Toehold region triggered CRISPR/Cas12a trans-cleavage for detection of uracil-DNA glycosylase activity.

DNA glycosylases are a group of enzymes that play a crucial role in the DNA repair process by recognizing and removing damaged or incorrect bases from DNA molecules, which maintains the integrity of the genetic information. The abnormal expression of uracil-DNA glycosylase (UDG), one of significant DNA glycosylases in the base-excision repair pathway, is linked to numerous diseases. Here, we proposed a simple UDG activity detection method based on toehold region triggered CRISPR/Cas12a trans-cleavage. The toehold region on hairpin DNA probe (HP) produced by UDG could induce the trans-cleavage of ssDNA with fluorophore and quencher, generating an obvious fluorescence signal. This protospacer adjacent motif (PAM)-free approach achieves remarkable sensitivity and specificity in detecting UDG, with a detection limit as low as 0.000368 U mL-1. Moreover, this method is able to screen inhibitors and measure UDG in complex biological samples. These advantages render it highly promising for applications in clinical diagnosis and drug discovery.

CRISPR-Hg: Rapid and visual detection of Hg2+ based on PCR coupled with CRISPR/Cas12a.

Mercury (Hg) is a notorious toxic heavy metal, causing neurotoxicity and liver damage, posing grave threats to human health and environmental safety. There is an urgent imperative for developing novel Hg2+ detection methods. In this work, we developed a CRISPR-based method for Hg2+ detection named CRISPR-Hg. A CRISPR/Cas12a system was employed and could be activated by the PCR product, generating fluorescence signals based on the trans-cleavage activity. CRISPR-Hg exhibited remarkable selectivity and specificity, achieving a detection limit of 10 pM and minimal interference with background signals. This approach has been successfully applied to detect Hg2+ in real samples, including water, soil, and mushroom. Ulteriorly, a portable device was devised to streamline the readout of fluorescence signals by a smartphone within 30 min. We offer an affordable, highly selective and visually interpretable method for Hg2+ detection, with the potential for broad application in Hg2+ monitoring for food safety and public health.

A Sensitive Technique Unravels the Kinetics of Activation and Trans-Cleavage of CRISPR-Cas Systems.

Activation of the CRISPR-Cas13a system requires the formation of a crRNA-Cas13a ribonucleoprotein (RNP) complex and the binding of an RNA activator to the RNP. These two binding processes play a crucial role in the performance of the CRISPR-Cas13a system. However, the binding kinetics remain poorly understood, and a main challenge is the lack of a sensitive method for real-time measurements of the dynamically formed active CRISPR-Cas13a enzyme. We describe here a new method to study the binding kinetics and report the rate constants (kon and koff) and dissociation constant (Kd) for the binding between Cas13a and its activator. The method is able to unravel and quantify the kinetics of binding and cleavage separately, on the basis of measuring the real-time trans-cleavage rates of the CRISPR-Cas system and obtaining the real-time concentrations of the active CRISPR-Cas ternary complex. We further discovered that once activated, the Cas13a system operates at a wide range of temperatures (7-37 °C) with fast trans-cleavage kinetics. The new method and findings are important for diverse applications of the Cas13a system, such as the demonstrated quantification of microRNA at ambient temperatures (e.g., 25 °C).

Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR-Cas12a.

The CRISPR-Cas12a system has emerged as a powerful tool for next-generation nucleic acid-based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA-enabled trans-cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by full-size RNA targets, although LbCas12a exhibits weaker trans-cleavage activity than AsCas12a on both single-stranded DNA and RNA substrates. Remarkably, we find that the RNA-activated Cas12a possesses higher specificity in recognizing mutated target sequences compared to DNA activation. Based on these findings, we develop the "Universal Nuclease for Identification of Virus Empowered by RNA-Sensing" (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM-less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay based on double-stranded DNA activation, demonstrating unrestricted target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a-based nucleic acid detection and further enhance the understanding of CRISPR-Cas biochemistry.

Improving trans-cleavage activity of CRISPR-Cas13a using engineered crRNA with a uridinylate-rich 5'-overhang.

