUUGUGAUUCUUA
AUUGCAAGGGGCAUCGUUUU.

### Claim 40 (depends on 31)

The composition of claim 31, wherein the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 397)
UAGCAAGUUAAAAUAAGGCUAGUCCG;
(SEQ ID NO: 1431)
UAGCAAGUUAAAAUAAGGCUUUGUCCG;
(SEQ ID NO: 1432)
CAGCGAGUUAAAUAAGGCUUAGUCCG;
(SEQ ID NO: 1433)
AGAAGCUACAAAGAUAAGGCUUCAUGCCG;
(SEQ ID NO: 1434)
GUUGCUACAAUAAGGCCGUCUGAAAAGAUGUGCCG;
and
(SEQ ID NO: 1435)
AGAGUUGCUACAAUAAGGCUUCUGAAAAGAAUGACCG.

### Claim 41 (depends on 31)

The composition of claim 31, wherein said nucleotide sequence of the duplex-forming segment of the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 395)
AAACAGCAUAGCAAGUUAAAAU;
(SEQ ID NO: 1436)
AAAUAACAUAGCAAGUUAAAAU;
(SEQ ID NO: 1437)
AAACAACACAGCAAGUUAAA;
(SEQ ID NO: 1438)
AAACAACACAGCGAGUUAAA;
(SEQ ID NO: 1439)
AAAUCUUGCAGAAGCUACAAAGAU;
(SEQ ID NO: 1440)
AAAUGAGAACCGUUGCUACAAU;
and
(SEQ ID NO: 1441)
AAAUGAGAGAGUUGCUACAAU.

### Claim 42 (depends on 31)

The composition of claim 31, wherein said nucleotide sequence of the duplex-forming segment of the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 562)
UAGCAAGUUAAAAU;
(SEQ ID NO: 1442)
CAGCGAGUUAAA;
(SEQ ID NO: 1443)
AGAAGCUACAAAGAU;
(SEQ ID NO: 1444)
GUUGCUACAAU;
and
(SEQ ID NO: 1445)
AGAGUUGCUACAAU.

### Claim 43 (depends on 31)

The composition of claim 31, wherein the activator-RNA comprises a nucleotide sequence having 90% nucleotide sequence identity with the tracrRNA sequence set forth in any one of SEQ ID NOs.: 478 and 1425-1430 over a stretch of at least 25 nucleotides.

### Claim 44 (depends on 31)

The composition of claim 31, wherein the activator-RNA comprises a nucleotide sequence having 90% nucleotide sequence identity with the tracrRNA sequence set forth in any one of SEQ ID NOs.: 397 and 1431-1435 over a stretch of at least 25 nucleotides.

### Claim 45 (depends on 31)

The composition of claim 31, wherein the activator-RNA comprises a nucleotide sequence having 100% nucleotide sequence identity with the tracrRNA sequence set forth in any one of SEQ ID NOs.: 478 and 1425-1430 over a stretch of at least 25 nucleotides.

### Claim 46 (depends on 31)

The composition of claim 31, wherein the activator-RNA comprises a nucleotide sequence having 100% nucleotide sequence identity with the tracrRNA sequence set forth in any one of SEQ ID NOs.: 397 and 1431-1435 over a stretch of at least 25 nucleotides.

### Claim 47 (depends on 31)

The composition of claim 31, further comprising a buffering agent.