Structural basis of negative regulation of CRISPR-Cas7-11 by TPR-CHAT.

CRISPR‒Cas7-11 is a Type III-E CRISPR-associated nuclease that functions as a potent RNA editing tool. Tetratrico-peptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) acts as a regulatory protein that interacts with CRISPR RNA (crRNA)-bound Cas7-11 to form a CRISPR-guided caspase complex (Craspase). However, the precise modulation of Cas7-11's nuclease activity by TPR-CHAT to enhance its utility requires further study. Here, we report cryo-electron microscopy (cryo-EM) structures of Desulfonema ishimotonii (Di) Cas7-11-crRNA, complexed with or without the full length or the N-terminus of TPR-CHAT. These structures unveil the molecular features of the Craspase complex. Structural analysis, combined with in vitro nuclease assay and electrophoretic mobility shift assay, reveals that DiTPR-CHAT negatively regulates the activity of DiCas7-11 by preventing target RNA from binding through the N-terminal 65 amino acids of DiTPR-CHAT (DiTPR-CHATNTD). Our work demonstrates that DiTPR-CHATNTD can function as a small unit of DiCas7-11 regulator, potentially enabling safe applications to prevent overcutting and off-target effects of the CRISPR‒Cas7-11 system.

Harnessing noncanonical crRNA for highly efficient genome editing.

The CRISPR-Cas12a system is more advantageous than the widely used CRISPR-Cas9 system in terms of specificity and multiplexibility. However, its on-target editing efficiency is typically much lower than that of the CRISPR-Cas9 system. Here we improved its on-target editing efficiency by simply incorporating 2-aminoadenine (base Z, which alters canonical Watson-Crick base pairing) into the crRNA to increase the binding affinity between crRNA and its complementary DNA target. The resulting CRISPR-Cas12a (named zCRISPR-Cas12a thereafter) shows an on-target editing efficiency comparable to that of the CRISPR-Cas9 system but with much lower off-target effects than the CRISPR-Cas9 system in mammalian cells. In addition, zCRISPR-Cas12a can be used for precise gene knock-in and highly efficient multiplex genome editing. Overall, the zCRISPR-Cas12a system is superior to the CRISPR-Cas9 system, and our simple crRNA engineering strategy may be extended to other CRISPR-Cas family members as well as their derivatives.

Regulatory sequence-based discovery of anti-defense genes in archaeal viruses.

In silico identification of viral anti-CRISPR proteins (Acrs) has relied largely on the guilt-by-association method using known Acrs or anti-CRISPR associated proteins (Acas) as the bait. However, the low number and limited spread of the characterized archaeal Acrs and Aca hinders our ability to identify Acrs using guilt-by-association. Here, based on the observation that the few characterized archaeal Acrs and Aca are transcribed immediately post viral infection, we hypothesize that these genes, and many other unidentified anti-defense genes (ADG), are under the control of conserved regulatory sequences including a strong promoter, which can be used to predict anti-defense genes in archaeal viruses. Using this consensus sequence based method, we identify 354 potential ADGs in 57 archaeal viruses and 6 metagenome-assembled genomes. Experimental validation identified a CRISPR subtype I-A inhibitor and the first virally encoded inhibitor of an archaeal toxin-antitoxin based immune system. We also identify regulatory proteins potentially akin to Acas that can facilitate further identification of ADGs combined with the guilt-by-association approach. These results demonstrate the potential of regulatory sequence analysis for extensive identification of ADGs in viruses of archaea and bacteria.

Phosphorothioate-modified G-quadruplex as a signal-on dual-mode reporter for CRISPR/Cas12a-based portable detection of environmental pollutants.

