Cell Host & Microbe
·
2023
·
IF 30.3
Chao Liu,Rui Wang,Jie Li,Feiyue Cheng,Xian Shu,Huiwei Zhao,Qiong Xue,Haiying Yu,Aici Wu,Lingyun Wang,Sushu Hu,Yihan Zhang,Jun Yang,Hua Xiang,Ming Li
CRISPR RNAs (crRNAs) and Cas proteins work together to provide prokaryotes with adaptive immunity against genetic invaders like bacteriophages and plasmids. However, the coordination of crRNA production and cas expression remains poorly understood. Here, we demonstrate that widespread modulatory mini-CRISPRs encode cas-regulating RNAs (CreRs) that mediate autorepression of type I-B, I-E, and V-A Cas proteins, based on their limited complementarity to cas promoters. This autorepression not only reduces autoimmune risks but also responds to changes in the abundance of canonical crRNAs that compete with CreR for Cas proteins. Furthermore, the CreR-guided autorepression of Cas proteins can be alleviated or even subverted by diverse bacteriophage anti-CRISPR (Acr) proteins that inhibit Cas effectors, which, in turn, promotes the generation of new Cas proteins. Our findings reveal a general RNA-guided autorepression paradigm for diverse Cas effectors, shedding light on the intricate self-coordination of CRISPR-Cas and its transcriptional counterstrategy against Acr proteins.
研究领域:
分子生物学
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微生物学
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遗传学
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PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Songkuan Zhuang,Xiaojun Huang,Muyan Diao,Ruiling Dong,Yueyu Dong,Jun Liu,Yizhen Liu
Current one-pot CRISPR diagnostics necessitate meticulous control of nucleic acid hybridization parameters or extensive modification of CRISPR components to achieve complete enzymatic silencing, a fundamental bottleneck limiting their robustness and generalizability. Here, we resolve this challenge through dynamic crRNA reconfiguration, a paradigm-shifting strategy that exploits the intrinsic structural duality of CRISPR RNA. We present CONVERT (CRISPR One-Pot Nucleic acid detection Via Engineered crRNA Reconfiguration Technology), a universal platform where nontarget intact crRNA acts as a universal suppressor, achieving complete Cas12a inhibition during RPA (recombinase polymerase amplification) by irreversible enzyme sequestration. Target detection is initiated through programmable conversion to split crRNA activators, bypassing sequence-specific design constraints entirely. This crRNA conformational switching mechanism is implemented via photocleavable linker cleavage and subsequent assembly with presupplied truncated crRNA (tcrRNA), activating trans-cleavage signal amplification. Three transformative advances emerge: First, the endogenous crRNA engineering eliminates dependency on exogenous nucleic acid blockers or chemical modifications, reducing optimization costs and time. Second, near-total enzymatic suppression ensures zero cross-talk between amplification and detection phases. Third, the unified workflow enables contamination-resistant operation through spatiotemporal control, requiring no physical partitioning. Validated for Enterocytozoon bieneusi detection, the system demonstrates attomolar sensitivity of 1 aM, 100% diagnostic concordance with qPCR, and absolute specificity against related pathogens. By effectively decoupling CRISPR regulation from sequence-context constraints through rational crRNA structural plasticity, this work establishes a generalized framework for next-generation molecular diagnostics.
研究领域:
分子生物学
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生物工程
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诊断技术
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合成生物学
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核酸扩增
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PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2025
·
IF 10.5
Miao He,Jinhua Chen,MuRilege Chao,Wenting Cheng,Han Pan,Qiufeng Wang,Zhongyun Wang,Yang Xiang
mRNA vaccine had emerged as a novel and promising modality in cancer immunotherapy. As anti-tumor mRNA vaccines entered clinical trials, continuous monitoring of mRNA levels becomes particularly important. In this paper, a new method of collaborative detection of tumor mRNA vaccine by double Cas12a system based on split crRNA was created. Specifically, the target was extended based on the RCA ring template to generate RCA products and completed the first round of signal amplification; At this time, there were a lot of secondary structures in RCA products. Subsequently, the double Cas12a system with split crRNA bound to adjacent sites, reduced secondary structure of the DNA strand, stabilized the linearization state, and identified multiple sites at the same time, so as to achieve the second round of collaborative signal amplification. When there was no target in the system, the RCA reaction cannot be triggered. In this method, the entire detection process was completed by one-pot method. The detection limit was as low as 0.2pM, and it showed a good linear relationship in the range of 0–100 pM, which provided a new method for the screening and pharmacokinetics study of tumor mRNA vaccines.
