Long-Chain Fluorescent Probe for Straightforward and Nondestructive Staining Mitochondria in Fixed Cells and Tissues.

Normally, small-molecule fluorescent probes dependent on the mitochondrial membrane potential (MMP) are invalid for fixed cells and tissues, which limits their clinical applications when the fixation of pathological specimens is imperative. Given that mitochondrial morphology is closely associated with disease, we developed a long-chain mitochondrial probe for fixed cells and tissues, DMPQ-12, by installing a C12-alkyl chain into the quinoline moiety. In fixed cells stained with DMPQ-12, filament mitochondria and folded cristae were observed with confocal and structural illumination microscopy, respectively. In titration test with three major phospholipids, DMPQ-12 exhibited a stronger binding force to mitochondria-exclusive cardiolipin, revealing its targeting mechanism. Moreover, mitochondrial morphological changes in the three lesion models were clearly visualized in fixed cells. Finally, by DMPQ-12, three kinds of mitochondria with different morphologies were observed in situ in fixed muscle tissues. This work breaks the conventional concept that organic fluorescent probes only stain mitochondria with normal membrane potentials and opens new avenues for comprehensive mitochondrial investigations in research and clinical settings.

CILF: CRISPR/Cas9 based integration of large DNA fragments in Saccharomyces cerevisiae.

Genome integration technology has markedly expedited the construction of cell factories. However, its application is currently limited by the inefficient integration of large DNA fragments. Here, we report a CRISPR/Cas9 based integration of large DNA fragments (CILF) method to efficiently integrate large DNA fragments in Saccharomyces cerevisiae. In this approach, a fusion protein, Cas9-Brex27-FadR, was employed for the targeted delivery of donor plasmid to double-strand breaks (DSBs), while simultaneously recruiting Rad51 to enhance the efficiency of homologous recombination (HR). Our findings demonstrate that this method can achieve an integration efficiency of 98% for 10 kb DNA fragments and nearly 80% for 40 kb DNA fragments at a single site, using donor plasmids with 1000 bp homology arms (HAs) and 12 FadR binding sites (BSs). The CILF technique significantly enriches the synthetic biology toolbox of S. cerevisiae, offering significant potential to propel advancements in both synthetic biology and metabolic engineering.

Identification of active ingredients in Naomaitai capsules using high-resolution mass spectrometry unite molecular network analysis and prediction of their action mechanisms.

RATIONALE: Although Naomaitai capsule (NMC) is widely used in clinical practice and has a good curative effect for cerebral infarction, its material basis and mechanism of action remain unclear. METHODS: In this study, ultra-high-performance liquid chromatography (UHPLC) coupled with quadrupole Orbitrap MS technology was used to analyse the in vivo and in vitro components of NMC, and the Global Natural Products Social Molecular Networking website was used to further analyse the components of NMC. Next, systems biology approaches were employed to investigate the mechanism of action of NMC. Finally, molecular docking technology was used to verify the network pharmacological results. RESULTS: In total, 177 compounds were identified in vitro, including 65 terpenoids, 62 flavonoids, 25 organic acids and 11 quinones. 64 compounds were identified in the blood of mice, and the main active components included ginkgolide C, ginkgolide A, ligustilide, tanshinone IIB, olmelin, emodin and puerarin. The main targets in vivo included TP53, SRC, STAT3, PIK3CA and PIK3R1. CONCLUSIONS: In conclusion, this study has revealed that NMC acts on multiple targets in the body through various active components, exerting synergistic effects in the treatment of CI. Its mechanism of action may involve inhibiting neuronal apoptosis, oxidative stress and inflammatory responses as well as reducing cerebral vascular permeability and promoting cerebral vascular regeneration.

Ultrahigh-performance liquid chromatography Q-Exactive-Orbitrap mass spectrometry and molecular network analysis alongside network pharmacology to elucidate the active constituents and mechanism of action of Huahong tablet in treating pelvic inflammatory disease.