BACKGROUND: Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-powered biosensor with a G-quadruplex (G4) reporter offer the benefits of simplicity and sensitivity, making them extensively utilized in detection applications. However, these biosensors used for monitoring pollutants in environmental water samples may face the problem of high background signal and easy interference due to the "signal-off" output. It is obvious that a biosensor based on the CRISPR/Cas12a system and G4 with a "signal on" output mode needs to be designed for detecting environmental pollutants. RESULTS: By using phosphorothioate-modified G4 as a reporter and catalytic hairpin assembly (CHA) integrated with Cas12a as an amplification strategy, a "signal-on" colorimetric/photothermal biosensor (psG4-CHA/Cas) for portable detection of environmental pollutants was developed. With the help of functional nucleotides, the target pollutant (kanamycin or Pb2+) triggers a CHA reaction to produce numerous double-strand DNA, which can activate Cas12a's trans-cleavage activity. The active Cas12a cleaves locked DNA to release caged psG-rich sequences. Upon binding hemin, the psG-rich sequence forms a psG4/hemin complex, facilitating the oxidation of the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into the blue photothermal agent (oxTMB). The smartphone was employed for portable colorimetric detection of kanamycin and Pb2+. The detection limits were found to be 100 pM for kanamycin and 50 pM for Pb2+. Detection of kanamycin and Pb2+ was also carried out using a portable thermometer with a detection limit of 10 pM for kanamycin and 8 pM for Pb2+. SIGNIFICANCE: Sensitive, selective, simple and robust detection of kanamycin and Pb2+ in environmental water samples is achieved with the psG4-CHA/Cas system. This system not only provides a new perspective on the development of efficient CRISPR/Cas12a-based "signal-on" designs, but also has a promising application for safeguarding human health and environmental monitoring.

Deciphering the Substrate Specificity Reveals that CRISPR-Cas12a Is a Bifunctional Enzyme with Both Endo- and Exonuclease Activities.

The class 2 CRISPR-Cas9 and CRISPR-Cas12a systems, originally described as adaptive immune systems of bacteria and archaea, have emerged as versatile tools for genome-editing, with applications in biotechnology and medicine. However, significantly less is known about their substrate specificity, but such knowledge may provide instructive insights into their off-target cleavage and previously unrecognized mechanism of action. Here, we document that the Acidaminococcus sp. Cas12a (AsCas12a) binds preferentially, and independently of crRNA, to a suite of branched DNA structures, such as the Holliday junction (HJ), replication fork and D-loops, compared with single- or double-stranded DNA, and promotes their degradation. Further, our study revealed that AsCas12a binds to the HJ, specifically at the crossover region, protects it from DNase I cleavage and renders a pair of thymine residues in the HJ homologous core hypersensitive to KMnO4 oxidation, suggesting DNA melting and/or distortion. Notably, these structural changes enabled AsCas12a to resolve HJ into nonligatable intermediates, and subsequently their complete degradation. We further demonstrate that crRNA impedes HJ cleavage by AsCas12a, and that of Lachnospiraceae bacterium Cas12a, without affecting their DNA-binding ability. We identified a separation-of-function variant, which uncouples DNA-binding and DNA cleavage activities of AsCas12a. Importantly, we found robust evidence that AsCas12a endonuclease also has 3'-to-5' and 5'-to-3' exonuclease activity, and that these two activities synergistically promote degradation of DNA, yielding di- and mononucleotides. Collectively, this study significantly advances knowledge about the substrate specificity of AsCas12a and provides important insights into the degradation of different types of DNA substrates.

RNA targeting and cleavage by the type III-Dv CRISPR effector complex.

CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1-5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes6-8. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors9,10, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.

Multiplexed in-situ mutagenesis driven by a dCas12a-based dual-function base editor.

Mutagenesis driving genetic diversity is vital for understanding and engineering biological systems. However, the lack of effective methods to generate in-situ mutagenesis in multiple genomic loci combinatorially limits the study of complex biological functions. Here, we design and construct MultiduBE, a dCas12a-based multiplexed dual-function base editor, in an all-in-one plasmid for performing combinatorial in-situ mutagenesis. Two synthetic effectors, duBE-1a and duBE-2b, are created by amalgamating the functionalities of cytosine deaminase (from hAPOBEC3A or hAID*Δ ), adenine deaminase (from TadA9), and crRNA array processing (from dCas12a). Furthermore, introducing the synthetic separator Sp4 minimizes interference in the crRNA array, thereby facilitating multiplexed in-situ mutagenesis in both Escherichia coli and Bacillus subtilis. Guided by the corresponding crRNA arrays, MultiduBE is successfully employed for cell physiology reprogramming and metabolic regulation. A novel mutation conferring streptomycin resistance has been identified in B. subtilis and incorporated into the mutant strains with multiple antibiotic resistance. Moreover, surfactin and riboflavin titers of the combinatorially mutant strains improved by 42% and 15-fold, respectively, compared with the control strains with single gene mutation. Overall, MultiduBE provides a convenient and efficient way to perform multiplexed in-situ mutagenesis.

