# US 11,306,305 B2 · Tuning CRISPR/Cas9 activity with chemically modified nucleotide substitutions

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,306,305 B2
- Document ID: US11306305B2
- Status: Granted
- Kind: B2
- Application number: 16/327,605
- Filed: 2017-09-22
- Published: 2019-06-20
- Granted: 2022-04-19
- Earliest claimed: 2016-09-23
- Assignees: Board of Trustees of Southern Illinois University; The Royal Institution for the Advancement of Learning/Mcgill University
- Applicants: Board of Trustees of Southern Illinois University; The Royal Institution for the Advancement of Learning/McGill University
- Inventors: Keith Gagnon; Masad Damha; Elise Malek-Adamian; Maryam Habibian; Daniel Timothy O'Reilly; Zachery Kartje
- Web page: https://patentgrep.com/patent/US11306305B2

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- 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 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 15/113: 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 15/90: Stable introduction of foreign DNA into chromosome
- C12N 15/907: {in mammalian cells}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/315: Phosphorothioates
- C12N 2310/318: where the PO2 is completely replaced, e.g. MMI or formacetal
- C12N 2310/319: linked by 2'-5' linkages, i.e. having a free 3'-position
- C12N 2310/3231: having an additional ring, e.g. LNA, ENA
- C12N 2310/344: Position-specific modifications, e.g. on every purine, at the 3'-end
- C12N 2310/346: having a combination of backbone and sugar modifications
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)

## Abstract

The present disclosure provides CRISPR/Cas9 ribonucleoprotein compositions comprising chemically modified CRISPR RNA (crRNA) guide and trans-acting CRISPR RNA (tracrRNA) components. Methods of using the disclosed CRISPR/Cas9 ribonucleoprotein compositions are also provided.

## Claims

### Claim 1

A composition comprising a Cas9 protein, a first oligonucleotide comprising a first region having a 5′ end and a 3′ end and a second region, and a second oligonucleotide comprising a first region and a second region, wherein said second region of said first oligonucleotide interacts with said first region of said second oligonucleotide, and said second region of said second oligonucleotide interacts with said Cas9 protein, wherein all nucleotides of said first oligonucleotide are 2′-deoxyribonucleotides or 2′-deoxyribonucleotide analogs.

### Claim 2 (depends on 1)

The composition of claim 1, wherein said first oligonucleotide comprises the chemical structure of crRNA_K.

### Claim 3 (depends on 1)

The composition of claim 1, wherein said first oligonucleotide comprises at least a first 2′-fluoroarabinonucleotide.

### Claim 4 (depends on 1)

The composition of claim 1, wherein said Cas9 protein comprises at least a first mutation that inactivates said Cas9 protein.

### Claim 5 (depends on 4)

The composition of claim 4, wherein said Cas9 protein comprises a D10A or a H840A mutation.

### Claim 6 (depends on 5)

The composition of claim 5, wherein said Cas9 protein comprises a D10A and a H840A mutation.

### Claim 7 (depends on 1)

A method of binding a double-stranded DNA without cleaving said double-stranded DNA, comprising contacting said double-stranded DNA with the composition of claim 1.

### Claim 8 (depends on 7)

The method of claim 7, wherein said first oligonucleotide comprises at least a first 2′-fluoroarabinonucleotide.

### Claim 9 (depends on 1)

A method of inhibiting transcription of a double-stranded DNA, comprising contacting said double-stranded DNA with the composition of claim 1.

### Claim 10 (depends on 9)

The method of claim 9, wherein said first oligonucleotide comprises at least a first 2′-fluoroarabinonucleotide.

### Claim 11 (depends on 9)

The method of claim 9, wherein said double-stranded DNA is located in a cell.

### Claim 12 (depends on 1)

A method of activating transcription of a double-stranded DNA, comprising contacting said double-stranded DNA with the composition of claim 1.

### Claim 13 (depends on 12)

The method of claim 12, wherein said first oligonucleotide comprises at least a first 2′-fluoroarabinonucleotide.

### Claim 14 (depends on 12)

The method of claim 12, wherein said double-stranded DNA is located in a cell.