The engieering of Cas13a crRNA to enhance its binding affinity with the Cas enzyme or target is a promising method of improving the collateral cleavage efficiency of CRISPR-Cas13a systems, thereby amplifying the sensitivity of nucleic acid detection. An examination of the top-performing engineered crRNA (24 nt 5'7U LbuCas13a crRNA, where the 5'-end was extended using 7-mer uridinylates) and optimized conditions revealed an increased rate of LbuCas13a-mediated collateral cleavage activity that was up to seven-fold higher than that of the original crRNA. Particularly, the 7-mer uridinylates extension to crRNA was determined to be spacer-independent for enhancing the LbuCas13a-mediacted collateral cleavage activity, and also benefited the LwaCas13a system. The improved trans-cleavage activity was explained by the interactions between crRNA and LbuCas13a at the molecular level, i.e. the 5'-overhangs were anchored in the cleft formed between the Helical-1 and HEPN2 domains with the consequence of more stable complex, and experimentally verified. Consequently, the improved CRISPR-Cas13a system detected the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA with a sensitivity of 2.36 fM that was 160-times higher than that of the original system. Using isothermal amplification via reverse transcription-recombinase polymerase amplification (RT-RPA), the system was capable to detect SARS-CoV-2 with attomolar sensitivity and accurately identified the SARS-CoV-2 Omicron variant (20/21 agreement) in clinical samples within 40 min.

CRISPR/Cas12a integrated electrochemiluminescence biosensor for pufferfish authenticity detection based on NiCo2O4 NCs@Au as a coreaction accelerator.

Meat adulteration has brought economic losses, health risks, and religious concerns, making it a pressing global issue. Herein, combining the high amplification efficiency of polymerase chain reaction (PCR) and the accurate recognition of CRISPR/Cas12, a sensitive and reliable electrochemiluminescence (ECL) biosensor was developed for the detection of pufferfish authenticity using NiCo2O4 NCs@Au-ABEI as nanoemitters. In the presence of target DNA, the trans-cleavage activity of CRISPR/Cas12a is activated upon specific recognition by crRNA, and then it cleaves dopamine-modified single stranded DNA (ssDNA-DA), triggering the ECL signal from the "off" to "on" state. However, without target DNA, the trans-cleavage activity of CRISPR/Cas12a is silenced. By rationally designing corresponding primers and crRNA, the biosensor was applied to specific identification of four species of pufferfish. Furthermore, as low as 0.1 % (w/w) adulterate pufferfish in mixture samples could be detected. Overall, this work provides a simple, low-cost and sensitive approach to trace pufferfish adulteration.

PAM-Engineered Toehold Switches as Input-Responsive Activators of CRISPR-Cas12a for Sensing Applications.

The RNA-programmed CRISPR effector protein Cas12a has emerged as a powerful tool for gene editing and molecular diagnostics. However, additional bio-engineering strategies are required to achieve control over Cas12a activity. Here, we show that Toehold Switch DNA hairpins, presenting a rationally designed locked protospacer adjacent motif (PAM) in the loop, can be used to control Cas12a in response to molecular inputs. Reconfiguring the Toehold Switch DNA from a hairpin to a duplex conformation through a strand displacement reaction provides an effective means to modulate the accessibility of the PAM, thereby controlling the binding and cleavage activities of Cas12a. Through this approach, we showcase the potential to trigger downstream Cas12a activity by leveraging proximity-based strand displacement reactions in response to target binding. By utilizing the trans-cleavage activity of Cas12a as a signal transduction method, we demonstrate the versatility of our approach for sensing applications. Our system enables rapid, one-pot detection of IgG antibodies and small molecules with high sensitivity and specificity even within complex matrices. Besides the bioanalytical applications, the switchable PAM-engineered Toehold Switches serve as programmable tools capable of regulating Cas12a-based targeting and DNA processing in response to molecular inputs and hold promise for a wide array of biotechnological applications.

Sensing of trans-cleavage activity of CRISPR/Cas12a for detection of Salmonella.

