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Customization of aptamer to develop CRISPR/Cas12a-derived ultrasensitive biosensor.

Wenping Xing1, Qian Li1, Cong Han2

  • 1State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai University, Tianjin, 300350, PR China.

Talanta
|February 4, 2023
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Summary

Researchers discovered a "SET" effect where aptamer sequence tuning balances target affinity and CRISPR/Cas12a activation for sensitive SARS-CoV-2 S1 detection. This optimized aptasensor enables rapid point-of-care testing in complex samples.

Keywords:
AptamerCRISPR/Cas12a systemElectrochemical biosensorSpike proteinTrans-cleavage

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Area of Science:

  • Biotechnology and Biosensing
  • Molecular Biology and Diagnostics
  • CRISPR-based Detection Systems

Background:

  • CRISPR/Cas systems offer versatile biosensing, with aptamer-CRISPR/Cas sensors detecting non-nucleic acid targets.
  • Optimizing aptamer sequences for CRISPR/Cas12a activation, balancing target affinity and cleavage efficiency, remains underexplored.

Purpose of the Study:

  • To develop novel aptamers targeting SARS-CoV-2 spike protein 1 (S1).
  • To investigate the relationship between aptamer sequence, target affinity, and CRISPR/Cas12a trans-cleavage activity.
  • To establish an optimized aptasensor for sensitive and specific COVID-19 antigen detection.

Main Methods:

  • Development and screening of aptamers against SARS-CoV-2 S1 protein.
  • Surface Plasmon Resonance (SPR) for affinity measurements.
  • CRISPR/Cas12a trans-cleavage assays to evaluate activation efficiency.
  • Fabrication and testing of electrochemical biosensors.

Main Results:

  • nM-level affinity aptamers for S1 were generated.
  • A 'Sequence Essential Trans-cleavage activity' (SET) effect was identified, linking aptamer sequence to Cas12a activation.
  • An optimized 27-nt aptamer demonstrated high affinity, specificity, and efficient CRISPR/Cas12a activation.
  • The electrochemical aptasensor achieved a low detection limit of 1.5 pg/mL for S1 and detected multiple SARS-CoV-2 variants.

Conclusions:

  • Aptamer sequence optimization is crucial for efficient CRISPR/Cas12a activation, requiring a balance between target affinity and cleavage ability.
  • The developed CRISPR/Cas12a-aptasensor provides a sensitive, specific, and robust platform for COVID-19 antigen detection.
  • This approach offers a scalable and universal strategy for point-of-care testing in various biological fluids.