1
|
Yuan J, Liao YS, Zhang TC, Tang YQ, Yu P, Liu YN, Cai DJ, Yu SG, Zhao L. Integrating Bulk RNA and Single-Cell Sequencing Data Unveils Efferocytosis Patterns and ceRNA Network in Ischemic Stroke. Transl Stroke Res 2025; 16:733-746. [PMID: 38678526 DOI: 10.1007/s12975-024-01255-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2024] [Revised: 03/17/2024] [Accepted: 04/18/2024] [Indexed: 05/01/2024]
Abstract
Excessive inflammatory response following ischemic stroke (IS) injury is a key factor affecting the functional recovery of patients. The efferocytic clearance of apoptotic cells within ischemic brain tissue is a critical mechanism for mitigating inflammation, presenting a promising avenue for the treatment of ischemic stroke. However, the cellular and molecular mechanisms underlying efferocytosis in the brain after IS and its impact on brain injury and recovery are poorly understood. This study explored the roles of inflammation and efferocytosis in IS with bioinformatics. Three Gene Expression Omnibus Series (GSE) (GSE137482-3 m, GSE137482-18 m, and GSE30655) were obtained from NCBI (National Center for Biotechnology Information) and GEO (Gene Expression Omnibus). Differentially expressed genes (DEGs) were processed for GSEA (Gene Set Enrichment Analysis), GO (Gene Ontology Functional Enrichment Analysis), and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses. Efferocytosis-related genes were identified from the existing literature, following which the relationship between Differentially Expressed Genes (DEGs) and efferocytosis-related genes was examined. The single-cell dataset GSE174574 was employed to investigate the distinct expression profiles of efferocytosis-related genes. The identified hub genes were verified using the dataset of human brain and peripheral blood sample datasets GSE56267 and GSE122709. The dataset GSE215212 was used to predict competing endogenous RNA (ceRNA) network, and GSE231431 was applied to verify the expression of differential miRNAs. At last, the middle cerebral artery (MCAO) model was established to validate the efferocytosis process and the expression of hub genes. DEGs in two datasets were significantly enriched in pathways involved in inflammatory response and immunoregulation. Based on the least absolute shrinkage and selection operator (LASSO) analyses, we identified hub efferocytosis-related genes (Abca1, C1qc, Ptx3, Irf5, and Pros1) and key transcription factors (Stat5). The scRNA-seq analysis showed that these hub genes were mainly expressed in microglia and macrophages which are the main cells with efferocytosis function in the brain. We then identified miR-125b-5p as a therapeutic target of IS based on the ceRNA network. Finally, we validated the phagocytosis and clearance of dead cells by efferocytosis and the expression of hub gene Abca1 in MCAO mice models.
Collapse
Affiliation(s)
- Jing Yuan
- Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu, 610075, Sichuan Province, China
| | - Yu-Sha Liao
- Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu, 610075, Sichuan Province, China
| | - Tie-Chun Zhang
- Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu, 610075, Sichuan Province, China
| | - Yu-Qi Tang
- Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu, 610075, Sichuan Province, China
| | - Pei Yu
- Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu, 610075, Sichuan Province, China
| | - Ya-Ning Liu
- Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu, 610075, Sichuan Province, China
| | - Ding-Jun Cai
- Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu, 610075, Sichuan Province, China
| | - Shu-Guang Yu
- Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu, 610075, Sichuan Province, China
| | - Ling Zhao
- Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine, Chengdu, 610075, Sichuan Province, China.
| |
Collapse
|
2
|
Zhang S, Xu D, Li F, Wang J. CRISPR-based non-nucleic acid detection. Trends Biotechnol 2025:S0167-7799(25)00139-8. [PMID: 40368676 DOI: 10.1016/j.tibtech.2025.04.012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2025] [Revised: 04/13/2025] [Accepted: 04/17/2025] [Indexed: 05/16/2025]
Abstract
Characterization of clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) trans-cleavage activities has initiated the era of next-generation CRISPR diagnostics. By using the trans-cleavage reaction for signal output, CRISPR systems have been engineered to detect non-nucleic acids (NNAs), including ions, inorganic small molecules, organic compounds, proteins, and bacteria. Diverse strategies are being used to specifically recognize NNAs and regulate Cas trans-cleavage activities, via generation or depletion of output signals. In this review, we introduce the principles and advantages of CRISPR-based NNA detection. We then classify CRISPR-based NNA detection strategies into three classes: the generation or depletion of free activators, synthesis of crRNAs, and reconstruction of active Cas effectors. Finally, we discuss the challenges and potential strategies to advance both clinical and nonclinical applications of CRISPR-based NNA detection.
Collapse
Affiliation(s)
- Shanshan Zhang
- School of Life Sciences, Huaibei Normal University, Huaibei, Anhui 235000, China
| | - Dayong Xu
- School of Life Sciences, Huaibei Normal University, Huaibei, Anhui 235000, China.
| | - Feng Li
- School of Life Sciences, Huaibei Normal University, Huaibei, Anhui 235000, China.
| | - Jin Wang
- School of Life Sciences, Huaibei Normal University, Huaibei, Anhui 235000, China; Tolo Biotechnology Co., Ltd, Wuxi, Jiangsu 214100, China.
| |
Collapse
|
3
|
He P, Jiang H, Zhu J, Hu M, Song P. Identification and validation of the inflammatory response-related LncRNAs as diagnostic biomarkers for acute ischemic stroke. Sci Rep 2025; 15:13818. [PMID: 40258919 PMCID: PMC12012103 DOI: 10.1038/s41598-025-98101-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2024] [Accepted: 04/09/2025] [Indexed: 04/23/2025] Open
Abstract
Ischemic stroke is one of the leading causes of deaths and disability, which is linked to inflammation. In this study, we aimed to identify inflammation-related lncRNAs as diagnostic biomarkers of acute ischemic stroke (AIS). A competing endogenous RNAs (ceRNA) network was established through whole transcriptome analysis. Gene expression datasets from the GEO database were analyzed to identify differentially expressed genes (DEGs), miRNAs and lncRNAs. Inflammation-related DEGs were determined through the intersection of the DEGs of the inflammation-related gene set from Genecards. Multiple databases like lncBase and Targetscan were analyzed to establish a ceRNA network. Several hub genes and sub-networks were obtained from a protein to protein (PPI) network. In addition, the candidate lncRNAs derived from the subnetwork were validated using mice MCAO model and clinical samples. Finally, a network comprising 20 lncRNAs, 26 miRNAs, and 43 inflammatory genes was analyzed, leading to the identification of MALAT1, SNHG8, and GAS5 as potential diagnostic biomarkers. Knockdown of MALAT1 and GAS5 resulted in decreased neurological severity score and inflammation response in mice MCAO model, indicating that these genes were significant diagnostic biomarkers for distinguishing AIS from healthy controls. These findings show that circulating MALAT1 and GAS5 have the potential to serve as clinical diagnostic biomarkers of AIS associated with inflammation.
Collapse
Affiliation(s)
- Peidong He
- Renmin Hospital of Wuhan University, Wuhan, 430060, China
| | - Hongxiang Jiang
- Department of Neurosurgery, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuchang Distict, Wuhan, 430060, Hubei Province, China
| | - Jiangrui Zhu
- Renmin Hospital of Wuhan University, Wuhan, 430060, China
| | - Min Hu
- Department of Gynecology and Obstetrics, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
| | - Ping Song
- Department of Neurosurgery, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuchang Distict, Wuhan, 430060, Hubei Province, China.
| |
Collapse
|
4
|
Cao L, Chen W, Kang W, Lei C, Nie Z. Engineering stimuli-responsive CRISPR-Cas systems for versatile biosensing. Anal Bioanal Chem 2025; 417:1699-1711. [PMID: 39601843 DOI: 10.1007/s00216-024-05678-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2024] [Revised: 11/11/2024] [Accepted: 11/15/2024] [Indexed: 11/29/2024]
Abstract
The precise target recognition and nuclease-mediated effective signal amplification capacities of CRISPR-Cas systems have attracted considerable research interest within the biosensing field. Guided by insights into their structural and biochemical mechanisms, researchers have endeavored to engineer the key biocomponents of CRISPR-Cas systems with stimulus-responsive functionalities. By the incorporation of protein/nucleic acid engineering techniques, a variety of conditional CRISPR-Cas systems whose activities depend on the presence of target triggers have been established for the efficient detection of diverse types of non-nucleic acid analytes. In this review, we summarized recent research progress in engineering Cas proteins, guide RNA, and substrate nucleic acids to possess target analyte-responsive abilities for diverse biosensing applications. Furthermore, we also discussed the challenges and future possibilities of the stimulus-responsive CRISPR-Cas systems in versatile biosensing.
Collapse
Affiliation(s)
- Linxin Cao
- State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha, 410082, Hunan, China
| | - Wenhui Chen
- State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha, 410082, Hunan, China
| | - Wenyuan Kang
- Key Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education & Laboratory of Tropical Medicinal Plant Chemistry of Hainan Province, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, 571158, Hainan, China
| | - Chunyang Lei
- State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha, 410082, Hunan, China.
| | - Zhou Nie
- State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha, 410082, Hunan, China.
| |
Collapse
|
5
|
Miceli F, Bracaglia S, Sorrentino D, Porchetta A, Ranallo S, Ricci F. MAIGRET: a CRISPR-based immunoassay that employs antibody-induced cell-free transcription of CRISPR guide RNA strands. Nucleic Acids Res 2025; 53:gkaf238. [PMID: 40156855 PMCID: PMC11952961 DOI: 10.1093/nar/gkaf238] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2024] [Revised: 03/08/2025] [Accepted: 03/17/2025] [Indexed: 04/01/2025] Open
Abstract
Here we report on the development of a CRISPR-based assay for the sensitive and specific detection of antibodies and antigens directly in complex sample matrices. The assay, called Molecular Assay based on antibody-Induced Guide-RNA Enzymatic Transcription (MAIGRET), is based on the use of a responsive synthetic DNA template that triggers the cell-free in vitro transcription of a guide RNA strand upon recognition of a specific target antibody. Such transcribed guide RNA activates the DNA collateral activity of the Cas12a enzyme, leading to the downstream cleavage of a fluorophore/quencher-labeled reporter and thus resulting in an increase in the measured fluorescence signal. We have used MAIGRET for the detection of six different antibodies with high sensitivity (detection limit in the picomolar range) and specificity (no signal in the presence of non-target antibodies). MAIGRET can also be adapted to a competitive approach for the detection of specific antigens. With MAIGRET, we significantly expand the scope and applicability of CRISPR-based sensing approaches to potentially enable the measurement of any molecular target for which an antibody is available.
Collapse
Affiliation(s)
- Francesca Miceli
- Department of Chemical Sciences and Technologies, University of Rome, Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy
| | - Sara Bracaglia
- Department of Chemical Sciences and Technologies, University of Rome, Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy
| | - Daniela Sorrentino
- Department of Chemical Sciences and Technologies, University of Rome, Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy
- Department of Mechanical and Aerospace Engineering and of Bioengineering, University of California at Los Angeles, 420 Westwood Plaza, Los Angeles, CA 90095, United States
| | - Alessandro Porchetta
- Department of Chemical Sciences and Technologies, University of Rome, Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy
- Istituto Nazionale Biostrutture e Biosistemi, INBB, Via dei Carpegna, 00165 Rome, Italy
| | - Simona Ranallo
- Department of Chemical Sciences and Technologies, University of Rome, Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy
- Istituto Nazionale Biostrutture e Biosistemi, INBB, Via dei Carpegna, 00165 Rome, Italy
| | - Francesco Ricci
- Department of Chemical Sciences and Technologies, University of Rome, Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome, Italy
- Istituto Nazionale Biostrutture e Biosistemi, INBB, Via dei Carpegna, 00165 Rome, Italy
| |
Collapse
|
6
|
Wang K, Liu S, Zhou S, Qileng A, Wang D, Liu Y, Chen C, Lei C, Nie Z. Ligand-Responsive Artificial Protein-Protein Communication for Field-Deployable Cell-Free Biosensing. Angew Chem Int Ed Engl 2025; 64:e202416671. [PMID: 39558180 DOI: 10.1002/anie.202416671] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2024] [Revised: 11/12/2024] [Accepted: 11/18/2024] [Indexed: 11/20/2024]
Abstract
Natural protein-protein communications, such as those between transcription factors (TFs) and RNA polymerases/ribosomes, underpin cell-free biosensing systems operating on the transcription/translation (TXTL) paradigm. However, their deployment in field analysis is hampered by the delayed response (hour-level) and the complex composition of in vitro TXTL systems. For this purpose, we present a de novo-designed ligand-responsive artificial protein-protein communication (LIRAC) by redefining the connection between TFs and non-interacting CRISPR/Cas enzymes. By rationally designing a chimeric DNA adaptor and precisely regulating its binding affinities to both proteins, LIRAC immediately transduces target-induced TF allostery into rapid CRISPR/Cas enzyme activation within a homogeneous system. Consequently, LIRAC obviates the need for RNA/protein biosynthesis inherent to conventional TXTL-based cell-free systems, substantially reducing reaction complexity and time (from hours to 10 minutes) with improved sensitivity and tunable dynamic range. Moreover, LIRAC exhibits excellent versatility and programmability for rapidly and sensitively detecting diverse contaminants, including antibiotics, heavy metal ions, and preservatives. It also enables the creation of a multi-protein communication-based tristate logic for the intelligent detection of multiple contaminants. Integrated with portable devices, LIRAC has been proven effective in the field analysis of environmental samples and personal care products, showcasing its potential for environmental and health monitoring.
