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Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases
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CRISPR in MOF Formulation with Enhanced Stability, Activity, and Altered PAM Specificity for Broad-Spectrum Diagnosis

Tathagata Pal1, Zilong Liu1, Meera G Nair2

  • 1Department of Bioengineering, University of California, Riverside, CA, 92521, USA.

Advanced Science (Weinheim, Baden-Wurttemberg, Germany)
|November 24, 2025
PubMed

Insights

A new CRISPR-based diagnostic platform, CRISPR-FLEXMO, offers rapid, broad-spectrum detection of sepsis-causing bacteria. Encapsulated for stability and utilizing a flexible PAM site, it provides accurate results even under challenging conditions.

Area of Science:

  • Biotechnology
  • Molecular Diagnostics
  • Infectious Disease Research

Background:

  • Sepsis is a critical global health issue requiring rapid, broad-spectrum diagnostics.
  • Current CRISPR-based sepsis diagnostics are limited by narrow PAM compatibility and poor enzyme stability.
  • A need exists for robust, field-deployable diagnostic solutions for sepsis detection.

Purpose of the Study:

  • To develop a modular CRISPR diagnostic platform, CRISPR-FLEXMO, for stable and specific detection of sepsis-causing bacteria.
  • To overcome limitations of existing CRISPR assays by integrating a PAM-relaxed Cas12a variant with metal-organic framework (MOF) encapsulation.
  • To achieve broad-spectrum detection of both Gram-negative and Gram-positive pathogens with high sensitivity and specificity.

Main Methods:

  • Developed CRISPR-FLEXMO by combining a PAM-relaxed Cas12a variant (K607R) with manganese-coordinated MOF (Mn-MOF) encapsulation.
  • Targeted a conserved region upstream of the Shine-Dalgarno sequence in the 16S rRNA gene with a universal TTCC PAM.
  • Evaluated enzyme stability under various stresses and tested assay sensitivity and specificity in bacterial lysates and human serum samples.

Main Results:

  • CRISPR-FLEXMO demonstrated broad-spectrum detection of Gram-negative and Gram-positive bacteria using a single primer pair.
  • The K607R variant exhibited enhanced cleavage activity, while Mn-MOF encapsulation preserved enzyme functionality under stress.
  • The assay achieved a limit of detection of 10 CFU mL⁻¹ and showed 100% sensitivity and specificity in clinical serum samples.
  • The platform maintained over 78% activity after 12 weeks of room-temperature storage.

Conclusions:

  • CRISPR-FLEXMO is a stable, specific, and sensitive CRISPR-based diagnostic platform for sepsis detection.
  • The modular design and enhanced stability make it suitable for field deployment in infectious disease diagnostics.
  • This technology represents a significant advancement for next-generation sepsis diagnostics and combating infectious diseases.
Keywords:
CRISPR assayCas12a variantMOFPAM‐altered detectionsepsis diagnosis

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