A Novel CRISPR/Cas12a-Mediated Ratiometric Dual-Signal Electrochemical Biosensor for Ultrasensitive and Reliable Detection of Circulating Tumor Deoxyribonucleic Acid.

Circulating tumor DNA (ctDNA) holds great promise as a noninvasive biomarker for cancer diagnosis, treatment, and prognosis. However, the accurate and specific quantification of low-abundance ctDNA in serum remains a significant challenge. This study introduced, for the first time, a novel exponential amplification reaction (EXPAR)-assisted CRISPR/Cas12a-mediated ratiometric dual-signal electrochemical biosensor for ultrasensitive and reliable detection of ctDNA. To implement the dual-signal strategy, a signal unit (ssDNA-MB@Fc/UiO-66-NH2) was prepared, consisting of methylene blue-modified ssDNA as the biogate to encapsulate ferrocene signal molecules within UiO-66-NH2 nanocarriers. The presence of target ctDNA KRAS triggered EXPAR amplification, generating numerous activators for Cas12a activation, resulting in the cleavage of ssDNA-P fully complementary to the ssDNA-MB biogate. Due to the inability to form a rigid structure dsDNA (ssDNA-MB/ssDNA-P), the separation of ssDNA-MB biogate from the UiO-66-NH2 surface was hindered by electrostatic interactions. Consequently, the supernatant collected after centrifugation exhibited either no or only a weak presence of Fc and MB signal molecules. Conversely, in the absence of the target ctDNA, the ssDNA-MB biogate was open, leading to the leakage of Fc signal molecules. This clever ratiometric strategy with Cas12a as the "connector", reflecting the concentration of ctDNA KRAS based on the ratio of the current intensities of the two electroactive signal molecules, enhanced detection sensitivity by at least 60-300 times compared to single-signal strategies. Moreover, this strategy demonstrated satisfactory performance in ctDNA detection in complex human serum, highlighting its potential for cancer diagnosis.

Highly sensitive and selective demethylase FTO detection using a DNAzyme-mediated CRISPR/Cas12a signal cascade amplification electrochemiluminescence biosensor with C-CN/PCNV heterojunction as emitter.

Fat mass and obesity-associated protein (FTO) has gained attention as the first RNA N6-methyladenosine (m6A) modification eraser due to its overexpression being associated with various cancers. In this study, an electrochemiluminescence (ECL) biosensor for the detection of demethylase FTO was developed based on DNAzyme-mediated CRISPR/Cas12a signal cascade amplification system and carboxylated carbon nitride nanosheets/phosphorus-doped nitrogen-vacancy modified carbon nitride nanosheets (C-CN/PCNV) heterojunction as the emitter. The biosensor was constructed by modifying the C-CN/PCNV heterojunction and a ferrocene-tagged probe (ssDNA-Fc) on a glassy carbon electrode. The presence of FTO removes the m6A modification on the catalytic core of DNAzyme, restoring its cleavage activity and generating activator DNA. This activator DNA further activates the trans-cleavage ability of Cas12a, leading to the cleavage of the ssDNA-Fc and the recovery of the ECL signal. The C-CN/PCNV heterojunction prevents electrode passivation and improves the electron-hole recombination, resulting in significantly enhanced ECL signal. The biosensor demonstrates high sensitivity with a low detection limit of 0.63 pM in the range from 1.0 pM to 100 nM. Furthermore, the biosensor was successfully applied to detect FTO in cancer cell lysate and screen FTO inhibitors, showing great potential in early clinical diagnosis and drug discovery.

A CRISPR/Cas12a-Based System for Sensitive Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales.

