Journal: Advanced Science

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

doi: 10.1002/advs.202513439

Figure Lengend Snippet: Schematic of the CRISPR‐FLXEMO diagnostic platform integrating the PAM‐flexible Cas12a K607R variant with squaric acid‐based Mn‐MOF encapsulation for stable and broad‐spectrum sepsis diagnostics. Upon Mn‐MOF disassembly, the released Cas12a‐crRNA RNP targets a conserved 16S rRNA region containing a universal TTCC PAM and adjacent spacer. The Cas12a K607R variant exhibits enhanced fluorescence via trans ‐cleavage, enabling sensitive detection of both Gram‐negative and Gram‐positive bacteria.

Article Snippet: Synthetic bacterial 16S rRNA gene fragments were ordered from Twist Bioscience (San Francisco, CA).

Techniques: CRISPR, Diagnostic Assay, Variant Assay, Encapsulation, Fluorescence, Bacteria

Journal: Advanced Science

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

doi: 10.1002/advs.202513439

Figure Lengend Snippet: Cas12a K607R variant exhibits enhanced cis ‐ and trans ‐cleavage activity compared to wild‐type (WT) Cas12a. a) Schematic of cis ‐cleavage assay using a 371 bp PCR amplicon generated with universal primers targeting a conserved region upstream of the Shine‐Dalgarno sequence within the 16S rRNA gene. Cleavage at the TTCC PAM site yields 236 bp and 135 bp fragments. Gel electrophoresis confirms superior cleavage efficiency of Cas12a K607R over WT Cas12a, as indicated by more prominent cleavage bands. b) Schematic of trans ‐cleavage assay, wherein activated Cas12a cleaves a circular single‐stranded DNA (ssDNA) reporter probe upon target recognition. Time‐dependent degradation of ssDNA reveals faster and more extensive cleavage by Cas12a K607R than WT Cas12a. c) Kinetic fluorescence measurements show markedly higher signal output for Cas12a K607R (+) compared to WT Cas12a (+), with minimal background in negative controls. d) Endpoint fluorescence intensities and visual readouts further highlight the robust signal generation enabled by Cas12a K607R, supporting its suitability for diagnostics. Error bars represent the standard deviation obtained in three parallel experiments ( n = 3).

Article Snippet: Synthetic bacterial 16S rRNA gene fragments were ordered from Twist Bioscience (San Francisco, CA).

Techniques: Variant Assay, Activity Assay, Cleavage Assay, Amplification, Generated, Sequencing, Nucleic Acid Electrophoresis, Fluorescence, Standard Deviation

Journal: Advanced Science

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

doi: 10.1002/advs.202513439

Figure Lengend Snippet: Clinical validation of the MOF‐encapsulated Cas12a K607R CRISPR‐Cas12a system for detection of bacterial DNA in patient serum samples. a) Schematic overview of the diagnostic workflow: patient sample collection, MOF disintegration, isothermal amplification using RPA with universal primers targeting the 16S rRNA gene, followed by CRISPR activation and fluorescence‐based detection. b) Quantitative PCR (qPCR) amplification curves for 15 patient samples (P1–P15) and 3 healthy controls (H1–H3), ctrl: purchased human serum as no target control. c) Endpoint PCR product of the clinical samples, showing strong bands for patient‐derived DNA. d) RPA products used for CRISPR detection, demonstrating compatibility and consistent amplification across patient samples. e) Fluorescence signal (F/F 0 ) output of the MOF‐CRISPR assay confirms positive detection in all patient samples and negligible background in negative controls (Fluorescence image of the reaction product in the inset). f) ROC analysis indicating high diagnostic performance. g) Confusion matrix comparing CRISPR results with reference diagnosis (qPCR): 15 true positives (TP), 3 true negatives (TN), and no false positives (FP) or false negatives (FN), demonstrating 100% sensitivity and specificity. F/F 0 represents the normalized fluorescence signal, where F is the measured fluorescence intensity at each condition, and F 0 is the baseline fluorescence in the absence of target DNA. Error bars represent the standard deviation obtained in three parallel experiments ( n = 3).

Article Snippet: Synthetic bacterial 16S rRNA gene fragments were ordered from Twist Bioscience (San Francisco, CA).

Techniques: Biomarker Discovery, CRISPR, Diagnostic Assay, Amplification, Activation Assay, Fluorescence, Real-time Polymerase Chain Reaction, Control, Derivative Assay, Standard Deviation