DETECTION ASSAYS
The present disclosure provides improved detection (e.g., diagnostic) assays that utilize a Cas protein collateral cleavage activity.
1 - 18 . (canceled)
19 . An engineered composition comprising:
(a) a Cas protein having collateral cleavage activity that is thermostable at temperatures above at least 50° C.; and
(b) at least one guide capable of forming a complex with the thermostable Cas protein.
20 . The composition of claim 19 , wherein the Cas protein is a Cas12 protein.
21 . The composition of claim 20 , wherein the Cas12 protein is a Cas12a.
22 . The composition of claim 21 , wherein the Cas12a protein has an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NO: 15, 18, 83, 95, 99 and 101.
23 . The composition of claim 22 , wherein the Cas12a protein has an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 15.
24 . The composition of claim 19 , wherein the Cas protein is a Cas13 protein.
25 . The composition of claim 19 , wherein the collateral cleavage activity is thermostable above a temperature of at least 55° C.
26 . A method of detecting a target nucleic acid sequence in a sample comprising: contacting the sample with a mixture comprising:
(a) a Cas protein with collateral cleavage activity that is thermostable at temperatures above at least 50° C.;
(b) a guide polynucleotide that specifically hybridizes with the target nucleic acid sequence and can form a complex with the thermostable Cas protein; and
(c) a detectably labeled nucleic acid probe susceptible to collateral cleavage activity of the Cas protein,
wherein the Cas protein collateral cleavage activity is activated when the guide polynucleotide hybridizes with the target; and
wherein cleavage of the detectably labeled nucleic acid probe indicates presence of the target nucleic acid sequence in the sample.
27 . The method of claim 26 , wherein the Cas protein is a Cas12 protein.
28 . The method of claim 27 , wherein the Cas12 protein is a Cas12a.
29 . The method of claim 28 , wherein the Cas12a protein has an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NO: 15, 18, 83, 95, 99 and 101.
30 . The method of claim 29 , wherein the Cas12a protein has an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 15.
31 . The method of claim 26 , wherein the Cas protein is a Cas13 protein.
32 . The method of claim 26 , wherein the target sequence to be detected has been amplified by an amplification method.
33 . The method of claim 32 , 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.
34 . The method of claim 33 , wherein the step of amplifying utilizes a thermostable nucleic acid polymerase.
35 . The method of claim 33 , wherein the steps of amplifying and contacting are performed in a single vessel.
36 . The method of claim 26 , wherein the collateral cleavage activity is thermostable above a temperature of at least 55° C.
37 . In a method of detecting a target nucleic acid in a sample, using a Cas protein with collateral cleavage activity, the improvement that comprises:
using a Cas protein whose collateral cleavage activity is thermostable
38 . The improvement of claim 37 , wherein the method of detecting a target nucleic acid in a sample is conducted in a single vessel.
39 . The improvement of claim 37 , wherein the Cas protein is a Cas12 protein.
40 . The improvement of claim 39 , wherein the Cas12 protein is a Cas12a.
41 . The improvement of claim 40 , wherein the Cas12a protein has an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NO: 15, 18, 83, 95, 99 and 101.
42 . The improvement of claim 41 , wherein the Cas12a protein has an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 15.
43 . The improvement of claim 37 , wherein the Cas protein is a Cas13 protein.
44 . The improvement of claim 37 , wherein the thermostable collateral cleavage activity is thermostable above a temperature of at least 50° C.
45 . The improvement of claim 37 , wherein the collateral cleavage activity is thermostable above a temperature of at least 55° C.
46 . The improvement of claim 37 , wherein the target sequence to be detected has been amplified by an amplification method.
47 . The improvement of claim 46 , 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.