Salmonella typhimurium (S. typhi) a predominant foodborne pathogen, significantly impacting global public health. Therefore, timely diagnosis is imperative to safeguard overall human health. To address this, we developed a novel CRISPR/Cas12a-mediated electrochemical detection system (biosensor) for targeting the SifA gene of S. typhi. To construct the biosensor, we utilized a screen-printed gold electrode (SPGE) as an electrochemical transducer and CRISPR/Cas12a for detection of SifA gene of S. typhi. The developed electrochemical biosensor exhibited an exceptional detection limit of 0.634 ± 0.029 pM, which was determined through differential pulse voltammetry (DPV) by utilizing a potentiostat. We compared the fabricated biosensor with gold standard RT-PCR and the visual detection limit of SifA was found to be 10 µM (in spiked buffer samples). The lower detection limit of fabricated biosensor provides an upper edge over the RT-PCR. Further, the fabricated biosensor also has the potential to serve as a rapid, stable, efficient, and early detection tool for S. typhi, offering promising advancements in diagnostic realms.

A New RNA-Dependent Cas12g Nuclease.

The development of RNA-targeting CRISPR-Cas systems represents a major step forward in the field of gene editing and regulation. RNA editing presents a viable alternative to genome editing in certain scenarios as it offers a reversible and manageable approach, reducing the likelihood of runaway mutant variants. One of the most promising applications is in the treatment of genetic disorders caused by mutations in RNA molecules. In this study, we investigate a previously undescribed Cas12g nuclease which was found in metagenomes from promising thermophilic microbial communities during the expedition to the Republic of North Ossetia-Alania in 2020. The method outlined in this study can be applied to other Cas orthologs and variants, leading to a better understanding of the CRISPR-Cas system and its enzymatic activities. The cis-cleavage activity of the new type V-G Cas effector was indicated by in vitro RNA cleavage experiments. While CRISPR-Cas systems are known for their high specificity, there is still a risk of unintended cleavage of nontargeted RNA molecules. Ultimately, the search for new genome editing tools and the study of their properties will remove barriers to research in this area. With continued research and development, we may be able to unlock their full potential.

DNA Probes for Cas12a-Based Assay with Fluorescence Anisotropy Enhanced Due to Anchors and Salts.

CRISPR/Cas12a is a potent biosensing tool known for its high specificity in DNA analysis. Cas12a recognizes the target DNA and acquires nuclease activity toward single-stranded DNA (ssDNA) probes. We present a straightforward and versatile approach to transforming common Cas12a-cleavable DNA probes into enhancing tools for fluorescence anisotropy (FA) measurements. Our study involved investigating 13 ssDNA probes with linear and hairpin structures, each featuring fluorescein at one end and a rotation-slowing tool (anchor) at the other. All anchors induced FA changes compared to fluorescein, ranging from 24 to 110 mr. Significant FA increases (up to 180 mr) were obtained by adding divalent metal salts (Mg2+, Ca2+, Ba2+), which influenced the rigidity and compactness of the DNA probes. The specific Cas12a-based recognition of double-stranded DNA (dsDNA) fragments of the bacterial phytopathogen Erwinia amylovora allowed us to determine the optimal set (probe structure, anchor, concentration of divalent ion) for FA-based detection. The best sensitivity was obtained using a hairpin structure with dC10 in the loop and streptavidin located near the fluorescein at the stem in the presence of 100 mM Mg2+. The detection limit of the dsDNA target was equal to 0.8 pM, which was eight times more sensitive compared to the common fluorescence-based method. The enhancing set ensured detection of single cells of E. amylovora per reaction in an analysis based on CRISPR/Cas12a with recombinase polymerase amplification. Our approach is universal and easy to implement. Combining FA with Cas12a offers enhanced sensitivity and signal reliability and could be applied to different DNA and RNA analytes.

CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of dumbbell DNA for specific detection of human 8-oxoguanine DNA glycosylase activity.

