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

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.