Application of cas protein, method for detecting target nucleic acid molecule and kit

Abstract

The present invention provides use of a Case 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, characterized by adding a guide RNA, a Cas protein, a nucleic acid probe and a buffer into the system containing the target nucleic acid molecules to be detected, and then detecting the nucleic acid probe. 
     
     
         2 . The method for detecting target nucleic acid molecules of  claim 1 , characterized in that the Cas protein is Cas12a or a Cas protein having an activity similar to the collateral single-stranded DNA cleavage activity of Cas12a;
 the Cas12a is preferably one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a; the Cas12a is preferably LbCas12a.   
     
     
         3 . The method for detecting target nucleic acid molecules of  claim 1 , characterized in that the guide RNA refers to an RNA that directs the Cas protein to specifically bind to the target DNA. 
     
     
         4 . The method for detecting target nucleic acid molecules of  claim 1 , characterized in that the nucleic acid probe is a single-stranded DNA; the single-stranded DNA is preferably a fluorescently labelled single-stranded DNA; and the single-stranded DNA is preferably a fluorescent probe labelled with a fluorescent group HEX at the 5′ end and a quenching group BHQ1 at the 3′ end; preferably,
 the detection method of the nucleic acid probe is preferably a fluorescence detection method; and the fluorescence detection method is preferably detection method using a microplate reader or a fluorescence spectrophotometer. 
 
     
     
         5 . The method for detecting target nucleic acid molecules of any one of  claims 1  to  4 , characterized in that the target nucleic acid molecules to be detected in the reaction system of the target nucleic acid molecules to be detected are obtained by amplification. 
     
     
         6 . The method for detecting target nucleic acid molecules of  claim 5 , characterized in that the detection method can detect pathogenic microorganisms, gene mutations or specific target DNA. 
     
     
         7 . The method of  claim 1 , characterized in that the Cas protein comprises Cas12b (i.e., C2c1). 
     
     
         8 . A use of a Cas protein in a method for detecting target nucleic acid molecules. 
     
     
         9 . The use of  claim 8 , characterized in that when the target DNA, the guide RNA and the Cas protein form a ternary complex, the complex cleaves other single-stranded DNA molecules in the system; preferably, the guide RNA refers to RNA that directs the Cas protein to specifically bind to the target DNA. 
     
     
         10 . A kit for detecting target nucleic acid molecules, characterized in that the kit comprises a guide RNA, a Cas protein, and a nucleic acid probe. 
     
     
         11 . A detection system for detecting target nucleic acid molecules, characterized in that the system comprises:
 (a) a Cas protein, which is Cas12a or a Cas protein having an activity similar to the collateral single-stranded DNA cleavage activity of Cas12a;   (b) a guide RNA, which directs the Cas protein to specifically bind to target nucleic acid molecules; and   (c) a nucleic acid probe, which is a single-stranded DNA;   wherein the target nucleic acid molecule is a target DNA.   
     
     
         12 . The detection system of  claim 11 , characterized in that the Cas protein having an activity similar to the collateral single-stranded DNA cleavage activity of Cas12a is selected from the group consisting of: Cas12b (i.e., C2c1). 
     
     
         13 . The detection system of  claim 11 , characterized in that the nucleic acid probe comprises a single-stranded DNA with a detectable label. 
     
     
         14 . A kit for detecting target nucleic acid molecules, characterized in that the kit comprises:
 i) a first container and a Cas protein located in the first container, the Cas protein is Cas12a or a Cas protein having an activity similar to the collateral single-strand DNA cleavage activity of Cas12a;   ii) optionally a second container and a guide RNA located in the second container, the guide RNA directs the Cas protein to specifically bind to the target nucleic acid molecules;   iii) a third container and a nucleic acid probe located in the third container; and   iv) optionally a fourth container and a buffer located in the fourth container;   wherein the target nucleic acid molecule is a target DNA.   
     
     
         15 . A method for detecting the presence or absence of target nucleic acid molecules in a sample, characterized by comprising the steps of:
 (a) providing a detection system for detecting target nucleic acid molecules according to  claim 11 , and the detection system further contains a sample to be detected; and   (b) detecting whether the nucleic acid probe in the detection system is cleaved by the Cas protein, the cleavage is a trans-cleavage of the collateral single-stranded DNA;   wherein, if the nucleic acid probe is cleaved by the Cas protein, it indicates that the target nucleic acid molecule is present in the sample; and if the nucleic acid probe is not cleaved by the Cas protein, it indicates that the target nucleic acid molecule is not present in the sample.   
     
