# US 10,415,059 B2 · Using truncated guide RNAs (tru-gRNAs) to increase specificity for RNA-guided genome editing

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- Number: US 10,415,059 B2
- Document ID: US10415059B2
- Status: Granted
- Kind: B2
- Application number: 15/430,218
- Filed: 2017-02-10
- Published: 2017-06-01
- Granted: 2019-09-17
- Earliest claimed: 2013-03-15
- Assignees: The General Hospital Corporation
- Applicants: The General Hospital Corporation
- Inventors: J. Keith Joung; Jeffry D. Sander; Morgan Maeder; Yanfang Fu
- Web page: https://patentgrep.com/patent/US10415059B2

## CPC

- C12N 15/907: {in mammalian cells}
- C07K 14/005: from viruses
- C07K 14/195: from bacteria
- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12N 9/1007: {Methyltransferases (general) (2.1.1.)}
- C12N 9/16: acting on ester bonds (3.1)
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/224: {Class 1 CAS enzyme complex, e.g. multi-enzyme}
- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/1031: {mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/85: for animal cells
- C12Y 301/21004: Type II site-specific deoxyribonuclease (3.1.21.4)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/01: containing a localisation/targetting motif
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2710/00033: Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
- C12N 2770/00033: Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12Y 114/11: with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
- C12Y 201/01: Methyltransferases (2.1.1)
- C12Y 301/00: Hydrolases acting on ester bonds (3.1)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES

## Abstract

Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems, using truncated guide RNAs (tru-gRNAs).

## Claims

### Claim 1

A method of RNA-guided genome editing in a cell, the method comprising contacting the cell with a guide RNA (gRNA) that includes a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a target genomic sequence, wherein the gRNA comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:2404, SEQ ID NO:2407, or SEQ ID NO: 2408, and wherein in the presence of a S. pyogenes Cas9 nuclease, the gRNA complementarity region binds and directs the Cas9 nuclease to the target genomic sequence, and the Cas9 nuclease edits the target genomic sequence.

### Claim 2

A Streptococcus pyogenes gRNA molecule that includes a complementarity region at the 5′ end of the gRNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a target genomic sequence, wherein the target genomic sequence is immediately 5′ of a protospacer adjacent motif, wherein the gRNA is a single gRNA or a CRISPR RNA (crRNA), and wherein in the presence of an S. pyogenes Cas9 nuclease, the gRNA complementarity region binds and directs the Cas9 nuclease to the target genomic sequences, and the Cas9 nuclease edits the target genomic sequence.

### Claim 3 (depends on 2)

The gRNA of claim 2, wherein the complementarity region of the gRNA consists of 17 nucleotides.

### Claim 4 (depends on 2)

The gRNA of claim 2, comprising a ribonucleic acid consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:2404, SEQ ID NO:2407, or SEQ ID NO: 2408.

### Claim 5 (depends on 2)

The gRNA of claim 2, comprising a ribonucleic acid that includes one or more uracil (U) at the 3′ end of the molecule.

### Claim 6 (depends on 2)

The gRNA of claim 2, comprising a ribonucleic acid that includes one or more additional nucleotides at the 5′ end of the RNA molecule that is not complementary to the target genomic sequence.

### Claim 7 (depends on 2)

The gRNA of claim 2, comprising a ribonucleic acid that includes one, two, or three additional nucleotides at the 5′ end of the RNA molecule that are not complementary to the target genomic sequence.

### Claim 8 (depends on 2)

The gRNA of claim 2, wherein the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of a target genomic sequence.

### Claim 9 (depends on 2)

The gRNA of claim 2, wherein the complementarity region is complementary to 18 consecutive nucleotides of the complementary strand of a target genomic sequence.

### Claim 10 (depends on 2)

A DNA molecule encoding the gRNA of claim 2.

### Claim 11 (depends on 10)

A vector comprising the DNA molecule of claim 10.

### Claim 12 (depends on 11)

A host cell expressing the vector of claim 11.

### Claim 13 (depends on 12)

The host cell of claim 12, wherein the cell is a eukaryotic cell.

### Claim 14 (depends on 13)

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

### Claim 15 (depends on 2)

The gRNA of claim 2, wherein the complementarity region of the gRNA consists of 18 nucleotides.

### Claim 16 (depends on 2)

The gRNA of claim 2, and wherein the gRNA retains the ability to form a complex with a Cas9 nuclease or a catalytically inactive Cas9 (dCas9) nuclease.

### Claim 17

A complex comprising:
a Streptococcus pyogenes Cas9 nuclease and
a Streptococcus pyogenes gRNA molecule that includes a complementarity region at the 5′ end of the gRNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a target genomic sequence, wherein the target genomic sequence is immediately 5′ of a protospacer adjacent motif,
wherein the gRNA is a single gRNA or a CRISPR RNA (crRNA), and
wherein in the presence of the S. pyogenes Cas9 nuclease, the gRNA complementary region binds and directs the Cas9 nuclease to the target genomic sequence, and the Cas9 nuclease edits the target genomic sequence.

### Claim 18 (depends on 17)

The complex of claim 17, wherein the Cas9 nuclease is a dCas9 nuclease.

### Claim 19 (depends on 17)

The complex of claim 17, wherein the complementarity region of the gRNA consists of 17 nucleotides.

### Claim 20 (depends on 17)

The complex of claim 17, wherein the gRNA comprises a ribonucleic acid consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:2404, SEQ ID NO:2407, or SEQ ID NO: 2408.

### Claim 21 (depends on 17)

The complex of claim 17, wherein the gRNA includes one or more uracil (U) at the 3′ end of the molecule.

### Claim 22 (depends on 17)

The complex of claim 17, wherein the gRNA includes one or more additional nucleotides at the 5′ end of the gRNA that is not complementary to the target genomic sequence.

### Claim 23 (depends on 17)

The complex of claim 17, wherein the gRNA includes one,
two, or three additional nucleotides at the 5′ end of the RNA molecule that are not complementary to the target genomic sequence.

### Claim 24 (depends on 17)

The complex of claim 17, wherein the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of a target genomic sequence.

### Claim 25 (depends on 17)

The complex of claim 17, wherein the complementarity region is complementary to 18 consecutive nucleotides of the complementary strand of a target genomic sequence.

### Claim 26 (depends on 17)

The complex of claim 17, wherein the complementarity region of the gRNA consists of 18 nucleotides.

### Claim 27 (depends on 17)

A DNA molecule encoding the complex of claim 17.

### Claim 28 (depends on 27)

A vector comprising the DNA molecule of claim 27.

### Claim 29 (depends on 28)

A host cell expressing the vector of claim 28.

### Claim 30 (depends on 29)

The host cell of claim 29, wherein the cell is a eukaryotic cell.

### Claim 31 (depends on 30)

The host cell of claim 30, wherein the cell is a mammalian cell.