CRISPR in MOF Formulation with Enhanced Stability, Activity, and Altered PAM Specificity for Broad‐Spectrum Diagnosis of Bacterial Sepsis

CRISPR‐FLEXMO (CRISPR with flexible PAM in MOF encapsulation) integrates a PAM‐relaxed Cas12a K607R enzyme with a manganese‐coordinated MOF for broad‐spectrum diagnosis of bacterial sepsis. The MOF enhances activity and preserves functionality under heat, solvent, and chaotropic stress, supporting long‐term room‐temperature storage. The platform delivers robust performance in patient samples of varied etiology and effectively distinguishes bacterial from viral infections. Abstract Sepsis is a life‐threatening condition caused by polymicrobial infections and remains a global health emergency that requires rapid and broad‐spectrum diagnostics. Existing CRISPR‐based assays face two major limitations that restrict their application for sepsis: narrow protospacer adjacent motif (PAM) site compatibility and poor enzyme stability under clinical and environmental stresses. A modular diagnostic platform is presented, CRISPR‐FLEXMO (CRISPR with flexible PAM in metal‐organic framework encapsulation, MOF), which integrates a PAM‐relaxed Cas12a variant (K607R) with a manganese‐coordinated MOF (Mn‐MOF) for stable and specific detection of sepsis‐causing bacteria. The system targets a conserved region upstream of the Shine‐Dalgarno sequence in the 16S rRNA gene containing a universal TTCC PAM, enabling broad‐spectrum detection with a single universal primer pair across Gram‐negative and Gram‐positive pathogens. The K607R variant shows enhanced cis‐ and trans‐cleavage activity, while Mn‐MOF encapsulation maintains enzyme functionality under ambient, thermal, and chaotropic stress. The assay detects as low as 10 CFU mL−1 in bacterial lysates following amplification and achieves 100% sensitivity and specificity in serum samples from 15 sepsis patients and 3 healthy individuals, with no cross‐reactivity to six respiratory viruses. The platform retains over 78% activity after 12 weeks of room‐temperature storage, offering a field‐deployable CRISPR diagnostic solution for next‐generation infectious disease detection. CRISPR-FLEXMO (CRISPR with flexible PAM in MOF encapsulation) integrates a PAM-relaxed Cas12a K607R enzyme with a manganese-coordinated MOF for broad-spectrum diagnosis of bacterial sepsis. The MOF enhances activity and preserves functionality under heat, solvent, and chaotropic stress, supporting long-term room-temperature storage. The platform delivers robust performance in patient samples of varied etiology and effectively distinguishes bacterial from viral infections. Abstract Sepsis is a life-threatening condition caused by polymicrobial infections and remains a global health emergency that requires rapid and broad-spectrum diagnostics. Existing CRISPR-based assays face two major limitations that restrict their application for sepsis: narrow protospacer adjacent motif (PAM) site compatibility and poor enzyme stability under clinical and environmental stresses. A modular diagnostic platform is presented, CRISPR-FLEXMO (CRISPR with flexible PAM in metal-organic framework encapsulation, MOF), which integrates a PAM-relaxed Cas12a variant (K607R) with a manganese-coordinated MOF (Mn-MOF) for stable and specific detection of sepsis-causing bacteria. The system targets a conserved region upstream of the Shine-Dalgarno sequence in the 16S rRNA gene containing a universal TTCC PAM, enabling broad-spectrum detection with a single universal primer pair across Gram-negative and Gram-positive pathogens. The K607R variant shows enhanced cis - and trans -cleavage activity, while Mn-MOF encapsulation maintains enzyme functionality under ambient, thermal, and chaotropic stress. The assay detects as low as 10 CFU mL −1 in bacterial lysates following amplification and achieves 100% sensitivity and specificity in serum samples from 15 sepsis patients and 3 healthy individuals, with no cross-reactivity to six respiratory viruses. The platform retains over 78% activity after 12 weeks of room-temperature storage, offering a field-deployable CRISPR diagnostic solution for next-generation infectious disease detection. Advanced Science, EarlyView.