Extended DNA-sensing GRNAS

Patent metadata

Field	Value
Publication number	US-9340800-B2
Application number	US-201414326361-A
Country	US
Kind code	B2
Filing date	Jul 8, 2014
Priority date	Sep 6, 2013
Publication date	May 17, 2016
Grant date	May 17, 2016

Abstract

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity and/or improving the specificity of RNA-programmable endonucleases, such as Cas9. For example, provided are guide RNAs (gRNAs) that are engineered to exist in an “on” or “off” state, which control the binding and hence cleavage activity of RNA-programmable endonucleases. Some aspects of this disclosure provide gRNAs that modulate the activity of an RNA-programmable endonuclease based on the presence or absence of an extended DNA (xDNA).

First claim

What is claimed is:

1. An extended DNA (xDNA)-sensing single-guide RNA (sgRNA) comprising:
(1) a domain that binds a Cas9 protein, comprising a sgRNA backbone sequence forming a first stem-loop structure; and
(2) a DNA-targeting domain, comprising
(i) a guide region comprising a sequence of at least 10 contiguous nucleotides that is 100% complementary to a first region of a target DNA sequence;
(ii) a guide block region comprising a sequence of at least 10 contiguous nucleotides that is 100% complementary to a nucleotide sequence of the guide region (i); and
(iii) an xDNA sensor region that hybridizes to a second region of the target DNA,
wherein the domain that binds a Cas9 protein (1) and the DNA-targeting domain (2) do not overlap, and wherein the guide region (i) and the guide block region (ii) do not overlap.

2. The xDNA-sensing sgRNA of claim 1 , wherein region (i) comprises a sequence of at least 15 contiguous nucleotides that is 100% complementary to the first region of the target DNA sequence, region (ii) comprises a sequence of at least 15 contiguous nucleotides that is 100% complementary to the nucleotide sequence of the guide region (i), and wherein the xDNA sensor region comprises at least 10 nucleotides.

3. The xDNA-sensing sgRNA of claim 1 , wherein the DNA-targeting domain (2) forms a second stem-loop structure in which the stem comprises a sequence of the guide block region (ii) hybridized to part or all of the guide region (i), and the loop is formed by part or all of the sequence of the xDNA sensor region (iii).

4. The xDNA-sensing sgRNA of claim 3 , wherein the guide block region (ii) and the xDNA sensor region (iii) are both either 5′ or 3′ to the guide region (i).

5. The xDNA-sensing sgRNA of claim 3 , wherein the second stem-loop structure forms in the absence of the target DNA that hybridizes to the xDNA sensor region (iii).

6. The xDNA-sensing sgRNA of claim 3 , wherein binding of the target DNA to the xDNA sensor region (iii) results in the unfolding of the second stem-loop structure, or prevents the formation of the second stem-loop structure, such that the guide block region (ii) does not hybridize to the guide region (i).

7. The xDNA-sensing sgRNA of claim 1 , wherein the sgRNA binds a Cas9 protein, and wherein the guide region (i) binds the target DNA when the xDNA sensor region (iii) binds the target DNA.

8. A complex comprising the xDNA-sensing sgRNA of claim 1 and a Cas9 protein.

9. The complex of claim 8 further comprising a target DNA.

10. An isolated polynucleotide encoding the xDNA-sensing sgRNA of claim 1 .

11. A vector comprising the polynucleotide of claim 10 .

12. A vector for recombinant expression comprising a polynucleotide encoding a xDNA-sensing gRNA of claim 1 and optionally a polynucleotide encoding a Cas9 protein.

13. An isolated cell comprising a genetic construct for expressing the xDNA-sensing sgRNA of claim 1 .

14. A method for site specific DNA cleavage comprising contacting a DNA with a complex comprising Cas9 and the xDNA-sensing sgRNA of claim 1 , wherein the binding of the complex to the DNA results in DNA cleavage.

15. The method of claim 14 , wherein the DNA is in a cell.

16. The method of claim 15 , wherein the cell is in vitro.

17. The method of claim 15 , wherein the cell is a eukaryotic cell in an individual.

18. The method of claim 17 , wherein the individual is a human.

19. The xDNA-sensing sgRNA of claim 1 , wherein the stem loop structure of the sgRNA backbone comprises
(a) a sequence that is homologous to a tracrRNA sequence; and
(b) a sequence that is homologous to a crRNA sequence,

wherein the sequences of (a) and (b) hybridize to form the stem of the stem-loop structure, and wherein the 5′-end of the tracrRNA is connected to the 3′-end of the crRNA sequence via a polynucleotide linker that forms the loop of the stem-loop structure.

20. The xDNA-sensing sgRNA of claim 1 , wherein the sgRNA backbone sequence comprises a nucleotide sequence that is at least 90% identical the entire length of to the nucleotide sequence 5′-GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC-3′ (nucleotides 53-107 of SEQ ID NO: 9) and/or at least 90% identical the entire length of to the nucleotide sequence 5′-GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAU-3′ (nucleotides 22-59 of SEQ ID NO: 8).

21. The xDNA-sensing sgRNA of claim 1 , wherein the stem loop of the sgRNA backbone is formed by the sequence 5′-GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAU-3′ (residues 53-90 of SEQ ID NO: 9).

22. The xDNA-sensing sgRNA of claim 1 , wherein the guide block region (ii) is positioned 5′ of the guide region (i).

23. The xDNA-sensing sgRNA of claim 1 , wherein the xDNA sensor region (iii) is positioned 5′ of the guide region (i).

24. The xDNA-sensing sgRNA of claim 1 , wherein the guide block region (ii) and the xDNA sensor region (iii) are positioned 5′ of the guide region (i).

25. The xDNA-sensing sgRNA of claim 1 , wherein the first region and the second region of the target DNA do not overlap.

26. The xDNA-sensing sgRNA of claim 1 , wherein the guide region (i) comprises at least 15 contiguous nucleotides that are 100% complementary to a nucleic acid sequence of the first region of the target DNA sequence.

27. The xDNA-sensing sgRNA of claim 1 , wherein the guide region (i) comprises at least 20 contiguous nucleotides that are 100% complementary to a nucleic acid sequence of the first region of the target DNA sequence.

28. The xDNA-sensing sgRNA of claim 1 , wherein the guide region (i) comprises at least 25 contiguous nucleotides that are 100% complementary to a nucleic acid sequence of the first region of the target DNA sequence.

29. The xDNA-sensing sgRNA of claim 1 , wherein the target DNA sequence is a genomic DNA sequence.

30. The xDNA-sensing sgRNA of claim 1 , wherein the guide region (i), the guide block region (ii) and the xDNA sensor region (iii) do not overlap.

Assignees

• Harvard College

Inventors

• Liu David R
• Hu Johnny Hao

Classifications

• C12N15/63

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• C12N15/102 Primary

  Mutagenizing nucleic acids · CPC title

• A61K48/00

  Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy · CPC title

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title

• C12N2320/53

  reducing unwanted side-effects · CPC title