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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.