Collapse
Affiliation(s)
- Ke Wang
- State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha, 410082, P. R. China
| | - Siqian Liu
- State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha, 410082, P. R. China
| | - Shuqi Zhou
- State Key Laboratory of Membrane Biology, Beijing Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, 100084, P. R. China
| | - Aori Qileng
- Key Laboratory for Biobased Materials and Energy of Ministry of Education, College of Materials and Energy, South China Agricultural University, Guangzhou, 510642, P. R. China
| | - Dingyi Wang
- State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, P. R. China
| | - Yingju Liu
- Key Laboratory for Biobased Materials and Energy of Ministry of Education, College of Materials and Energy, South China Agricultural University, Guangzhou, 510642, P. R. China
| | - Chunlai Chen
- State Key Laboratory of Membrane Biology, Beijing Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, 100084, P. R. China
| | - Chunyang Lei
- State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha, 410082, P. R. China
| | - Zhou Nie
- State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Biomacromolecular Chemical Biology, Hunan University, Changsha, 410082, P. R. China
| |
Collapse
|
7
|
Wang H, Zhang J, Liu Z, Chen M, Ji G, Liu L, Chang Z, Wang Y, Gao Z, Shi H. CRISPR-Cas14a and allosteric transcription factors empowered cell-free electrochemical biosensor for highly sensitive and stable detection of progesterone in multiple scenarios. Biosens Bioelectron 2025; 268:116919. [PMID: 39522471 DOI: 10.1016/j.bios.2024.116919] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2024] [Accepted: 11/05/2024] [Indexed: 11/16/2024]
Abstract
In this study, a cell-free electrochemical assay based on allosteric transcription factors (aTFs) and CRISPR-Cas14a was developed for the detection of progesterone in trace samples. This electrochemical biosensor helps to overcome the drawbacks of the traditional fluorescence assay based on the CRISPR-Cas system and aTFs combined for non-nucleic acid targets that is poorly effective for the detection of colored samples. By comparing and optimizing the concentration and length of the probes in the straight chain and hairpin structure, the sensor performance was improved. In addition, different sgRNA from other studies was designed to overcome the effect of sequence folding in the space region on Cas14a activation. Based on these optimization results, we constructed an electrochemical sensor for progesterone quantification in the range of 66.7pM to 3.33 × 10-1μM. This method requires only 2 μL of sample and does not necessitate complex pretreatment steps, with detection completed within 1.5 h. The method has been successfully applied to food, environmental, and biological samples, with recovery rates between 82.65% and 109%. This suggests that CRISPR and allosteric transcription factor-powered electrochemical detection methods have significant potential for use in the field of small molecule detection under various scenarios.
Collapse
Affiliation(s)
- Haoran Wang
- School of Public Health, Hebei Medical University, Shijiazhuang, 050017, China; Military Medical Science Academy, Academy of Military Science, Tianjin, 300050, China; Hebei Key Laboratory of Environment and Human Health, Hebei Medical University, Shijiazhuang, 050017, China
| | - Jiangshan Zhang
- Military Medical Science Academy, Academy of Military Science, Tianjin, 300050, China; Department of Toxicology and Health Inspection and Quarantine, School of Public Health, Tianjin Medical University, Tianjin, 300070, China
| | - Zesheng Liu
- Military Medical Science Academy, Academy of Military Science, Tianjin, 300050, China
| | - Mengmeng Chen
- Military Medical Science Academy, Academy of Military Science, Tianjin, 300050, China
| | - Guangna Ji
- Military Medical Science Academy, Academy of Military Science, Tianjin, 300050, China; Department of Toxicology and Health Inspection and Quarantine, School of Public Health, Tianjin Medical University, Tianjin, 300070, China
| | - Linyuan Liu
- Military Medical Science Academy, Academy of Military Science, Tianjin, 300050, China
| | - Zhuxin Chang
- Military Medical Science Academy, Academy of Military Science, Tianjin, 300050, China; Department of Toxicology and Health Inspection and Quarantine, School of Public Health, Tianjin Medical University, Tianjin, 300070, China
| | - Yu Wang
- Military Medical Science Academy, Academy of Military Science, Tianjin, 300050, China.
| | - Zhixian Gao
- Military Medical Science Academy, Academy of Military Science, Tianjin, 300050, China.
| | - Hongmei Shi
- School of Public Health, Hebei Medical University, Shijiazhuang, 050017, China; Hebei Key Laboratory of Environment and Human Health, Hebei Medical University, Shijiazhuang, 050017, China.
| |
Collapse
|
8
|
Kalantary-Charvadeh A, Morovat S, Aslani S, Ziamajidi N, Emami Razavi A, Abbasalipourkabir R. The role of long non-coding RNA LINC00839 in oral squamous cell carcinoma based on bioinformatics and experimental research. Sci Rep 2024; 14:31817. [PMID: 39738469 PMCID: PMC11686358 DOI: 10.1038/s41598-024-82922-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2024] [Accepted: 12/10/2024] [Indexed: 01/02/2025] Open
Abstract
This study explores the role of LINC00839 and its potential interaction with the miR-195-5p/cyclin E1 (CCNE1) axis in oral squamous cell carcinoma (OSCC). Using The Cancer Genome Atlas, we analyzed lncRNA, miRNA, and mRNA sequencing data for OSCC. Different online tools were applied to detect lncRNA-related miRNAs and their target mRNAs, forming a lncRNA/miRNA/mRNA axis. Co-expression analysis determined the correlation between lncRNA and mRNA expression. Afterward, protein-protein interaction network and functional enrichment analyses disclosed the biological activity of target genes. The expression and correlations of LINC00839, miR-195-5p, and CCNE1 were examined in 30 pairs of OSCC and noncancerous tissues. A Chi-square test was used to determine clinicopathological associations, and ROC analysis estimated diagnostic value. A total of 66 differentially expressed lncRNAs, 80 miRNAs, and 1149 mRNAs were identified in OSCC versus non-tumor samples. After filtering lncRNAs based on novelty, and predicting lncRNA-miRNA, and miRNA-mRNA interactions, the LINC00839/miR-195-5p/CCNE1 axis was discovered. RT-qPCR showed upregulation of LINC00839 and CCNE1 was accompanied by the downregulation of miR-195-5p. A significant positive correlation was observed between LINC00839 and CCNE1 mRNA expression, along with a significant negative correlation between LINC00839 and miR-195-5p expression. Moreover, increased LINC00839 was associated with tumor grade and lymph node status, while decreased miR-195-5p was correlated with lymph, depth, and vascular invasion (p < 0.05). The combined ROC curve demonstrated a significant area under the curve of 0.93. This discovery reveals a novel regulatory mechanism underlying OSCC tumorigenesis and may provide effective diagnosis and potential therapeutic targets to cure this devastating cancer.
Collapse
Affiliation(s)
- Ashkan Kalantary-Charvadeh
- Department of Clinical Biochemistry, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
- Student Research Committee, Hamadan University of Medical Sciences, Hamadan, Iran
| | - Saman Morovat
- Department of Medical Genetics and Molecular Biology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Somayeh Aslani
- Department of Clinical Biochemistry, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
| | - Nasrin Ziamajidi
- Department of Clinical Biochemistry, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
| | - Amirnader Emami Razavi
- Iran National Tumor Bank, Cancer Institute of Iran, Tehran University of Medical Sciences, Tehran, Iran
| | - Roghayeh Abbasalipourkabir
- Department of Clinical Biochemistry, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran.
| |
Collapse
|
9
|
Kang R, Li R, Mjengi J, Abbas Z, Song Y, Zhang L. A tiny sample rapid visual detection technology for imidacloprid resistance in Aphis gossypii by CRISPR/Cas12a. THE SCIENCE OF THE TOTAL ENVIRONMENT 2024; 951:175712. [PMID: 39181260 DOI: 10.1016/j.scitotenv.2024.175712] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/09/2024] [Revised: 08/19/2024] [Accepted: 08/21/2024] [Indexed: 08/27/2024]
Abstract
Insecticide resistance monitoring is essential for guiding chemical pest control and resistance management policies. Currently, rapid and effective technology for monitoring the resistance of tiny insects in the field is absent. Aphis gossypii Glover is a typical tiny insect, and one of the most frequently reported insecticide-resistant pests. In this study, we established a novel CRISPR/Cas12a-based rapid visual detection approach for detecting the V62I and R81T mutations in the β1 subunit of the nAChR in A. gossypii, to reflect target-site resistance to imidacloprid. Based on the nAChR β1 subunit gene in A. gossypii, the V62I/R81T-specific RPA primers and crRNAs were designed, and the ratio of 10 μM/2 μM/10 μM for ssDNA/Cas12a/crRNA was selected as the optimal dosage for the CRISPR reaction, ensuring that Cas12a only accurately recognizes imidacloprid-resistance templates. Our data show that the field populations of resistant insects possessing V62I and R81T mutations to imidacloprid can be accurately identified within one hour using the RPA-CRISPR/Cas12a detection approach under visible blue light at 440-460 nm. The protocol for RPA-CRISPR detection necessitates a single less than 2 mm specimen of A. gossypii tissues to perform RPA-CRISPR detection, and the process only requires a container at 37 °C and a portable blue light at 440-460 nm. Our research represents the first application of RPA-CRISPR technology in insecticide resistance detection, offers a new method for the resistance monitoring of A. gossypii or other tiny insects, helps delay the development of resistance to imidacloprid, improves the sustainability of chemical control, and provides theoretical guidance for managing pest resistance.
Collapse
Affiliation(s)
- Rujing Kang
- Department of Entomology, College of Plant Protection, China Agricultural University, Beijing 100193, China
| | - Ren Li
- Department of Entomology, College of Plant Protection, China Agricultural University, Beijing 100193, China
| | - Juma Mjengi
- Department of Entomology, College of Plant Protection, China Agricultural University, Beijing 100193, China
| | - Zohair Abbas
- Department of Entomology, College of Plant Protection, China Agricultural University, Beijing 100193, China
| | - Yihong Song
- Department of Plant Biosecurity and MOA Key Laboratory of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing 100193, China
| | - Lei Zhang
- Department of Entomology, College of Plant Protection, China Agricultural University, Beijing 100193, China.
| |
Collapse
|
10
|
Xi Q, Yang G, He X, Zhuang H, Li L, Lin B, Wang L, Wang X, Fang C, Chen Q, Yang Y, Yu Z, Zhang H, Cai W, Li Y, Shen H, Liu L, Zhang R. M 6A-mediated upregulation of lncRNA TUG1 in liver cancer cells regulates the antitumor response of CD8 + T cells and phagocytosis of macrophages. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2400695. [PMID: 38981064 PMCID: PMC11425850 DOI: 10.1002/advs.202400695] [Citation(s) in RCA: 9] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/19/2024] [Revised: 06/11/2024] [Indexed: 07/11/2024]
Abstract
Tumor immune evasion relies on the crosstalk between tumor cells and adaptive/innate immune cells. Immune checkpoints play critical roles in the crosstalk, and immune checkpoint inhibitors have achieved promising clinical effects. The long non-coding RNA taurine-upregulated gene 1 (TUG1) is upregulated in hepatocellular carcinoma (HCC). However, how TUG1 is upregulated and the effects on tumor immune evasion are incompletely understood. Here, METTL3-mediated m6A modification led to TUG1 upregulation is demonstrated. Knockdown of TUG1 inhibited tumor growth and metastasis, increased the infiltration of CD8+ T cells and M1-like macrophages in tumors, promoted the activation of CD8+ T cells through PD-L1, and improved the phagocytosis of macrophages through CD47. Mechanistically, TUG1 regulated PD-L1 and CD47 expressions by acting as a sponge of miR-141 and miR-340, respectively. Meanwhile, TUG1 interacted with YBX1 to facilitate the upregulation of PD-L1 and CD47 transcriptionally, which ultimately regulated tumor immune evasion. Clinically, TUG1 positively correlated with PD-L1 and CD47 in HCC tissues. Moreover, the combination of Tug1-siRNA therapy with a Pdl1 antibody effectively suppressed tumor growth. Therefore, the mechanism of TUG1 in regulating tumor immune evasion is revealed and can inform existing strategies targeting TUG1 for enhancing HCC immune therapy and drug development.
Collapse
Affiliation(s)
- Qing Xi
- Department of Gastroenterology and HepatologyThe First Affiliated Hospital of Guangdong Pharmaceutical UniversityGuangzhou510080China
- School of Biomedical Sciences and EngineeringSouth China University of TechnologyGuangzhou511442China
| | - Guangze Yang
- Laboratory of Immunology and InflammationDepartment of ImmunologyKey Laboratory of Immune Microenvironment and Diseases of Educational Ministry of ChinaTianjin Medical UniversityTianjin300070China
| | - Xue He
- Laboratory of Immunology and InflammationDepartment of BiotechnologySchool of Life Sciences and BiopharmaceuticsGuangdong Provincial Key Laboratory of Advanced Drug DeliveryGuangdong Provincial Engineering Center of Topical Precise Drug Delivery SystemGuangdong Pharmaceutical UniversityGuangzhou51006China
| | - Hao Zhuang
- Department of Hepatobiliopancreatic SurgeryThe Affiliated Cancer Hospital of Zhengzhou University & Henan Cancer HospitalZhengzhou450008China
| | - Li Li
- Laboratory of Immunology and InflammationDepartment of BiotechnologySchool of Life Sciences and BiopharmaceuticsGuangdong Provincial Key Laboratory of Advanced Drug DeliveryGuangdong Provincial Engineering Center of Topical Precise Drug Delivery SystemGuangdong Pharmaceutical UniversityGuangzhou51006China
| | - Bing Lin
- Laboratory of Immunology and InflammationDepartment of BiotechnologySchool of Life Sciences and BiopharmaceuticsGuangdong Provincial Key Laboratory of Advanced Drug DeliveryGuangdong Provincial Engineering Center of Topical Precise Drug Delivery SystemGuangdong Pharmaceutical UniversityGuangzhou51006China
| | - Lingling Wang
- Laboratory of Immunology and InflammationDepartment of BiotechnologySchool of Life Sciences and BiopharmaceuticsGuangdong Provincial Key Laboratory of Advanced Drug DeliveryGuangdong Provincial Engineering Center of Topical Precise Drug Delivery SystemGuangdong Pharmaceutical UniversityGuangzhou51006China
| | - Xianyang Wang
- Laboratory of Immunology and InflammationDepartment of BiotechnologySchool of Life Sciences and BiopharmaceuticsGuangdong Provincial Key Laboratory of Advanced Drug DeliveryGuangdong Provincial Engineering Center of Topical Precise Drug Delivery SystemGuangdong Pharmaceutical UniversityGuangzhou51006China
| | - Chunqiang Fang
- Laboratory of Immunology and InflammationDepartment of BiotechnologySchool of Life Sciences and BiopharmaceuticsGuangdong Provincial Key Laboratory of Advanced Drug DeliveryGuangdong Provincial Engineering Center of Topical Precise Drug Delivery SystemGuangdong Pharmaceutical UniversityGuangzhou51006China
| | - Qiurui Chen
- Department of BioscienceSchool of Life Sciences and BiopharmaceuticsGuangdong Pharmaceutical UniversityGuangzhou51006China
| | - Yongjie Yang
- Laboratory of Immunology and InflammationDepartment of BiotechnologySchool of Life Sciences and BiopharmaceuticsGuangdong Provincial Key Laboratory of Advanced Drug DeliveryGuangdong Provincial Engineering Center of Topical Precise Drug Delivery SystemGuangdong Pharmaceutical UniversityGuangzhou51006China
| | - Zhaoan Yu
- Laboratory of Immunology and InflammationDepartment of BiotechnologySchool of Life Sciences and BiopharmaceuticsGuangdong Provincial Key Laboratory of Advanced Drug DeliveryGuangdong Provincial Engineering Center of Topical Precise Drug Delivery SystemGuangdong Pharmaceutical UniversityGuangzhou51006China
| | - Hao Zhang
- Laboratory of Immunology and InflammationDepartment of BiotechnologySchool of Life Sciences and BiopharmaceuticsGuangdong Provincial Key Laboratory of Advanced Drug DeliveryGuangdong Provincial Engineering Center of Topical Precise Drug Delivery SystemGuangdong Pharmaceutical UniversityGuangzhou51006China
| | - Wenqian Cai
- Laboratory of Immunology and InflammationDepartment of BiotechnologySchool of Life Sciences and BiopharmaceuticsGuangdong Provincial Key Laboratory of Advanced Drug DeliveryGuangdong Provincial Engineering Center of Topical Precise Drug Delivery SystemGuangdong Pharmaceutical UniversityGuangzhou51006China
| | - Yan Li
- Laboratory of Immunology and InflammationDepartment of BiotechnologySchool of Life Sciences and BiopharmaceuticsGuangdong Provincial Key Laboratory of Advanced Drug DeliveryGuangdong Provincial Engineering Center of Topical Precise Drug Delivery SystemGuangdong Pharmaceutical UniversityGuangzhou51006China
| | - Han Shen
- Department of BioscienceSchool of Life Sciences and BiopharmaceuticsGuangdong Pharmaceutical UniversityGuangzhou51006China
| | - Li Liu
- Department of RadiologyThe University of Texas Southwestern Medical Center5323 Harry Hines Blvd.DallasTX75390USA
| | - Rongxin Zhang
- Laboratory of Immunology and InflammationDepartment of BiotechnologySchool of Life Sciences and BiopharmaceuticsGuangdong Provincial Key Laboratory of Advanced Drug DeliveryGuangdong Provincial Engineering Center of Topical Precise Drug Delivery SystemGuangdong Pharmaceutical UniversityGuangzhou51006China
| |
Collapse
|
11
|
Ali N, Vora C, Mathuria A, Kataria N, Mani I. Advances in CRISPR-Cas systems for gut microbiome. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2024; 208:59-81. [PMID: 39266188 DOI: 10.1016/bs.pmbts.2024.07.008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/14/2024]
Abstract
CRISPR-Cas technology has revolutionized microbiome research by enabling precise genetic manipulation of microbial communities. This review explores its diverse applications in gut microbiome studies, probiotic development, microbiome diagnostics, pathogen targeting, and microbial community engineering. Engineered bacteriophages and conjugative probiotics exemplify CRISPR-Cas's capability for targeted bacterial manipulation, offering promising strategies against antibiotic-resistant infections and other gut-related disorders. CRISPR-Cas systems also enhance probiotic efficacy by improving stress tolerance and colonization in the gastrointestinal tract. CRISPR-based techniques in diagnostics enable early intervention by enabling fast and sensitive pathogen identification. Furthermore, CRISPR-mediated gene editing allows tailored modification of microbial populations, mitigating risks associated with horizontal gene transfer and enhancing environmental and health outcomes. Despite its transformative potential, ethical and regulatory challenges loom large, demanding robust frameworks to guide its responsible application. This chapter highlights CRISPR-Cas's pivotal role in advancing microbiome research toward personalized medicine and microbial therapeutics while emphasizing the imperative of balanced ethical deliberations and comprehensive regulatory oversight.
