Nucleic acid detection using type III CRISPR complex
Inventors
Wiedenheft, Blake A. • SANTIAGO-FRANGOS, Andrew • NEMUDRAIA, Anna A. • NEMUDRYI, Artem A.
Assignees
Montana State University Bozeman
Publication Number
US-11814689-B2
Publication Date
2023-11-14
Expiration Date
2042-07-21
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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.
Core Innovation
The disclosure introduces systems and methods that utilize an engineered type III CRISPR complex for programmatically capturing and detecting target nucleic acids, such as RNA or DNA, within a sample that can be a complex mixture. The engineered system improves detection sensitivity by employing a guide sequence that is complementary to the target nucleic acid, enabling targeted capture. This capture is followed by concentration of the target–CRISPR complex, which amplifies the detectable activity produced by the complex, such as polymerase activity that generates linear or cyclic oligonucleotides.
The invention addresses the problem that detecting target nucleic acids of interest in complex mixtures is challenging due to low concentrations and high background noise from other substances, which diminishes signal-to-noise ratio. Existing molecular methods like PCR improve sensitivity via amplification but require complex, time-consuming laboratory processes that are not ideal for rapid or field-deployable diagnostics.
In the disclosed methods, the engineered type III CRISPR complex captures and concentrates the target nucleic acid without prior amplification. The bound complex can then be used to trigger detectable activities, such as generating cyclic oligoadenylates (cA3, cA4) that activate ancillary nucleases (such as Can1, Can2, or NucC), which in turn act on reporter molecules to produce measurable signals (e.g., fluorescence). This programmable and amplification-free approach enhances sensitivity and is shown effective for applications like direct detection of viral RNA, including detection from nasopharyngeal swabs.
Claims Coverage
The independent claim covers a method for programmatically capturing and detecting target nucleic acids using an engineered type III CRISPR complex. Four main inventive features are specified.
Engineered type III CRISPR complex for nucleic acid capture
The inventive method employs an engineered type III CRISPR-Cas complex with a CRISPR guide sequence that is complementary to the target nucleic acid in a sample. The complex facilitates specific capture of the target nucleic acid within the sample.
Concentration of captured nucleic acid to amplify detectable activity
After initial capture, the method includes concentrating the complex-bound target nucleic acid, thereby amplifying any detectable activity produced by the engineered type III CRISPR-Cas complex. This amplification is performed without requiring nucleic acid amplification techniques such as PCR.
Detection based on activity of the concentrated engineered complex
Detection is achieved by monitoring the activity generated by the captured and concentrated engineered type III CRISPR-Cas complex. The detectable activity may be the generation of a linear or cyclic oligonucleotide (e.g., cA3 or cA4), which is used as a signal for the presence of the target nucleic acid.
Programmability of the CRISPR complex and compatible ancillary readouts
The method utilizes a CRISPR guide engineered for target specificity and enables compatibility with various ancillary nucleases (such as Can1, Can2, NucC) that are activated by the generated oligonucleotide products. These nucleases can act on reporter systems, such as fluorescence-based reporters, thereby facilitating a wide range of detection modalities.
Collectively, the inventive features provide a programmable, amplification-free, and sensitive method for the detection of target nucleic acids in complex mixtures, leveraging an engineered type III CRISPR complex integrated with concentration and detection strategies.
Stated Advantages
The method increases the sensitivity of nucleic acid detection without the need for prior amplification steps such as PCR.
It enables direct detection of target nucleic acids from complex mixtures, including clinical samples such as nasopharyngeal swabs, without complex laboratory processing.
The approach allows for rapid and simplified workflows suitable for point-of-care or deployable diagnostics.
The system is programmable: guide sequences can be engineered for desired targets, and detection readouts can be tailored by choosing different ancillary nucleases and reporters.
Use of ancillary nucleases that do not degrade their activator preserves detection signals and further improves sensitivity over other CRISPR-based methods.
Documented Applications
Direct detection of viral RNA from nasopharyngeal swab samples, including detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic RNA.
Detection and quantification of viral RNA in biological samples for infectious disease diagnostics.
Sequence-specific capture, concentration, and enrichment of RNA from complex biological mixtures, with subsequent sequencing (Capture & Seq and Cleavage & Seq approaches).
Detection of small RNAs (such as microRNAs, siRNAs, or piRNAs) directly from patient samples including saliva, urine, or blood.
Use in lateral flow assay readouts and other reporter-based detection systems for rapid, field-usable diagnostics.
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