Antimicrobial-resistant (AMR) bacteria pose a significant global health threat, and bacteria that produce New Delhi metallo-ß-lactamase (NDM) are particularly concerning due to their resistance to most ß-lactam antibiotics, including carbapenems. The emergence and spread of NDM-producing genes in food-producing animals highlight the need for a fast and accurate method for detecting AMR bacteria. We therefore propose a PCR-coupled CRISPR/Cas12a-based fluorescence assay that can detect NDM-producing genes (blaNDM) in bacteria. Thanks to its designed gRNA, this CRISPR/Cas12a system was able to simultaneously cleave PCR amplicons and ssDNA-FQ reporters, generating fluorescence signals. Our method was found to be highly specific when tested against other foodborne pathogens that do not carry blaNDM and also demonstrated an excellent capability to distinguish single-nucleotide polymorphism. In the case of blaNDM-1 carrying E. coli, the assay performed exceptionally well, with a detection limit of 2.7 × 100 CFU/mL: 100 times better than conventional PCR with gel electrophoresis. Moreover, the developed assay detected AMR bacteria in food samples and exhibited enhanced performance compared to previously published real-time PCR assays. Thus, this novel PCR-coupled CRISPR/Cas12a-based fluorescence assay has considerable potential to improve current approaches to AMR gene detection and thereby contribute to mitigating the global threat of AMR.

Paper-based loop-mediated isothermal amplification and CRISPR integrated platform for on-site nucleic acid testing of pathogens.

We report the development and initial validation of a paper-based nucleic acid testing platform that integrates Loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR) technology, referred to as PLACID (Paper-based LAMP-CRISPR Integrated Diagnostics). LAMP eliminates the need for thermal cycling, resulting in simplified instrumentation, and the CRISPR-associated protein (Cas 12a) system eliminates false positive signals from LAMP products, resulting in highly selective and sensitive assays. We optimized the assay to perform both amplification and detection entirely on paper, eliminating the need for complex fluid handling steps and lateral flow assay transfers. Additionally, we engineered a smartphone-operated system that includes a low-powered, non-contact IR heating chamber to actuate paper-based LAMP and CRISPR reactions and enable the detection of fluorescent signals from the paper. The platform demonstrates high specificity and sensitivity in detecting nucleic acid targets with a limit of detection of 50 copies/µL. We integrate an equipment-free sample preparation separation technology designed to streamline the preparation of crude samples prior to nucleic acid testing. The practical utility of our platform is demonstrated by the successful detection of spiked SARS-CoV-2 RNA fragments in saliva, E. Coli in soil, and pathogenic E. Coli in clinically fecal samples of infected patients. Furthermore, we demonstrate that the paper-based LAMP CRISPR chips employed in our assays possess a shelf life of several weeks, establishing them as viable candidates for on-site diagnostics.

High-Throughput µPAD with Cascade Signal Amplification through Dual Enzymes for arsM in Paddy Soil.

The arsM gene is a critical biomarker for the potential risk of arsenic exposure in paddy soil. However, on-site screening of arsM is limited by the lack of high-throughput point-of-use (POU) methods. Here, a multiplex CRISPR/Cas12a microfluidic paper-based analytical device (µPAD) was constructed for the high-throughput POU analysis of arsM, with cascade amplification driven by coupling crRNA-enhanced Cas12a and horseradish peroxidase (HRP)-modified probes. First, seven crRNAs were designed to recognize arsM, and their LODs and background signal intensities were evaluated. Next, a step-by-step iterative approach was utilized to develop and optimize coupling systems, which improved the sensitivity 32 times and eliminated background signal interference. Then, ssDNA reporters modified with HRP were introduced to further lower the LOD to 16 fM, and the assay results were visible to the naked eye. A multiplex channel microfluidic paper-based chip was developed for the reaction integration and simultaneous detection of 32 samples and generated a recovery rate between 87.70 and 114.05%, simplifying the pretreatment procedures and achieving high-throughput POU analysis. Finally, arsM in Wanshan paddy soil was screened on site, and the arsM abundance ranged from 1.05 × 106 to 6.49 × 107 copies/g; this result was not affected by the environmental indicators detected in the study. Thus, a coupling crRNA-based cascade amplification method for analyzing arsM was constructed, and a microfluidic device was developed that contains many more channels than previous paper chips, greatly improving the analytical performance in paddy soil samples and providing a promising tool for the on-site screening of arsM at large scales.

KP177R-based visual assay integrating RPA and CRISPR/Cas12a for the detection of African swine fever virus.

