NUCLEIC ACID SEQUENCE DETECTION BY MEASURING FREE MONORIBONUCLEOTIDES GENERATED BY ENDONUCLEASE COLLATERAL CLEAVAGE ACTIVITY
Researchers at the Chan Zuckerberg Biohub have developed an improved method to detect target nucleic acid sequences in a biological sample, NANCI™.
CRISPR-associated protein (Cas) technology is a sequence-specific tool that has revolutionized gene editing. In addition to site-specific cis-cleavage of target DNA, some Cas proteins, when bound to target DNA, can promiscuously cleave other nucleic acid molecules that do not contain the target sequence. This trans-cleavage activity is referred to as Cas “collateral activity” and has been used in development of diagnostic biosensor assays. However, assay readout for current Cas collateral activity platforms is done using FRET (Fluorescence Resonance Energy Transfer), limiting feasibility in low-resource settings.
Stage of Research
The inventors have developed an improved system for detection of predetermined nucleic acid sequences. Their method, NANCI™ (exoNuclease-Assisted endoNuclease Cleavage Indicator) couples target DNA detection to a luminescent readout and is robust, sensitive, specific, and may be used for a variety of biological samples. Upon binding of a CRISPR associated protein (Cas)-gRNA complex to its target DNA, the Cas trans-cleavage activity is activated, and the enzyme cleaves an oligonucleotide probe at one or more internal sites, producing unblocked fragments amenable to exonuclease degradation. An exonuclease is introduced to degrade the probe into monoribonucleotides, which are converted to ATP and consumed in a luciferase reaction to generate light. In the absence of target DNA, the Cas remains inactive, the probe blocked, and no monoribonucleotides are available for the luciferase reaction. The inventors demonstrate the sensitivity of NANCI™ in target gene detection to as low as 500 pM.
Applications
- Detection of low abundance sequences in biological samples, including pathogen detection in direct clinical samples
- Detection of antimicrobial resistance sequences, mutations and SNP analysis
- Adaptation as a biosensing platform for sensitive detection of diverse small molecules or proteins of interest
Advantages
- Modular platform for any nucleic acid target sequence recognition, with choice of endonucleases, probe substrates, and exonucleases
- Implemented as a homogenous assay, without requiring separation steps
- More sensitive and lacks interference from background autofluorescence in comparison to prior methods
- Signal can be detected in the absence of an excitation source, compatible with commercially available luciferase-based kits, making use in low-resource settings more feasible
Stage of Development
Research – in vitro
Publications
PCT Publication No. WO2021173587
Keywords
Biomolecules, biosensor system, CRISPR, Diagnosis, Nuclease
Technology Reference
CZB-148
