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Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation.

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Summary

A new bioluminescent nucleic acid sensor (LUNAS) platform enables rapid, highly sensitive detection of nucleic acids. This CRISPR-based system integrates with isothermal amplification for point-of-care diagnostics, including SARS-CoV-2 RNA detection.

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Area of Science:

  • Biotechnology
  • Molecular Diagnostics
  • Biosensing

Background:

  • CRISPR and isothermal amplification show promise for point-of-care diagnostics.
  • Current methods often require external equipment or complex procedures.
  • Need for rapid, sensitive, and user-friendly nucleic acid detection.

Purpose of the Study:

  • To develop a bioluminescent nucleic acid sensor (LUNAS) platform.
  • To integrate LUNAS with isothermal amplification for enhanced sensitivity and speed.
  • To demonstrate LUNAS for rapid SARS-CoV-2 RNA detection.

Main Methods:

  • Developed LUNAS using dCas9-based probes and split NanoLuc luciferase complementation for target dsDNA detection.
  • Integrated LUNAS with recombinase polymerase amplification (RPA) in a one-pot assay.
  • Incorporated a calibrator luciferase for ratiometric readout and real-time monitoring via digital camera.

Main Results:

  • Achieved attomolar sensitivity using the integrated RPA-LUNAS assay.
  • Demonstrated a rapid, one-pot assay for nucleic acid detection.
  • Successfully developed an RT-RPA-LUNAS assay for SARS-CoV-2 RNA detection without RNA isolation.
  • Detected SARS-CoV-2 in patient samples with viral loads of ~200 cp/μL within ~20 minutes.

Conclusions:

  • The LUNAS platform offers a sensitive and rapid method for nucleic acid detection.
  • RPA-LUNAS is a promising tool for point-of-care infectious disease diagnostics.
  • The developed assay simplifies SARS-CoV-2 detection for COVID-19 diagnosis.