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 a target nucleic acid molecule that contains a target sequence of interest and is in a sample, the method comprising steps of:
(a) providing a mixture including:
a sample that might contain the target sequence of interest;
a Cas12 protein having collateral cleavage activity that is increased when the Cas12 protein is complexed with a guide RNA that is specifically hybridized with the target sequence of interest;
a guide RNA that specifically hybridizes with the target sequence of interest wherein, if the sample contains the target sequence of interest and the guide RNA hybridizes therewith, the collateral cleavage activity of the Cas12 protein is increased;
a nucleic acid probe susceptible to the collateral cleavage activity, so that the nucleic acid probe has a first uncleaved state and a second cleaved state, and it transitions from the first uncleaved state to the second cleaved state when cleaved by the Cas12 protein, the nucleic acid probe being detectably labeled so that its transition from the first state to the second state is detectable;
a buffer;
(b) incubating the mixture under conditions sufficient for specific hybridization of the guide RNA with the target sequence of interest; wherein if specific hybridization occurs the Cas12 protein collateral cleavage activity is increased and the Cas12 protein cleaves the nucleic acid probe so that the nucleic acid probe transitions from the first state to the second state; and
(c) determining extent of transition achieved during the step of incubating;
(d) comparing the achieved extent of transition with a background level to determine whether the target nucleic acid sequence of interest is present in the sample,
wherein extent of cleavage greater than the background level, indicates presence of the target nucleic acid sequence of interest in the sample.
2. 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; and C2c1.
3. The method of claim 1 , wherein the target nucleic acid molecule to be detected has been amplified by an amplification method.
4. The method of claim 3 , wherein the target nucleic acid molecule has 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.
5. The method of claim 3 , wherein the target nucleic acid molecule has been amplified using primers comprising a protospacer adjacent motif (PAM) sequence.
6. The method of claim 5 , wherein the amplification product comprises a PAM sequence upstream or downstream of the guide RNA's target site.
7. The method of claim 6 , wherein the PAM site is −20nt to +20nt upstream or downstream of the target site.
8. The method of claim 1 , wherein the nucleic acid probe comprises a fluorescent group at the 5′ end and a quenching group at the 3′ end.
9. The method of claim 8 , wherein determining whether the probe has transitioned from the first state to the second state comprises detecting emitted fluorescence.
10. The method of claim 9 , wherein the step of detecting fluorescence comprises using a microplate reader or a fluorescence spectrophotometer.
11. The method of claim 1 , wherein determining whether the probe has transitioned from the first state to the second state comprises detecting a difference in size.
12. The method of claim 8 , wherein the fluorescent group is HEX and the quenching group is BHQ1.