Methods of specifically labeling nucleic acids using CRISPR/Cas

• US 10,640,810 B2
• Filed: 10/18/2017
• Issued: 05/05/2020
• Est. Priority Date: 10/19/2016
• Status: Active Grant

• Current Assignee: Drexel University, Temple University of The Commonwealth System of Higher Education, The Wistar Institute
• Original Assignee: Drexel University
• Inventors: Xiao, Ming, Riethman, Harold C., Hu, Wenhui, McCaffrey, Jennifer
• Primary Examiner(s): Leith, Nancy J
• Application Number:
  US15/787,319
• Publication Number:
  US 20180105867A1
• Time in Patent Office: 930 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53

Abstract

Provided herein are methods of detecting a target nucleic acid sequence. In one embodiment, the method includes contacting genomic DNA with a guide RNA having a portion complementary to the target sequence in the genomic DNA and with Cas9 nickase to produce a single-strand break in the genomic DNA at a specific location adjacent to the target sequence. The method further includes contacting the nicked DNA with a polymerase and fluorescently labeled nucleotide. The fluorescently labeled nucleotide is incorporated into the nicked DNA at the specific location and the target nucleic acid sequence is detected via fluorescent label.

Claims

1. A method of labeling a nucleic acid sequence comprising:

a) contacting genomic DNA with a guide RNA having a seed sequence which is complementary to a target sequence in the genomic DNA and with Cas9 nickase to produce a nick in the genomic DNA at a specific location adjacent to the target sequence; and

contacting the nicked DNA with a polymerase and fluorescently labeled nucleotide, wherein the fluorescently labeled nucleotide is incorporated into the nicked genomic DNA at the specific location; and

b) contacting the nicked genomic DNA with a motif-specific nicking endonuclease thereby producing a second nick in the nicked genomic DNA at a motif sequence; and

contacting the nicked genomic DNA with a polymerase and second fluorescently labeled nucleotide of the same color or different color, wherein the second fluorescently labeled nucleotide is incorporated into the nicked genomic DNA at the motif sequence location;

wherein nick-labeled DNA is produced; and

wherein the nick-labeled DNA is detected via fluorescent label.

2. The method of claim 1, wherein step b) is performed before step a).

3. The method of claim 1, wherein a pattern of fluorescence provided by the Cas9 nickase is used as a barcode to identify the location or identity of the genomic DNA.

4. The method of claim 1, wherein a pattern of fluorescence provided by the motif-specific nicking endonuclease is used as a barcode to identify the location or identity of the genomic DNA.

5. The method of claim 1, wherein a pattern of fluorescence provided by the Cas9 nickase and the motifi-specific nicking endonuclease is used as a barcode to identify the location or identity of the genomic DNA.

6. The method according to claim 1, wherein the guide RNA comprises a crRNA and a tracrRNA.

7. The method according to claim 1, wherein the guide RNA is a single gRNA sequence.

8. The method according to claim 1, where the guide RNA and the Cas9 nickase are contacted with each other to form a complex, prior to contacting with the genomic DNA.

9. The method according to claim 1, wherein the Cas9 nickase is Cas9 D10A or H840A.

10. The method according to claim 1, wherein the genomic DNA is contacted with multiple guide RNAs, each guide RNA having a seed sequence complementary to a different target sequence in the genomic DNA, wherein each target nucleic acid sequence is detected via the same or different fluorescent label, thus providing a barcode of a portion of the genomic DNA.

11. The method of claim 1, wherein the guide RNA targets a repeated sequence.

12. The method according to claim 1, wherein the target sequence is a viral nucleic acid sequence.

13. The method according to claim 1, wherein the target sequence is a sequence adjacent to a translocation breakpoint.

14. The method according to claim 1, further comprising repairing the nick-labeled DNA with a ligase.

15. The method according to claim 1, further comprising imaging the nick-labeled DNA in a nanochannel.

16. The method according to claim 15, comprising transporting the nick-labeled DNA into a nanochannel and maintaining the DNA in elongated form in the nanochannel.

17. The method according to claim 1, further comprising staining the DNA backbone.

18. The method according to claim 1, wherein the motif-specific nicking endonuclease is Nt.BspQI.

19. The method according to claim 1, wherein the target sequence is present in more than one location in the genomic DNA.

20. The method according to claim 1, further comprising one or more of:

i. treating the DNA with RNAse;

ii. treating the DNA with protease;

iii. Removing labeled free nucleotides;

iv. Linearizing the DNA; and

v. Fragmenting the DNA.