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THRUST: translesion synthesis-driven hierarchical regulation using a template-activator construct for Cas12a

Lulu Qin1, Wen-Jin Wang2, Xinyi Xia1

  • 1School of Pharmacy, Nanjing Medical University Nanjing Jiangsu 211166 China.

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We developed a new CRISPR/Cas12a regulation strategy called THRUST. This economical method precisely controls Cas12a activity for advanced biosensing and molecular diagnostics.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Diagnostics

Background:

  • CRISPR/Cas12a is a powerful tool for biosensing and molecular diagnostics due to its specific recognition and trans-cleavage activity.
  • Current crRNA-based Cas12a regulation methods are complex, costly, and require multiple components.

Purpose of the Study:

  • To develop a novel, economical, and precise CRISPR/Cas12a regulation strategy.
  • To enhance the CRISPR/Cas12a system for improved molecular diagnostics.

Main Methods:

  • Developed translesion synthesis-driven hierarchical regulation using a template-activator construct for Cas12a activity (THRUST).
  • Utilized a bifunctional template-activator construct (TAC) serving as a T7 RNA polymerase transcriptional template and a Cas12a activator.
  • Engineered TAC with deoxyuridine (dU) lesions and apurinic/apyrimidinic (AP) sites to control transcription and crRNA length.
  • Strategically positioned transcriptional regulatory units for hierarchical regulation of Cas12a.

Main Results:

  • THRUST enables precise control over crRNA length and Cas12a activation.
  • Demonstrated the efficacy of THRUST in "Dim down" and "Light up" biosensing platforms.
  • Successfully applied THRUST in an aggregation-induced emission lateral flow test for molecular diagnostics.

Conclusions:

  • THRUST offers a powerful, economical, and hierarchical regulation strategy for CRISPR/Cas12a.
  • This approach simplifies CRISPR/Cas12a system complexity and reduces costs.
  • THRUST significantly enriches the regulatory toolbox for CRISPR/Cas12a-based molecular diagnostics.