     
         16 . The method of  claim 15 , characterized in that the nucleic acid amplification method is 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. 
     
     
         17 . The method of  claim 15 , characterized in that, when the PAM sequence is absent at the upstream or downstream of the target site (in the range of from −20 nt to +20 nt, preferably in the range of from −15 nt to +15 nt, more preferably in the range of from −10 nt to +10 nt), nucleic acid amplification was carried out using primers introduced with PAM. 
     
     
         18 . The method of  claim 17 , characterized in that the primer introduced with PAM has a structure of formula I from 5′ to 3′:
   P1-P2-P3   (I)
 
 wherein, 
 P1 is a 5′ segment sequence complementary or non-complementary to the sequence of the target nucleic acid molecule at the 5′ end; 
 P2 is a PAM sequence; and 
 P3 is a 3′ segment sequence complementary to the sequence of the target nucleic acid molecule at the 3′ end. 
 
     
     
         19 . The method of  claim 15 , characterized in that, when the PAM sequence is present at the upstream or downstream of the target site (in the range of from −20 nt to +20 nt, preferably in the range of from −15 nt to +15 nt, more preferably in the range of from −10 nt to +10 nt), primers with or without a PAM sequence can be used, and the amplified amplification product contains the PAM sequence. 
     
     
         20 . A use of a Cas protein, characterized by being used for the preparation of a detection reagent or kit for detecting target nucleic acid molecules based on a collateral single-stranded DNA cleavage, wherein the Cas protein is Cas12a or a Cas protein having an activity similar to the collateral single-strand DNA cleavage activity of Cas12a. 
     
     
         21 . The use of  claim 20 , characterized in that the Cas protein having an activity similar to the collateral single-stranded DNA cleavage activity of Cas12a is selected from the group consisting of: Cas12b (or C2c1). 
     
     
         22 . A method for detecting target nucleic acid molecules comprising steps of:
 (a) providing a mixture including:
 a Cas12 protein having collateral cleavage activity; 
 a guide RNA operative with the Cas12 protein; 
 a detectably labeled nucleic acid probe susceptible to the collateral cleavage activity; 
 a buffer; and 
 a system in which target nucleic acid molecules are to be detected; and 
   (b) detecting cleavage of the nucleic acid probe.   
     
     
         23 . The method of  claim 22 , wherein the guide RNA specifically binds to the target nucleic acid molecule(s). 
     
     
         24 . The method of  claim 22 , wherein the Cas12 protein is selected from the group consisting of: FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a, Lb4Cas12a; LbCas12a and Cas12b (i.e., C2c1). 
     
     
         25 . The method of  claim 22 , wherein the target nucleic acid molecules to be detected have been amplified. 
     
     
         26 . The method of  claim 25 , wherein the target nucleic acid molecules have been amplified by a 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. 
     
     
         27 . The method of  claim 25 , wherein the target nucleic acid molecules have been amplified using primers comprising a PAM sequence. 
     
     
         28 . The method of  claim 27 , wherein the amplification product comprises a PAM sequence upstream or downstream of the guide RNA's target site. 
     
     
         29 . The method of  claim 28 , wherein the PAM site is −20 nt to +20 nt upstream or downstream of the target site. 
     
     
         30 . The method of  claim 22 , wherein the nucleic acid is detectably labeled with a fluorescent label. 
     
     
         31 . The method of  claim 30 , wherein the fluorescent label comprises a fluorescent group HEX at the 5′ end and a quenching group BHQ1 at the 3′ end. 
     
     
         32 . The method of  claim 22 , wherein detecting cleavage of the nucleic acid probe comprises detecting fluorescence. 
     
     
         33 . The method of  claim 30 , wherein the step of detecting fluorescence comprises using a microplate reader or a fluorescence spectrophotometer. 
     
     
         34 . A combination comprising: a Cas12 protein having collateral cleavage activity;
 a guide RNA operative with the Cas12 protein and directed to a sequence in a target nucleic acid;   a detectably labeled nucleic acid probe; and   a buffer.   
     
     
         35 . The combination of  claim 34 , further comprising a sample in which the target nucleic acid is to be detected.