CAS9-based Isothermal Method of Detection of Specific DNA Sequence

• US 20150211058A1
• Filed: 01/28/2015
• Published: 07/30/2015
• Est. Priority Date: 01/29/2014
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Carstens, Carsten-Peter
• Granted Patent: US 9,850,525 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

The present invention relates to an isothermal method for detecting in a sample a target nucleic acid strand.

Claims

1. An isothermal method for detecting in a sample a target nucleic acid strand having (i) a first CAS-targeted site having a first target sequence and a first PAM site and (ii) a second CAS-targeted site having a second target sequence and a second PAM site, the first target sequence being different from the second target sequence, the method comprising the steps of:

(a) contacting a sample suspected to contain the target nucleic acid strand with;

a first CAS9 mutant having a single-strand nicking activity,

a first guide RNA (gRNA) targeting the first CAS-targeted site,

a strand-displacing nucleic acid polymerase, and

nucleotides,

under conditions allowing

nicking of the target nucleic acid strand by the CAS9 mutant at the first CAS targeted cite, and

strand-displacing by the strand-displacing nucleic acid polymerase to create one or more copies of a section of the target nucleic acid strand, each copy containing the second CAS-targeted site;

(b) contacting said one or more copies with;

a second CAS9 mutant having a single-strand nicking activity,

a second gRNA targeting the second CAS-targeted site,

a strand-displacing nucleic acid polymerase,

nucleotides, and

one or more circular probes, each having a tag region and a CAS region that is complementary to the second CAS-targeted site,

under conditions allowing

hybridizing of said one or more copies to said one or more circular probes to generate one or more annealed copies,

nicking of said one or more annealed copies by the second CAS9 mutant at the second CAS-targeted cite, and

strand-displacing by the strand-displacing nucleic acid polymerase to create one or more extension products of said one or more annealed copies, each product containing a detecting region that is complementary to the tag region and recognizable by a detecting agent; and

(c) detecting presence of the one or more extension products using the detecting agent, whereby the presence of the one or more extension products is an indicator of the presence of the target nucleic acid strand in the sample.

2. The method of claim 1, wherein steps (a) and (b) are carried out by incubating a reaction mixture containing

(i) the sample,

(ii) the first CAS9 mutant,

(iii) the first gRNA,

(iv) the second CAS9 mutant,

(v) the second gRNA,

(vi) the strand-displacing nucleic acid polymerase,

(vii) the nucleotides, and

(viii) the one or more circular probes

under conditions permitting:

(i) nicking the target nucleic acid strand by the first CAS9 mutant at the first CAS targeted cite,

(ii) strand displacing by the strand-displacing nucleic acid polymerase to create said one or more copies,

(iii) hybridizing said one or more copies to said one or more circular probes to generate said one or more annealed copies,

(iv) nicking said one or more annealed copies by the second CAS9 mutant at the second CAS-targeted cite, and

(v) strand displacing by the strand-displacing nucleic acid polymerase to create said one or more extension products.

3. The method of claim 1, wherein the first or second CAS9 mutant is a D10A or H839A mutant version of SEQ ID NO.:

1.

4. The method of claim 1, wherein the strand-displacing nucleic acid polymerase is a φ

29 DNA polymerase.

5. The method of claim 1, wherein the detecting agent is a nucleotide probe.

6. The method of claim 5, wherein the nucleotide probe is a molecular beacon probe or a Yin-Yang probe that is labeled with a fluorophore and a quencher.

7. The method of claim 6, wherein detecting step is carried out by measuring the fluorescent signal emitted upon hybridization of the nucleotide probe to the detecting region.

8. The method of claim 1, wherein said one or more copies contain the first PAM site, or said one or more extension products contain the second CAS-targeted site.

9. The method of claim 1, wherein the target nucleic acid strand is on one strand of a genomic DNA of a microorganism or a cell of a subject.

10. The method of claim 9, wherein the sample contains the microorganism or a cell of the subject and the method further comprises lysing the microorganism or the cell before step (a) or (b).

11. The method of claim 8, wherein the target nucleic acid strand contains a mutation of the subject.

12. The method of claim 11, wherein the mutation is a translocation or an inversion.

13. The method of claim 9, wherein the subject is an animal.

14. The method of claim 13, wherein the animal is a human.

15. The method of claim 9, wherein the microorganism is selected from the group consisting of a virus, a bacterium, and a fungus.

16. The method of claim 9, wherein the microorganism is a pathogen.

17. An in vitro, cell-free composition comprising:

a first CAS9 mutant having a single-strand nicking activity,

a first gRNA, and

one or more reagents selected from the group consisting of a strand-displacing nucleic acid polymerase, nucleotides, a second CAS9 mutant having a single-strand nicking activity, a second gRNA, a detecting agent, and a circular probe.

wherein the first gRNA and the second gRNA target a first CAS-targeted site and a second CAS-targeted site, respectively, the first target sequence being different from the second target sequence, and

wherein the circular probe has (i) a CAS region that is substantially complementary to the second CAS-targeted site and (ii) a tag region the complement of which is recognizable by the detecting agent.

18. The composition of claim 17, wherein the detecting agent is a nucleotide probe.

19. The composition of claim 18, wherein the nucleotide probe is a molecular beacon probe or a Yin-Yang probe that is labeled with a fluorophore and a quencher.

20. A kit comprising

a first CAS9 mutant having a single-strand nicking activity,

a first gRNA, and

one or more reagents selected from the group consisting of a strand-displacing nucleic acid polymerase, nucleotides, a second CAS9 mutant having a single-strand nicking activity, a second gRNA, a detecting agent, and a circular probe.

wherein the first gRNA and the second gRNA target a first CAS-targeted site and a second CAS-targeted site, respectively, the first target sequence being different from the second target sequence, and

wherein the circular probe has (i) a CAS region that is substantially complementary to the second CAS-targeted site and (ii) a tag region the complement of which is recognizable by the detecting agent.