A PAM-free Cas12a system with double-stranded substrate positioning-unwinding (dsPU-Cas12a), wherein "bubble" structures formed by unpaired base pairs release partial single-stranded target strand as a toehold, providing a high-performance tool for clinical gene point mutation detection.
Abstract
The CRISPR/Cas12a system holds great promise for nucleic acid detection, but its strict dependence on the protospacer adjacent motif (PAM) severely limits its application in gene point mutation analysis, with fewer than 2% of known mutation sites naturally harboring adjacent PAM sequences. Herein, we developed a PAM-free Cas12a system with double-stranded substrate positioning-unwinding (dsPU-Cas12a), wherein "bubble" structures formed by unpaired base pairs release partial single-stranded target strand as a toehold, and excess auxiliary strands induce local unwinding of double-stranded DNA to facilitate R-loop formation. After optimization, the dsPU-Cas12a system achieved an ultra-low limit of detection of 0.013% for gene point mutations, with excellent linearity over the mutation abundance range of 0-10%. Furthermore, it exhibited robust feasibility and accuracy in detecting the JAK2 V617F mutation in blood samples from patients with myeloproliferative neoplasms. This simple and universal strategy overcomes the sequence limitation of Cas12a, providing a high-performance tool for clinical gene point mutation detection.
The potential of terminally engineered PAM-less dsDNA as a structural handle for programming Cas12a activity is highlighted and useful insight is provided for the design of CRISPR-based biosensing strategies.
Traditional CRISPR-Cas12a mutation detection systems are limited by poor single-base specificity, target-specific crRNA redesign, and insufficient sensitivity for low-abundance mutations, restricting their clinical liquid biopsy applications. Herein, we developed a crRNA-universal, sensitive and specific CRISPR-Cas12a detection platform, termed DESIC (double-end blocker and split-input mediated CRISPR-Cas12a system), for single-base mutation detection. The DESIC system adopts two key structural designs: double-end blocker (DEB) and duplicated split-input (SIN). The DEB spatially isolates crRNA recognition and target-binding regions, enabling universal detection of various mutation sites without crRNA redesign. The SIN strategy amplifies thermodynamic differences from single-base mismatches, greatly improving single-nucleotide discrimination. We targeted four prevalent pancreatic cancer KRAS mutations (G12D, G12R, G12V, Q61H) and optimized the system to achieve optimal discrimination. The optimized DESIC system exhibited ultra-low limits of detection down to 0.01% mutant allele fraction with reliable linear quantitative performance. Clinical validation using 15 pairs of pancreatic cancer tissue and peripheral blood samples confirmed that DESIC results were highly consistent with gold-standard NGS data. With a flexible modular design, this low-cost, easy-operated platform can be readily extended to multiple tumor mutations, holding great potential for tumor liquid biopsy and early molecular diagnosis.
Shi-Zhen Li, Yangwei Liao, Xiaoxiang Wang et al.· Biosensors & bioelectronics· 0 citations
A one-pot self-primer isothermal exponential amplification reaction (SP-EXPAR) combined with a CRISPR/Cas14a assay was developed for detecting KRAS G12C and G12D and demonstrated 100% sensitivity and 100% specificity compared with DNA sequencing.
Guozhi Yang, Yaqin Chen, Wenyong Zhao et al.· Analytical Methods· 0 citations
Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has become a powerful genome-editing tool that uses RNA–DNA pairing to cleave target DNA with protospacer adjacent motif (PAM) sequences. While its primary function is well-studied, secondary activities remain poorly understood, causing unintended off-target effects. This study reports for the first time that Cas9 specifically cleaves the 5′ overhang of the non-target strand (NTS) in target double-stranded DNA. This specific cleavage requires an additional PAM element at the NTS 5′ region, is mediated by Cas9’s RuvC domain, and is regulated by the HNH domain. It depends on the exact positioning of the NTS 5′ end, but not on the overhang homopolymer sequence or overhang length. Adequate single-guide RNA–DNA complementarity is also essential. This discovery potentially advances our understanding of Cas9’s enzymatic versatility to enhance genome-editing precision and efficacy and offers new nucleic acid detection strategies. Based on this cleavage, we developed a sensitive assay for Severe Acute Respiratory Syndrome Coronavirus 2 pseudovirus down to 2.4 copies μL−1, demonstrated extremely high sensitivity in diagnostic applications.
CRISPR/Cas12a has emerged as a powerful tool for nucleic acid detection; however, its clinical utility is severely hampered by intrinsic limitations in single-nucleotide variant (SNV) discrimination, reliance on pre-processed single-stranded DNA (ssDNA) templates, and cumbersome multi-step workflows. Here, we report a novel molecular design principle by engineering crRNAs with site-specific dual mismatches (positions 12 and 14 relative to the PAM), which we systematically demonstrate to drastically enhance the SNV discrimination capability of Cas12a. Leveraging this breakthrough, we developed FOCUS (Fast One-step CRISPR-based Universalizable SNV detection system), an all-in-one CRISPR sensing platform that enables isothermal detection of SNVs from double-stranded DNA (dsDNA) by integrating amplification and detection in a single reaction system. FOCUS achieved attomolar-level sensitivity (13.15 aM) and ultrafast readout (< 20 min) for distinguishing the highly homologous survival motor neuron 1 (SMN1) and SMN2 genes-the gold standard challenge for SNV genotyping in spinal muscular atrophy (SMA) diagnostics. To validate its clinical translatability, FOCUS was successfully adapted to a low-cost, equipment-free assay using lateral flow strips and UV visualization, facilitating point-of-care testing (POCT). In a comprehensive validation across 175 clinical samples, FOCUS exhibited 100% diagnostic concordance with gold-standard methods for SMA (21 samples), high-risk HPV 16/18 (27 samples), Staphylococcus aureus (20 samples), and SARS-CoV-2 (107 samples). Collectively, our study establishes a generalizable engineering strategy for Cas12a crRNAs and presents FOCUS as a robust, versatile, and field-deployable solution for precision SNV genotyping, underscoring the translational medicine value of FOCUS in molecular diagnostics.
Miaojin Zhou, K. Du, Mingjun Jiang et al.· ACS Sensors· 0 citations
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) system enables sensitive and specific biomolecular detection due to its programmability, high fidelity, and signal amplification. Herein, a novel universal CRISPR/Cas12a-G4 DNAzyme-TMB (Cas-GT) enzymatic biosensing platform was constructed. This platform regulates the catalytic function of G4 DNAzyme through the trans-cleavage activity of Cas12a, achieving an "off/on" response of the TMB-H2O2 enzymatic signal for nucleic acid, protein, and metal ion targets. In proof-of-concept experiments, the detection limit of Cas-GT for in vitro transcribed SARS-CoV-2 RNA reached as low as 100 aM and it distinguished clinical positive from negative patients with good diagnostic performance (AUC = 0.9420). It is also suitable for protein targets, enabling quantitative analysis of prostate-specific antigen (PSA) within the range of 0-100 ng mL-1, with results highly consistent with clinical chemiluminescence immunoassay (CLIA). For small-molecule targets, Cas-GT exhibited good universality, achieving quantitative detection of Hg2+ within the range of 0.06-4 ng mL-1, with recovery rates of 93%-107.4% in spiked river water samples, showing no significant difference from ICP-MS (P = 0.9968). In summary, Cas-GT is a simple, sensitive, rapid, and label-free enzymatic biosensing platform with significant potential for clinical biomarker detection and environmental pollution monitoring.
Huisi Qin, Jing-Xuan Liu, Yang Luo et al.· In Analysis· 0 citations
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