Granted Patent B2

US 12,435,330 · App. 17/767,777 · Granted Oct 7, 2025

Inventors: David R. Liu (Cambridge, MA); Andrew Vito Anzalone (Cambridge, MA); James William Nelson (Cambridge, MA); Peter J. Chen (Cambridge, MA)

Assignees: The Broad Institute, Inc.; President and Fellows of Harvard College

C12N15/102C12N9/127C12N9/22C12N15/111C07K2319/85C12N2310/20C12Y207/07048

Abstract

Claims (20)

1. A fusion protein comprising a nucleic acid-programmable RNA binding protein and an RNA-dependent RNA polymerase.

2. The fusion protein of claim 1 , wherein the fusion protein when complexed to an RNA prime editing guide RNA (rpegRNA) is capable of appending a single-stranded RNA sequence to a target RNA.

3. The fusion protein of claim 2 , wherein the single-stranded RNA sequence is appended to the 3′ terminus of the target RNA or to a 3′ terminus which is formed upon cleavage of the target RNA by the fusion protein at a cut site.

4. The fusion protein of claim 2 , wherein the single-stranded RNA sequence is polymerized by the RNA-dependent RNA polymerase using the rpegRNA as a template.

5. The fusion protein of claim 2 , wherein the rpegRNA comprises the following structure: 5′-[spacer sequence]-[scaffold sequence]-[template sequence]-3′, wherein the spacer sequence anneals to the target RNA at a complementary protospacer sequence, the scaffold sequence binds the rpegRNA to the nucleic acid-programmable RNA binding protein of the fusion protein, and the template sequence provides an RNA template for synthesis of the single-stranded RNA sequence by the RNA-dependent RNA polymerase of the fusion protein.

6. The fusion protein of claim 1 , wherein the nucleic acid-programmable RNA binding protein is a Cas13 protein.

7. The fusion protein of claim 6 , wherein the Cas13 protein is a Cas13a, Cas13b, or Cas13d protein.

8. The fusion protein of claim 6 , wherein the Cas13 protein is nuclease inactive.

9. The fusion protein of claim 6 , wherein the Cas13 protein has an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 1.

10. The fusion protein of claim 1 , wherein the RNA-dependent RNA polymerase is capable of polymerizing a single-stranded RNA sequence using an rpegRNA as a template.

11. The fusion protein of claim 1 , wherein the RNA-dependent RNA polymerase comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-7.

12. The fusion protein of claim 1 , wherein the fusion protein has the structure: NH 2 -[RNA-dependent RNA polymerase]-[nucleic acid-programmable RNA binding protein]-COOH or NH 2 -[nucleic acid-programmable RNA binding protein]-[RNA-dependent RNA polymerase]-COOH, wherein “]-[” represents a linker sequence.

13. The fusion protein of claim 12 , wherein the linker sequence has an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-23.

14. An RNA prime editor complex for appending a single-stranded RNA sequence to a target RNA comprising a fusion protein of claim 1 and an rpegRNA.

15. The RNA prime editor complex of claim 14 , wherein the rpegRNA is capable of programming the fusion protein to bind to the target RNA.

16. The RNA prime editor complex of claim 14 , wherein the nucleic acid-programmable RNA binding protein of the fusion protein comprises a nuclease activity which cleaves the target RNA at a cut site upon binding of the complex thereto.

17. The RNA prime editor complex of claim 14 , wherein the nucleic acid-programmable RNA binding protein of the fusion protein is catalytically inactive.

18. A method for appending a desired single-stranded RNA sequence to the 3′ end of a target RNA, the method comprising contacting the target RNA with an RNA prime editor complex of claim 14 .

19. The fusion protein of claim 1 , wherein the fusion protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 9-13.

20. An RNA prime editor complex for appending a single-stranded RNA sequence to a target RNA comprising: (i) a first fusion protein comprising a catalytically inactive nucleic acid-programmable RNA binding protein and an RNA-dependent RNA polymerase; (ii) a second fusion protein comprising a catalytically active nucleic acid-programmable RNA binding protein that is capable of cleaving the target RNA to generate a free 3′ terminus; (iii) an rpegRNA that directs the first fusion protein to a first locus in the target RNA; and (iv) a guide RNA that directs the second fusion protein to a second locus in the target RNA.

Assignments (8)

Continuity (2)

References Cited (400)