RATIONALE: Huahong tablet, a commonly used clinical Chinese patent medicine, shows good efficacy in treating pelvic inflammation and other gynaecological infectious diseases. However, the specific composition of Huahong tablets, which are complex herbal formulations, remains unclear. Therefore, this study aims to identify the active compounds and targets of Huahong tablets and investigate their mechanism of action in pelvic inflammatory diseases. METHODS: We utilised ultrahigh-performance liquid chromatography Q-Exactive-Orbitrap mass spectrometry and the relevant literature to identify the chemical components of Huahong tablets. The GNPS database was employed to further analyse and speculate on the components. Potential molecular targets of the active ingredients were predicted using the SwissTargetPrediction website. Protein-protein interaction analysis was conducted using the STRING database, with visualisation in Cytoscape 3.9.1. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the DAVID database. Additionally, a traditional Chinese medicine-ingredient-target-pathway network was constructed using Cytoscape 3.10.1. Molecular docking validation was carried out to investigate the interaction between core target and specific active ingredient. RESULTS: A total of 66 chemical components were identified, and 41 compounds were selected as potential active components based on the literature and the TCMSP database. Moreover, 38 core targets were identified as key targets in the treatment of pelvic inflammatory diseases with Huahong tablets. GO and KEGG enrichment analysis revealed 986 different biological functions and 167 signalling pathways. CONCLUSION: The active ingredients in Huahong tablets exert therapeutic effects on pelvic inflammatory diseases by acting on multiple targets and utilising different pathways. Molecular docking confirmed the high affinity between the specific active ingredients and disease targets.

Multiplexed CRISPR-Based Nucleic Acid Detection Using a Single Cas Protein.

Thanks to its ease, speed, and sensitivity, CRISPR-based nucleic acid detection has been increasingly explored for molecular diagnostics. However, one of its major limitations is lack of multiplexing capability because the detection relies on the trans-cleavage activity of the Cas protein, which necessitates the use of multiple orthogonal Cas proteins for multiplex detection. Here we report the development of a multiplexed CRISPR-based nucleic acid detection system with single-nucleotide resolution using a single Cas protein (Cas12a). This method, termed as CRISPR-TMSD, integrates the toehold-mediated strand displacement (TMSD) reaction, and the cis-cleavage activity of the Cas protein and multiplexed detection are achieved using a single Cas protein owing to the use of target-specific reporters. A set of computational simulation toolkits was used to design the TMSD reporter, allowing for highly sensitive and specific identification of target sequences. In combination with the recombinase polymerase amplification (RPA), the detection limit can reach as low as 1 copy/µL. As proof of concept, CRISPR-TMSD was subsequently used to detect an oncogenic gene and SARS-CoV-2 RNA with a single-nucleotide resolution. This work represents a conceptually new strategy for designing a CRISPR-based diagnostic system and has great potential to expand the application of CRISPR-based diagnostics.

PI3K/AKT pathway-related microRNA variants in childhood acute lymphoblastic leukemia.

BACKGROUND: Dysregulation of microRNAs (miRNAs) targeting genes in the PI3K/Akt pathway has been implicated in the pathogenesis of childhood acute lymphoblastic leukemia (ALL). However, the impact of genetic variants in these miRNAs on ALL susceptibility has not been extensively explored in the Chinese population. METHODS: To address this gap, we conducted a case-control study to evaluate the association between genetic variants in five PI3K/AKT pathway-related miRNAs (miR-149, miR-126, miR-492, miR-612, and miR-423) and childhood ALL susceptibility in the Chinese population. Additionally, we investigated the effects of the rs2292832 mutation on ALL cell proliferation and apoptosis. RESULTS: Our analyses revealed that the miR-149 rs2292832 mutant heterozygous CT genotype was more frequent in the control group than in the ALL cases, indicating a protective effect against ALL (adjusted odds ratio [OR] = 0.78, 95% confidence interval [CI] = 0.63-0.97, p = .024). Stratification analyses further revealed that the miR-149 rs2292832 CC genotype was associated with an increased risk of childhood ALL in subgroups of older children, females, those with parents who never smoked or drank alcohol, those living in painted houses, those with B-ALL, and those with high-risk ALL. Finally, we observed that the rs2292832 mutation inhibited ALL cell proliferation and induced apoptosis (p = .001), providing a potential mechanism by which this genetic variant may influence ALL susceptibility. CONCLUSION: Our study highlights the significant association between the miR-149 rs2292832 genetic variant and childhood ALL susceptibility in the Chinese population. These findings expand our understanding of the complex genetic landscape underlying ALL and have implications for the development of personalized therapeutic strategies.

Ratiometric and Discriminative Visualization of Autophagy and Apoptosis with a Single Fluorescent Probe Based on the Aggregation/Monomer Principle.

