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.
Abstract
Precise and programmable regulation of CRISPR-Cas12a activity is essential for advancing controllable nucleic acid diagnostics, yet the structural determinants governing Cas12a activation by short PAM-less double-stranded DNA (dsDNA) remain largely unexplored. This study systematically investigates the effects of the terminal architectures of short PAM-less dsDNA on Cas12a trans-cleavage activity. By profiling a series of dsDNA constructs bearing distinct 5'/3' overhang configurations, a 5' dual-overhang motif was identified as a highly effective structural inhibitor that suppresses Cas12a activation. Kinetic fluorescence assays combined with computational structural modeling indicated that this inhibition arises from steric constraints imposed by the 5' terminal architecture. Leveraging this structure-guided regulatory mechanism, an amplification-free CRISPR-Cas12a assay was developed for the direct detection of oncogenic microRNAs miR-155 and miR-21, achieving femtomolar sensitivity without reverse transcription. The assay was further evaluated in human serum samples spiked with target miRNAs, supporting its proof-of-concept performance in a more complex matrix. Collectively, these findings highlight the potential of terminally engineered PAM-less dsDNA as a structural handle for programming Cas12a activity and provide useful insight for the design of CRISPR-based biosensing strategies.
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.
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.
Zhujun Liu, Jinjin Wang, Zhengguang Yang et al.· Biosensors & bioelectronics· 0 citations
RAPID (RNA/DNA Advanced chimeric, PAM-independent, Integrated Nicking, Diagnostics), a nick-tuned, PAM-duplex-mediated platform for PAM-independent RNA and DNA detection and applies it to detect single-point mutations in ssDNA and RNA substrates, a challenge for traditional Cas12 and Cas13 systems.
I. Iwe, Frank X. Liu, A. Corsano et al.· Nucleic Acids Research· 0 citations
CRISPR-Cas12a has emerged as a powerful tool in molecular diagnostics, owing to its robust signal amplification and compact crRNA design. However, its uncontrolled enzymatic activity often hampers application in streamlined one-pot assays. Although existing temporal or spatial regulation strategies can mitigate this issue, they typically introduce operational complexity or increased cost. Here, we designed a Thermally regulated, Oligonucleotide-mediated one-Pot System for CRISPR-Cas12a (TOPS-CRISPR), which employs a programmable inhibition strategy based on complementary RNA blockers with tunable length and binding sites, enabling efficient and reversible steric inhibition of the LbCas12a-crRNA ribonucleoprotein (RNP) complex, resolving the inherent contradiction between amplification and cleavage in one-pot assay. TOPS-CRISPR not only is operational simple and cost-effective but also achieves over 60-fold higher sensitivity than conventional one-pot platforms. We demonstrated the clinical applicability of TOPS-CRISPR by accurately detecting Brucella and Streptococcus in both spiked and clinical samples. Moreover, the system integrates seamlessly with rapid sample processing, lyophilized reagents, and miniaturized workflows, enabling field-deployable pathogen identification within 50 min.
Shusen Ji, Bin Wang, Yi Yan et al.· Biosensors & bioelectronics· 0 citations
Abstract Selective and tunable regulation of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a activity enables on-demand control, yet current strategies remain hindered by nonspecific regulation and limited tunability. Inspired by proximity effect, we present a Cas-regulation-targeting chimera (CasTAC) strategy that employs CRISPR RNA (crRNA) as a proximity mediator to carry phosphorothioate regulators to interfere with catalytic or recognition domains of Cas12a and consequently suppress its activity. This crRNA-induced proximity approach can effectively eliminate nonspecific interaction between phosphorothioate regulators and proteins within complex multi-enzyme systems, thereby enabling selective control over CRISPR/Cas12a activity. Furthermore, CRISPR/Cas12a activity can be finely tuned to different inhibitory levels by varying the number of phosphorothioate regulators. The CasTAC strategy also improves nuclease resistance and single-nucleotide discrimination, offering potential advances in the sensitivity of molecular diagnostics and the accuracy of gene editing. Notably, the CasTAC balances the kinetics of nucleic acid amplification and CRISPR cleavage, facilitating efficient product accumulation and resolving compatibility issues in one-pot assays. As a proof of concept, we develop a one-pot, one-step recombinase polymerase amplification–CasTAC assay that achieves over 1000-fold higher detection sensitivity than the conventional one-pot recombinase polymerase amplification−CRISPR/Cas12a assay. The CasTAC strategy provides a versatile framework for fine–tuning Cas activity and advances CRISPR technology toward refined and context-adaptable functionality.
The CRISPR/Cas12a system is widely used in nucleic-acid diagnostics because Cas12a couples guide-directed recognition of a cis-target with indiscriminate collateral trans-cleavage of ssDNA. Controlling these activities through DNA structure can tune diagnostic signals. G-quadruplexes (G4s) are strong structural regulators, yet their ability to modulate Cas12a activation and collateral cleavage remains undefined. Here we focused on the G4 scaffold (TGGG)n and tested it with LbCas12a and AsCas12a by real-time cleavage assays, circular dichroism, FRET and denaturing PAGE. In the cis position, compact (TGGG)n G4s activated Cas12a; for the most stable (TGGG)5, kcat/KM was 5.9×104 and 8.9×104 M−1 s−1 for LbCas12a and AsCas12a, respectively. K+ reduced cis-activation rates by approximately 10-fold, depending on scaffold and temperature. FRET and denaturing PAGE showed that productive cis-recognition involves G4 unfolding followed by target-strand cleavage. In the trans position, compact G4s fully resisted collateral reporter cleavage. Core disruption and G-rich non-G4 controls restored reporter cleavage, showing that resistance depends on G4 architecture rather than guanine content. These data define a compact G4 scaffold that remains functionally trans-resistant while retaining guide-dependent cis-target competence. Thus, compact G4 folding provides a programmable structural mechanism for separating Cas12a activation from reporter cleavage, opening a route to signal-gated diagnostic designs. Graphical abstract
I. Safenkova, Maria V. Kamionskaya, A. V. Samokhvalov et al.· bioRxiv· 0 citations