Human 8-oxoguanine DNA glycosylase (hOGG1) is an essential enzyme that recognizes and removes 8-oxoguanine (8-oxoG), a common DNA oxidative damage caused by reactive oxygen species, to maintain genomic integrity of living organisms. Abnormal expression of hOGG1 has been proved to be associated with different diseases such as cancer and neurogenerative disorders, making it a potential biomarker and therapeutic target. In this study, we report the development of  a novel strategy for detecting hOGG1 activity based on CRISPR/Cas12a trans-cleavage triggered by cleavage ligation of a dumbbell DNA probe (DBP) designed with a 3' overhang and an 8-oxoG modification. When hOGG1 is present, it cleaves the DBP at the 8-oxoG site, forming a 5' phosphate termini and exposing a single-strand region allowing complementary to the 3' overhang. After hybridization, the 3' and 5' termini in the juxtaposition are ligated by T4 DNA ligase, leading to a closed DBP for CRISPR/Cas12a-crRNA to recognize and initiate the trans-cleavage of the surrounding ssDNAs with fluorophore and quencher. The method achieves a limit of detection (LOD) with 370 µU/mL and high selectivity. Furthermore, it demonstrates a good compatibility for detecting hOGG1 activity in cell lysates, suggesting a good performance for further application in disease diagnosis and scientific research.

CRISPR/Cas-Powered Amplification-Free Detection of Nucleic Acids: Current State of the Art, Challenges, and Futuristic Perspectives.

CRISPR/Cas system is becoming an increasingly influential technology that has been repositioned in nucleic acid detection. A preamplification step is usually required to improve the sensitivity of CRISPR/Cas-based detection. The striking biological features of CRISPR/Cas, including programmability, high sensitivity and sequence specificity, and single-base resolution. More strikingly, the target-activated trans-cleavage could act as a biocatalytic signal transductor and amplifier, thereby empowering it to potentially perform nucleic acid detection without a preamplification step. The reports of such work are on the rise, which is not only scientifically significant but also promising for futuristic end-user applications. This review started with the introduction of the detection methods of nucleic acids and the CRISPR/Cas-based diagnostics (CRISPR-Dx). Next, we objectively discussed the pros and cons of preamplification steps for CRISPR-Dx. We then illustrated and highlighted the recently developed strategies for CRISPR/Cas-powered amplification-free detection that can be realized through the uses of ultralocalized reactors, cascade reactions, ultrasensitive detection systems, or others. Lastly, the challenges and futuristic perspectives were proposed. It can be expected that this work not only makes the researchers better understand the current strategies for this emerging field, but also provides insight for designing novel CRISPR-Dx without a preamplification step to win practicable use in the near future.

A Critical Study on DNA Probes Attached to Microplate for CRISPR/Cas12 Trans-Cleavage Activity.

CRISPR/Cas12-based biosensors are emerging tools for diagnostics. However, their application of heterogeneous formats needs the efficient detection of Cas12 activity. We investigated DNA probes attached to the microplate surface and cleaved by Cas12a. Single-stranded (ss) DNA probes (19 variants) and combined probes with double-stranded (ds) and ssDNA parts (eight variants) were compared. The cleavage efficiency of dsDNA-probes demonstrated a bell-shaped dependence on their length, with a cleavage maximum of 50%. On the other hand, the cleavage efficiency of ssDNA probes increased monotonously, reaching 70%. The most effective ssDNA probes were integrated with fluorescein, antibodies, and peroxidase conjugates as reporters for fluorescent, lateral flow, and chemiluminescent detection. Long ssDNA probes (120-145 nt) proved the best for detecting Cas12a trans-activity for all of the tested variants. We proposed a test system for the detection of the nucleocapsid (N) gene of SARS-CoV-2 based on Cas12 and the ssDNA-probe attached to the microplate surface; its fluorescent limit of detection was 0.86 nM. Being united with pre-amplification using recombinase polymerase, the system reached a detection limit of 0.01 fM, thus confirming the effectiveness of the chosen ssDNA probe for Cas12-based biosensors.

Comparison of Single-Stranded DNA Probes Conjugated with Magnetic Particles for Trans-Cleavage in Cas12a-Based Biosensors.

