IP Library › Granted Patent US 12,428,638
Granted Patent B2
US 12,428,638 · App. 17/815,621 · Granted Sep 30, 2025

Three-component CRISPR/Cas complex system and uses thereof

Inventors: Haoyi Wang (Beijing, CN); Albert Cheng (Bar Harbor, ME); Nathaniel Jillette (Bar Harbor, ME)
Assignee: The Jackson Laboratory
C12N15/11C12N9/22C12N15/111C12N15/907C12N2310/20C12N2310/531C12N2800/80
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Quick Facts
Patent No.
US 12,428,638
App. No.
17/815,621
Granted
Sep 30, 2025
Kind
B2
Abstract

The invention described herein provides compositions and reagents for assembling a tripartite complex at a specific location of a target DNA. The invention also provides methods for using the complex to, for example, label a specific genomic locus, to regulate the expression of a target gene, or to create a gene regulatory network.

Claims (22)

1. A method comprising delivering to a cell a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA) encoding an RNA that comprises: (a) a DNA-targeting sequence that is complementary to a target polynucleotide sequence in the cell; (b) a Cas9-binding sequence; and (c) at least 5 tandem copies of a PUF domain-Binding Sequence (PBS).

2. The method of claim 1 , wherein the DNA-targeting sequence is complementary to the target polynucleotide sequence over about 12-22 nucleotides.

3. The method of claim 1 , wherein the RNA further comprises a linker sequence linking the DNA-targeting sequence to the Cas9-binding sequence.

4. The method of claim 1 , wherein the Cas9-binding sequence forms a hairpin structure.

5. The method of claim 1 , wherein the Cas9-binding sequence has a length of about 37-47 nucleotides.

6. The method of claim 1 , wherein each of the at least 5 tandem copies of the PBS has a length of about 8 nucleotides.

7. The method of claim 1 , wherein the RNA comprises a PBS of the sequence 5′-UGUAUGUA-3′ that can be bound by the PUF domain PUF (3-2).

8. The method of claim 1 , wherein the RNA comprises a PBS of the sequence 5′-UUGAUAUA-3′ that can be bound by the PUF domain PUF (6-2/7-2).

9. The method of claim 1 , wherein the RNA comprises 5-35 tandem copies of identical PUF domain-Binding Sequences.

10. The method of claim 1 , wherein the RNA comprises 5-35 tandem copies of different PUF domain-Binding Sequences.

11. The method of claim 1 , wherein the RNA comprises 5-10 tandem copies of identical PUF domain-Binding Sequences.

12. The method of claim 1 , wherein the RNA comprises 5-10 tandem copies of different PUF domain-Binding Sequences.

13. The method of claim 1 , wherein the tandem copies of the PBS are separated from each other by a spacer sequence.

14. The method of claim 1 , further comprising delivering to the cell:

(a) a Cas9 protein or a polynucleotide encoding a Cas9 protein; and

(b) a polynucleotide encoding a PUF domain.

15. The method of claim 14 , wherein the Cas9 protein is a wild-type Cas9 protein.

16. The method of claim 14 , wherein the Cas9 protein is a nuclease-deficient dCas9 protein.

17. The method of claim 14 , wherein the PUF domain is fused to an effector domain.

18. The method of claim 17 , wherein the effector domain is a fluorescent protein.

19. The method of claim 18 , wherein the method further comprises imaging the cell.

20. The method of claim 19 , wherein the cell is a live cell.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 2, 2022
From: WANG, HAOYI; CHENG, ALBERT; JILLETTE, NATHANIEL
To: THE JACKSON LABORATORY
Reel/Frame 061632/0091 →
Continuity (5)
Division 15702944 · Sep 13, 2017
Continuation PCTUS2016021491 · Mar 9, 2016
Provisional Application 62221249 · Sep 21, 2015
Provisional Application 62132644 · Mar 13, 2015
Related Publication 20230071712A1 · Mar 9, 2023
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