Optimized small guide rnas and methods of use

Patent metadata

Field	Value
Publication number	US-2016289673-A1
Application number	US-201415025217-A
Country	US
Kind code	A1
Filing date	Sep 29, 2014
Priority date	Sep 27, 2013
Publication date	Oct 6, 2016
Grant date	—

Abstract

Methods, compositions, and kits are provided herein for CRISPER/Cas-mediated nucleic acid detection or modification.

First claim

1 . A small guide RNA molecule comprising from 5′ to 3′:
a binding region, comprising between about 5 and about 50 nucleotides;
a 5′ hairpin region, comprising:
fewer than four consecutive uracil nucleotides; or
a length of at least 31 nucleotides; and

a 3′ hairpin region; and
a transcription termination sequence,
wherein the small guide RNA is configured to form a complex with a small guide RNA-mediated nuclease, the complex having increased stability or activity relative to a complex containing a small guide RNA-mediated nuclease and a small guide RNA comprising at least 95% identity to SEQ ID NO:1 or a complement thereof.

2 . The small guide RNA molecule of claim 1 , wherein the 5′ hairpin region comprises fewer than four consecutive uracil nucleotides and a length of at least 31 nucleotides.

3 . The small guide RNA molecule of claim 1 , wherein the small guide RNA molecule further comprises an additional hairpin region designed to interact with a protein or small-molecule to conditionally stabilize the secondary and/or tertiary structure of the small guide RNA molecule.

4 . The small guide RNA molecule of claim 1 , comprising at least 95% identity to SEQ ID NOs:2, 3, or 4, or a complement thereof.

5 . A composition for nucleic acid modification or detection comprising any of the small guide RNA molecules of claims 1 - 4 , the composition further comprising a small guide RNA-mediated nuclease, wherein the small guide RNA and the small guide RNA-mediated nuclease form a complex having increased stability or activity relative to a complex containing a small guide RNA comprising at least 95% identity to SEQ ID NO:1 or a complement thereof.

6 . The composition of claim 5 , wherein the composition is nuclease defective, thereby forming a complex configured to bind to, but not cleave or nick, a target nucleic acid substantially complementary to the binding region of the small guide RNA.

7 .- 9 . (canceled)

10 . The composition of claim 6 , wherein the nuclease defective composition comprises a labeled Cas9 protein.

11 .- 12 . (canceled)

13 . The composition of claim 5 , wherein the composition has nuclease activity, thereby forming a complex configured to bind and cleave a target nucleic acid sequence substantially complementary to the binding region of the small guide RNA.

14 . The composition of claim 13 , wherein the small guide RNA-mediated nuclease has nicking activity, but is substantially defective at catalyzing double stranded breaks in the target sequence.

15 . (canceled)

16 . An expression cassette comprising a promoter operably linked to a nucleic acid encoding any of the small guide RNAs of claims 1 - 4 .

17 . The expression cassette of claim 16 , wherein the promoter is an RNA polymerase III promoter.

18 .- 19 . (canceled)

20 . An expression cassette comprising a promoter operably linked to a nucleic acid encoding a small guide RNA-mediated nuclease, wherein:
the promoter is a weak mammalian promoter as compared to the human elongation factor 1 promoter (EF1A), wherein the weak mammalian promoter is selected from the group consisting of a ubiquitin C promoter, a phosphoglycerate kinase 1 promoter (PGK), and a TetOn promoter in the absence of an inducer; and the nucleic acid further encodes a one or two nuclear localization sequences.

21 .- 24 . (canceled)

25 . A host cell comprising any one of the small guide RNAs of claims 1 - 4 .

26 .- 29 . (canceled)

30 . A method of detecting a target nucleic acid sequence in a cell, the method comprising:
introducing into the cell:
(a) one or more small guide RNAs, each small guide RNA specific for the target nucleic acid sequence; and
(b) a labeled nuclease-deficient small guide RNA-mediated nuclease, thereby forming a labeled RNA:nuclease complex; and
(ii) incubating the cell to allow the labeled RNA:nuclease complex to localize to the target nucleic acid sequence; and (iii) detecting the presence, absence, or quantity of labeled complex in the nucleus of the cell, thereby detecting the target nucleic acid.

31 . The method of claim 30 , the method further comprising introducing at least 5 different small guide RNAs, each specific for a different portion of the target nucleic acid sequence.

32 . The method of claim 30 , wherein the one or more small guide RNAs are specific for a repeated target sequence.

33 .- 35 . (canceled)

36 . The method of claim 30 , wherein the introducing comprises:
forming a complex between:
a first labeled nuclease-deficient small guide RNA-mediated nuclease and one or more small guide RNAs, to form a first labeled complex; and
a second labeled nuclease deficient small guide RNA-mediated nuclease and one or more small guide RNAs, to form a second labeled complex; and
contacting the cell with the first and second complexes.

37 . (canceled)

38 . The method of claim 36 , wherein each labeled complex is specific for a different target nucleic acid sequence or specific for a different region of a chromosome.

39 . The method of claim 36 , wherein each labeled complex is labeled with a different label, and the method comprises detecting the presence, absence, or quantity of each labeled complex in the nucleus of the cell.

40 . A method of modifying a target nucleic acid sequence in a cell, the method comprising:
introducing into the cell:
(a) a small guide RNA of claims 1 - 4 , the small guide RNA specific for the target nucleic acid sequence; and
(b) a small guide RNA-mediated nuclease, thereby forming a small guide RNA:nuclease complex; and
(ii) incubating the cell to allow the small guide RNA:nuclease complex to bind to and cleave or nick the target nucleic acid, thereby modifying the target nucleic acid sequence in the cell.

41 .- 42 . (canceled)

43 . The method of claim 40 , wherein the method further comprises
(i) introducing into the cell:
(a) a pair of small guide RNAs of claims 1 - 4 , each small guide RNA specific for a target nucleic acid, wherein the pair of small guide RNAs bind to target nucleic acids on a chromosome and flank a nucleic acid region of interest; and
(b) a small guide RNA-mediated nuclease, thereby forming a pair of small guide RNA:nuclease complexes; and
(ii) incubating the cell to allow the small guide RNA:nuclease complexes to localize to the target nucleic acid sequence, thereby creating nicks or double stranded breaks that flank the nucleic acid region of interest; and (iii) incubating the cell to allow non homologous end joining (NHEJ) or homologous DNA repair (HDR) to occur, thereby reducing heterozygosity in the cell or deleting at least a portion of the nucleic acid region of interest.

44 . The method of claim 43 , wherein the method further comprises introducing into the cell a heterologous nucleic acid that contains regions of substantial homology to the nucleic acid region of interest, thereby incorporating at least a portion of the heterologous nucleic acid into the nucleic acid region of interest.

45 . A kit comprising an sgRNA of any one of claims 1 - 4 , and a labeled nuclease defective sgRNA-mediated nuclease.

46 .- 47 . (canceled)

Assignees

• Univ California

Inventors

• Huang Bo
• Chen Baohui
• Qi Lei

Classifications

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title

• C12N2310/531

  Stem-loop; Hairpin · CPC title

• C12N15/11 Primary

  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

• C12N2800/80

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