Biosensors based on endonuclease Cas12 provide high specificity in pathogen detection. Sensitive detection using Cas12-based assays can be achieved using trans-cleaved DNA probes attached to simply separated carriers, such as magnetic particles (MPs). The aim of this work was to compare polyA, polyC, and polyT single-stranded (ss) DNA with different lengths (from 10 to 145 nt) as trans-target probes were immobilized on streptavidin-covered MPs. Each ssDNA probe was labeled using fluorescein (5') and biotin (3'). To compare the probes, we used guide RNAs that were programmed for the recognition of two bacterial pathogens: Dickeya solani (causing blackleg and soft rot) and Erwinia amylovora (causing fire blight). The Cas12 was activated by targeting double-stranded DNA fragments of D. solani or E. amylovora and cleaved the MP-ssDNA conjugates. The considered probes demonstrated basically different dependencies in terms of cleavage efficiency. PolyC was the most effective probe when compared to polyA or polyT probes of the same length. The minimal acceptable length for the cleavage follows the row: polyC < polyT < polyA. The efficiencies of polyC and polyT probes with optimal length were proven for the DNA targets' detection of D. solani and E. amylovora. The regularities found can be used in Cas12a-based detection of viruses, bacteria, and other DNA/RNA-containing analytes.

Combining virtual screening with cis-/trans-cleavage enzymatic assays effectively reveals broad-spectrum inhibitors that target the main proteases of SARS-CoV-2 and MERS-CoV.

The main protease (Mpro) of SARS-CoV-2 is essential for viral replication, which suggests that the Mpro is a critical target in the development of small molecules to treat COVID-19. This study used an in-silico prediction approach to investigate the complex structure of SARS-CoV-2 Mpro in compounds from the United States National Cancer Institute (NCI) database, then validate potential inhibitory compounds against the SARS-CoV-2 Mpro in cis- and trans-cleavage proteolytic assays. Virtual screening of ∼280,000 compounds from the NCI database identified 10 compounds with highest site-moiety map scores. Compound NSC89640 (coded C1) showed marked inhibitory activity against the SARS-CoV-2 Mpro in cis-/trans-cleavage assays. C1 strongly inhibited SARS-CoV-2 Mpro enzymatic activity, with a half maximal inhibitory concentration (IC50) of 2.69 µM and a selectivity index (SI) of >74.35. The C1 structure served as a template to identify structural analogs based on AtomPair fingerprints to refine and verify structure-function associations. Mpro-mediated cis-/trans-cleavage assays conducted with the structural analogs revealed that compound NSC89641 (coded D2) exhibited the highest inhibitory potency against SARS-CoV-2 Mpro enzymatic activity, with an IC50 of 3.05 µM and a SI of >65.57. Compounds C1 and D2 also displayed inhibitory activity against MERS-CoV-2 with an IC50 of <3.5 µM. Thus, C1 shows potential as an effective Mpro inhibitor of SARS-CoV-2 and MERS-CoV. Our rigorous study framework efficiently identified lead compounds targeting the SARS-CoV-2 Mpro and MERS-CoV Mpro.

Framework-Hotspot Enhanced Trans Cleavage of CRISPR-Cas12a for Clinical Samples Detection.

The trans-cleavage property of CRISPR-Cas12a system makes it an excellent tool for disease diagnosis. Nevertheless, most methods based on CRISPR-Cas system still require pre-amplification of the target to achieve the desired detection sensitivity. Here we generate Framework-Hotspot reporters (FHRs) with different local densities to investigate their effect on trans-cleavage activity of Cas12a. We find that the cleavage efficiency increases and the cleavage rate accelerates with increasing reporter density. We further construct a modular sensing platform with CRISPR-Cas12a-based target recognition and FHR-based signal transduction. Encouragingly, this modular platform enables sensitive (100 fM) and rapid (<15 min) detection of pathogen nucleic acids without pre-amplification, as well as detection of tumor protein markers in clinical samples. The design provides a facile strategy for enhanced trans cleavage of Cas12a, which accelerates and broadens its applications in biosensing.

RNA-Dependent RNA Targeting by CRISPR-Cas Systems: Characterizations and Applications.

Genome editing technologies that are currently available and described have a fundamental impact on the development of molecular biology and medicine, industrial and agricultural biotechnology and other fields. However, genome editing based on detection and manipulation of the targeted RNA is a promising alternative to control the gene expression at the spatiotemporal transcriptomic level without complete elimination. The innovative CRISPR-Cas RNA-targeting systems changed the conception of biosensing systems and also allowed the RNA effectors to be used in various applications; for example, genomic editing, effective virus diagnostic tools, biomarkers, transcription regulations. In this review, we discussed the current state-of-the-art of specific CRISPR-Cas systems known to bind and cleave RNA substrates and summarized potential applications of the versatile RNA-targeting systems.

