IMPROVED DETECTION ASSAYS
The present disclosure provides improved detection (e.g., diagnostic) assays that utilize a Cas protein collateral cleavage activity.
1 . A detection method comprising steps of:
contacting a CRISPR-Cas complex comprising:
a Cas protein with collateral cleavage activity that is thermostable at temperatures above at least 60-65° C.; and
a guide RNA selected or engineered to be complementary to a target sequence; with
a sample potentially comprising a nucleic acid of the target sequence.
2 . The method of claim 1 , wherein the step of contacting comprises contacting the CRISRP-Cas complex and sample with a reporter susceptible to cleavage by the Cas protein collateral activity.
3 . The method of claim 1 , wherein the step of contacting comprises incubating for a period of time above the temperature.
4 . The method of claim 1 , further comprising a step of amplifying nucleic acid present in the sample.
5 . The method of claim 4 , wherein the step of amplifying utilizes a thermostable nucleic acid polymerase.
6 . The method of claim 4 , wherein the steps of amplifying and contacting are performed in a single vessel.
7 . The method of claim 1 , wherein the Cas protein is a Cas12 protein.
8 . The method of claim 7 , wherein the Cas protein has an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 15.
9 . The method of claim 7 , wherein the Cas protein has an amino acid sequence having at least 80%, sequence identity to any one of SEQ ID Nos. 3-21, 33-47, 51-56, 68-178, and 274-283.
10 . The method of claim 1 , wherein the Cas protein has an amino acid sequence having 80%, sequence identity to any one of SEQ ID Nos. 1-283.
11 . In a method of performing a detection assay utilizing a Cas protein with collateral cleavage activity, the improvement that comprises utilizing a Cas protein with thermostable collateral cleavage activity.
12 . The improvement of claim 11 , wherein the Cas protein is a Cas12 protein.
13 . The improvement of claim 12 , wherein the Cas protein has an amino acid sequence that is at least 80% identical to that of SEQ ID NO: 15.
14 . The improvement of claim 12 , wherein the Cas protein has an amino acid sequence having at least 80%, sequence identity to any one of SEQ ID Nos. 3-21, 33-47, 51-56, 68-178, and 274-283.
15 . The improvement of claim 11 , wherein a method of performing a detection assay is conducted in a single reaction vessel.
16 . The improvement of claim 11 , wherein the thermostable collateral cleavage activity is thermostable above a temperature of about 60° C.
17 . The improvement of claim 11 , wherein the thermostable collateral cleavage activity is thermostable above a temperature of about 65° C.
18 . The improvement of claim 11 , wherein the Cas protein has an amino acid sequence having at least 80% sequence identity to any one of SEQ ID Nos. 1-283.