研究领域:
癌症免疫治疗
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分子诊断
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CRISPR技术
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生物分析化学
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药代动力学
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核酸扩增
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PubMed
高影响因子
NUCLEIC ACIDS RESEARCH
·
2026
·Vol. 54
·期 5
·
IF 15
Wang Zhongzhong,Wu Yi,Wang Zixuan,Zhang Simeng,Liu Hao,Nie You,Chen Kexin,Huang Yanjun,Zhou Yu,Cao Yuanyuan,Sun Lin,Hao Rongzhang
CRISPR–Cas12a has transformative potential in molecular diagnostics owing to its robust signal amplification, but its sustained activity state severely limits temporal programmability and precise nuclease control in complex detection workflows. Here, we demonstrate that the conserved crRNA scaffold secondary structure itself can be repurposed as a reversible and programmable conformational switch to regulate Cas12a activity. By introducing short complementary DNA blockers of tunable length, we achieved length-dependent disruption and remodeling of scaffold secondary structure, shifting LbCas12a into an inactive conformation. Scaffold structure was subsequently reinstated through either single or cooperative strand displacement activation, enabling time-resolved and on-demand restoration of Cas12a activity. The conserved scaffold ensures intrinsic assay universality, while its programmable rewiring markedly improves SNVs discrimination and enables compatibility with one-pot isothermal amplification assays, delivering analytical sensitivity comparable to conventional two-step assays. This regulatory framework was further demonstrated in the detection of Klebsiella pneumoniae and Mycobacterium tuberculosis. By validating the crRNA scaffold as a practical and programmable switch for Cas12a activity control, this work establishes a universal and reversible framework for scaffold rewiring to modulate CRISPR nucleases and offers mechanistic insight to guide future assay engineering.
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高影响因子
NUCLEIC ACIDS RESEARCH
·
2025
·Vol. 53
·期 21
·
IF 13.1
Xiao Yao,Zhao Rujian,Bao Yin,Lu Baiyang,Jiang Yanfang,Tang Yidan,Li Bingling
The Cas12a–crRNA system possesses inherent sequence-specific recognition of double-stranded DNA/single-stranded DNA (dsDNA/ssDNA) coupled with trans-cleavage activity toward ssDNA, making it a powerful tool for nucleic acid diagnostics. However, its application beyond nucleic acid targets remains challenging, limiting its potential as a universal detection platform. In this study, we systematically explore the key parameters governing the activation of a Cas12a-split crRNA system and established a comprehensive set of design guidelines. Building on these findings, we developed CASCADE (Cas12a-Assisted Split crRNA Complex for Analysis and Detection of Diverse Entities), an adaptable detection platform that extends Cas12a’s application beyond nucleic acids. Using microRNA as a model, we validated the system’s sensitivity, specificity, and mismatch discrimination capability. Additionally, we successfully demonstrated its capability for non-nucleic acid target detection by detecting tobramycin, kanamycin, biotin, and tetracycline repressor protein, confirming its sensitivity and specificity. Finally, by integrating a lateral flow assay (LFA), we enhanced the portability of CASCADE, enabling user-friendly, on-site detection. This work expands the application scope of the Cas12a system and offers a promising strategy for point-of-care diagnostics or environmental monitoring.
