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Direct microRNA detection via topologically engineered CRISPR/Cas12a cascade amplification assay.

Meng Shen1, Peiying Zhang1, Lihua Ding1

  • 1College of Public Health, Zhengzhou University, Zhengzhou 450001, China.

Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy
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Summary

This study presents a novel CRISPR/Cas12a biosensor for direct microRNA detection without amplification. The assay achieves high sensitivity and specificity, enabling accurate quantification of miRNA-21 in cancer cells.

Keywords:
CRISPR/Cas12aCascadeRNATarget-amplification-free

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Biosensing

Background:

  • CRISPR/Cas12a is a potent biosensing tool, but direct RNA detection is challenging due to its DNA-targeting nature.
  • Existing methods often require RNA amplification steps like reverse transcription.

Purpose of the Study:

  • To develop a one-pot CRISPR/Cas12a assay for direct microRNA detection without amplification.
  • To enable sensitive and specific quantification of microRNA-21.

Main Methods:

  • Utilized a molecular switch probe (MSP) to recognize miRNA-21 and activate Cas12a.
  • Incorporated an amplifier probe (AMP) for a self-driven cascade signal amplification.
  • Developed a one-pot assay for direct RNA detection.

Main Results:

  • Achieved a low detection limit of 2.88 pM for miRNA-21.
  • Demonstrated high accuracy (95.5%-108.6% recovery) and precision (2.35%-7.18% RSD).
  • Showcased excellent specificity against homologous miRNAs and successfully quantified elevated miRNA-21 in lung cancer cells.

Conclusions:

  • The developed CRISPR/Cas12a biosensor offers a simple, reliable method for direct RNA detection.
  • This approach bypasses the need for target amplification, streamlining the detection process.
  • The assay shows potential for clinical applications, such as cancer diagnostics.