Introduction: Early detection of the virus in the environment or in infected pigs is a critical step to stop African swine fever virus (ASFV) transmission. The p22 protein encoded by ASFV KP177R gene has been shown to have no effect on viral replication and virulence and can serve as a molecular marker for distinguishing field virus strains from future candidate KP177R deletion vaccine strains. Methods: This study established an ASFV detection assay specific for the highly conserved ASFV KP177R gene based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12 reaction system. The KP177R gene served as the initial template for the RPA reaction to generate amplicons, which were recognized by guide RNA to activate the trans-cleavage activity of Cas12a protein, thereby leading to non-specific cleavage of single-stranded DNA as well as corresponding color reaction. The viral detection in this assay could be determined by visualizing the results of fluorescence or lateral flow dipstick (LFD) biotin blotting for color development, and was respectively referred to as fluorescein-labeled RPA-CRISPR/Cas12a and biotin-labeled LFD RPA-CRISPR/Cas12a. The clinical samples were simultaneously subjected to the aforementioned assay, while real-time quantitative PCR (RT-qPCR) was employed as a control for determining the diagnostic concordance rate between both assays. Results: The results showed that fluorescein- and biotin-labeled LFD KP177R RPA-CRISPR/Cas12a assays specifically detected ASFV, did not cross-react with other swine pathogens including PCV2, PEDV, PDCoV, and PRV. The detection assay established in this study had a limit of detection (LOD) of 6.8 copies/µL, and both assays were completed in 30 min. The KP177R RPA-CRISPR/Cas12a assay demonstrated a diagnostic coincidence rate of 100% and a kappa value of 1.000 (p < 0.001), with six out of ten clinical samples testing positive for ASFV using both KP177R RPA-CRISPR/Cas12a and RT-qPCR, while four samples tested negative in both assays. Discussion: The rapid, sensitive and visual detection assay for ASFV developed in this study is suitable for field application in swine farms, particularly for future differentiation of field virus strains from candidate KP177R gene-deleted ASFV vaccines, which may be a valuable screening tool for ASF eradication.

Allosteric Activator-Regulated CRISPR/Cas12a System Enables Biosensing and Imaging of Intracellular Endogenous and Exogenous Targets.

Sensors designed based on the trans-cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the "on-off-on" mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans-cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially "blocked" state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the "RESET" effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.

Insights into the inhibition of protospacer integration via direct interaction between Cas2 and AcrVA5.

Spacer acquisition step in CRISPR-Cas system involves the recognition and subsequent integration of protospacer by the Cas1-Cas2 complex in CRISPR-Cas systems. Here we report an anti-CRISPR protein, AcrVA5, and reveal the mechanisms by which it strongly inhibits protospacer integration. Our biochemical data shows that the integration by Cas1-Cas2 was abrogated in the presence of AcrVA5. AcrVA5 exhibits low binding affinity towards Cas2 and acetylates Cas2 at Lys55 on the binding interface of the Cas2 and AcrVA5 N-terminal peptide complex to inhibit the Cas2-mediated endonuclease activity. Moreover, a detailed structural comparison between our crystal structure and homolog structure shows that binding of AcrVA5 to Cas2 causes steric hindrance to the neighboring protospacer resulting in the partial disassembly of the Cas1-Cas2 and protospacer complex, as demonstrated by electrophoretic mobility shift assay. Our study focuses on this mechanism of spacer acquisition inhibition and provides insights into the biology of CRISPR-Cas systems.

Dissecting the CRISPR Cas1-Cas2 Protospacer Binding and Selection Mechanism by Using Molecular Dynamics Simulations.

