CIS-blocked guide RNA

Patent metadata

Field	Value
Publication number	US-9932566-B2
Application number	US-201514820521-A
Country	US
Kind code	B2
Filing date	Aug 6, 2015
Priority date	Aug 7, 2014
Publication date	Apr 3, 2018
Grant date	Apr 3, 2018

Abstract

This invention discloses reagents and methods for increasing specificity and efficiency of RNA-guided genome editing.

First claim

What is claimed is:

1. A guide RNA comprising an RNA molecule or a group of RNA molecules, wherein the guide RNA comprises:
a guide sequence complementary to a target site,
a protein-binding sequence with tracrRNA activity, and
a blocking sequence,
wherein the guide sequence is linked to the blocking sequence, optionally by a linker, wherein the guide sequence is complementary to a target sequence, and the blocking sequence is complementary to at least a portion of the guide sequence such that the blocking sequence competes with the target site and an off-target site for hybridization to the guide sequence, and wherein:
(i) hybridization of the guide sequence to the target site is thermodynamically favored over competing hybridization of the guide sequence to the blocking sequence, and
(ii) hybridization of the guide sequence to the blocking sequence is thermodynamically, favored over competing hybridization of the guide sequence to the off-target site.

2. The guide RNA of claim 1 , comprising a linker between the guide sequence and the blocking sequence.

3. The guide RNA of claim 1 , wherein the guide sequence is 12-25 nucleotides long.

4. The guide RNA of any of claim 1 , wherein the blocking sequence is 5-20 nucleotides lone.

5. The guide RNA of claim 1 , wherein the guide RNA is a single-guide RNA comprising the guide sequence and the region with tracrRNA activities.

6. The guide RNA of claim 1 , wherein the guide sequence, the blocking sequence, or both include one or more modified nucleotides.

7. The guide RNA of claim 2 , wherein the linker is 4-20 nucleotides long.

8. The guide RNA of claim 2 , wherein the linker includes one or modified nucleotides.

9. The guide RNA of claim 2 , wherein the linker includes one or more spacers selected from the group consisting of a hexanediol spacer, a TEG spacer, a 3-amino-1-propanol (C3) spacer, and a polyA spacer.

10. The guide RNA of claim 1 , wherein said portion of the guide sequence is a 5′ portion, a middle portion, or a 3′ portion.

11. A kit comprising a CRISPR protein or a nucleic acid encoding the CRISPR protein and a guide RNA of claim 1 .

12. The kit of claim 11 , wherein the CRISPR protein is a type II CRISPR protein.

13. A vector encoding the guide RNA of claim 1 .

14. The vector of claim 13 , further encoding a CRISPR protein.

15. A method for cutting a target nucleic acid with a CRISPR protein or for binding a target nucleic acid with a CRISPR protein, comprising:
contacting the target nucleic acid with a CRISPR protein and a guide RNA comprising RNA molecule or a group of RNA molecules, wherein the guide RNA comprises:
a guide sequence complementary to the target nucleic acid,
a protein-binding sequence having tracrRNA activity, and
a blocking sequence weakly base-paired with at least a portion of the guide sequence,
wherein the guide sequence is linked to the blocking sequence, optionally by a linker,

melting the weakly base-paired blocking sequence from the guide sequence when the blocked guide sequence recognizes the target nucleic acid; and
hybridizing the target nucleic acid with the guide sequence, wherein the hybridizing of the target nucleic acid is thermodynamically favored over binding of off-target sites, to result in a double-strand break or a single-strand break in the target nucleic acid or to result in binding of the target nucleic acid by the CRISPR protein.

16. The method of claim 15 , wherein the target nucleic acid is located in a cell or embryo and the method comprises introducing a nucleic acid that expresses the CRISPR protein into the cell.

17. The method of claim 16 , wherein the target nucleic acid is located in a cell or an embryo, and the method comprises introducing a vector that expresses, the CRISPR protein and the guide RNA into the cell or the embryo.

18. The method of claim 15 , wherein the target nucleic acid is a genomic DNA of a microorganism or a cell of a subject.

19. The method of claim 15 , wherein the CRISPR protein is a type II CRISPR protein.

Assignees

• Agilent Technologies Inc

Inventors

• Kennedy Andrew
• Ryan Daniel E

Classifications

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/902

  using homologous recombination · CPC title

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12Y301/04

  Phosphoric diester hydrolases (3.1.4) · CPC title