Autophagy and apoptosis play a central role in maintaining homeostasis in mammals. Therefore, discriminative visualization of the two cellular processes is an important and challenging task. However, fluorescent probes enabling ratiometric visualization of both autophagy and apoptosis with different sets of fluorescence signals have not been developed yet. In this work, we constructed a versatile single fluorescent probe (NKLR) based on the aggregation/monomer principle for the ratiometric and discriminative visualization of autophagy and apoptosis. NKLR can simultaneously perform two-color imaging of RNA (deep red channel) and lysosomes (yellow channel) in aggregation and monomer states, respectively. During autophagy, NKLR migrated from cytoplasmic RNA and nuclear RNA to lysosomes, showing enhanced yellow emission and sharply decreased deep red fluorescence. Moreover, this migration process was reversible upon the recovery of autophagy. Comparatively, during apoptosis, NKLR immigrated from lysosomes to RNA, and the yellow emission decreased and even disappeared, while the fluorescence of the deep red channel slightly increased. Overall, autophagy and apoptosis could be discriminatively visualized via the fluorescence intensity ratios of the two channels. Meanwhile, the cells in three different states (healthy, autophagic, apoptotic) could be distinguished by three point-to-point fluorescence images via the localization and emission color of NKLR. Therefore, the probe NKLR can serve as a desirable molecular tool to reveal the in-depth relation between autophagy and apoptosis and facilitate the study on the two cellular processes.

Ultra-specific nucleic acid testing by target-activated nucleases.

Specific and sensitive detection of nucleic acids is essential to clinical diagnostics and biotechnological applications. Currently, amplification steps are necessary for most detection methods due to the low concentration of nucleic acid targets in real samples. Although amplification renders high sensitivity, poor specificity is prevalent because of the lack of highly accurate precise strategies, resulting in significant false positives and false negatives. Nucleases exhibit high catalytic activity for nucleic acid cleavage which is regulated in a programmable manner. This review focuses on the latest progress in nucleic acid testing methods based on the target-activated nucleases. It summarizes the property of enzymes such as CRISPR/Cas, Argonautes, and some gene-editing irrelevant nucleases, which have been leveraged to create highly specific and sensitive nucleic acid testing tools. We elaborate on recent advances in the field of nuclease-mediated DNA recognition techniques for nucleic acid detection, and discuss its future applications and challenges in molecular diagnostics.

Efficient pose and motion estimation of non-cooperative target based on LiDAR.

In on-orbit servicing missions, autonomous close proximity operations require knowledge of the target's pose and motion parameters. Due to the lack of prior information about the non-cooperative target in an unknown environment, the pose and motion estimation of an uncooperative target is a challenging task. In this paper, a relative position and attitude estimation method is proposed using consecutive point clouds. First, a fast plane detection method is used to extract the global features of non-cooperative targets. Compared with some other local feature-detection methods, the method mentioned in this paper is faster. Then a two-stage angle adjustment method and iterative closest point algorithm are used to register the two adjacent point clouds. Finally, an unscented Kalman filter is designed to estimate the relative pose and motion parameters (velocity and angular velocity) of the target. Experiments show that the proposed measurement method of pose and motion parameters has acceptable accuracy and good stability.

Endothelial progenitor cells improve the quality of transplanted hematopoietic stem cells and maintain longer term effects in mice.

Engraftment of hematopoietic stem cells (HSCs) is a pre-requisite for the success of hematopoietic stem cell transplantation (HSCT). Fetal blood cell (FBC)-derived endothelial progenitor cells (EPCs) are known to facilitate HSC reconstitution in the early phase. However, longer term effects on HSCs remain unclear. The purpose of this study was to evaluate the effect of EPCs on the quality of transplanted hematopoietic stem cells in mouse HSCT model. BALB/c mice were randomly divided into four groups, namely, control, total body irradiation only, HSCT, and HSCT + EPCs (with infusion of 5 × 105 EPCs). Mice was sacrificed on days 7, 14, 21, and 28 post-HSCT for the analysis of the bone marrow pathology by H&E staining, measurement of c-kit+sca-1+, c-kit+, apoptosis, and necrosis by flow cytometry as well as colony formation assay. Secondary transplantation involved the injection of transplanted BALB/c-derived HSCs into new TBI-treated BALB/c mice. Compared with HSCT, EPCs infusion promoted the differentiation and reduced apoptosis of transplanted HSCs, possibly through promotion of vascular repair of the bone marrow microenvironment via differentiation into the bone marrow endothelial cells. Significantly, EPCs' effect on HSCs was maintained for a long period as demonstrated using a secondary transplantation approach. These data revealed EPCs improved the quality and quantity of transplanted HSCs and maintained their effects over the longer term, suggesting a novel approach to improve HSCT efficiency and outcomes.