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高影响因子
POULTRY SCIENCE
·
2024
·
IF 3.8
Sha-Sha Chen,Yong-Lei Yang,Hong-Yun Wang,Tian-Kui Guo,Riaz-M Azeem,Chun-Wei Shi,Gui-Lian Yang,Hai-Bin Huang,Yan-Long Jiang,Jian-Zhong Wang,Xin Cao,Nan Wang,Yan Zeng,Wen-Tao Yang,Chun-Feng Wang
ABSTRACT Avian influenza virus (AIV) subtype H9N2 has significantly threatened the poultry business in recent years by having become the predominant subtype in flocks of chickens, ducks, and pigeons. In addition, the public health aspects of H9N2 AIV pose a significant threat to humans. Early and rapid diagnosis of H9N2 AIV is therefore of great importance. In this study, a new method for the detection of H9N2 AIV based on fluorescence intensity was successfully established using CRISPR/Cas13a technology. The Cas13a protein was first expressed in a prokaryotic system and purified using nickel ion affinity chromatography, resulting in a high-purity Cas13a protein. The best RPA(Recombinase polymerase amplification) primer pairs and crRNA were designed and screened, successfully constructing the detection of H9N2 AIV based on CRISPR/Cas13a technology. Optimal concentration of Cas13a and crRNA was determined to optimise the constructed assay. The sensitivity of the optimised detection system is excellent, with a minimum detection limit of 10° copies/μL and didn't react with other avian susceptible viruses, with excellent specificity. The detection method provides the basis for the field detection of the H9N2 AIV.
研究领域:
分子生物学
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病毒学
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生物技术
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公共卫生
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PubMed
高影响因子
MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Feifei Zhou,Yingzhan Fan,Qinrui Cao,Yingying Yuan,Yingxiao Chen,Lingling Liu,Jingwen Mai,Nandi Chen,Tianxing Ji,Dou Wang,Chenchen Ge
Dual-function DNA probe (dfDNA) enables a split crRNA/Cas12a (SCas12a) assay. • The dfDNA acts as both a Cas12a activator and a fluorescent reporter. • The dfDNA activates both the cis - and trans -cleavage activities of Cas12a. • The dfDNA/SCas12a assay detects microRNA-21 (miR-21) without amplification. • Bead-assisted dfDNA format improves the sensitivity for miR-21 detection. MicroRNA-21 (miR-21) is a promising non-invasive biomarker for tumor diagnosis, but its short length and low abundance make amplification-free detection challenging. Here, we developed a dual-function DNA probe (dfDNA)-enabled split crRNA-Cas12a (SCas12a) system for miR-21 detection. This design separates crRNA into a constant scaffold fragment (sc-crRNA) and a variable spacer fragment (sp-crRNA), and miR-21 functionally replaces the spacer fragment to activate Cas12a in the presence of dfDNA. The dfDNA acts as both a target-recognition probe and a fluorescent reporter: it hybridizes with miR-21, activates Cas12a, and presents a single-stranded fluorescent overhang that can be cleaved after Cas12a activation. Because target recognition and signal reporting are integrated within a single probe, dfDNA positions the activated Cas12a complex close to the cleavable overhang, improving signal conversion compared with the conventional single-stranded DNA reporter and fully complementary double-stranded DNA probe format. Under optimized conditions, the limit of detection (LOD) with the homogeneous ssDNA reporter-based assay was 347.9 pM for miR-21, whereas the homogeneous dfDNA-based assay reduced the LOD to 19.8 pM. When transferred to a magnetic bead (MB)-assisted format, the dfDNA-based system achieved an LOD of 1.1 pM, supporting that the localized reaction accelerates the trans -cleavage kinetics of Cas12a. The MB-assisted dfDNA assay achieves comparable or even superior sensitivity for miR-21 detection compared with previously reported split crRNA-based fluorescence methods. Further optimization, including target enrichment or signal amplification strategies, is needed before this method can be reliably used for low-abundance miRNAs in clinical serum samples. Download: Download high-res image (88KB) Download: Download full-size image
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高影响因子
METHODS
·
2021
·
IF 3.608
Menglu Hu,Debin Zhu,Xiaoming Zhou
The construction of a rapid, simple, and specific nucleic acid detection platform is of great significance to the control of the large-scale spread of infectious diseases. We have recently established a magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system (termed M−CDC), which effectively integrates the advantages of CRISPR/Cas12a, magnetic beads-based separation, and AuNP bioprobe to provide a simple and specific biosensing platform for nucleic acid assay. The M−CDC method is compatible with point-of-care testing and enables the detection of nucleic acid samples in less than an hour without relying on expensive and complex instruments. In this paper, step-by-step instructions for M−CDC assay, including recombinase polymerase amplification (RPA)/reverse transcription-polymerase chain reaction (RT-RPA) of DNA or RNA, Cas12a-mediated target recognition and cleavage, and subsequent magnetic beads-mediated colorimetric readouts are provided. In addition, the protocol for the expression and purification of Lachnospiraceae bacterium -Cas12a (LbCas12a) protein, the design and synthesis of high-efficient crRNA, and the preparation of AuNP bioprobe are also offered.