Collapse
Affiliation(s)
- Namra Ali
- Department of Microbiology, Gargi College, University of Delhi, New Delhi, India
| | - Chaitali Vora
- Department of Biosciences and Biomedical Engineering, Indian Institute of Technology, Indore, India
| | - Anshu Mathuria
- Department of Biochemistry, Sri Venkateswara College, University of Delhi, New Delhi, India
| | - Naina Kataria
- Department of Biochemistry, Sri Venkateswara College, University of Delhi, New Delhi, India
| | - Indra Mani
- Department of Microbiology, Gargi College, University of Delhi, New Delhi, India.
| |
Collapse
|
12
|
Kehrli J, Husser C, Ryckelynck M. Fluorogenic RNA-Based Biosensors of Small Molecules: Current Developments, Uses, and Perspectives. BIOSENSORS 2024; 14:376. [PMID: 39194605 DOI: 10.3390/bios14080376] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/05/2024] [Revised: 07/14/2024] [Accepted: 07/19/2024] [Indexed: 08/29/2024]
Abstract
Small molecules are highly relevant targets for detection and quantification. They are also used to diagnose and monitor the progression of disease and infectious processes and track the presence of contaminants. Fluorogenic RNA-based biosensors (FRBs) represent an appealing solution to the problem of detecting these targets. They combine the portability of molecular systems with the sensitivity and multiplexing capacity of fluorescence, as well as the exquisite ligand selectivity of RNA aptamers. In this review, we first present the different sensing and reporting aptamer modules currently available to design an FRB, together with the main methodologies used to discover modules with new specificities. We next introduce and discuss how both modules can be functionally connected prior to exploring the main applications for which FRB have been used. Finally, we conclude by discussing how using alternative nucleotide chemistries may improve FRB properties and further widen their application scope.
Collapse
Affiliation(s)
- Janine Kehrli
- Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR 9002, F-67000 Strasbourg, France
| | - Claire Husser
- Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR 9002, F-67000 Strasbourg, France
| | - Michael Ryckelynck
- Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR 9002, F-67000 Strasbourg, France
| |
Collapse
|
13
|
Song R, Chen Z, Xiao H, Wang H. The CRISPR-Cas system in molecular diagnostics. Clin Chim Acta 2024; 561:119820. [PMID: 38901631 DOI: 10.1016/j.cca.2024.119820] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2024] [Revised: 06/14/2024] [Accepted: 06/17/2024] [Indexed: 06/22/2024]
Abstract
Robust, sensitive, and rapid molecular detection tools are essential prerequisites for disease diagnosis and epidemiological control. However, the current mainstream tests necessitate expensive equipment and specialized operators, impeding the advancement of molecular diagnostics. The CRISPR-Cas system is an integral component of the bacterial adaptive immune system, wherein Cas proteins recognize PAM sequences by binding to CRISPR RNA, subsequently triggering DNA or RNA cleavage. The discovery of the CRISPR-Cas system has invigorated molecular diagnostics. With further in-depth research on this system, its application in molecular diagnosis is flourishing. In this review, we provide a comprehensive overview of recent research progress on the CRISPR-Cas system, specifically focusing on its application in molecular diagnosis.
Collapse
Affiliation(s)
- Rao Song
- Department of Pharmacy, Sichuan Clinical Research Center for Cancer, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, Affiliated Cancer Hospital of University of Electronic Science and Technology of China, Chengdu 610041, China
| | - Zhongyi Chen
- Department of Pathology, Suining Central Hospital, Suining 629000, China
| | - Hongtao Xiao
- Department of Pharmacy, Sichuan Clinical Research Center for Cancer, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, Affiliated Cancer Hospital of University of Electronic Science and Technology of China, Chengdu 610041, China
| | - Haojun Wang
- Department of Pathology, Suining Central Hospital, Suining 629000, China.
| |
Collapse
|
14
|
Pathmendra P, Park Y, Enguita FJ, Byrne JA. Verification of nucleotide sequence reagent identities in original publications in high impact factor cancer research journals. NAUNYN-SCHMIEDEBERG'S ARCHIVES OF PHARMACOLOGY 2024; 397:5049-5066. [PMID: 38194106 PMCID: PMC11166861 DOI: 10.1007/s00210-023-02846-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/27/2023] [Accepted: 11/09/2023] [Indexed: 01/10/2024]
Abstract
Human gene research studies that describe wrongly identified nucleotide sequence reagents have been mostly identified in journals of low to moderate impact factor, where unreliable findings could be considered to have limited influence on future research. This study examined whether papers describing wrongly identified nucleotide sequences are also published in high-impact-factor cancer research journals. We manually verified nucleotide sequence identities in original Molecular Cancer articles published in 2014, 2016, 2018, and 2020, including nucleotide sequence reagents that were claimed to target circRNAs. Using keywords identified in some 2018 and 2020 Molecular Cancer papers, we also verified nucleotide sequence identities in 2020 Oncogene papers that studied miRNA(s) and/or circRNA(s). Overall, 3.8% (251/6647) and 4.0% (47/1165) nucleotide sequences that were verified in Molecular Cancer and Oncogene papers, respectively, were found to be wrongly identified. Wrongly identified nucleotide sequences were distributed across 18% (91/500) original Molecular Cancer papers, including 38% (31/82) Molecular Cancer papers from 2020, and 40% (21/52) selected Oncogene papers from 2020. Original papers with wrongly identified nucleotide sequences were therefore unexpectedly frequent in two high-impact-factor cancer research journals, highlighting the risks of employing journal impact factors or citations as proxies for research quality.
Collapse
Affiliation(s)
- Pranujan Pathmendra
- School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Camperdown, NSW, 2050, Australia
| | - Yasunori Park
- School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Camperdown, NSW, 2050, Australia
| | - Francisco J Enguita
- Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028, Lisbon, Portugal
| | - Jennifer A Byrne
- School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Camperdown, NSW, 2050, Australia.
- NSW Health Statewide Biobank, NSW Health Pathology, Camperdown, NSW, 2050, Australia.
| |
Collapse
|
15
|
Burbano DA, Kiattisewee C, Karanjia AV, Cardiff RAL, Faulkner ID, Sugianto W, Carothers JM. CRISPR Tools for Engineering Prokaryotic Systems: Recent Advances and New Applications. Annu Rev Chem Biomol Eng 2024; 15:389-430. [PMID: 38598861 DOI: 10.1146/annurev-chembioeng-100522-114706] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/12/2024]
Abstract
In the past decades, the broad selection of CRISPR-Cas systems has revolutionized biotechnology by enabling multimodal genetic manipulation in diverse organisms. Rooted in a molecular engineering perspective, we recapitulate the different CRISPR components and how they can be designed for specific genetic engineering applications. We first introduce the repertoire of Cas proteins and tethered effectors used to program new biological functions through gene editing and gene regulation. We review current guide RNA (gRNA) design strategies and computational tools and how CRISPR-based genetic circuits can be constructed through regulated gRNA expression. Then, we present recent advances in CRISPR-based biosensing, bioproduction, and biotherapeutics across in vitro and in vivo prokaryotic systems. Finally, we discuss forthcoming applications in prokaryotic CRISPR technology that will transform synthetic biology principles in the near future.
Collapse
Affiliation(s)
- Diego Alba Burbano
- Department of Chemical Engineering, University of Washington, Seattle, Washington, USA
- Molecular Engineering & Sciences Institute and Center for Synthetic Biology, University of Washington, Seattle, Washington, USA;
| | - Cholpisit Kiattisewee
- Department of Chemical Engineering, University of Washington, Seattle, Washington, USA
- Molecular Engineering & Sciences Institute and Center for Synthetic Biology, University of Washington, Seattle, Washington, USA;
| | - Ava V Karanjia
- Department of Chemical Engineering, University of Washington, Seattle, Washington, USA
- Molecular Engineering & Sciences Institute and Center for Synthetic Biology, University of Washington, Seattle, Washington, USA;
| | - Ryan A L Cardiff
- Molecular Engineering & Sciences Institute and Center for Synthetic Biology, University of Washington, Seattle, Washington, USA;
| | - Ian D Faulkner
- Department of Chemical Engineering, University of Washington, Seattle, Washington, USA
- Molecular Engineering & Sciences Institute and Center for Synthetic Biology, University of Washington, Seattle, Washington, USA;
| | - Widianti Sugianto
- Department of Chemical Engineering, University of Washington, Seattle, Washington, USA
- Molecular Engineering & Sciences Institute and Center for Synthetic Biology, University of Washington, Seattle, Washington, USA;
| | - James M Carothers
- Department of Chemical Engineering, University of Washington, Seattle, Washington, USA
- Molecular Engineering & Sciences Institute and Center for Synthetic Biology, University of Washington, Seattle, Washington, USA;
| |
Collapse
|
16
|
Saleh EAM, Ali E, Muxamadovna GM, Kassem AF, Kaur I, Kumar A, Jabbar HS, Alwaily ER, Elawady A, Omran AA. CRISPR/Cas-based colorimetric biosensors: a promising tool for the diagnosis of bacterial foodborne pathogens in food products. ANALYTICAL METHODS : ADVANCING METHODS AND APPLICATIONS 2024; 16:3448-3463. [PMID: 38804827 DOI: 10.1039/d4ay00578c] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/29/2024]
Abstract
Some physical phenomena and various chemical substances newly introduced in nanotechnology have allowed scientists to develop valuable devices in the field of food sciences. Regarding such progress, the identification of foodborne pathogenic microorganisms is an imperative subject nowadays. These bacterial species have been found to cause severe health impacts after food ingestion and can result in high mortality in acute cases. The rapid detection of foodborne bacterial species at low concentrations is in high demand in recent diagnostics. CRISPR/Cas-mediated biosensors possess the potential to overcome several challenges in classical assays such as complex pretreatments, long turnaround time, and insensitivity. Among them, colorimetric nanoprobes based on the CRISPR strategy afford promising devices for POCT (point-of-care testing) since they can be visualized with the naked eye and do not require diagnostic apparatus. In this study, we briefly classify and discuss the working principles of the different CRISPR/Cas protein agents that have been employed in biosensors so far. We assess the current status of the CRISPR system, specifically focusing on colorimetric biosensing platforms. We discuss the utilization of each Cas effector in the detection of foodborne pathogens and examine the restrictions of the existing technology. The challenges and future opportunities are also indicated and addressed.
