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DNA Robots for CRISPR/Cas12a Activity Management and Universal Platforms for Biosensing.

Xinyi Xia1, Qiutong Chen1, Tongshan Zuo1

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

Analytical Chemistry
|January 13, 2024
PubMed
Summary

We developed a novel DNA Robots for Enzyme Activity Management (DREAM) strategy to precisely control CRISPR/Cas12a systems for enhanced biosensing and molecular diagnostics.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genomics

Background:

  • CRISPR/Cas12a is a powerful genome editing tool used in biosensing and diagnostics.
  • Controlling Cas12a's trans-cleavage activity often requires complex methods.
  • Simple modifications for precise Cas12a regulation are limited.

Purpose of the Study:

  • To develop a novel, simple regulatory mechanism for CRISPR/Cas12a activity.
  • To enhance the precision and selectivity of Cas12a in biosensing applications.
  • To create a versatile platform for molecular diagnostics.

Main Methods:

  • Introduced DNA robots (DREAM strategy) with AP site or nick activators to regulate Cas12a.
  • Investigated Cas12a regulation mechanisms based on varying binding affinities.
  • Developed a modular biosensor for base excision repair enzymes using DNA robots.
  • Integrated multi-signal outputs (fluorescence, colorimetry, lateral flow) and logic sensing circuits.

Main Results:

  • The DREAM strategy precisely regulated Cas12a activity through differential binding affinities.
  • Enhanced Cas12a selectivity for identifying base mismatches.
  • Successfully developed a modular biosensor for base excision repair enzymes.
  • Constructed a multi-signal output platform and logic sensing circuits for simultaneous detection.

Conclusions:

  • The DREAM strategy offers a new approach for programmable CRISPR/Cas12a biosensing.
  • This method enables portable, specific, and user-friendly molecular diagnostics.
  • The strategy holds significant potential for advancing molecular diagnostic tools.