Evaluation of the effects of preconditioning regimens on hepatic veno-occlusive disease in mice after hematopoietic stem cell transplantation.

Pre-conditioning regimens before hematopoietic stem cell transplantation (HSCT), such as total body irradiation (TBI) or busulfan/cyclophosphamide (BU/CY), are associated with hepatic veno-occlusive disease (HVOD). However, the mechanism of these regimens on hepatic veno-occlusive disease remains unclear. The aim of this study is to evaluate the effect of TBI or BU/CY on HVOD in mice after HSCT. Mice received TBI or BU/CY followed by HSCT. Analysis of liver pathology and function, and platelet aggregation were performed. Both these regimens caused damage to liver sinusoid endothelial cells, leading to loss of normal structural integrity of liver sinusoid, abnormal liver function, fibrin deposition, inflammatory cells infiltration and platelet aggregation. No differences of liver function in these regimens were observed. Increased hepatic lipid droplets, mitochondrial swelling and higher incidence of HVOD were observed in BU/CY. In conclusion, both TBI and BU/CY caused damage to liver sinusoid endothelial cells and occurrence of HVOD with higher incidence for BU/CY. Meanwhile, inflammation and platelet activation was also observed, suggesting targeting them maybe beneficial in the prophylaxis of HVOD.

On Hierarchical Multi-UAV Dubins Traveling Salesman Problem Paths in a Complex Obstacle Environment.

This article aims to solve a hierarchical multi-UAV Dubins traveling salesman problem (HMDTSP). Optimal hierarchical coverage and multi-UAV collaboration are achieved by the proposed approaches in a 3-D complex obstacle environment. A multi-UAV multilayer projection clustering (MMPC) algorithm is presented to reduce the cumulative distance from multilayer targets to corresponding cluster centers. A straight-line flight judgment (SFJ) was developed to reduce the calculation of obstacle avoidance. An improved adaptive window probabilistic roadmap (AWPRM) algorithm is addressed to plan obstacle-avoidance paths. The AWPRM improves the feasibility of finding the optimal sequence based on the proposed SFJ compared with a traditional probabilistic roadmap. To solve the solution to TSP with obstacles constraints, the proposed sequencing-bundling-bridging (SBB) framework combines the bundling ant colony system (BACS) and homotopic AWPRM. An obstacle-avoidance optimal curved path is constructed with a turning radius constraint based on the Dubins method and followed up by solving the TSP sequence. The results of simulation experiments indicated that the proposed strategies can provide a set of feasible solutions for HMDTSPs in a complex obstacle environment.

From germline genome to highly fragmented somatic genome: genome-wide DNA rearrangement during the sexual process in ciliated protists.

Genomes are incredibly dynamic within diverse eukaryotes and programmed genome rearrangements (PGR) play important roles in generating genomic diversity. However, genomes and chromosomes in metazoans are usually large in size which prevents our understanding of the origin and evolution of PGR. To expand our knowledge of genomic diversity and the evolutionary origin of complex genome rearrangements, we focus on ciliated protists (ciliates). Ciliates are single-celled eukaryotes with highly fragmented somatic chromosomes and massively scrambled germline genomes. PGR in ciliates occurs extensively by removing massive amounts of repetitive and selfish DNA elements found in the silent germline genome during development of the somatic genome. We report the partial germline genomes of two spirotrich ciliate species, namely Strombidium cf. sulcatum and Halteria grandinella, along with the most compact and highly fragmented somatic genome for S. cf. sulcatum. We provide the first insights into the genome rearrangements of these two species and compare these features with those of other ciliates. Our analyses reveal: (1) DNA sequence loss through evolution and during PGR in S. cf. sulcatum has combined to produce the most compact and efficient nanochromosomes observed to date; (2) the compact, transcriptome-like somatic genome in both species results from extensive removal of a relatively large number of shorter germline-specific DNA sequences; (3) long chromosome breakage site motifs are duplicated and retained in the somatic genome, revealing a complex model of chromosome fragmentation in spirotrichs; (4) gene scrambling and alternative processing are found throughout the core spirotrichs, offering unique opportunities to increase genetic diversity and regulation in this group. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00213-x.