研究领域:
生物技术
·
分子生物学
·
诊断方法
·
纳米技术
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DOI
PubMed
高影响因子
ACS Sensors
·
2022
·
IF 9.618
Jiahao Li,Lina Tang,Tingxian Li,Kun Li,Yulin Zhang,Wei Ni,Meng-Meng Xiao,Youyun Zhao,Zhi-Yong Zhang,Guo-Jun Zhang
The path toward field-effect transistor (FET) application from laboratory to clinic has delivered a compelling push in the biomedical domain, yet ultrasensitive and timely pathogen identification without PCR remains a long-lasting challenge. Herein, we create a generic check station termed “CRISPR-FET”, first incorporating the CRISPR/Cas13a system within the FET modality, for accelerated and unamplified detection of viral RNA. Unlike conventional FETs bearing target-specific receptors, this sensor holds three unique advancements: (i) an ingenious sensing mechanism is used, which converts the signal of a large-sized analyte into an on-chip cleavage response of an immobilized CRISPR reporter, enabling signal generation events to occur all within the Debye length; (ii) the multipurpose inspection of the CoV ORF1ab, CoV N gene, and HCV RNA unveils the potential for “one-for-all” scalable FET-based molecular diagnostics; and (iii) it is shown that Cas13a-crRNAs targeting different sites of the viral genome can be deployed in tandem to amplify the FET response, empowering the detection limit down to 1.56 aM, which is a world-record level of sensitivity in the FET for direct viral gene sensing. Notably, a brilliant clinical applicability was made in distinguishing HCV-infected patients from normal controls. Overall, this study sheds new insights into FET-based nucleic acid sensing technology and invokes a vision for its possible future roles in diagnosis of various viral diseases.
研究领域:
生物医学工程
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分子诊断
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纳米技术
·
病毒学
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2023
·
IF 7.4
Liping Xia,Juxin Yin,Jianjian Zhuang,Weihong Yin,Zheyu Zou,Ying Mu
Rapid and sensitive pathogen detection methods are critical for disease diagnosis and treatment. RPA-CRISPR/Cas12 systems have displayed remarkable potential in pathogen detection. A self-priming digital PCR chip is a powerful and attractive tool for nucleic detection. However, the application of the RPA-CRISPR/Cas12 system to the self-priming chip still has great challenges due to the problems of protein adsorption and two-step detection mode of RPA-CRISPR/Cas12. In this study, an adsorption-free self-priming digital chip was developed and a direct digital dual-crRNAs (3D) assay was established based on the chip for ultrasensitive detection of pathogens. This 3D assay combined the advantages of rapid amplification of RPA, specific cleavage of Cas12a, accurate quantification of digital PCR, and point-of-care testing (POCT) of microfluidics, enabling accurate and reliable digital absolute quantification of Salmonella in POCT. Our method can provide a good linear relationship of Salmonella detection in the range from 2.58 × 101 to 2.58 × 104 cells/mL with a limit of detection ∼0.2 cells/mL within 30 min in a digital chip by targeting the invA gene of Salmonella. Moreover, the assay could directly detect Salmonella in milk without nucleic acid extraction. Therefore, the 3D assay has the significant potential to provide accurate and rapid pathogen detection in POCT. This study provides a powerful nucleic detection platform and facilitates the application of CRISPR/Cas-assisted detection and microfluidic chips.
