Abstract: The disclosure relates to engineered systems and methods for detecting target nucleic acid in a sample, which may be a complex mixture. The systems and methods may improve sensitivity of target nucleic acid detection by enhancing signal generation. For example, signal generation may be enhanced through programmable capture and concentration of the target nucleic acid using an engineered type III CRISPR complex. Various ancillary nucleases such as Can1, Can2, and NucC are identified and may be used for detection. For example, binding of the engineered type III CRISPR complex may produce products that activate the identified ancillary nucleases. Different activators trigger changes in the substrate specificity of these nucleases. The activated nucleases may be used to detect programmatic detection of the target nucleic in the sample. The systems and methods are shown to detect viral RNA directly from nasopharyngeal swab samples.

Abstract: The disclosure relates to engineered systems and methods for detecting target nucleic acid in a sample, which may be a complex mixture. The systems and methods may improve sensitivity of target nucleic acid detection by enhancing signal generation. For example, signal generation may be enhanced through programmable capture and concentration of the target nucleic acid using an engineered type III CRISPR complex. Various ancillary nucleases such as Can1, Can2, and NucC are identified and may be used for detection. For example, binding of the engineered type III CRISPR complex may produce products that activate the identified ancillary nucleases. Different activators trigger changes in the substrate specificity of these nucleases. The activated nucleases may be used to detect programmatic detection of the target nucleic in the sample. The systems and methods are shown to detect viral RNA directly from nasopharyngeal swab samples.

Abstract: The disclosure relates to engineered systems and methods for detecting target nucleic acid in a sample, which may be a complex mixture. The systems and methods may improve sensitivity of target nucleic acid detection by enhancing signal generation. For example, signal generation may be enhanced through programmable capture and concentration of the target nucleic acid using an engineered type III CRISPR complex. Various ancillary nucleases such as Can1, Can2, and NucC are identified and may be used for detection. For example, binding of the engineered type III CRISPR complex may produce products that activate the identified ancillary nucleases. Different activators trigger changes in the substrate specificity of these nucleases. The activated nucleases may be used to detect programmatic detection of the target nucleic in the sample. The systems and methods are shown to detect viral RNA directly from nasopharyngeal swab samples.

Abstract: The disclosure relates to an engineered type III CRISPR-Cas system for sensitive and sequence specific detection of nucleic acid in a sample. For example, the engineered type III CRISPR-Cas system may be implemented as an assay for testing SARS-CoV-2 virus (or other target nucleic acid in the sample) that can be performed quickly, such as in one hour or less. Nucleic acid recognition by type III systems may trigger Cas10-mediated nuclease activity and/or polymerase activity, which may generate pyrophosphates, protons and cyclic oligonucleotides. The nuclease activity and/or the one or more products of the Cas10-polymerase are detected using colorimetric, visible fluorometric, and/or instrumented fluorometric detection.

Abstract: The disclosure relates to an engineered type III CRISPR-Cas system for sensitive and sequence specific detection of nucleic acid in a sample. For example, the engineered type III CRISPR-Cas system may be implemented as an assay for testing SARS-CoV-2 virus (or other target nucleic acid in the sample) that can be performed quickly, such as in one hour or less. Nucleic acid recognition by type III systems may trigger Cas10-mediated nuclease activity and/or polymerase activity, which may generate pyrophosphates, protons and cyclic oligonucleotides. The nuclease activity and/or the one or more products of the Cas10-polymerase are detected using colorimetric, visible fluorometric, and/or instrumented fluorometric detection.