RNA-guided DNA nucleases and uses thereof

Patent metadata

Field	Value
Publication number	US-12391932-B2
Application number	US-202318329183-A
Country	US
Kind code	B2
Filing date	Jun 5, 2023
Priority date	Oct 9, 2015
Publication date	Aug 19, 2025
Grant date	Aug 19, 2025

Abstract

Provided herein are systems, methods, and compositions for the modification of target DNA sequences. More particularly, systems, methods, and compositions for cleaving a target DNA in eukaryotic cells with a guide RNA capable of hybridizing with a target sequence and an RNA-guided DNA nuclease are provided. Also provided are vectors and vector systems which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods for identifying and validating novel CRISPR systems.

First claim

What is claimed is:

1. A recombinant nucleic acid, comprising a heterologous promoter operably linked to a polynucleotide encoding a CRISPR enzyme with the amino acid sequence of SEQ ID NO: 80.

2. The recombinant nucleic acid of claim 1 , wherein the CRISPR enzyme is encoded by a nucleotide sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 93, 510-514, and 760-764.

3. The recombinant nucleic acid of claim 1 , further comprising at least one polynucleotide encoding a guide RNA, wherein the guide RNA forms a complex with the CRISPR enzyme.

4. The recombinant nucleic acid of claim 3 , wherein the at least one polynucleotide encoding a guide RNA is operably linked to a second promoter.

5. The recombinant nucleic acid of claim 1 , further comprising at least one polynucleotide encoding a donor polynucleotide.

6. The recombinant nucleic acid of claim 5 , wherein the at least one polynucleotide encoding a donor polynucleotide is operably linked to a second promoter.

7. The recombinant nucleic acid of claim 1 , wherein the polynucleotide encoding the CRISPR enzyme further encodes at least one nuclear localization signal (NLS).

8. A vector comprising the recombinant nucleic acid of claim 1 .

9. A eukaryotic cell comprising the recombinant nucleic acid of claim 1 .

10. A non-naturally occurring system for sequence-specific modification of a target nucleic acid sequence, the system comprising
(a) one or more guide RNAs comprising a nucleotide sequence complementary to the target nucleic acid sequence in an endogenous sequence of a eukaryotic cell or a DNA molecule encoding the one or more guide RNAs comprising a nucleotide sequence complementary to the target nucleic acid sequence in an endogenous sequence of a eukaryotic cell, and
(b) a CRISPR enzyme having the amino acid sequence of SEQ ID NO: 80 or polynucleotide encoding the CRISPR enzyme,
wherein the one or more guide RNAs and the CRISPR enzyme do not naturally occur together.

11. The system of claim 10 , wherein the polynucleotide encoding the CRISPR enzyme comprises a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 93, 510-514, and 760-764.

12. The system of claim 10 , wherein the target nucleic acid sequence comprises a coding nucleic acid sequence.

13. The system of claim 10 , wherein the target nucleic acid sequence comprises an endogenous gene.

14. The system of claim 10 , wherein the system comprises a divalent cation.

15. The system of claim 10 , wherein the guide RNA or a DNA molecule encoding a guide RNA and the polynucleotide encoding the CRISPR enzyme are provided on a single nucleic acid molecule.

16. The system of claim 10 , wherein the guide RNA is an Agrobacterium vector.

17. The system of claim 10 , further comprising a donor polynucleotide.

18. The system of claim 17 , wherein the donor polynucleotide comprises a coding nucleic acid sequence.

19. The system of claim 17 , wherein the donor polynucleotide comprises a promoter.

20. The system of claim 10 , wherein the CRISPR enzyme comprises one or more nuclear localization signals.

21. The system of claim 10 , wherein the target sequence is within a eukaryotic cell.

22. The system of claim 21 , wherein the eukaryotic cell is a plant cell.

23. A method for sequence-specific modification of a target nucleic acid sequence in a cell, comprising providing the system of claim 10 to a cell that comprises the target nucleic acid sequence.

24. The method of claim 23 , wherein the cell is a plant cell.

25. A method for sequence-specific modification of a target nucleic acid sequence in a eukaryotic cell, comprising providing to the cell (a) a guide RNA, and (b) a CRISPR enzyme comprising the amino acid sequence of SEQ ID NO: 80, whereby the target nucleic acid sequence is modified.

26. The method of claim 25 , wherein:
(a) the guide RNA is provided by expressing in the cell a recombinant DNA molecule encoding the guide RNA;
(b) the CRISPR enzyme is provided by expressing in the cell a recombinant DNA molecule encoding the CRISPR enzyme; or
(c) both (a) and (b).

27. The method of claim 25 , wherein:
(a) the guide RNA is provided by contacting the cell with a composition comprising the guide RNA or a recombinant DNA molecule encoding the guide RNA;
(b) the CRISPR enzyme is provided by contacting the cell with a composition comprising the CRISPR enzyme or a recombinant DNA molecule encoding the CRISPR enzyme; or
(c) the CRISPR enzyme is complexed with the guide RNA and is provided to the cell as a particle.

28. The method of claim 25 , wherein the CRISPR enzyme comprises one or more nuclear localization signals.

29. The method of claim 25 , wherein the recombinant DNA molecule encoding the CRISPR enzyme comprises a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 93, 510-514, and 760-764.

30. The method of claim 25 , wherein the target nucleic acid sequence comprises a coding nucleic acid sequence.

31. The method of claim 25 , wherein the target nucleic acid sequence comprises (a) an endogenous nuclear gene of the cell or of an organelle in the cell; or (b) an endogenous organellar gene of the cell.

32. The method of claim 25 , further comprising providing a donor polynucleotide to the cell.

33. The method of claim 32 , wherein the donor polynucleotide comprises a coding nucleic acid sequence.

34. The method of claim 25 , wherein the cell is a plant cell.

35. A method of selectively modulating transcription of at least one target DNA in a eukaryotic cell comprising contacting the eukaryotic cell with:
(a) a guide RNA or a DNA encoding a guide RNA, wherein the guide RNA further comprises:
(i) a first segment comprising a nucleotide sequence that is complementary to the target DNA; and
(ii) a second segment that interacts with a CRISPR enzyme; and

(b) a CRISPR enzyme comprising the amino acid sequence of SEQ ID NO: 80, wherein components (a) and (b) are located on same or different vectors, wherein the guide RNA and the CRISPR enzyme form a complex in the eukaryotic cell, and wherein the complex selectively modulates transcription of the target DNA.

Assignees

• Monsanto Technology Llc

Inventors

• Chittoor Jaishree M
• Nagy Ervin
• Turner Keith H

Classifications

• C12N2800/80

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

• C12N15/902

  using homologous recombination · 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

• C07K2319/60

  containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] · CPC title

• C07K2319/09

  containing a nuclear localisation signal · CPC title