Collapse
Affiliation(s)
- Ebraheem Abdu Musad Saleh
- Department of Chemistry, College of Science and Humanities in Al-Kharj, Prince Sattam Bin Abdulaziz University, Al-Kharj, 11942, Saudi Arabia.
| | - Eyhab Ali
- Al-Zahraa University for Women, Karbala, Iraq
| | | | - Asmaa F Kassem
- Department of Chemistry, College of Science and Humanities in Al-Kharj, Prince Sattam Bin Abdulaziz University, Al-Kharj, 11942, Saudi Arabia
- Chemistry of Natural and Microbial Products Department, Pharmaceutical and Drug Industries Research Institute, National Research Centre, Dokki, Cairo 12622, Egypt
| | - Irwanjot Kaur
- Department of Biotechnology and Genetics, Jain (Deemed-to-be) University, Bengaluru, Karnataka-560069, India
- Department of Allied Healthcare and Sciences, Vivekananda Global University, Jaipur, Rajasthan-303012, India
| | - Abhinav Kumar
- Department of Nuclear and Renewable Energy, Ural Federal University Named After the First President of Russia Boris Yeltsin, Yekaterinburg 620002, Russia
| | - Hijran Sanaan Jabbar
- Department of Chemistry, College of Science, Salahaddin University-Erbil, Erbil, Kurdistan Region, Iraq
| | - Enas R Alwaily
- Microbiology Research Group, College of Pharmacy, Al-Ayen University, Thi-Qar, Iraq
| | - Ahmed Elawady
- College of Technical Engineering, The Islamic University of Al Diwaniyah, Al Diwaniyah, Iraq
- College of Technical Engineering, The Islamic University, Najaf, Iraq
- College of Technical Engineering, The Islamic University of Babylon, Babylon, Iraq
| | - Alaa A Omran
- Department of Engineering, AL-Nisour University College, Baghdad, Iraq
| |
Collapse
|
17
|
Li D, Liang P, Ling S, Wu Y, Lv B. An optimized microRNA detection platform based on PAM formation-regulated CRISPR/Cas12a activation. Int J Biol Macromol 2024; 266:130848. [PMID: 38521316 DOI: 10.1016/j.ijbiomac.2024.130848] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2023] [Revised: 03/01/2024] [Accepted: 03/11/2024] [Indexed: 03/25/2024]
Abstract
MicroRNAs (miRNAs) have emerged as biomarkers for the diagnosis and prognosis of various diseases, such as cancer. Recent advancements in CRISPR/Cas12a-based biosensors in combination with hybridization chain reaction (HCR) make it a promising approach for miRNA detection. To increase the compatibility of HCR and CRISPR/Cas12a, we compared two design strategies of hairpin DNA in HCR. The results showed that different arrangements of the protospacer sequence and protospacer adjacent motif (PAM) in the hairpin DNA could affect the sensing performance. The "PAM Formation" strategy, by which the duplex PAM sites are absent in the hairpin DNA and present in the long duplex DNA after HCR, exhibited advantages in detection sensitivity. By optimizing the probe sequences and reaction conditions, we developed a miRNA detection platform. With the same crRNA, this platform enables the identification of different miRNAs by simply replacing the loop region of the target recognition probe. In addition, the proposed platform can detect single-stranded DNA and distinguishing single or multiple base mutations in the target strand. The application of discriminating the target miRNA expression levels from different cell lines validated the reliability and practicability of the sensor platform, indicating its potential applications in early clinical accurate diagnosis of cancers.
Collapse
Affiliation(s)
- Dawei Li
- Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of State Forestry and Grassland Administration on Subtropical Forest Biodiversity Conservation, College of Life Sciences, Nanjing Forestry University, Nanjing 210037, China
| | - Pengda Liang
- Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of State Forestry and Grassland Administration on Subtropical Forest Biodiversity Conservation, College of Life Sciences, Nanjing Forestry University, Nanjing 210037, China
| | - Shen Ling
- Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of State Forestry and Grassland Administration on Subtropical Forest Biodiversity Conservation, College of Life Sciences, Nanjing Forestry University, Nanjing 210037, China
| | - Yapeng Wu
- Co-Innovation Center for Sustainable Forestry in Southern China, Key Laboratory of State Forestry and Grassland Administration on Subtropical Forest Biodiversity Conservation, College of Life Sciences, Nanjing Forestry University, Nanjing 210037, China
| | - Bei Lv
- Key Lab of Innovative Applications of Bioresources and Functional Molecules of Jiangsu Province, College of Life Science and Chemistry, Jiangsu Second Normal University, Nanjing 210013, China.
| |
Collapse
|
18
|
Zhong J, Wang C, Zhang D, Yao X, Zhao Q, Huang X, Lin F, Xue C, Wang Y, He R, Li XY, Li Q, Wang M, Zhao S, Afridi SK, Zhou W, Wang Z, Xu Y, Xu Z. PCDHA9 as a candidate gene for amyotrophic lateral sclerosis. Nat Commun 2024; 15:2189. [PMID: 38467605 PMCID: PMC10928119 DOI: 10.1038/s41467-024-46333-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2023] [Accepted: 02/23/2024] [Indexed: 03/13/2024] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. To identify additional genetic factors, we analyzed exome sequences in a large cohort of Chinese ALS patients and found a homozygous variant (p.L700P) in PCDHA9 in three unrelated patients. We generated Pcdhα9 mutant mice harboring either orthologous point mutation or deletion mutation. These mice develop progressive spinal motor loss, muscle atrophy, and structural/functional abnormalities of the neuromuscular junction, leading to paralysis and early lethality. TDP-43 pathology is detected in the spinal motor neurons of aged mutant mice. Mechanistically, we demonstrate that Pcdha9 mutation causes aberrant activation of FAK and PYK2 in aging spinal cord, and dramatically reduced NKA-α1 expression in motor neurons. Our single nucleus multi-omics analysis reveals disturbed signaling involved in cell adhesion, ion transport, synapse organization, and neuronal survival in aged mutant mice. Together, our results present PCDHA9 as a potential ALS gene and provide insights into its pathogenesis.
Collapse
Affiliation(s)
- Jie Zhong
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100101, China
| | - Chaodong Wang
- Department of Neurology, Xuanwu Hospital, Capital Medical University, National Clinical Research Center for Geriatric Disease, Beijing, 100053, China.
| | - Dan Zhang
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100101, China
| | - Xiaoli Yao
- Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China
| | - Quanzhen Zhao
- Department of Neurology, West China Hospital, Sichuan University, Chengdu, 610041, China
| | - Xusheng Huang
- Department of Neurology, The First Medical Center, Chinese PLA General Hospital, Beijing, 100853, China
| | - Feng Lin
- Department of Neurology, Fujian Medical University Union Hospital, Fuzhou, 350001, China
| | - Chun Xue
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100101, China
| | - Yaqing Wang
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100101, China
| | - Ruojie He
- Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China
| | - Xu-Ying Li
- Department of Neurology, Xuanwu Hospital, Capital Medical University, National Clinical Research Center for Geriatric Disease, Beijing, 100053, China
| | - Qibin Li
- Shenzhen Clabee Biotechnology Incorporation, Shenzhen, 518057, China
| | - Mingbang Wang
- Shanghai Key Laboratory of Birth Defects, Division of Neonatology, Children's Hospital of Fudan University, National Center for Children's Health, Shanghai, 201102, China
| | - Shaoli Zhao
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100101, China
| | - Shabbir Khan Afridi
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100101, China
| | - Wenhao Zhou
- Shanghai Key Laboratory of Birth Defects, Division of Neonatology, Children's Hospital of Fudan University, National Center for Children's Health, Shanghai, 201102, China
| | - Zhanjun Wang
- Department of Neurology, Xuanwu Hospital, Capital Medical University, National Clinical Research Center for Geriatric Disease, Beijing, 100053, China
| | - Yanming Xu
- Department of Neurology, West China Hospital, Sichuan University, Chengdu, 610041, China.
| | - Zhiheng Xu
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China.
- University of Chinese Academy of Sciences, Beijing, 100101, China.
| |
Collapse
|
19
|
Sinha S, Molina Vargas A, Arantes P, Patel A, O’Connell M, Palermo G. Unveiling the RNA-mediated allosteric activation discloses functional hotspots in CRISPR-Cas13a. Nucleic Acids Res 2024; 52:906-920. [PMID: 38033317 PMCID: PMC10810222 DOI: 10.1093/nar/gkad1127] [Citation(s) in RCA: 15] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2023] [Revised: 10/25/2023] [Accepted: 11/09/2023] [Indexed: 12/02/2023] Open
Abstract
Cas13a is a recent addition to the CRISPR-Cas toolkit that exclusively targets RNA, which makes it a promising tool for RNA detection. It utilizes a CRISPR RNA (crRNA) to target RNA sequences and trigger a composite active site formed by two 'Higher Eukaryotes and Prokaryotes Nucleotide' (HEPN) domains, cleaving any solvent-exposed RNA. In this system, an intriguing form of allosteric communication controls the RNA cleavage activity, yet its molecular details are unknown. Here, multiple-microsecond molecular dynamics simulations are combined with graph theory to decipher this intricate activation mechanism. We show that the binding of a target RNA acts as an allosteric effector, by amplifying the communication signals over the dynamical noise through interactions of the crRNA at the buried HEPN1-2 interface. By introducing a novel Signal-to-Noise Ratio (SNR) of communication efficiency, we reveal critical allosteric residues-R377, N378, and R973-that rearrange their interactions upon target RNA binding. Alanine mutation of these residues is shown to select target RNA over an extended complementary sequence beyond guide-target duplex for RNA cleavage, establishing the functional significance of these hotspots. Collectively our findings offer a fundamental understanding of the Cas13a mechanism of action and pave new avenues for the development of highly selective RNA-based cleavage and detection tools.
Collapse
Affiliation(s)
- Souvik Sinha
- Department of Bioengineering, University of California Riverside, Riverside, CA , 92521, USA
| | - Adrian M Molina Vargas
- Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA
- Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA
- Department of Biomedical Genetics, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA
| | - Pablo R Arantes
- Department of Bioengineering, University of California Riverside, Riverside, CA , 92521, USA
| | - Amun Patel
- Department of Bioengineering, University of California Riverside, Riverside, CA , 92521, USA
| | - Mitchell R O’Connell
- Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA
- Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA
| | - Giulia Palermo
- Department of Bioengineering, University of California Riverside, Riverside, CA , 92521, USA
- Department of Chemistry, University of California Riverside, Riverside, CA, 92521, USA
| |
Collapse
|
20
|
Kim Y, Nam D, Lee ES, Kim S, Cha BS, Park KS. Aptamer-Based Switching System for Communication of Non-Interacting Proteins. BIOSENSORS 2024; 14:47. [PMID: 38248424 PMCID: PMC10812979 DOI: 10.3390/bios14010047] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/18/2023] [Revised: 01/09/2024] [Accepted: 01/10/2024] [Indexed: 01/23/2024]
Abstract
Biological macromolecules, such as DNA, RNA, and proteins in living organisms, form an intricate network that plays a key role in many biological processes. Many attempts have been made to build new networks by connecting non-communicable proteins with network mediators, especially using antibodies. In this study, we devised an aptamer-based switching system that enables communication between non-interacting proteins. As a proof of concept, two proteins, Cas13a and T7 RNA polymerase (T7 RNAP), were rationally connected using an aptamer that specifically binds to T7 RNAP. The proposed switching system can be modulated in both signal-on and signal-off manners and its responsiveness to the target activator can be controlled by adjusting the reaction time. This study paves the way for the expansion of biological networks by mediating interactions between proteins using aptamers.
Collapse
Affiliation(s)
| | | | | | | | | | - Ki Soo Park
- Department of Biological Engineering, College of Engineering, Konkuk University, Seoul 05029, Republic of Korea; (Y.K.); (D.N.); (E.S.L.); (S.K.); (B.S.C.)
| |
Collapse
|
21
|
Khodr R, Husser C, Ryckelynck M. Direct fluoride monitoring using a fluorogenic RNA-based biosensor. Methods Enzymol 2024; 696:85-107. [PMID: 38658090 DOI: 10.1016/bs.mie.2023.12.019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/26/2024]
Abstract
Fluorinated compounds, whether naturally occurring or from anthropogenic origin, have been extensively exploited in the last century. Degradation of these compounds by physical or biochemical processes is expected to result in the release of fluoride. Several fluoride detection mechanisms have been previously described. However, most of these methods are not compatible with high- and ultrahigh-throughput screening technologies, lack the ability to real-time monitor the increase of fluoride concentration in solution, or rely on costly reagents (such as cell-free expression systems). Our group recently developed "FluorMango" as the first completely RNA-based and direct fluoride-specific fluorogenic biosensor. To do so, we merged and engineered the Mango-III light-up RNA aptamer and the fluoride-specific aptamer derived from a riboswitch, crcB. In this chapter, we explain how this RNA-based biosensor can be produced in large scale before providing examples of how it can be used to quantitatively detect (end-point measurement) or monitor in real-time fluoride release in complex biological systems by translating it into measurable fluorescent signal.
Collapse
Affiliation(s)
- Radi Khodr
- Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR, Strasbourg, France
| | - Claire Husser
- Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR, Strasbourg, France
| | - Michael Ryckelynck
- Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR, Strasbourg, France.
| |
Collapse
|
22
|
Misra G, Qaisar S, Singh P. CRISPR-based therapeutic targeting of signaling pathways in breast cancer. Biochim Biophys Acta Mol Basis Dis 2024; 1870:166872. [PMID: 37666438 DOI: 10.1016/j.bbadis.2023.166872] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2023] [Revised: 08/24/2023] [Accepted: 09/01/2023] [Indexed: 09/06/2023]
Abstract
Breast cancer remains a leading cause of death for women worldwide, and new treatment strategies are needed. There are innumerable anomalous genes that are responsible for the multi-factorial carcinogenesis pathway. Although several disease-causing mutations have been detected, therapy frequently focuses on attenuating the manifestation of the disease rather than harmonizing the mutation in the target area. The advent of CRISPR-Cas9 technology has revolutionized genome editing, allowing for precise and efficient manipulation of gene expression. The purpose of this review paper is to summarize recent progress in the use of CRISPR-based approaches to target key signaling pathways associated with breast cancer progression. The first section introduces basic concepts of CRISPR technology, focusing on its application in genome editing and transcriptional regulation followed by an overview of aspects involving complex signaling pathways in breast cancer such as P13K/AKT/mTOR, EPK/MAPK and Wnt/β catenin. An extensive literature search using PubMed and Google Scholar is performed for information retrieval. Further, the role of CRISPR-based interventions in regulating gene expression revealed, altered pathway activity and potential therapeutic consequences are discussed. This review will be a valuable addition to providing comprehensive knowledge of CRISPR-Cas-mediated therapeutic targeting in breast cancer.
Collapse
Affiliation(s)
- Gauri Misra
- National Institute of Biologicals, Noida 201309, UP, India.
| | - Sidra Qaisar
- National Institute of Biologicals, Noida 201309, UP, India
| | | |
Collapse
|
23
|
Shi P, Wu X. Programmable RNA targeting with CRISPR-Cas13. RNA Biol 2024; 21:1-9. [PMID: 38764173 PMCID: PMC11110701 DOI: 10.1080/15476286.2024.2351657] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Revised: 04/20/2024] [Accepted: 05/01/2024] [Indexed: 05/21/2024] Open
Abstract
The RNA-targeting CRISPR-Cas13 system has enabled precise engineering of endogenous RNAs, significantly advancing our understanding of RNA regulation and the development of RNA-based diagnostic and therapeutic applications. This review aims to provide a summary of Cas13-based RNA targeting tools and applications, discuss limitations and challenges of existing tools and suggest potential directions for further development of the RNA targeting system.
