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New CRISPR-based diagnostics utilize novel enzymes for rapid, single-step detection of SARS-CoV-2. This advancement offers accurate, scalable testing directly from nasal swabs in under 30 minutes.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Infectious Disease Diagnostics

Background:

  • The COVID-19 pandemic underscored the need for accessible diagnostics.
  • Current CRISPR diagnostics require separate amplification steps, limiting clinical utility.
  • Novel Cas enzymes active at higher temperatures are needed for streamlined detection.

Purpose of the Study:

  • To discover novel Cas12 enzymes for improved CRISPR-based diagnostics.
  • To develop a single-step, real-time nucleic acid detection method.
  • To enable rapid, multiplexed detection of SARS-CoV-2.

Main Methods:

  • Discovery and characterization of two novel Cas12 enzymes (SLK9, SLK5-2) with 60°C activity.
  • Integration with Loop-mediated Isothermal Amplification (LAMP) for real-time detection (real-time SLK).
  • Development of direct sample testing (real-time SLK Direct) and multiplexed assays.

Main Results:

  • Real-time SLK achieved 100% positive and negative percent agreement with RT-qPCR in clinical samples.
  • Real-time SLK Direct enabled testing without nucleic acid extraction.
  • Multiplexed assays detected SARS-CoV-2 and a human control with sensitivity down to 5 copies/μL in under 30 minutes.

Conclusions:

  • Novel Cas12 enzymes enable a simplified, single-step CRISPR diagnostic.
  • The developed real-time SLK platform offers accurate and rapid SARS-CoV-2 detection.
  • This technology holds potential for widespread, low-cost diagnostic deployment.