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Aptamer assisted CRISPR-Cas12a strategy for small molecule diagnostics.

Chenqi Niu1, Chuyi Wang1, Fan Li1

  • 1MOE Key Laboratory for Industrial Biocatalysis, Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China.

Biosensors & Bioelectronics
|April 11, 2021
PubMed
Summary

A new Molecular Radar diagnostic strategy uses CRISPR-Cas12a to detect small molecules like adenosine-5'-triphosphate (ATP) rapidly at point-of-care settings. This aptamer-based assay offers a sensitive and specific method for molecular diagnostics.

Keywords:
ATPAptamerCRISPR-Cas12aCleavage kineticsMolecular diagnostics

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Area of Science:

  • Molecular diagnostics
  • Biosensor technology
  • CRISPR-Cas systems

Background:

  • CRISPR-Cas systems are crucial for disease management but primarily target nucleic acids.
  • Existing biosensors often lack versatility for small molecule detection.
  • Point-of-care (POC) diagnostics require rapid, sensitive, and convenient assays.

Purpose of the Study:

  • To develop a versatile diagnostic strategy for small molecule detection using CRISPR-Cas12a.
  • To adapt this strategy into a rapid biosensor for POC and on-site molecular diagnostics.
  • To demonstrate the strategy's efficacy using adenosine-5 prime -triphosphate (ATP) as a model target.

Main Methods:

  • Developed the Molecular Radar (Random Molecular Aptamer-Dependent CRISPR-Assist Reporter) strategy.
  • Utilized a CRISPR Cas12a-assisted fluorescence reporter system with aptamers and F-Q probes.
  • Optimized the assay for rapid detection at 37 °C within 25 minutes.

Main Results:

  • Achieved specific and sensitive detection of ATP.
  • Established a linear detection range from 25 to 500 μM for ATP.
  • Determined a low detection limit of 104 nM for ATP.
  • Observed a significant reduction in fluorescence signal upon target molecule binding.

Conclusions:

  • The Molecular Radar strategy is a versatile and rapid method for small molecule detection.
  • The assay shows significant potential for adaptation into POC and on-site biosensors.
  • This work expands the understanding of CRISPR-Cas12a applications in molecular diagnostics.