研究领域:
分子生物学
·
生物技术
·
微生物学
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分析化学
·
即时诊断
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PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2022
·
IF 9.221
Kai Zhang,Meina Yang,Helin Sun,Xueyin Wang,Hao Li,Jinxiang Han,Zhongwen Zhang
Using Peptide nucleic acid (PNA), this work provides a unique approach for controlling CRISPR/Cas13a activity by RNA Inhibitory PNA (RIP). This method reduces the action of CRISPR/Cas13a by inhibiting the binding of crRNA to CRISPR/Cas13a, which was subsequently utilized to detect metalloproteinase-2 (MMP-2) activity. We carefully constructed a PNA sequence that includes a peptide sequence (Gly-Pro-Leu-Gly-Val-Arg-Gly) that is particularly cleaved by MMP-2 in the middle. When MMP-2 is not present in the system, the PNA/crRNA complex reduces crRNA binding to CRISPR/Cas13a, hence inhibiting CRISPR/Cas13a cleavage activity. When MMP-2 is present in the system, it cleaves the peptide sequence (Gly-Pro-Leu-Gly-Val-Arg-Gly) selectively and so prevents PNA from binding to crRNA, enabling crRNA to reach the "free" form. Then, in the presence of an RNA activator, crRNA may bind to CRISPR/Cas13a and stimulate its activity. CRISPR/Cas13a triggered in this manner may precisely cleave the DNA sequence containing modified -UU- bases on the electrode surface . Thus, the ferrocene (Fc) is changed away from the electrode surface at one end of the DNA , enabling for the recovery of the Electrochemiluminescence (ECL) signal from the two-dimensional material transformed on the electrode surface. As a result, MMP-2 activity was detected utilizing this technique of inhibiting crRNA activity with a detection limit of 15.56 fM. Our suggested method of inhibiting CRISPR/Cas13a activity via PNA has a broad variety of potential therapeutic applications in gene editing and the development of CRISPR/Cas13a-based biosensors.
研究领域:
分子生物学
·
基因编辑
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生物传感技术
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高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2026
·
IF 8.3
Wenji Bao,Ailing Su,Gerile Aodeng,Lu Ga,Lan Yu,Jun Ai
A highly sensitive colorimetric sensing platform was developed based on the trans-cleavage activity of CRISPR/Cas12a and the controllable aggregation of gold nanoparticles (AuNPs) for rapid and visual detection of the foodborne pathogen Staphylococcus aureus. Following PCR or RPA, the target DNA was specifically recognized by the Cas12a/crRNA complex, thereby activating its trans-cleavage activity. This process led to the degradation of linker single-stranded DNA (ssDNA) responsible for crosslinking AuNPs probes, resulting in significant changes in both UV–vis absorption signals and solution color. Three different crRNA sequences were systematically evaluated, revealing distinct efficiencies in target recognition and signal amplification. Among them, the crRNA-3 mediated system exhibited superior analytical performance, achieving an ultralow detection limit of 1.14 fM and a good linear response over a low concentration range. Furthermore, the proposed platform demonstrated excellent anti-interference capability in complex matrices, including serum, milk, bovine serum albumin (BSA), and phosphate-buffered saline (PBS), with recovery rates ranging from 83.2% to 125.9%. This work provides a robust and visual tool for the on-site detection of S. aureus and offers new insights into the design of portable CRISPR-based diagnostic platforms.
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高影响因子
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