Optimized small guide RNAs and methods of use

Patent metadata

Field	Value
Publication number	US-10822606-B2
Application number	US-201415025217-A
Country	US
Kind code	B2
Filing date	Sep 29, 2014
Priority date	Sep 27, 2013
Publication date	Nov 3, 2020
Grant date	Nov 3, 2020

Abstract

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

First claim

What is claimed is:

1. A method of genome imaging a target nucleic acid sequence in a living cell nucleus, the method comprising:
(i) introducing into the living cell:
(a) one or more small guide RNAs (sgRNAs) comprising sequences of SEQ ID NO:4, SEQ ID NO:8, and/or SEQ ID NO:17, wherein each of the sgRNAs is specific for the target nucleic acid sequence in the living cell nucleus; and
(b) a first labeled nuclease-deficient Cas9 (dCas9), thereby forming a first labeled complex comprising at least one of the sgRNAs and the first labeled dCas9; and

(ii) incubating the living cell to allow the first labeled complex to localize to the target nucleic acid sequence in the living cell nucleus; and
(iii) imaging the presence or quantity of the first labeled complex in the nucleus of the living cell, thereby imaging the target nucleic acid.

2. The method of claim 1 , wherein the one or more sgRNAs comprise at least 2 different sgRNAs, each specific for a different portion of the target nucleic acid sequence.

3. The method of claim 1 , wherein the one or more sgRNAs are specific for a repeated target sequence.

4. The method of claim 1 , wherein the introducing further comprises forming a second labeled complex comprising the one or more sgRNAs and a second labeled dCas9; and
contacting the living cell with the second labeled complex.

5. The method of claim 4 , wherein the first and second labeled complexes are specific for a different target nucleic acid sequence in the living cell nucleus or specific for a different region of a chromosome in the living cell nucleus.

6. The method of claim 4 , wherein the first and second labeled complexes are labeled with a different label, and the method comprises imaging the presence or quantity of the first and second labeled complexes in the nucleus of the living cell, thereby imaging the target nucleic acid.

7. A method of genome imaging a target nucleic acid sequence in a living cell nucleus, the method comprising:
(i) introducing into the living cell:
(a) one or more small guide RNAs (sgRNAs) comprising a sequence of SEQ ID NO:4 and/or SEQ ID NO:8, wherein each of the sgRNAs is specific for the target nucleic acid sequence in the living cell nucleus; and
(b) a first labeled nuclease-deficient Cas9 (dCas9), thereby forming a first labeled complex comprising at least one of the sgRNAs and the first labeled dCas9; and
(ii) incubating the living cell to allow the first labeled complex to localize to the target nucleic acid sequence in the living cell nucleus; and
(iii) imaging the presence or quantity of the first labeled complex in the nucleus of the living cell, thereby imaging the target nucleic acid.

8. The method of claim 1 , wherein the one or more sgRNAs comprise SEQ ID NO:4.

9. The method of claim 1 , wherein the first labeled dCas9 comprises a Cas9 protein containing a mutation at one or more of the following residues:
D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, or A987.

Assignees

• Univ California

Inventors

• Huang Bo
• Chen Baohui
• Qi Lei
• Gilbert Luke

Classifications

• C12N9/22 Primary

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

• C12N2310/10

  Type of nucleic acid · CPC title

• C12N2310/20

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

• C12N15/111

  General methods applicable to biologically active non-coding nucleic acids · CPC title

• C12Y301/00

  Hydrolases acting on ester bonds (3.1) · CPC title