Application of CAS protein, method for detecting target nucleic acid molecule and kit
The present invention provides a use of a Cas protein, and a method and a kit for detecting target nucleic acid molecules. The method for detecting target nucleic acid molecules comprises adding a guide RNA, a Cas12a, and a nucleic acid probe into a reaction system containing target nucleic acid molecules to be detected, and detecting it after the reaction is completed.
1. A method for detecting target nucleic acid molecules comprising a target sequence, the method comprising steps of:
1) Adding (a) a guide RNA comprising a target site that specifically hybridizes with the target sequence;
(b) a Cas12 protein having collateral cleavage activity, wherein the collateral cleavage activity of the Cas 12 protein is activated when specific hybridization of the guide RNA and the target sequence occurs;
(c) a detectably labeled nucleic acid probe susceptible to the collateral cleavage activity of the Cas12 protein; and
(d) a buffer into a system containing the target nucleic acid molecules to be detected, and
2) then detecting cleavage of the nucleic acid probe.
2. The method of claim 1 , wherein the target nucleic acid molecules to be detected have been amplified.
3. The method of claim 2 , wherein the target nucleic acid molecules have been amplified by an amplification method selected from the group consisting of: PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification, rolling circle amplification, HDA, SPIA, NEAR, TMA and SMAP2.
4. The method of claim 2 , wherein the target nucleic acid molecules are amplified using primers comprising a protospacer adjacent motif (PAM) sequence.
5. The method of claim 4 , wherein the amplification product comprises a PAM sequence upstream or downstream of the target site of the guide RNA.
6. The method of claim 5 , wherein the PAM site is-20nt to +20nt upstream or downstream of the target site of the guide RNA.
7. The method of claim 1 , wherein detecting cleavage of the nucleic acid probe comprises a fluorescence detection method.
8. The method of claim 7 , wherein the fluorescence detection method comprises a detection method using a microplate reader or a fluorescence spectrophotometer.
9. The method of claim 1 , wherein the detectably labeled nucleic acid probe comprises a fluorescent group at the 5′ end and a quenching group at the 3′ end.
10. The method of claim 9 , wherein the fluorescent group is HEX and the quenching group is BHQ1.
11. The method of claim 1 , wherein the Cas12 protein is selected from the group consisting of: FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a; LbCas12a and C2c1.
12. The method of claim 1 , wherein the target nucleic acid molecules comprise a sequence found in a pathogen.
13. The method of claim 1 , wherein the target nucleic acid molecules comprise a sequence found in a human cancer.
14. A detection system comprising:
(a) target nucleic acid molecules comprising a target sequence of interest;
(b) a Cas12 protein having collateral cleavage activity;
(c) a guide RNA comprising a target site that specifically hybridizes with the target sequence of interest, wherein the collateral cleavage activity is activated when the specific hybridization occurs;
(d) a detectably labeled nucleic acid probe susceptible to the collateral cleavage activity of the Cas12 protein; and
(e) buffer.
15. The detection system of claim 14 , wherein the Cas12 protein is selected from the group consisting of: FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a and C2c1.
16. The detection system of claim 14 , wherein the target nucleic acid molecules to be detected have been amplified by an amplification method.
17. The detection system of claim 16 , wherein the target nucleic acid molecules have been amplified by an amplification method selected from the group consisting of: PCR amplification, LAMP amplification, RPA amplification, ligase chain reaction, branched DNA amplification, NASBA, SDA, transcription-mediated amplification, rolling circle amplification, HDA, SPIA, NEAR, TMA and SMAP2.
18. The detection system of claim 16 , wherein the target nucleic acid molecules are amplified using primers comprising a protospacer adjacent motif (PAM) sequence.
19. The detection system of claim 18 , wherein the amplification product comprises a PAM sequence upstream or downstream of a target site of the guide RNA.
20. The detection system of claim 19 , wherein the PAM site is −20nt to +20nt upstream or downstream of the target site of the guide RNA.
21. The detection system of claim 14 , wherein the target nucleic acid molecules comprise a sequence found in a pathogen.
22. The detection system of claim 14 , wherein the target nucleic acid molecules comprise a sequence found in a human cancer.
23. The detection system of claim 14 , wherein the detectably labeled nucleic acid probe comprises a fluorescent group at the 5′ end and a quenching group at the 3′ end.
24. The detection system of claim 23 , wherein the fluorescent is HEX and the quenching group is BHQ1.
25. A kit for detecting target nucleic acid molecules comprising a target sequence, characterized in that the kit comprises:
i) a first container and a Cas12 protein located in the first container, wherein the Cas12 protein has collateral cleavage activity;
ii) a second container and a guide RNA located in the second container, wherein the guide RNA that specifically hybridizes with the target sequence, wherein the collateral cleavage activity is activated when the specific hybridization occurs;
iii) a third container and a detectably labeled nucleic acid probe located in the third container, wherein the detectably labeled nucleic acid probe is susceptible to the collateral cleavage activity of the Cas12 protein; and
iv) a fourth container and a buffer located in the fourth container.