An extraction-free one-pot assay for rapid detection of Klebsiella pneumoniae by combining RPA and CRISPR/Cas12a.

Klebsiella pneumoniae poses a significant threat to global public health. Traditional clinical diagnostic methods, such as bacterial culture and microscopic identification, are not suitable for point-of-care testing. In response, based on the suboptimal protospacer adjacent motifs, this study develops an extraction-free one-pot assay, named EXORCA (EXtraction-free One-pot RPA-CRISPR/Cas12a assay), designed for the immediate, sensitive and efficient detection of K. pneumoniae. The EXORCA assay can be completed within approximately 30 min at a constant temperature and allows for the visualization of results either through a fluorescence reader or directly by the naked eye under blue light. The feasibility of the assay was evaluated using twenty unextracted clinical samples, achieving a 100% (5/5) positive predictive value and a 100% (15/15) negative predictive value in comparison to qPCR. These results suggest that the EXORCA assay holds significant potential as a point-of-care testing tool for the rapid identification of pathogens, such as K. pneumonia.

Genome-wide identification of ATP-binding cassette transporter B subfamily, focusing on its structure, evolution and rearrangement in ciliates.

ATP-binding cassette subfamily B (ABCB) has been implicated in various essential functions such as multidrug resistance, auxin transport and heavy metal tolerance in animals and plants. However, the functions, the genomic distribution and the evolutionary history have not been characterized systematically in lower eukaryotes. As a lineage of highly specialized unicellular eukaryotes, ciliates have extremely diverse genomic features including nuclear dimorphism. To further understand the genomic structure and evolutionary history of this gene family, we investigated the ABCB gene subfamily in 11 ciliates. The results demonstrate that there is evidence of substantial gene duplication, which has occurred by different mechanisms in different species. These gene duplicates show consistent purifying selection, suggesting functional constraint, in all but one species, where positive selection may be acting to generate novel function. We also compare the gene structures in the micronuclear and macronuclear genomes and find no gene scrambling during genome rearrangement, despite the abundance of such scrambling in two of our focal species. These results lay the foundation for future analyses of the function of these genes and the mechanisms responsible for their evolution across diverse eukaryotic lineages.

Protein engineering of invertase for enhancing yeast dough fermentation under high-sucrose conditions.

During yeast dough fermentation, such as the high-sucrose bread-making process, the yeast cells are subjected to considerable osmotic stress, resulting in poor outcomes. Invertase is important for catalyzing the irreversible hydrolysis of sucrose to free glucose and fructose, and decreasing the catalytic activity of the invertase may reduce the glucose osmotic stress on the yeast. In this study, we performed structural design and site-directed mutagenesis (SDM) on the Saccharomyces cerevisiae invertase (ScInV) in an Escherichia coli expression system to study the catalytic activity of ScInV mutants in vitro. In addition, we generated the same mutation sites in the yeast endogenous genome and tested their invertase activity in yeast and dough fermentation ability. Our results indicated that appropriately reduced invertase activity of yeast ScInV can enhance dough fermentation activity under high-sucrose conditions by 52%. Our systems have greatly accelerated the engineering of yeast endogenous enzymes both in vitro and in yeast, and shed light on future metabolic engineering of yeast.

Comparative analysis of single-cell genome sequencing techniques toward the characterization of germline and somatic genomes in ciliated protists.

Ciliated protists contain both germline micronucleus (MIC) and somatic macronucleus (MAC) in a single cytoplasm. Programmed genome rearrangements occur in ciliates during sexual processes, and the extent of rearrangements varies dramatically among species, which lead to significant differences in genomic architectures. However, genomic sequences remain largely unknown for most ciliates due to the difficulty in culturing and in separating the germline from the somatic genome in a single cell. Single-cell whole genome amplification (WGA) has emerged as a powerful technology to characterize the genomic heterogeneity at the single-cell level. In this study, we compared two single-cell WGA, multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC) in characterizing the germline and somatic genomes in ciliates with different genomic architectures. Our results showed that: 1) MALBAC exhibits strong amplification bias towards MAC genome while MDA shows bias towards MIC genome of ciliates with extensively fragmented MAC genome; 2) both MDA and MALBAC could amplify MAC genome more efficiently in ciliates with moderately fragmented MAC genome. Moreover, we found that more sample replicates could help to obtain more genomic data. Our work provides a reference for selecting the appropriate method to characterize germline and somatic genomes of ciliates.

