Click-chemistry-mediated modulation of CRISPR-Cas12a activity through activator modification.

Cas-regulation-targeting chimera enables selective and tunable control of CRISPR/Cas12a

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.

Chemical additives-enhanced CRISPR/Cas12a-based RNA detection.

The CRISPR/Cas12a system has revolutionized molecular diagnostics due to its RNA-guided trans-cleavage activity, enabling programmable and highly accurate nucleic acid detection. However, most Cas12a-based assays are optimized for DNA targets, while direct RNA detection constrained by limited sensitivity, typically at the nanomolar level. Existing strategies to improve the performance of RNA analysis often rely on additional DNA activators or complex auxiliary systems. Here, we report a simple yet effective chemical additive-based strategy that overcomes these limitations. This chemical additives-enhanced CRISPR/Cas12a-based RNA detection (CARD) enables femtomolar-level RNA detection using only a single crRNA, without the need for DNA activators, reverse transcription, or strand-displacement reactions. Notably, this approach can be adapted to single-stranded DNA, enabling ssDNA detection at attomolar levels. Collectively, CARD provides a straightforward, amplification-free, and highly sensitive diagnostic framework that might be readily extended to other CRISPR/Cas systems for ultrasensitive nucleic acid diagnostics.

TOPS-CRISPR: Thermally-regulated and oligonucleotide-mediated one-pot CRISPR-Cas12a assay for ultra-sensitive and rapid on-site diagnostics.

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.

Biphasic spatiotemporal regulation of Cas12a substrate cleavage enables one-pot autocatalytic CRISPR biosensing of non-nucleic-acid targets.

One-pot autocatalytic CRISPR biosensing offers a promising route for signal amplification without nucleic acid pre-amplification, but its efficiency is limited by an intrinsic readout-amplification conflict: the ssDNA reporter required for signal output competes with the autocatalytic mediator required for feedback amplification. Here, we report a biphasic spatiotemporal regulation strategy (BS-Cas12a system) to resolve this substrate competition for one-pot autocatalytic CRISPR biosensing of non-nucleic-acid targets. Mechanistic studies revealed that Cas12a preferentially cleaved the ssDNA reporter over the autocatalytic circular mediator, leading to insufficient mediator linearization and impaired autocatalytic amplification. A glycerol/water biphasic system was therefore constructed to spatially delay reporter access to activated Cas12a, allowing preferential cleavage of the autocatalytic mediator and subsequent generation of additional Cas12a activators. This biphasic system achieved a 3-fold increase in the autocatalytic amplification efficiency. By integrating an aptamer-mediated target-to-activator conversion module, non-nucleic-acid recognition was programmably converted into Cas12a activation. Using sulfadimethoxine as a model target, the platform achieved a detection limit of 65 pM and showed good recoveries in fish samples. The system was further extended to Cd2+ and thrombin detection with limits of detection of 0.65 nM and 11.63 pM, respectively. Moreover, by replacing the fluorescent reporter with a FAM/biotin-labeled reporter, a lateral-flow readout was achieved. This work provides a kinetic-regulation strategy for modular one-pot Cir DNA autocatalytic biosensing with an independent and exchangeable reporter and expands CRISPR-based detection toward diverse non-nucleic-acid targets.

Programmable Allosteric DNAzyme Coupled with CRISPR/Cas12a System for Multiplexed and Sensitive Detection of Extracellular Vesicle Derived MicroRNAs

Signal logic gate mediated by a controllable CRISPR/Cas12a system for simultaneous detection of DNA mutation and methylation.

Early detection of both genetic mutations and epigenetic modifications is critical for cancer diagnosis, but current methods often require separate assays or suffer from bisulfite-induced DNA damage. Here, we present a controllable CRISPR/Cas12a-based signal logic gate that enables simultaneous detection of KRAS G12C mutation and Septin9 promoter methylation in a single reaction. The strategy converts target information into two distinct ssDNA activators (T1 for methylation and T2 for mutation) via orthogonal enzymatic cascades (GlaI- and FEN1-mediated cleavage followed by strand displacement amplification). By limiting crRNA concentrations, the CRISPR/Cas12a trans-cleavage activity produces three well-resolved fluorescence kinetic states: low (mutation only), medium (methylation only), and high (both targets), respectively, which could be distinguished using three predefined threshold values. Validation with clinical samples from colorectal cancer patients and healthy controls showed complete concordance with Sanger sequencing and qPCR. And the proposed method successfully detected Septin9 methylation in peripheral blood. This isothermal, single-tube, bisulfite-free strategy offers a simple and reliable platform for simultaneous genetic and epigenetic analysis in point-of-care cancer screening.