IP Library › Granted Patent US 12,297,433
Granted Patent B2
US 12,297,433 · App. 18/449,850 · Granted May 13, 2025

Targeted trans-splicing using CRISPR/Cas13

Inventor: Jacob Borrajo (Cambridge, MA)
Assignee: Massachusetts Institute of Technology
C12N15/113C12N9/22C12N2310/20
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Quick Facts
Patent No.
US 12,297,433
App. No.
18/449,850
Granted
May 13, 2025
Kind
B2
Abstract

This disclosure provides compositions and methods of using these compositions to mediate a targeted trans-splicing event on a pre-mRNA in a cell.

Claims (22)

1. A system for targeting a pre-mRNA nucleic acid for trans-splicing, the system comprising:

(i) a CRISPR/Cas 13 polypeptide comprising two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) endoRNAse domains or a variant thereof;

(ii) a nucleic acid guide that recognizes the target pre-mRNA and directs the CRISPR/Cas 13 polypeptide to the pre-mRNA; and

(ii) a trans-splicing RNA template comprising a splice donor and/or acceptor.

2. The system of claim 1 , wherein the nucleic acid guide is an RNA guide.

3. The system of claim 1 , wherein the nucleic acid guide is a DNA guide.

4. The system of claim 1 , wherein the system comprises one nucleic acid guide.

5. The system of claim 1 , wherein the system comprises more than one nucleic acid guide.

6. The system of claim 5 , wherein the more than one nucleic acid guides target one nucleic acid of interest.

7. The system of claim 5 , wherein the more than one nucleic acid guides target multiple nucleic acids of interest.

8. The system of claim 1 , wherein the nucleic acid guide targets a splice acceptor (SA) site.

9. The system of claim 1 , wherein the nucleic acid guide targets a splice donor (SD) site.

10. The system of claim 1 , wherein the nucleic acid guide targets a region near a splice site.

11. The system of claim 9 , wherein the nucleic acid guide targets a region within 200 nucleotides of a splice site.

12. The system of claim 1 , wherein the Cas polypeptide is a nuclease-inactive Cas polypeptide.

13. The system of claim 1 , comprising more than one trans-splicing RNA template.

14. The system of claim 1 , wherein the trans-splicing RNA template comprises the splice acceptor.

15. The system of claim 1 , wherein the trans-splicing RNA template comprises the splice donor.

16. The system of claim 1 , wherein the trans-splicing RNA template comprises a sequence that binds to the nucleic acid.

17. The system of claim 1 , comprising one or more polynucleotides encoding the CRISPR/Cas polypeptide, the nucleic acid-guide, and/or the trans-splicing RNA template.

18. The system of claim 17 , comprising a polynucleotide encoding the CRISPR/Cas polypeptide and a polynucleotide encoding the nucleic acid-guide and the trans-splicing RNA template.

19. The system of claim 17 , comprising one or more vectors comprising the one or more polynucleotides.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Nov 15, 2023
From: BORRAJO, JACOB
To: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 065566/0211 →
Continuity (4)
Continuation 16994230 · Aug 14, 2020
Provisional Application 62985633 · Mar 5, 2020
Provisional Application 62888210 · Aug 16, 2019
Related Publication 20240035031A1 · Feb 1, 2024
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