Cas1 and Cas2 are highly conserved proteins among the clustered regularly interspaced short palindromic repeat Cas (CRISPR-Cas) systems and play a crucial role in protospacer selection and integration. According to the double-forked CRISPR Cas1-Cas2 complex, we conducted extensive all-atom molecular dynamics simulations to investigate the protospacer DNA binding and recognition. Our findings revealed that single-point amino acid mutations in Cas1 or in Cas2 had little impact on the binding of the protospacer, both in the binding and precatalytic states. In contrast, multiple-point amino acid mutations, particularly G74A, P80L, and V89A mutations on Cas2 and Cas2' proteins (m-multiple1 system), significantly affected the protospacer binding and selection. Notably, mutations on Cas2 and Cas2' led to an increased number of hydrogen bonds (#HBs) between Cas2&Cas2' and the dsDNA in the m-multiple1 system compared with the wild-type system. And the strong electrostatic interactions between Cas1-Cas2 and the protospacer DNA (psDNA) in the m-multiple1 system again suggested the increase in the binding affinity of protospacer acquisition. Specifically, mutations in Cas2 and Cas2' can remotely make the protospacer adjacent motif complementary (PAMc) sequences better in recognition by the two active sites, while multiple mutations K211E, P202Q, P212L, R138L, V134A, A286T, P282H, and P294H on Cas1a/Cas1b&Cas1a'/Cas1b' (m-multiple2 system) decrease the #HBs and the electrostatic interactions and make the PAMc worse in recognition compared with the wild-type system.

Ratiometric CRISPR/Cas12a-Triggered CHA System Coupling with the MSRE to Detect Site-Specific DNA Methylation.

The precise determination of DNA methylation at specific sites is critical for the timely detection of cancer, as DNA methylation is closely associated with the initiation and progression of cancer. Herein, a novel ratiometric fluorescence method based on the methylation-sensitive restriction enzyme (MSRE), CRISPR/Cas12a, and catalytic hairpin assembly (CHA) amplification were developed to detect site-specific methylation with high sensitivity and specificity. In detail, AciI, one of the commonly used MSREs, was employed to distinguish the methylated target from nonmethylated targets. The CRISPR/Cas12a system was utilized to recognize the site-specific target. In this process, the protospacer adjacent motif and crRNA-dependent identification, the single-base resolution of Cas12a, can effectively ensure detection specificity. The trans-cleavage ability of Cas12a can convert one target into abundant activators and can then trigger the CHA reaction, leading to the accomplishment of cascaded signal amplification. Moreover, with the structural change of the hairpin probe during CHA, two labeled dyes can be spatially separated, generating a change of the Förster resonance energy transfer signal. In general, the proposed strategy of tandem CHA after the CRISPR/Cas12a reaction not only avoids the generation of false-positive signals caused by the target-similar nucleic acid but can also improve the sensitivity. The use of ratiometric fluorescence can eradicate environmental effects by self-calibration. Consequently, the proposed approach had a detection limit of 2.02 fM. This approach could distinguish between colorectal cancer and precancerous tissue, as well as between colorectal patients and healthy people. Therefore, the developed method can serve as an excellent site-specific methylation detection tool, which is promising for biological and disease studies.

Chemical control of CRISPR/Cpf1 editing via orthogonal activation and deactivation of crosslinked crRNA.

Through the integration of CRISPR/Cpf1 with optogenetics and a reduction-responsive motif, we have developed a photoactivatable cross-linked crRNA that enables precise genome editing upon light exposure. This system also allows for termination of editing activity through external application of reducing agent. The dual-stimuli-responsive CRISPR/Cpf1 editing process operates in a unique OFF → ON → OFF sequence, making it a valuable tool for investigating time-sensitive biological events.

Development of a CRISPR/Cas12a-mediated aptasensor for Mpox virus antigen detection.

The emergence and rapid spread of Mpox (formerly monkeypox) have caused significant societal challenges. Adequate and appropriate diagnostics procedures are an urgent necessity. Herein, we discover a pair of aptamers through the systematic evolution of ligands by exponential enrichment (SELEX) that exhibit high affinity and bind to different sites towards the A29 protein of the Mpox virus. Subsequently, we propose a facile, sensitive, convenient CRISPR/Cas12a-mediated aptasensor for detecting the A29 antigen. The procedure employs the bivalent aptamers recognition, which induces the formation of a proximity switch probe and initiates subsequent cascade strand displacement reactions, then triggers CRISPR/Cas12a DNA trans-cleavage to achieve the sensitive detection of Mpox. Our method enables selective and ultrasensitive evaluation of the A29 protein within the range of 1 ng mL-1 to 1 µg mL-1, with a limit of detection (LOD) at 0.28 ng mL-1. Moreover, spiked A29 protein recovery exceeds 96.9%, while the detection activity remains above 91.9% after six months of storage at 4 °C. This aptasensor provides a novel avenue for exploring clinical diagnosis in cases involving Mpox as facilitating development in various analyte sensors.