Programmable nuclease compositions and methods of use thereof

Abstract

Described herein are devices, systems, fluidic devices, kits, and methods for detection of target nucleic acids associated with diseases, cancers, genetic disorders, a genotype, a phenotype, or ancestral origin. The devices, systems, fluidic devices, kits, and methods may comprise reagents of a guide nucleic acid targeting a target nucleic acid, a programmable nuclease, and a single stranded detector nucleic acid with a detection moiety. The target nucleic acid of interest may be indicative of a disease, and the disease may be communicable diseases, or of a cancer or genetic disorder. The target nucleic acid of interest may be indicative of a genotype, a phenotype, or ancestral origin.

1 .- 96 . (canceled) 
     
     
         97 . A cartridge for detecting a target nucleic acid, the cartridge comprising:
 (a) a first chamber for receiving a sample comprising nucleic acids; and   (b) a second chamber fluidically connected by a first valve to the first chamber;   wherein: (i) the second chamber comprises a programmable nuclease, a guide nucleic acid, and a reporter; (ii) the programmable nuclease is effective to form an activated complex with the guide nucleic acid upon binding of the guide nucleic acid to the target nucleic acid; (iii) the reporter comprises a nucleic acid and a detection moiety; and (iv) the nucleic acid of the reporter is a cleavage substrate of the activated complex.   
     
     
         98 . The cartridge of  claim 97 , wherein the second chamber further comprises a polymerase. 
     
     
         99 . The cartridge of  claim 97 , further comprising a third chamber, wherein the third chamber (i) is fluidically connected by the first valve to the first chamber, (ii) is fluidically connected by a second valve to the second chamber, and (iii) is disposed between the first chamber and the second chamber. 
     
     
         100 . The cartridge of  claim 99 , wherein the third chamber comprises a polymerase. 
     
     
         101 . The cartridge of  claim 97 , wherein the first chamber comprises one or more cell lysis reagents. 
     
     
         102 . The cartridge of  claim 97 , wherein the programmable nuclease is a programmable Cas12 nuclease. 
     
     
         103 . The cartridge of  claim 97 , wherein the programmable nuclease is a programmable Cas13 nuclease. 
     
     
         104 . The cartridge of  claim 97 , wherein the programmable nuclease is a programmable Cas14 nuclease. 
     
     
         105 . The cartridge of  claim 97 , further comprising a detection region fluidically connected to the second chamber. 
     
     
         106 . The cartridge of  claim 97 , wherein the reporter is immobilized to a surface within the second chamber. 
     
     
         107 . The cartridge of  claim 97 , wherein the nucleic acid of the reporter comprises at least one ribonucleotide or at least one deoxyribonucleotide. 
     
     
         108 . The cartridge of  claim 97 , wherein the reporter is a hybrid reporter comprising at least one ribonucleotide and at least one deoxyribonucleotide. 
     
     
         109 . The cartridge of  claim 97 , further comprising a plurality of second chambers, wherein each second chamber (i) comprises a programmable nuclease, a guide nucleic acid, and a reporter, (ii) is fluidically connected by the first valve to the first chamber, and (iii) comprises a different guide nucleic acid for detecting a different target nucleic acid. 
     
     
         110 . The cartridge of  claim 97 , wherein the cartridge is in contact with a device comprising an actuator configured to move fluid from the first chamber to the second chamber. 
     
     
         111 . The cartridge of  claim 97 , wherein the cartridge is in contact with a device comprising a detector configured to detect a signal of the detection moiety. 
     
     
         112 . The cartridge of  claim 97 , wherein the first valve comprises a pneumatic valve, a sliding valve, a rotating valve, an electro-kinetic valve, a vacuum valve, a capillary valve, a pinch valve, a phase-change valve, or a burst valve. 
     
     
         113 . A method of detecting a target nucleic acid, the method comprising:
 (a) introducing a sample comprising the target nucleic acid into the first chamber of the cartridge of  claim 97 ;   (b) flowing the contents of the first chamber into the second chamber;   (c) forming an activated complex comprising (i) the programmable nuclease, (ii) the guide nucleic acid, and (iii) the target nucleic acid or an amplicon thereof,   (d) cleaving the reporter with the activated complex; and   (e) detecting the detection moiety, thereby detecting the target nucleic acid.   
     
     
         114 . The method of  claim 113 , further comprising amplifying the target nucleic acid in a third chamber fluidically connected to and disposed between the first and second chambers. 
     
     
         115 . The method of  claim 113 , further comprising amplifying the target nucleic acid in the second chamber. 
     
     
         116 . The method of  claim 113 , further comprising flowing the contents of the second chamber into a detection region of the cartridge prior to the detecting step. 
     
     
         117 . The method of  claim 113 , further comprising lysing the sample prior to the flowing step.