Collapse
Affiliation(s)
- Peiguo Shi
- Department of Medicine and Department of Systems Biology, Columbia University Irving Medical Center, New York, NY, USA
| | - Xuebing Wu
- Department of Medicine and Department of Systems Biology, Columbia University Irving Medical Center, New York, NY, USA
| |
Collapse
|
24
|
Zhou J, Ren XM, Wang X, Li Z, J Xian C. Recent advances and challenges of the use of the CRISPR/Cas system as a non-nucleic acid molecular diagnostic. Heliyon 2023; 9:e22767. [PMID: 38076202 PMCID: PMC10703615 DOI: 10.1016/j.heliyon.2023.e22767] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2023] [Revised: 10/26/2023] [Accepted: 11/19/2023] [Indexed: 02/28/2025] Open
Abstract
The clustered regularly interspaced short palindromic DNA sequence repeats (CRISPR) and CRISPR-associated (Cas) (CRISPR/Cas) systems are currently applied not only as a gene editing tool but also as a novel molecular diagnostic technique. The CRISPR/Cas systems have emerged as an efficient molecular diagnostic system that can detect nucleic acids, proteins and small molecule compounds, by converting a non-nucleic acid into a nucleic acid signal of Cas-identifiable and keeping inherent properties of high sensitivity and specificity. While its multiple advantages for nucleic acid detection have been widely published in excellent reviews, there have been no systematic analyses and reviews on the principles and characteristics of CRISPR/Cas-based diagnostic systems for non-nucleic acids. The present work reviewed the basic process, principles, characteristics, strategies, recent advances, and challenges of CRISPR/Cas-based molecular diagnostic methods for detecting non-nucleic acids, which may provide a basis or some references for future development and application as molecular diagnostic tools.
Collapse
Affiliation(s)
- Jian Zhou
- Department of Laboratory Medicine, The First Affiliated Hospital of Xi'an Medical University, Xi'an, 710077, China
| | - Xue-mei Ren
- Department of Laboratory Medicine, The First Affiliated Hospital of Xi'an Medical University, Xi'an, 710077, China
| | - Xin Wang
- Department of Laboratory Medicine, The First Affiliated Hospital of Xi'an Medical University, Xi'an, 710077, China
| | - Zhuo Li
- Department of Laboratory Medicine, The First Affiliated Hospital of Xi'an Medical University, Xi'an, 710077, China
| | - Cory J Xian
- UniSA Clinical & Health Science, University of South Australia, Adelaide, SA 5001, Australia
| |
Collapse
|
25
|
Wu Y, Chang D, Chang Y, Zhang Q, Liu Y, Brennan JD, Li Y, Liu M. Nucleic Acid Enzyme-Activated CRISPR-Cas12a With Circular CRISPR RNA for Biosensing. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2023; 19:e2303007. [PMID: 37294164 DOI: 10.1002/smll.202303007] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/21/2023] [Revised: 06/01/2023] [Indexed: 06/10/2023]
Abstract
clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are increasingly used in biosensor development. However, directly translating recognition events for non-nucleic acid targets by CRISPR into effective measurable signals represents an important ongoing challenge. Herein, it is hypothesized and confirmed that CRISPR RNAs (crRNAs) in a circular topology efficiently render Cas12a incapable of both site-specific double-stranded DNA cutting and nonspecific single-stranded DNA trans cleavage. Importantly, it is shown that nucleic acid enzymes (NAzymes) with RNA-cleaving activity can linearize the circular crRNAs, activating CRISPR-Cas12a functions. Using ligand-responsive ribozymes and DNAzymes as molecular recognition elements, it is demonstrated that target-triggered linearization of circular crRNAs offers great versatility for biosensing. This strategy is termed as "NAzyme-Activated CRISPR-Cas12a with Circular CRISPR RNA (NA3C)." Use of NA3C for clinical evaluation of urinary tract infections using an Escherichia coli-responsive RNA-cleaving DNAzyme to test 40 patient urine samples, providing a diagnostic sensitivity of 100% and specificity of 90%, is further demonstrated.
Collapse
Affiliation(s)
- Yunping Wu
- School of Environmental Science and Technology, Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), Dalian University of Technology, Dalian POCT Laboratory, Dalian, 116024, China
| | - Dingran Chang
- Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S4K1, Canada
| | - Yangyang Chang
- School of Environmental Science and Technology, Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), Dalian University of Technology, Dalian POCT Laboratory, Dalian, 116024, China
| | - Qiang Zhang
- School of Bioengineering, Dalian University of Technology, Dalian, 116024, China
| | - Yi Liu
- Department of Neurology, Dalian Municipal Central Hospital, Affiliated Hospital of Dalian Medical University, Dalian, 116033, China
| | - John D Brennan
- Biointerfaces Institute, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S4O3, Canada
| | - Yingfu Li
- Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S4K1, Canada
| | - Meng Liu
- School of Environmental Science and Technology, Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), Dalian University of Technology, Dalian POCT Laboratory, Dalian, 116024, China
| |
Collapse
|
26
|
Wisztorski M, Aboulouard S, Roussel L, Duhamel M, Saudemont P, Cardon T, Narducci F, Robin YM, Lemaire AS, Bertin D, Hajjaji N, Kobeissy F, Leblanc E, Fournier I, Salzet M. Fallopian tube lesions as potential precursors of early ovarian cancer: a comprehensive proteomic analysis. Cell Death Dis 2023; 14:644. [PMID: 37775701 PMCID: PMC10541450 DOI: 10.1038/s41419-023-06165-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2023] [Revised: 09/08/2023] [Accepted: 09/20/2023] [Indexed: 10/01/2023]
Abstract
Ovarian cancer is the leading cause of death from gynecologic cancer worldwide. High-grade serous carcinoma (HGSC) is the most common and deadliest subtype of ovarian cancer. While the origin of ovarian tumors is still debated, it has been suggested that HGSC originates from cells in the fallopian tube epithelium (FTE), specifically the epithelial cells in the region of the tubal-peritoneal junction. Three main lesions, p53 signatures, STILs, and STICs, have been defined based on the immunohistochemistry (IHC) pattern of p53 and Ki67 markers and the architectural alterations of the cells, using the Sectioning and Extensively Examining the Fimbriated End Protocol. In this study, we performed an in-depth proteomic analysis of these pre-neoplastic epithelial lesions guided by mass spectrometry imaging and IHC. We evaluated specific markers related to each preneoplastic lesion. The study identified specific lesion markers, such as CAVIN1, Emilin2, and FBLN5. We also used SpiderMass technology to perform a lipidomic analysis and identified the specific presence of specific lipids signature including dietary Fatty acids precursors in lesions. Our study provides new insights into the molecular mechanisms underlying the progression of ovarian cancer and confirms the fimbria origin of HGSC.
Collapse
Affiliation(s)
- Maxence Wisztorski
- Univ.Lille, Inserm, CHU Lille, U-1192 - Laboratoire Protéomique Réponse Inflammatoire Spectrométrie de Masse - PRISM, F-59000, Lille, France
| | - Soulaimane Aboulouard
- Univ.Lille, Inserm, CHU Lille, U-1192 - Laboratoire Protéomique Réponse Inflammatoire Spectrométrie de Masse - PRISM, F-59000, Lille, France
| | - Lucas Roussel
- Univ.Lille, Inserm, CHU Lille, U-1192 - Laboratoire Protéomique Réponse Inflammatoire Spectrométrie de Masse - PRISM, F-59000, Lille, France
| | - Marie Duhamel
- Univ.Lille, Inserm, CHU Lille, U-1192 - Laboratoire Protéomique Réponse Inflammatoire Spectrométrie de Masse - PRISM, F-59000, Lille, France
| | - Philippe Saudemont
- Univ.Lille, Inserm, CHU Lille, U-1192 - Laboratoire Protéomique Réponse Inflammatoire Spectrométrie de Masse - PRISM, F-59000, Lille, France
| | - Tristan Cardon
- Univ.Lille, Inserm, CHU Lille, U-1192 - Laboratoire Protéomique Réponse Inflammatoire Spectrométrie de Masse - PRISM, F-59000, Lille, France
| | - Fabrice Narducci
- Univ.Lille, Inserm, CHU Lille, U-1192 - Laboratoire Protéomique Réponse Inflammatoire Spectrométrie de Masse - PRISM, F-59000, Lille, France
- Department of Gynecology Oncology, Oscar Lambret Cancer Center, 59020, Lille, France
| | - Yves-Marie Robin
- Univ.Lille, Inserm, CHU Lille, U-1192 - Laboratoire Protéomique Réponse Inflammatoire Spectrométrie de Masse - PRISM, F-59000, Lille, France
- Department of Gynecology Oncology, Oscar Lambret Cancer Center, 59020, Lille, France
| | - Anne-Sophie Lemaire
- Univ.Lille, Inserm, CHU Lille, U-1192 - Laboratoire Protéomique Réponse Inflammatoire Spectrométrie de Masse - PRISM, F-59000, Lille, France
- Department of Gynecology Oncology, Oscar Lambret Cancer Center, 59020, Lille, France
| | - Delphine Bertin
- Univ.Lille, Inserm, CHU Lille, U-1192 - Laboratoire Protéomique Réponse Inflammatoire Spectrométrie de Masse - PRISM, F-59000, Lille, France
- Department of Gynecology Oncology, Oscar Lambret Cancer Center, 59020, Lille, France
| | - Nawale Hajjaji
- Univ.Lille, Inserm, CHU Lille, U-1192 - Laboratoire Protéomique Réponse Inflammatoire Spectrométrie de Masse - PRISM, F-59000, Lille, France
- Medical Oncology Department, Oscar Lambret Cancer Center, 59020, Lille, France
| | - Firas Kobeissy
- Department of Neurobiology, Center for Neurotrauma, Multiomics & Biomarkers (CNMB), MorehouseSchool of Medicine, Atlanta, GA, 30310, USA
- Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
| | - Eric Leblanc
- Univ.Lille, Inserm, CHU Lille, U-1192 - Laboratoire Protéomique Réponse Inflammatoire Spectrométrie de Masse - PRISM, F-59000, Lille, France.
- Department of Gynecology Oncology, Oscar Lambret Cancer Center, 59020, Lille, France.
| | - Isabelle Fournier
- Univ.Lille, Inserm, CHU Lille, U-1192 - Laboratoire Protéomique Réponse Inflammatoire Spectrométrie de Masse - PRISM, F-59000, Lille, France.
- Institut Universitaire de France, 75000, Paris, France.
| | - Michel Salzet
- Univ.Lille, Inserm, CHU Lille, U-1192 - Laboratoire Protéomique Réponse Inflammatoire Spectrométrie de Masse - PRISM, F-59000, Lille, France.
- Institut Universitaire de France, 75000, Paris, France.
| |
Collapse
|
27
|
Wu Y, Wang Y, Zhou J, Wang J, Zhan Q, Wang Q, Yang E, Jin W, Tong F, Zhao J, Hong B, Liu J, Kang C. Universal theranostic CRISPR/Cas13a RNA-editing system for glioma. Theranostics 2023; 13:5305-5321. [PMID: 37908718 PMCID: PMC10614676 DOI: 10.7150/thno.84429] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2023] [Accepted: 09/05/2023] [Indexed: 11/02/2023] Open
Abstract
Background: The CRISPR/Cas13a system offers the advantages of rapidity, precision, high sensitivity, and programmability as a molecular diagnostic tool for critical illnesses. One of the salient features of CRISPR/Cas13a-based bioassays is its ability to recognize and cleave the target RNA specifically. Simple and efficient approaches for RNA manipulation would enrich our knowledge of disease-linked gene expression patterns and provide insights into their involvement in the underlying pathomechanism. However, only a few studies reported the Cas13a-based reporter system for in vivo molecular diagnoses. Methods: A tiled crRNA pool targeting a particular RNA transcript was generated, and the optimally potential crRNA candidates were selected using bioinformatics modeling and in vitro biological validation methods. For in vivo imaging assessment of the anti-GBM effectiveness, we exploited a human GBM patient-derived xenograft model in nude mice. Results: The most efficient crRNA sequence with a substantial cleavage impact on the target RNA as well as a potent collateral cleavage effect, was selected. In the xenografted GBM rodent model, the Cas13a-based reporter system enabled us in vivo imaging of the tumor growth. Furthermore, systemic treatments using this approach slowed tumor progression and increased the overall survival time in mice. Conclusions: Our work demonstrated the clinical potential of a Cas13a-based in vivo imaging method for the targeted degradation of specific RNAs in glioma cells, and suggested the feasibility of a tailored approach like Cas13a for the modulation of diagnosis and treatment options in glioma.
Collapse
Affiliation(s)
- Ye Wu
- Tianjin Neurological Institute, Tianjin Medical University General Hospital, Key Laboratory of Post-neurotrauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin City, Tianjin 300052, China
- Department of Dermatovenereology, Tianjin Medical University General Hospital, Tianjin 300052, China
| | - Yunfei Wang
- Tianjin Neurological Institute, Tianjin Medical University General Hospital, Key Laboratory of Post-neurotrauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin City, Tianjin 300052, China
| | - Junhu Zhou
- Tianjin Neurological Institute, Tianjin Medical University General Hospital, Key Laboratory of Post-neurotrauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin City, Tianjin 300052, China
| | - Jianhao Wang
- Tianjin Neurological Institute, Tianjin Medical University General Hospital, Key Laboratory of Post-neurotrauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin City, Tianjin 300052, China
| | - Qi Zhan
- Tianjin Key Laboratory of Composite and Functional Materials, School of Materials Science and Engineering, Tianjin University, Tianjin, China
| | - Qixue Wang
- Tianjin Neurological Institute, Tianjin Medical University General Hospital, Key Laboratory of Post-neurotrauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin City, Tianjin 300052, China
| | - Eryan Yang
- Tianjin Neurological Institute, Tianjin Medical University General Hospital, Key Laboratory of Post-neurotrauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin City, Tianjin 300052, China
| | - Weili Jin
- Tianjin Neurological Institute, Tianjin Medical University General Hospital, Key Laboratory of Post-neurotrauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin City, Tianjin 300052, China
| | - Fei Tong
- Tianjin Neurological Institute, Tianjin Medical University General Hospital, Key Laboratory of Post-neurotrauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin City, Tianjin 300052, China
| | - Jixing Zhao
- Tianjin Neurological Institute, Tianjin Medical University General Hospital, Key Laboratory of Post-neurotrauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin City, Tianjin 300052, China
| | - Biao Hong
- Tianjin Neurological Institute, Tianjin Medical University General Hospital, Key Laboratory of Post-neurotrauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin City, Tianjin 300052, China
| | - Junrui Liu
- College of Pharmacy, Kunming Medical University, Yunnan, China
| | - Chunsheng Kang
- Tianjin Neurological Institute, Tianjin Medical University General Hospital, Key Laboratory of Post-neurotrauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin City, Tianjin 300052, China
| |
Collapse
|
28
|
Chakraborty S, Banerjee S. Multidimensional computational study to understand non-coding RNA interactions in breast cancer metastasis. Sci Rep 2023; 13:15771. [PMID: 37737288 PMCID: PMC10516999 DOI: 10.1038/s41598-023-42904-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2023] [Accepted: 09/15/2023] [Indexed: 09/23/2023] Open
Abstract
Metastasis is a major breast cancer hallmark due to which tumor cells tend to relocate to regional or distant organs from their organ of origin. This study is aimed to decipher the interaction among 113 differentially expressed genes, interacting non-coding RNAs and drugs (614 miRNAs, 220 lncRNAs and 3241 interacting drugs) associated with metastasis in breast cancer. For an extensive understanding of genetic interactions in the diseased state, a backbone gene co-expression network was constructed. Further, the mRNA-miRNA-lncRNA-drug interaction network was constructed to identify the top hub RNAs, significant cliques and topological parameters associated with differentially expressed genes. Then, the mRNAs from the top two subnetworks constructed are considered for transcription factor (TF) analysis. 39 interacting miRNAs and 1641 corresponding TFs for the eight mRNAs from the subnetworks are also utilized to construct an mRNA-miRNA-TF interaction network. TF analysis revealed two TFs (EST1 and SP1) from the cliques to be significant. TCGA expression analysis of miRNAs and lncRNAs as well as subclass-based and promoter methylation-based expression, oncoprint and survival analysis of the mRNAs are also done. Finally, functional enrichment of mRNAs is also performed. Significant cliques identified in the study can be utilized for identification of newer therapeutic interventions for breast cancer. This work will also help to gain a deeper insight into the complicated molecular intricacies to reveal the potential biomarkers involved with breast cancer progression in future.
