Tuning CRISPR/Cas9 activity with chemically modified nucleotide substitutions

Patent metadata

Field	Value
Publication number	US-12467045-B2
Application number	US-202217698390-A
Country	US
Kind code	B2
Filing date	Mar 18, 2022
Priority date	Sep 23, 2016
Publication date	Nov 11, 2025
Grant date	Nov 11, 2025

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.

First claim

What is claimed is:

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.

Assignees

• The Royal Institution For The Advancement Of Learning/Mcgill Univ
• Board Of Trustees Of Southern Illinois Univ

Inventors

• Gagnon Keith
• Damha Masad
• Malek-Adamian Elise
• Habibian Maryam
• O'Reilly Daniel
• Kartje Zachary

Classifications

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C12N15/907

  in mammalian cells · CPC title

• C12N15/11

  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} · CPC title

• C12N2310/3231

  having an additional ring, e.g. LNA, ENA · CPC title

• C12N2310/319

  linked by 2'-5' linkages, i.e. having a free 3'-position · CPC title