Impact of Divalent Metal Ions on Regulation of Trans-Cleavage Activity of CRISPR-Cas13a: A Combined Experimental and Computational Study.

CRISPR-LbuCas13a has emerged as a revolutionary tool for in vitro diagnosis. Similar to other Cas effectors, LbuCas13a requires Mg2+ to maintain its nuclease activity. However, the effect of other divalent metal ions on its trans-cleavage activity remains less explored. Herein, we addressed this issue by combining experimental and molecular dynamics simulation analysis. In vitro studies showed that both Mn2+ and Ca2+ could replace Mg2+ as cofactors of LbuCas13a. In contrast, Ni2+ , Zn2+ , Cu2+ , or Fe2+ inhibits the cis- and trans-cleavage activity, while Pb2+ does not affect it. Importantly, molecular dynamics simulations confirmed that calcium, magnesium, and manganese hydrated ions have a strong affinity to nucleotide bases, thus stabilizing the conformation of crRNA repeat region and enhancing the trans-cleavage activity. Finally, we showed that combination of Mg2+ and Mn2+ can further enhance the trans-cleavage activity to allow amplified RNA detection, revealing its potential advantage for in vitro diagnosis.

Cas12a/blocker DNA-based multiplex nucleic acid detection system for diagnosis of high-risk human papillomavirus infection.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins are an innovative tool in molecular diagnostics owing to their high specificity and modularity for target nucleic acid sequences. However, the sequence-indiscriminate trans-cleavage activity of the Cas protein renders multiplex detection challenging. In this study, we developed a Cas12a-based multiplex detection system by designing blocker DNA complementary to reporter DNA, which enables the simultaneous detection of two genes with a single Cas protein in a single reaction. As a proof of concept, we chose high-risk human papillomavirus (HPV) 16 and 18 as the model targets and incorporated recombinase polymerase amplification (RPA) and transcription reactions to achieve high accuracy and sensitivity. Using the proposed system, we detected the genes of both HPV 16 and 18 down to 1 aM within 80 min under isothermal conditions. We validated the performance of the system in detecting genomic DNA from various cell lines and clinical samples from cervical cancer patients with high specificity. The proposed system facilitated rapid multiplex detection of high-risk HPVs in a single reaction tube with only Cas12a, thus representing a more user-friendly and economical alternative to previous Cas protein-based multiplex detection assays. The proposed system has considerable potential for point-of-care testing and could be expanded to detect various nucleic acid biomarkers.

Engineering of DNA Structures Attached to Magnetic Particles for Effective Trans- and Cis-Cleavage in Cas12-Based Biosensors.

Sequence-specific endonuclease Cas12-based biosensors have rapidly evolved as a strong tool to detect nucleic acids. Magnetic particles (MPs) with attached DNA structures could be used as a universal platform to manipulate the DNA-cleavage activity of Cas12. Here, we propose nanostructures of trans- and cis-DNA targets immobilized on the MPs. The main advantage of the nanostructures is a rigid double-stranded DNA adaptor that distances the cleavage site from the MP surface to ensure maximum Cas12 activity. Adaptors with different lengths were compared by detecting the cleavage by fluorescence and gel electrophoresis of the released DNA fragments. The length-dependent effects for cleavage on the MPs' surface were found both for cis- and trans-targets. For trans-DNA targets with a cleavable 15-dT tail, the results showed that the optimal range of the adaptor length was 120-300 bp. For cis-targets, we varied the length and location of the adaptor (at the PAM or spacer ends) to estimate the effect of the MP's surface on the PAM-recognition process or R-loop formation. The sequential arrangement of an adaptor, PAM, and a spacer was preferred and required the minimum adaptor length of 3 bp. Thus, with cis-cleavage, the cleavage site can be located closer to the surface of the MPs than with trans-cleavage. The findings provide solutions for efficient Cas12-based biosensors using surface-attached DNA structures.