Cytoskeletal activation of NHE1 regulates cell volume and DNA methylation.

The regulation of mammalian cell volume is crucial for maintaining key cellular processes. Cells can rapidly respond to osmotic and hydrostatic pressure imbalances during environmental challenges, generating fluxes of water and ions that alter volume within minutes. While the role of ion pump and leak in cell volume regulation has been well-established, the role of the actomyosin cytoskeleton and its substantial interplay with ion transporters are still unclear. In this work, we discover a system of cell volume regulation controlled by cytoskeletal activation of ion transporters. Under hypotonic shock, NIH-3T3 and MCF-10A display a 20% secondary volume increase (SVI) following the initial regulatory volume decrease. We show that SVI is initiated by Ca 2+ influx through stretch activated channel Piezo1 and subsequent actomyosin remodeling. Rather than contracting cells, actomyosin triggers cell swelling by activating Na + -H + exchanger 1 (NHE1) through their co-binding partner ezrin. Cytoskeletal activation of NHE1 can be similarly triggered by mechanical stretch and attenuated by soft substrates. This mechanism is absent in certain cancer cell lines such as HT1080 and MDA-MB-231, where volume regulation is dominated by intrinsic response of ion transporters. Moreover, cytoskeletal activation of NHE1 during SVI induces nuclear deformation, leading to DNA demethylation and a significant, immediate transcriptomic response in 3T3 cells, a phenomenon that is absent in HT1080 cells. Overall, our findings reveal the central role of Ca 2+ and actomyosin-mediated mechanosensation in the regulation of ion transport, cell volume, DNA methylation, and transcriptomics.

Doublets of the unicellular organism Euplotes vannus (Alveolata, Ciliophora, Euplotida): the morphogenetic patterns of the ciliary and nuclear apparatuses associated with cell division.

Ciliated protists are one of the most diverse and highly differentiated group among unicellular organisms. Doublets occur in ciliates when two cells fuse into a single individual. Doublets contain two major cellular components (either cell in a doublet) and have traditionally been considered as developmental anomalies. Nevertheless, doublets can divide or even conjugate effectively, which may represent dispersal forms of the life stages. In addition, morphogenesis, as an important process in the life cycle, will provide important insights into the complex differentiation mechanism and various physiological phenomena. However, morphogenetic studies focusing on doublets of ciliates are very limited, which has become an obstacle to understand their complete life history. Here we isolated a doublet strain from the marine species Euplotes vannus (Müller, 1786) Diesing, 1850 and investigated its morphogenetic events during asexual reproduction. Our results indicate that: (1) the opisthe's oral primordium develops de novo beneath the cortex; (2) the frontoventral and transverse cirral anlagen, cirrus I/1, and marginal anlagen in both dividers develop de novo separately; (3) the dorsal kinety anlagen, the three rightmost ones of which produce three caudal cirri for the proter, occur within the parental structures in the mid-body region; (4) the opisthe acquires two caudal cirri, one from the end of each two rightmost kineties; and (5) there are two macronuclei and one micronucleus in the doublet and they divide amitotically and mitotically, respectively. Finally, we speculate that this special differentiation may be an adaptive form to adverse environments.

Rapid and Visual RPA-Cas12a Fluorescence Assay for Accurate Detection of Dermatophytes in Cats and Dogs.

Dermatophytosis, an infectious disease caused by several fungi, can affect the hair, nails, and/or superficial layers of the skin and is of global significance. The most common dermatophytes in cats and dogs are Microsporum canis and Trichophyton mentagrophytes. Wood's lamp examination, microscopic identification, and fungal culture are the conventional clinical diagnostic methods, while PCR (Polymerase Chain Reaction) and qPCR (Quantitative PCR) are playing an increasingly important role in the identification of dermatophytes. However, none of these methods could be applied to point-of-care testing (POCT). The recent development of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based diagnostic platform promises a rapid, accurate, and portable diagnostic tool. In this paper, we present a Cas12a-fluorescence assay to detect and differentiate the main dermatophytes in clinical samples with high specificity and sensitivity. The Cas12a-based assay was performed with a combination of recombinase polymerase amplification (RPA). The results could be directly visualized by naked eyes under blue light, and all tested samples were consistent with fungal culture and sequencing results. Compared with traditional methods, the RPA-Cas12a-fluorescence assay requires less time (about 30 min) and less complicated equipment, and the visual changes can be clearly observed with naked eyes, which is suitable for on-site clinical diagnosis.