Collapse
Affiliation(s)
- Sohini Chakraborty
- Department of Biotechnology, School of Biosciences and Technology, Vellore Institute of Technology, Vellore, 632014, Tamil Nadu, India
| | - Satarupa Banerjee
- Department of Biotechnology, School of Biosciences and Technology, Vellore Institute of Technology, Vellore, 632014, Tamil Nadu, India.
| |
Collapse
|
29
|
Chen S, Wang J, Zhang K, Ma B, Li X, Wei R, Nian H. LncRNA Neat1 targets NonO and miR-128-3p to promote antigen-specific Th17 cell responses and autoimmune inflammation. Cell Death Dis 2023; 14:610. [PMID: 37716986 PMCID: PMC10505237 DOI: 10.1038/s41419-023-06132-0] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2023] [Revised: 09/01/2023] [Accepted: 09/06/2023] [Indexed: 09/18/2023]
Abstract
Long non-coding RNAs (lncRNAs) interaction with RNA-Binding proteins (RBPs) plays an important role in immunological processes. The generation of antigen-specific Th17 cells is closely associated with autoimmune pathogenesis. However, the function of lncRNA-RBP interactions in the regulation of pathogenic Th17 cell responses during autoimmunity remains poorly understood. Here, we found that lncRNA Neat1, highly expressed in Th17 cells, promoted antigen-specific Th17 cell responses. Both global and CD4+ T cell-specific knockdown of Neat1 protected mice against the development of experimental autoimmune uveitis (EAU). Mechanistically, Neat1 regulated RNA-Binding protein NonO, thus relieving IL-17 and IL-23R from NonO-mediated transcriptional repression and supporting antigen-specific Th17 cell responses. In addition, Neat1 also modulated miR-128-3p/NFAT5 axis to increase the expression of IL-17 and IL-23R, leading to augmented Th17 cell responses. Our findings elucidate a previously unrecognized mechanistic insight into the action of Neat1 in promoting antigen-specific Th17 responses and autoimmunity, and may facilitate the development of therapeutic targets for T cell-mediated autoimmune diseases.
Collapse
Affiliation(s)
- Sisi Chen
- Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin, 300384, China
| | - Jiali Wang
- Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin, 300384, China
| | - Kailang Zhang
- Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin, 300384, China
| | - Binyun Ma
- Department of Medicine/Hematology, Keck School of Medicine of the University of Southern California, Los Angeles, CA, 90033, USA
| | - Xiaorong Li
- Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin, 300384, China
| | - Ruihua Wei
- Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin, 300384, China
| | - Hong Nian
- Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin, 300384, China.
| |
Collapse
|
30
|
Zhang R, Wang Y, Deng H, Zhou S, Wu Y, Li Y. Fast and bioluminescent detection of antibiotic contaminants by on-demand transcription of RNA scaffold arrays. Anal Chim Acta 2023; 1273:341538. [PMID: 37423654 DOI: 10.1016/j.aca.2023.341538] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2023] [Revised: 06/11/2023] [Accepted: 06/16/2023] [Indexed: 07/11/2023]
Abstract
Cell-free biosensors have inspired low-cost and field-applicable methods to detect antibiotic contaminants. However, the satisfactory sensitivity of current cell-free biosensors is mostly achieved by sacrificing the rapidity, which prolongs turnaround time by hours. Additionally, the software-based result interpretation provides an obstacle for delivering these biosensors to untrained individuals. Here, we present a bioluminescence-based cell-free biosensor, termed enhanced Bioluminescence sensing of Ligand-Unleashed RNA Expression (eBLUE). The eBLUE leveraged antibiotic-responsive transcription factors to regulate the transcription of RNA arrays that can serve as scaffolds for reassembling and activating multiple luciferase fragments. This process converted target recognition into an amplified bioluminescence response, enabling smartphone-based quantification of tetracycline and erythromycin directly in milk within 15 min. Moreover, the detection threshold of eBLUE can be easily tuned according to the maximum residue limits (MRLs) established by government agencies. Owing to this tunable nature, the eBLUE was further repurposed as an on-demand semi-quantification platform, allowing for fast (∼20 min) and software-free identification of safe and MRL-exceeding milk samples only by glancing over the smartphone photographs. Overall, the sensitivity, rapidity and user-friendliness of eBLUE demonstrate its potentials for practical applications, especially in resource-limited and household settings.
Collapse
Affiliation(s)
- Rui Zhang
- College of Life Sciences, South-Central Minzu University, Wuhan, 430074, PR China
| | - Yu Wang
- College of Life Sciences, South-Central Minzu University, Wuhan, 430074, PR China
| | - Haifeng Deng
- College of Life Sciences, South-Central Minzu University, Wuhan, 430074, PR China
| | - Shiwen Zhou
- College of Life Sciences, South-Central Minzu University, Wuhan, 430074, PR China
| | - Yunhua Wu
- College of Life Sciences, South-Central Minzu University, Wuhan, 430074, PR China
| | - Yong Li
- College of Life Sciences, South-Central Minzu University, Wuhan, 430074, PR China; Hubei Jiangxia Laboratory, Wuhan, 430200, PR China.
| |
Collapse
|
31
|
Li S, Li Z, Tan GY, Xin Z, Wang W. In vitro allosteric transcription factor-based biosensing. Trends Biotechnol 2023; 41:1080-1095. [PMID: 36967257 DOI: 10.1016/j.tibtech.2023.03.001] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2023] [Revised: 02/15/2023] [Accepted: 03/01/2023] [Indexed: 06/18/2023]
Abstract
A biosensor is an analytical device that converts a biological response into a measurable output signal. Bacterial allosteric transcription factors (aTFs) have been utilized as a novel class of recognition elements for in vitro biosensing, which circumvents the limitations of aTF-based whole-cell biosensors (WCBs) and helps to meet the increasing requirement of small-molecule biosensors for diverse applications. In this review, we summarize the recent advances related to the configuration of aTF-based biosensors in vitro. Particularly, we evaluate the advantages of aTFs for in vitro biosensing and highlight their great potential for the establishment of robust and easy-to-implement biosensing strategies. We argue that key technical innovations and generalizable workflows will enhance the pipeline for facile construction of diverse aTF-based small-molecule biosensors.
Collapse
Affiliation(s)
- Shanshan Li
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China
| | - Zilong Li
- State Key Laboratory of Microbial Resources, Institute of Microbiology, CAS, Beijing 100101, PR China
| | - Gao-Yi Tan
- State Key Laboratory of Bioreactor Engineering and School of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China
| | - Zhenguo Xin
- State Key Laboratory of Microbial Resources, Institute of Microbiology, CAS, Beijing 100101, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China
| | - Weishan Wang
- State Key Laboratory of Microbial Resources, Institute of Microbiology, CAS, Beijing 100101, PR China; University of Chinese Academy of Sciences, Beijing 100049, PR China.
| |
Collapse
|
32
|
Huang Z, Lyon CJ, Wang J, Lu S, Hu TY. CRISPR Assays for Disease Diagnosis: Progress to and Barriers Remaining for Clinical Applications. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2023; 10:e2301697. [PMID: 37162202 PMCID: PMC10369298 DOI: 10.1002/advs.202301697] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/18/2023] [Revised: 04/24/2023] [Indexed: 05/11/2023]
Abstract
Numerous groups have employed the special properties of CRISPR/Cas systems to develop platforms that have broad potential applications for sensitive and specific detection of nucleic acid (NA) targets. However, few of these approaches have progressed to commercial or clinical applications. This review summarizes the properties of known CRISPR/Cas systems and their applications, challenges associated with the development of such assays, and opportunities to improve their performance or address unmet assay needs using nano-/micro-technology platforms. These include rapid and efficient sample preparation, integrated single-tube, amplification-free, quantifiable, multiplex, and non-NA assays. Finally, this review discusses the current outlook for such assays, including remaining barriers for clinical or point-of-care applications and their commercial development.
Collapse
Affiliation(s)
- Zhen Huang
- National Clinical Research Center for Infectious DiseasesShenzhen Third People's HospitalSouthern University of Science and Technology29 Bulan RoadShenzhenGuangdong518112China
- Center for Cellular and Molecular DiagnosticsTulane University School of Medicine1430 Tulane AveNew OrleansLA70112USA
- Department of Biochemistry and Molecular BiologyTulane University School of Medicine1430 Tulane AveNew OrleansLA70112USA
| | - Christopher J. Lyon
- Center for Cellular and Molecular DiagnosticsTulane University School of Medicine1430 Tulane AveNew OrleansLA70112USA
- Department of Biochemistry and Molecular BiologyTulane University School of Medicine1430 Tulane AveNew OrleansLA70112USA
| | - Jin Wang
- Tolo Biotechnology Company Limited333 Guiping RoadShanghai200233China
| | - Shuihua Lu
- National Clinical Research Center for Infectious DiseasesShenzhen Third People's HospitalSouthern University of Science and Technology29 Bulan RoadShenzhenGuangdong518112China
| | - Tony Y. Hu
- Center for Cellular and Molecular DiagnosticsTulane University School of Medicine1430 Tulane AveNew OrleansLA70112USA
- Department of Biochemistry and Molecular BiologyTulane University School of Medicine1430 Tulane AveNew OrleansLA70112USA
| |
Collapse
|
33
|
Lu B, Guo Z, Zhong K, Osire T, Sun Y, Jiang L. State of the art in CRISPR/Cas system-based signal conversion and amplification applied in the field of food analysis. Trends Food Sci Technol 2023. [DOI: 10.1016/j.tifs.2023.04.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/05/2023]
|
34
|
Liu FX, Cui JQ, Wu Z, Yao S. Recent progress in nucleic acid detection with CRISPR. LAB ON A CHIP 2023; 23:1467-1492. [PMID: 36723235 DOI: 10.1039/d2lc00928e] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/18/2023]
Abstract
Recent advances in CRISPR-based biotechnologies have greatly expanded our capabilities to repurpose CRISPR for the development of molecular diagnostic systems. The key attribute that allows CRISPR to be widely utilized is its programmable and highly specific nature. In this review, we first illustrate the principle of the class 2 CRISPR nucleases for molecular diagnostics which originates from their immunologic defence systems. Next, we present the CRISPR-based schemes in the application of diagnostics with amplification-assisted or amplification-free strategies. By highlighting some of the recent advances we interpret how general bioengineering methodologies can be integrated with CRISPR. Finally, we discuss the challenges and exciting prospects for future CRISPR-based biosensing development. We hope that this review will guide the reader to systematically learn the start-of-the-art development of CRISPR-mediated nucleic acid detection and understand how to apply the CRISPR nucleases with different design concepts to more general applications in diagnostics and beyond.
Collapse
Affiliation(s)
- Frank X Liu
- Department of Mechanical and Aerospace Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
| | - Johnson Q Cui
- Department of Mechanical and Aerospace Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
| | - Zhihao Wu
- IIP-Advanced Materials, Interdisciplinary Program Office (IPO), Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong
| | - Shuhuai Yao
- Department of Mechanical and Aerospace Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
- Department of Chemical and Biological Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong
| |
Collapse
|
35
|
Shen Y, Hu K, Yuan M, Duan G, Guo Y, Chen S. Progress and bioapplication of CRISPR-based one-step, quantitative and multiplexed infectious disease diagnostics. J Appl Microbiol 2023; 134:lxad035. [PMID: 36813257 DOI: 10.1093/jambio/lxad035] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2022] [Revised: 01/06/2023] [Accepted: 02/21/2023] [Indexed: 02/24/2023]
Abstract
In Vitro Diagnosis (IVD) technology is able to accurately detect pathogens or biomarkers at an initial stage of disease, which works as an important toolbox for disease diagnosis. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system, as an emerging IVD method, plays a crucial role in the field of infectious disease detection due to its superior sensitivity and specificity. Recently, an increasing number of scientists have been devoted to improving the performance of CRISPR-based detection and on-site point-of-care testing (POCT) from extraction-free detection, amplification-free, modified Cas/crRNA complexes, quantitative assays, one-pot detection, and multiplexed platform. In this review, we describe the potential roles of these novel approaches and platforms in one-pot methods, quantitative molecular diagnostics as well as multiplexed detection. This review will not only help guide the full use of the CRISPR-Cas tools for quantification, multiplexed detection, POCT and as next-generation diagnostic biosensing platforms but also inspire new ideas, technological advances, and engineering strategies to address real-world challenges like the ongoing COVID-19 pandemic.
