IP Library › Granted Patent US 12,467,045
Granted Patent B2
US 12,467,045 · App. 17/698,390 · Granted Nov 11, 2025

Tuning CRISPR/Cas9 activity with chemically modified nucleotide substitutions

Inventors: Keith Gagnon (Carbondale, IL); Masad Damha (Montreal, CA); Elise Malek-Adamian (Ottawa, CA); Maryam Habibian (Montreal, CA); Daniel O'Reilly (Waterlooville, GB); Zachary Kartje (Valparaiso, IN)
Assignees: Board of Trustees of Southern Illinois University; The Royal Institution for the Advancement of Learning/McGill University
C12N15/102A61K48/00C12N9/22C12N15/11C12N15/113C12N15/90C12N15/907C12N2310/20C12N2310/315C12N2310/318C12N2310/319C12N2310/3231C12N2310/344C12N2310/346C12N2800/80
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Quick Facts
Patent No.
US 12,467,045
App. No.
17/698,390
Granted
Nov 11, 2025
Kind
B2
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 (31)

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 said first oligonucleotide:

a) comprises at least a first 2′-deoxyribonucleotide or 2′-deoxyribonucleotide analog in said second region or within about 12 nucleotides of said 5′ end of said first region; and

b) comprises at least two ribonucleotides or ribonucleotide analogs within about 8 nucleotides of the 3′ end of said first region,

wherein said second region of said first oligonucleotide comprises the chemical structure of crRNA-E2_J.

2 . The composition of claim 1 , wherein said second region of said first oligonucleotide comprises only 2′-deoxyribonucleotides or 2′-deoxyribonucleotide analogs.

3 . The composition of claim 1 , wherein said first oligonucleotide comprises at least a first 2′-fluoroarabinonucleotide in said second region or within about 12 nucleotides of said 5′ end of said first region.

4 . The composition of claim 1 , wherein said first oligonucleotide comprises a plurality of 2′-fluoroarabinonucleotides in said second region or within about 12 nucleotides of said 5′ end of said first region.

5 . The composition of claim 1 , wherein said first oligonucleotide comprises a combination of 2′-fluoroarabinonucleotides, 2′-deoxyribonucleotides and 2′-deoxyribonucleotide analogs in said second region or within about 12 nucleotides of said 5′ end of said first region.

6 . The composition of claim 1 , wherein said first oligonucleotide comprises a 2′-fluorinated ribonucleotide in said first region or within about 8 nucleotides of the 3′ end of said first region.

7 . The composition of claim 1 , wherein said first oligonucleotide is comprised of a mixture of 3′5′-linked ribonucleotides and 2′5′-linked ribonucleotides in said first region or within about 8 nucleotides of the 3′ end of said first region.

8 . The composition of claim 1 , wherein said first oligonucleotide is comprised of a mixture of 2′-fluorinated and 2′5′-linked ribonucleotides in said first region or within about 8 nucleotides of the 3′ end of said first region.

9 . The composition of claim 1 , wherein said first region of said first oligonucleotide comprises at least 6 ribonucleotides or ribonucleotide analogs.

10 . The composition of claim 9 , wherein said first region of said first oligonucleotide comprises only ribonucleotides or ribonucleotide analogs.

11 . The composition of claim 1 , wherein said second oligonucleotide comprises at least a first 2′-deoxyribonucleotide or 2′-deoxyribonucleotide analog.

12 . The composition of claim 11 , wherein said first region and said second region of said second oligonucleotide comprises at least a first 2′-deoxyribonucleotide or 2′-deoxyribonucleotide analog.

13 . The composition of claim 12 , wherein said first region and said second region of said second oligonucleotide comprises a plurality of 2′-deoxyribonucleotides or 2′-deoxyribonucleotide analogs.

14 . The composition of claim 13 , wherein said second oligonucleotide comprises the chemical structure of tracrR/DNA-C.

15 . The composition of claim 1 , wherein said first oligonucleotide and said second oligonucleotide are covalently linked.

16 . The composition of claim 1 , wherein said Cas9 protein is from Streptococcus pyogenes.

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

18 . The method of claim 17 , wherein said double-stranded DNA is present inside of a cell.

19 . The method of claim 18 , wherein said cell is comprised within a mammal.

20 . The method of claim 19 , wherein said mammal is a human subject.

21 . A method of mutating a double-stranded DNA segment, comprising contacting said double-stranded DNA segment with the composition of claim 1 .

22 . The method of claim 21 , wherein said double-stranded DNA segment is located in a cell.

23 . The method of claim 22 , wherein said double-stranded DNA segment is a genomic DNA segment.

24 . 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 said first oligonucleotide:

a) comprises at least a first 2′-deoxyribonucleotide or 2′-deoxyribonucleotide analog in said second region or within about 12 nucleotides of said 5′ end of said first region; and

b) comprises at least two ribonucleotides or ribonucleotide analogs within about 8 nucleotides of the 3′ end of said first region,

wherein said first oligonucleotide comprises the chemical structure of crRNA-E2_J.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 7, 2025
From: GAGNON, KEITH; DAMHA, MASAD; MALEK-ADAMIAN, ELISE; HABIBIAN, MARYAM; O'REILLY, DANIEL; KARTJE, ZACHARY
To: BOARD OF TRUSTEES OF SOUTHERN ILLINOIS UNIVERSITY; THE ROYAL INSTITUTION FOR THE ADVANCEMENT OF LEARNING/MCGILL UNIVERSITY
Reel/Frame 070145/0915 →
Continuity (3)
Division 16327605
Provisional Application 62398775 · Sep 23, 2016
Related Publication 20240229010A1 · Jul 11, 2024
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