Collapse
Affiliation(s)
- Yue Shen
- College of Public Health, Zhengzhou University, Zhengzhou 450000, China
| | - Kai Hu
- Laboratory Biosafety Technology Center, Henan Academy of Medical Sciences, Zhengzhou 450046, China
| | - Mingzhu Yuan
- College of Public Health, Zhengzhou University, Zhengzhou 450000, China
| | - Guangcai Duan
- College of Public Health, Zhengzhou University, Zhengzhou 450000, China
| | - Yongjun Guo
- Laboratory Biosafety Technology Center, Henan Academy of Medical Sciences, Zhengzhou 450046, China
| | - Shuaiyin Chen
- College of Public Health, Zhengzhou University, Zhengzhou 450000, China
| |
Collapse
|
36
|
Husser C, Vuilleumier S, Ryckelynck M. FluorMango, an RNA-Based Fluorogenic Biosensor for the Direct and Specific Detection of Fluoride. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2023; 19:e2205232. [PMID: 36436882 DOI: 10.1002/smll.202205232] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/25/2022] [Revised: 10/28/2022] [Indexed: 06/16/2023]
Abstract
Nucleic acids are not only essential actors of cell life but also extremely appealing molecular objects in the development of synthetic molecules for biotechnological application, such as biosensors to report on the presence and concentration of a target ligand by emission of a measurable signal. In this work, FluorMango, a fluorogenic ribonucleic acid (RNA)-based biosensor specific for fluoride is introduced. The molecule consists of two RNA aptamer modules, a fluoride-specific sensor derived from the crcB riboswitch which changes its structure upon interaction with the target ion, and the light-up RNA Mango-III that emits fluorescence when complexed with a fluorogen. The two modules are connected by an optimized communication module identified by ultrahigh-throughput screening, which results in extremely high fluorescence of FluorMango in the presence of fluoride, and background fluorescence in its absence. The value and efficiency of this biosensor by direct monitoring of defluorinase activity in living bacterial cells is illustrated, and the use of this new tool in future screening campaigns aiming at discovering new defluorinase activities is discussed.
Collapse
Affiliation(s)
- Claire Husser
- CNRS, Architecture et Réactivité de l'ARN, Université de Strasbourg, UPR 9002, 2 allée Konrad Roentgen, Strasbourg, 67000, France
| | - Stéphane Vuilleumier
- CNRS, Génétique Moléculaire, Génomique, Microbiologie, Université de Strasbourg, UMR 7156, 4 allée Konrad Roentgen, Strasbourg, 67000, France
| | - Michael Ryckelynck
- CNRS, Architecture et Réactivité de l'ARN, Université de Strasbourg, UPR 9002, 2 allée Konrad Roentgen, Strasbourg, 67000, France
| |
Collapse
|
37
|
Cryo-EM structure and protease activity of the type III-E CRISPR-Cas effector. Nat Microbiol 2023; 8:522-532. [PMID: 36702942 DOI: 10.1038/s41564-022-01316-4] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2022] [Accepted: 12/20/2022] [Indexed: 01/27/2023]
Abstract
The recently discovered type III-E CRISPR-Cas effector Cas7-11 shows promise when used as an RNA manipulation tool, but its structure and the mechanisms underlying its function remain unclear. Here we present four cryo-EM structures of Desulfonema ishimotonii Cas7-11-crRNA complex in pre-target and target RNA-bound states, and the cryo-EM structure of DiCas7-11-crRNA bound to its accessory protein DiCsx29. These data reveal structural elements for pre-crRNA processing, target RNA cleavage and regulation. Moreover, a 3' seed region of crRNA is involved in regulating RNA cleavage activity of DiCas7-11-crRNA-Csx29. Our analysis also shows that both the minimal mismatch of 4 nt to the 5' handle of crRNA and the minimal matching of the first 12 nt of the spacer by the target RNA are essential for triggering the protease activity of DiCas7-11-crRNA-Csx29 towards DiCsx30. Taken together, we propose that target RNA recognition and cleavage regulate and fine-tune the protease activity of DiCas7-11-crRNA-Csx29, thus preventing auto-immune responses.
Collapse
|
38
|
Kim ER, Joe C, Mitchell RJ, Gu MB. Biosensors for healthcare: current and future perspectives. Trends Biotechnol 2023; 41:374-395. [PMID: 36567185 DOI: 10.1016/j.tibtech.2022.12.005] [Citation(s) in RCA: 41] [Impact Index Per Article: 20.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2022] [Revised: 11/28/2022] [Accepted: 12/06/2022] [Indexed: 12/24/2022]
Abstract
Biosensors are utilized in several different fields, including medicine, food, and the environment; in this review, we examine recent developments in biosensors for healthcare. These involve three distinct types of biosensor: biosensors for in vitro diagnosis with blood, saliva, or urine samples; continuous monitoring biosensors (CMBs); and wearable biosensors. Biosensors for in vitro diagnosis have seen a significant expansion recently, with newly reported clustered regularly interspaced short palindromic repeats (CRISPR)/Cas methodologies and improvements to many established integrated biosensor devices, including lateral flow assays (LFAs) and microfluidic/electrochemical paper-based analytical devices (μPADs/ePADs). We conclude with a discussion of two novel groups of biosensors that have drawn great attention recently, continuous monitoring and wearable biosensors, as well as with perspectives on the commercialization and future of biosensors.
Collapse
Affiliation(s)
- Eun Ryung Kim
- Department of Biotechnology, Korea University, Anam-dong, Sungbuk-Gu, Seoul 02841, Republic of Korea
| | - Cheulmin Joe
- Department of Biotechnology, Korea University, Anam-dong, Sungbuk-Gu, Seoul 02841, Republic of Korea
| | - Robert J Mitchell
- Department of Biological Sciences, UNIST, Ulsan 44919, Republic of Korea
| | - Man Bock Gu
- Department of Biotechnology, Korea University, Anam-dong, Sungbuk-Gu, Seoul 02841, Republic of Korea.
| |
Collapse
|
39
|
Jiang W, Aman R, Ali Z, Mahfouz M. Bio-SCAN V2: A CRISPR/dCas9-based lateral flow assay for rapid detection of theophylline. Front Bioeng Biotechnol 2023; 11:1118684. [PMID: 36741753 PMCID: PMC9893010 DOI: 10.3389/fbioe.2023.1118684] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2022] [Accepted: 01/09/2023] [Indexed: 01/20/2023] Open
Abstract
Rapid, specific, and robust diagnostic strategies are needed to develop sensitive biosensors for small molecule detection, which could aid in controlling contamination and disease transmission. Recently, the target-induced collateral activity of Cas nucleases [clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases] was exploited to develop high-throughput diagnostic modules for detecting nucleic acids and small molecules. Here, we have expanded the diagnostic ability of the CRISPR-Cas system by developing Bio-SCAN V2, a ligand-responsive CRISPR-Cas platform for detecting non-nucleic acid small molecule targets. The Bio-SCAN V2 consists of an engineered ligand-responsive sgRNA (ligRNA), biotinylated dead Cas9 (dCas9-biotin), 6-carboxyfluorescein (FAM)-labeled amplicons, and lateral flow assay (LFA) strips. LigRNA interacts with dCas9-biotin only in the presence of sgRNA-specific ligand molecules to make a ribonucleoprotein (RNP). Next, the ligand-induced ribonucleoprotein is exposed to FAM-labeled amplicons for binding, and the presence of the ligand (small molecule) is detected as a visual signal [(dCas9-biotin)-ligRNA-FAM labeled DNA-AuNP complex] at the test line of the lateral flow assay strip. With the Bio-SCAN V2 platform, we are able to detect the model molecule theophylline with a limit of detection (LOD) up to 2 μM in a short time, requiring only 15 min from sample application to visual readout. Taken together, Bio-SCAN V2 assay provides a rapid, specific, and ultrasensitive detection platform for theophylline.
Collapse
|
40
|
Antropov DN, Stepanov GA. Molecular Mechanisms Underlying CRISPR/Cas-Based Assays for Nucleic Acid Detection. Curr Issues Mol Biol 2023; 45:649-662. [PMID: 36661529 PMCID: PMC9857636 DOI: 10.3390/cimb45010043] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2022] [Revised: 12/16/2022] [Accepted: 12/30/2022] [Indexed: 01/12/2023] Open
Abstract
Applied to investigate specific sequences, nucleic acid detection assays can help identify novel bacterial and viral infections. Most up-to-date systems combine isothermal amplification with Cas-mediated detection. They surpass standard PCR methods in detection time and sensitivity, which is crucial for rapid diagnostics. The first part of this review covers the variety of isothermal amplification methods and describes their reaction mechanisms. Isothermal amplification enables fast multiplication of a target nucleic acid sequence without expensive laboratory equipment. However, researchers aim for more reliable results, which cannot be achieved solely by amplification because it is also a source of non-specific products. This motivated the development of Cas-based assays that use Cas9, Cas12, or Cas13 proteins to detect nucleic acids and their fragments in biological specimens with high specificity. Isothermal amplification yields a high enough concentration of target nucleic acids for the specific signal to be detected via Cas protein activity. The second part of the review discusses combinations of different Cas-mediated reactions and isothermal amplification methods and presents signal detection techniques adopted in each assay. Understanding the features of Cas-based assays could inform the choice of an optimal protocol to detect different nucleic acids.
Collapse
|
41
|
Duan M, Li B, Zhao Y, Liu Y, Liu Y, Dai R, Li X, Jia F. A CRISPR/Cas12a-mediated, DNA extraction and amplification-free, highly direct and rapid biosensor for Salmonella Typhimurium. Biosens Bioelectron 2023; 219:114823. [PMID: 36308834 DOI: 10.1016/j.bios.2022.114823] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2022] [Revised: 10/13/2022] [Accepted: 10/14/2022] [Indexed: 11/19/2022]
Abstract
CRISPR/Cas-based biosensors were typically used for nucleic-acid targets detection and complex DNA extraction and amplification procedures were usually inevitable. Here, we report a CRISPR/Cas12a-mediated, DNA extraction and amplification-free, highly direct and rapid biosensor (abbreviated as "CATCHER") for Salmonella Typhimurium (S. Typhimurium) with a simple (3 steps) and fast (∼2 h) sensing workflow. Magnetic nanoparticle immobilized anti-S. Typhimurium antibody was worked as capture probe to capture the target and provide movable reaction interface. Colloidal gold labeled with anti-S. Typhimurium antibody and DNase I was used as detection probe to bridge the input target and output signal. First, in the presence of S. Typhimurium, an immuno-sandwich structure was formed. Second, DNase I in sandwich structure degraded the valid, complete activator DNA to invalid DNA fragments which can't trigger the trans-cleavage activity of Cas12a. Finally, the integrity of reporter DNA was preserved presenting a low fluorescence signal. Conversely, in the absence of S. Typhimurium, strong fluorescence recovery appeared owing to the cutting of reporter by activated Cas12a. Significantly, the proposed "CATCHER" showed satisfactory detection performance for S. Typhimurium with the limit of detection (LOD) of 7.9 × 101 CFU/mL in 0.01 M PBS and 6.31 × 103 CFU/mL in spiked chicken samples, providing a general platform for non-nucleic acid targets.
Collapse
Affiliation(s)
- Miaolin Duan
- College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China
| | - Bingyan Li
- College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China
| | - Yijie Zhao
- College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China
| | - Yana Liu
- College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China
| | - Yi Liu
- College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China
| | - Ruitong Dai
- College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China
| | - Xingmin Li
- College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China.
| | - Fei Jia
- Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR, 72701, USA.
| |
Collapse
|
42
|
Zhao L, Qiu M, Li X, Yang J, Li J. CRISPR-Cas13a system: A novel tool for molecular diagnostics. Front Microbiol 2022; 13:1060947. [PMID: 36569102 PMCID: PMC9772028 DOI: 10.3389/fmicb.2022.1060947] [Citation(s) in RCA: 37] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2022] [Accepted: 11/09/2022] [Indexed: 12/12/2022] Open
Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR) system is a natural adaptive immune system of prokaryotes. The CRISPR-Cas system is currently divided into two classes and six types: types I, III, and IV in class 1 systems and types II, V, and VI in class 2 systems. Among the CRISPR-Cas type VI systems, the CRISPR/Cas13a system has been the most widely characterized for its application in molecular diagnostics, gene therapy, gene editing, and RNA imaging. Moreover, because of the trans-cleavage activity of Cas13a and the high specificity of its CRISPR RNA, the CRISPR/Cas13a system has enormous potential in the field of molecular diagnostics. Herein, we summarize the applications of the CRISPR/Cas13a system in the detection of pathogens, including viruses, bacteria, parasites, chlamydia, and fungus; biomarkers, such as microRNAs, lncRNAs, and circRNAs; and some non-nucleic acid targets, including proteins, ions, and methyl groups. Meanwhile, we highlight the working principles of some novel Cas13a-based detection methods, including the Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) and its improved versions, Cas13a-based nucleic acid amplification-free biosensors, and Cas13a-based biosensors for non-nucleic acid target detection. Finally, we focus on some issues that need to be solved and the development prospects of the CRISPR/Cas13a system.
Collapse
Affiliation(s)
- Lixin Zhao
- Department of Biosafety, School of Basic Medicine, Army Medical University, Chongqing, China,Institute of Immunology, PLA, Army Medical University, Chongqing, China
| | - Minyue Qiu
- Department of Biosafety, School of Basic Medicine, Army Medical University, Chongqing, China,Institute of Immunology, PLA, Army Medical University, Chongqing, China
| | - Xiaojia Li
- Department of Biosafety, School of Basic Medicine, Army Medical University, Chongqing, China
| | - Juanzhen Yang
- Department of Biosafety, School of Basic Medicine, Army Medical University, Chongqing, China
| | - Jintao Li
- Department of Biosafety, School of Basic Medicine, Army Medical University, Chongqing, China,Institute of Immunology, PLA, Army Medical University, Chongqing, China,*Correspondence: Jintao Li,
| |
Collapse
|
43
|
Cheng X, Li Y, Kou J, Liao D, Zhang W, Yin L, Man S, Ma L. Novel non-nucleic acid targets detection strategies based on CRISPR/Cas toolboxes: A review. Biosens Bioelectron 2022; 215:114559. [DOI: 10.1016/j.bios.2022.114559] [Citation(s) in RCA: 80] [Impact Index Per Article: 26.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2022] [Revised: 06/20/2022] [Accepted: 07/07/2022] [Indexed: 12/26/2022]
|
44
|
Wang J, Yang X, Wang X, Wang W. Recent Advances in CRISPR/Cas-Based Biosensors for Protein Detection. Bioengineering (Basel) 2022; 9:512. [PMID: 36290480 PMCID: PMC9598526 DOI: 10.3390/bioengineering9100512] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2022] [Revised: 09/20/2022] [Accepted: 09/23/2022] [Indexed: 11/19/2022] Open
Abstract
CRISPR is an acquired immune system found in prokaryotes that can accurately recognize and cleave foreign nucleic acids, and has been widely explored for gene editing and biosensing. In the past, CRISPR/Cas-based biosensors were mainly applied to detect nucleic acids in the field of biosensing, and their applications for the detection of other types of analytes were usually overlooked such as small molecules and disease-related proteins. The recent work shows that CRISPR/Cas biosensors not only provide a new tool for protein analysis, but also improve the sensitivity and specificity of protein detections. However, it lacks the latest review to summarize CRISPR/Cas-based biosensors for protein detection and elucidate their mechanisms of action, hindering the development of superior biosensors for proteins. In this review, we summarized CRISPR/Cas-based biosensors for protein detection based on their mechanism of action in three aspects: antibody-assisted CRISPR/Cas-based protein detection, aptamer-assisted CRISPR/Cas-based protein detection, and miscellaneous CRISPR/Cas-based methods for protein detection, respectively. Moreover, the prospects and challenges for CRISPR/Cas-based biosensors for protein detection are also discussed.
Collapse
Affiliation(s)
- Jing Wang
- Collaborative Innovation Center of NPU, Shanghai 201100, China
- Institute of Medical Research, Northwestern Polytechnical University, 127 West Youyi Road, Xi’an 710072, China
- Research & Development Institute of Northwestern Polytechnical University in Shenzhen, 45 South Gaoxin Road, Shenzhen 518057, China
- Northwestern Polytechnical University Chongqing Technology Innovation Center, Chongqing 400000, China
| | - Xifang Yang
- Collaborative Innovation Center of NPU, Shanghai 201100, China
- Institute of Medical Research, Northwestern Polytechnical University, 127 West Youyi Road, Xi’an 710072, China
- Research & Development Institute of Northwestern Polytechnical University in Shenzhen, 45 South Gaoxin Road, Shenzhen 518057, China
- Northwestern Polytechnical University Chongqing Technology Innovation Center, Chongqing 400000, China
| | - Xueliang Wang
- Collaborative Innovation Center of NPU, Shanghai 201100, China
- Institute of Medical Research, Northwestern Polytechnical University, 127 West Youyi Road, Xi’an 710072, China
- Research & Development Institute of Northwestern Polytechnical University in Shenzhen, 45 South Gaoxin Road, Shenzhen 518057, China
- Northwestern Polytechnical University Chongqing Technology Innovation Center, Chongqing 400000, China
| | - Wanhe Wang
- Collaborative Innovation Center of NPU, Shanghai 201100, China
- Institute of Medical Research, Northwestern Polytechnical University, 127 West Youyi Road, Xi’an 710072, China
- Research & Development Institute of Northwestern Polytechnical University in Shenzhen, 45 South Gaoxin Road, Shenzhen 518057, China
- Northwestern Polytechnical University Chongqing Technology Innovation Center, Chongqing 400000, China
| |
Collapse
|
45
|
Chen K, Shen Z, Wang G, Gu W, Zhao S, Lin Z, Liu W, Cai Y, Mushtaq G, Jia J, Wan C(C, Yan T. Research progress of CRISPR-based biosensors and bioassays for molecular diagnosis. Front Bioeng Biotechnol 2022; 10:986233. [PMID: 36185462 PMCID: PMC9524266 DOI: 10.3389/fbioe.2022.986233] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2022] [Accepted: 08/10/2022] [Indexed: 11/13/2022] Open
Abstract
CRISPR/Cas technology originated from the immune mechanism of archaea and bacteria and was awarded the Nobel Prize in Chemistry in 2020 for its success in gene editing. Molecular diagnostics is highly valued globally for its development as a new generation of diagnostic technology. An increasing number of studies have shown that CRISPR/Cas technology can be integrated with biosensors and bioassays for molecular diagnostics. CRISPR-based detection has attracted much attention as highly specific and sensitive sensors with easily programmable and device-independent capabilities. The nucleic acid-based detection approach is one of the most sensitive and specific diagnostic methods. With further research, it holds promise for detecting other biomarkers such as small molecules and proteins. Therefore, it is worthwhile to explore the prospects of CRISPR technology in biosensing and summarize its application strategies in molecular diagnostics. This review provides a synopsis of CRISPR biosensing strategies and recent advances from nucleic acids to other non-nucleic small molecules or analytes such as proteins and presents the challenges and perspectives of CRISPR biosensors and bioassays.
Collapse
Affiliation(s)
- Kun Chen
- School of Life Sciences, Shanghai University, Shanghai, China
| | - Ziyi Shen
- School of Life Sciences, Shanghai University, Shanghai, China
| | - Guanzhen Wang
- School of Life Sciences, Shanghai University, Shanghai, China
- University and College Key Lab of Natural Product Chemistry and Application in Xinjiang, School of Chemistry and Environmental Science, Yili Normal University, Yining, China
| | - Wei Gu
- School of Life Sciences, Shanghai University, Shanghai, China
| | - Shengchao Zhao
- School of Life Sciences, Shanghai University, Shanghai, China
- University and College Key Lab of Natural Product Chemistry and Application in Xinjiang, School of Chemistry and Environmental Science, Yili Normal University, Yining, China
| | - Zihan Lin
- School of Life Sciences, Shanghai University, Shanghai, China
| | - Wei Liu
- University and College Key Lab of Natural Product Chemistry and Application in Xinjiang, School of Chemistry and Environmental Science, Yili Normal University, Yining, China
| | - Yi Cai
- Key Laboratory of Molecular Target & Clinical Pharmacology and The State & NMPA Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences and The Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou, China
| | - Gohar Mushtaq
- Center for Scientific Research, Faculty of Medicine, Idlib University, Idlib, Syria
| | - Jia Jia
- School of Life Sciences, Shanghai University, Shanghai, China
| | - Chunpeng (Craig) Wan
- Jiangxi Key Laboratory for Postharvest Technology and Nondestructive Testing of Fruits and Vegetables, College of Agronomy, Jiangxi Agricultural University, Nanchang, China
| | - Tingdong Yan
- School of Life Sciences, Shanghai University, Shanghai, China
| |
Collapse
|
46
|
Zhu C, Zhang F, Li H, Chen Z, Yan M, Li L, Qu F. CRISPR/Cas Systems Accelerating the Development of Aptasensors. Trends Analyt Chem 2022. [DOI: 10.1016/j.trac.2022.116775] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
|
47
|
Bhardwaj P, Kant R, Behera SP, Dwivedi GR, Singh R. Next-Generation Diagnostic with CRISPR/Cas: Beyond Nucleic Acid Detection. Int J Mol Sci 2022; 23:6052. [PMID: 35682737 PMCID: PMC9180940 DOI: 10.3390/ijms23116052] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2022] [Revised: 05/13/2022] [Accepted: 05/20/2022] [Indexed: 02/07/2023] Open
Abstract
The early management, diagnosis, and treatment of emerging and re-emerging infections and the rising burden of non-communicable diseases (NCDs) are necessary. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system has recently acquired popularity as a diagnostic tool due to its ability to target specific genes. It uses Cas enzymes and a guide RNA (gRNA) to cleave target DNA or RNA. The discovery of collateral cleavage in CRISPR-Cas effectors such as Cas12a and Cas13a was intensively repurposed for the development of instrument-free, sensitive, precise and rapid point-of-care diagnostics. CRISPR/Cas demonstrated proficiency in detecting non-nucleic acid targets including protein, analyte, and hormones other than nucleic acid. CRISPR/Cas effectors can provide multiple detections simultaneously. The present review highlights the technical challenges of integrating CRISPR/Cas technology into the onsite assessment of clinical and other specimens, along with current improvements in CRISPR bio-sensing for nucleic acid and non-nucleic acid targets. It also highlights the current applications of CRISPR/Cas technologies.
Collapse
Affiliation(s)
| | | | | | - Gaurav Raj Dwivedi
- ICMR-Regional Medical Research Centre, BRD Medical College Campus, Gorakhpur 273013, India; (P.B.); (R.K.); (S.P.B.)
| | - Rajeev Singh
- ICMR-Regional Medical Research Centre, BRD Medical College Campus, Gorakhpur 273013, India; (P.B.); (R.K.); (S.P.B.)
| |
Collapse
|
48
|
Ren Y, Yan C, Wu L, Zhao J, Chen M, Zhou M, Wang X, Liu T, Yi Q, Sun J. iUMRG: multi-layered network-guided propagation modeling for the inference of susceptibility genes and potential drugs against uveal melanoma. NPJ Syst Biol Appl 2022; 8:18. [PMID: 35610253 PMCID: PMC9130324 DOI: 10.1038/s41540-022-00227-8] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2022] [Accepted: 04/28/2022] [Indexed: 11/17/2022] Open
Abstract
Uveal melanoma (UM) is the most common primary malignant intraocular tumor. The use of precision medicine for UM to enable personalized diagnosis, prognosis, and treatment require the development of computer-aided strategies and predictive tools that can identify novel high-confidence susceptibility genes (HSGs) and potential therapeutic drugs. In the present study, a computational framework via propagation modeling on integrated multi-layered molecular networks (abbreviated as iUMRG) was proposed for the systematic inference of HSGs in UM. Under the leave-one-out cross-validation experiments, the iUMRG achieved superior predictive performance and yielded a higher area under the receiver operating characteristic curve value (0.8825) for experimentally verified SGs. In addition, using the experimentally verified SGs as seeds, genome-wide screening was performed to detect candidate HSGs using the iUMRG. Multi-perspective validation analysis indicated that most of the top 50 candidate HSGs were indeed markedly associated with UM carcinogenesis, progression, and outcome. Finally, drug repositioning experiments performed on the HSGs revealed 17 potential targets and 10 potential drugs, of which six have been approved for UM treatment. In conclusion, the proposed iUMRG is an effective supplementary tool in UM precision medicine, which may assist the development of new medical therapies and discover new SGs.
Collapse
Affiliation(s)
- Yueping Ren
- School of Biomedical Engineering, School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, 325027, P. R. China
| | - Congcong Yan
- School of Biomedical Engineering, School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, 325027, P. R. China
| | - Lili Wu
- Tibet Medical College, Beijing University of Chinese Medicine, Tibet, 850010, P. R. China
| | - Jingting Zhao
- School of Biomedical Engineering, School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, 325027, P. R. China
| | - Mingwei Chen
- Department of Human Anatomy, Harbin Medical University, Harbin, 150081, P. R. China
| | - Meng Zhou
- School of Biomedical Engineering, School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, 325027, P. R. China
| | - Xiaoyan Wang
- The Affiliated Ningbo Eye Hospital of Wenzhou Medical University, Ningbo, 315042, P. R. China
| | - Tonghua Liu
- Tibet Medical College, Beijing University of Chinese Medicine, Tibet, 850010, P. R. China.
| | - Quanyong Yi
- The Affiliated Ningbo Eye Hospital of Wenzhou Medical University, Ningbo, 315042, P. R. China.
| | - Jie Sun
- School of Biomedical Engineering, School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, 325027, P. R. China.
| |
Collapse
|
49
|
Long noncoding RNA NEAT1 promotes ferroptosis by modulating the miR-362-3p/MIOX axis as a ceRNA. Cell Death Differ 2022; 29:1850-1863. [PMID: 35338333 PMCID: PMC9433379 DOI: 10.1038/s41418-022-00970-9] [Citation(s) in RCA: 131] [Impact Index Per Article: 43.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2021] [Revised: 02/24/2022] [Accepted: 02/24/2022] [Indexed: 12/24/2022] Open
Abstract
Ferroptosis, a novel form of regulated cell death induced by iron-dependent lipid peroxidation, plays an essential role in the development and drug resistance of tumors. Long noncoding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) has been reported to be involved in the regulation of cell cycle, proliferation, apoptosis, and migration of tumor cells. However, the function and molecular mechanism of NEAT1 in regulating ferroptosis in tumors remain unclear. Here, we found that ferroptosis inducers erastin and RSL3 increased NEAT1 expression by promoting the binding of p53 to the NEAT1 promoter. Induced NEAT1 promoted the expression of MIOX by competitively binding to miR-362-3p. MIOX increased ROS production and decreased the intracellular levels of NADPH and GSH, resulting in enhanced erastin- and RSL3-induced ferroptosis. Importantly, overexpression of NEAT1 increased the anti-tumor activity of erastin and RSL3 by enhancing ferroptosis both in vitro and in vivo. Collectively, these data suggest that NEAT1 plays a novel and indispensable role in ferroptosis by regulating miR-362-3p and MIOX. Considering the clinical findings that HCC patients are insensitive to chemotherapy and immunotherapy, ferroptosis induction may be a promising therapeutic strategy for HCC patients with high NEAT1 expression.
Collapse
|
50
|
Zhuang J, Zhao Z, Lian K, Yin L, Wang J, Man S, Liu G, Ma L. SERS-based CRISPR/Cas assay on microfluidic paper analytical devices for supersensitive detection of pathogenic bacteria in foods. Biosens Bioelectron 2022; 207:114167. [PMID: 35325722 DOI: 10.1016/j.bios.2022.114167] [Citation(s) in RCA: 100] [Impact Index Per Article: 33.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2021] [Revised: 02/23/2022] [Accepted: 03/06/2022] [Indexed: 02/07/2023]
Abstract
Rapid and point-of-need (PON) detection of bacteria is crucial to directly provide rapid and reliable diagnostics information during on-site tests, allowing more room for taking proactive measures. By taking the multifaceted advantages of CRISPR/Cas12a and surface-enhanced Raman scattering (SERS), for the first time, we designed a recombinase polymerase amplification (RPA)-integrated microfluidic paper-based analytical device (μPAD), coined RPA-Cas12a-μPAD for supersensitive SERS detection. Single-stranded DNAs were designed to "pull down" SERS nanoprobes. The amplicons of the invA gene triggered the trans-cleavage of Cas12a, resulting in the indiscriminate shredding of linker ssDNA. Thus, the degree of aggregation of SERS nanoprobes was dependent on the concentration of Salmonella typhimurium (S. typhi), which was determined on a μPAD and monitored by a Raman spectrometer. The limit of detection for S. typhi was approximately 3-4 CFU/mL for spiked milk and meat samples with a dynamic detection range from 1 to 108 CFU/mL. The RPA-Cas12a-μPAD secured accurate tests for food samples in 45 min. This work expands the reach of CRISPR-based diagnostics (CRISPR-Dx) and provides a novel and robust bacterial PON detection platform.
Collapse
Affiliation(s)
- Jianwen Zhuang
- State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin Key Laboratory of Industry Microbiology, National and Local United Engineering Lab of Metabolic Control Fermentation Technology, China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China
| | - Zhiying Zhao
- State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin Key Laboratory of Industry Microbiology, National and Local United Engineering Lab of Metabolic Control Fermentation Technology, China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China
| | - Kai Lian
- School of Medicine, The Chinese University of Hong Kong, Shenzhen, 518172, China
| | - Lijuan Yin
- State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin Key Laboratory of Industry Microbiology, National and Local United Engineering Lab of Metabolic Control Fermentation Technology, China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China
| | - Jiajing Wang
- State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin Key Laboratory of Industry Microbiology, National and Local United Engineering Lab of Metabolic Control Fermentation Technology, China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China
| | - Shuli Man
- State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin Key Laboratory of Industry Microbiology, National and Local United Engineering Lab of Metabolic Control Fermentation Technology, China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China.
| | - Guozhen Liu
- School of Medicine, The Chinese University of Hong Kong, Shenzhen, 518172, China.
| | - Long Ma
- State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin Key Laboratory of Industry Microbiology, National and Local United Engineering Lab of Metabolic Control Fermentation Technology, China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China.
| |
Collapse
|