Electrochemical sensing technology based on a ligation-initiated LAMP-assisted CRISPR/Cas12a system for high-specificity detection of EGFR E746-A750 deletion mutation.

Epidermal growth factor receptor (EGFR) mutation status is pivotal in predicting the efficacy of tyrosine kinase inhibitor treatments against tumors. Among EGFR mutations, the E746-A750 deletion is particularly common and accurately quantifying it can guide targeted therapies. This study introduces a novel visual sensing technology using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system guided by ligation-initiated loop-mediated isothermal amplification (LAMP) to detect the del E746-A750 mutation in EGFR. Conventional LAMP primers were simplified by designing a pair of target-specific stem-loop DNA probes, enabling selective amplification of the target DNA. The CRISPR/Cas12a system was employed to identify the target nucleic acid and activate Cas12a trans-cleavage activity, thereby enhancing the specificity of the assay. Furthermore, the biosensor utilized high-performance nanomaterials such as triangular gold nanoparticles and graphdiyne, known for their large specific surface area, to enhance sensitivity effectively as a sensing platform. The proposed biosensor demonstrated outstanding specificity, achieving a low detection limit of 17 fM (S/N = 3). Consequently, this innovative strategy not only expands the application scope of CRISPR/Cas12a technology but also introduces a promising approach for clinical diagnostics in modern medicine.

CRISPR/Cas12a-drived electrochemiluminescence and fluorescence dual-mode magnetic biosensor for sensitive detection of Pseudomonas aeruginosa based on iridium(III) complex as luminophore.

The opportunistic human pathogen Pseudomonas aeruginosa (P. aeruginosa) poses a significant threat to human health, causing sepsis, inflammation, and pneumonia, so it is crucial to devise an expeditious detection platform for the P. aeruginosa. In this work, bis (2- (3, 5- dimethylphenyl) quinoline- C2, N') (acetylacetonato) iridium (III) Ir (dmpq)2 (acac) with excellent electrochemiluminescence (ECL) and fluorescence (FL) and magnetic nanoparticles were encapsulated in silica spheres. The luminescent units exhibited equal ECL and FL properties compared with single iridium complexes, and enabled rapid separation, which was of vital significance for the establishment of biosensors with effective detection. In addition, the luminescent units were further reacted with the DNA with quenching units to obtain the signal units, and the ECL/FL dual-mode biosensor was employed with the CRISPR/Cas12a system to further improve its specific recognition ability. The ECL detection linear range of as-proposed biosensor in this work was 100 fM-10 nM with the detection limit of 73 fM (S/N = 3), and FL detection linear range was 1 pM-10 nM with the detection limit of 0.126 pM (S/N = 3). Importantly, the proposed dual-mode biosensor exhibited excellent repeatability and stability in the detection of P. aeruginosa in real samples, underscoring its potential as an alternative strategy for infection prevention and safeguarding public health and safety in the future.

Dual amplification dynamic DNA network system for CRISPR/Cas12a based p53 gene detection.

BACKGROUND: It is estimated that over 50 % of human cancers are caused by mutations in the p53 gene. Early sensitive and accurate detection of the p53 gene is important for diagnosis of cancers in the early stage. However, conventional detection techniques often suffer from strict reaction conditions, or unsatisfied sensitivity, so we need to develop a new strategy for accurate detection of p53 gene with smart designability, multiple signal amplification in mild reaction conditions. RESULTS: In this study, CRISPR/Cas system is exploited in entropy-driven catalysis (EDC) and hybridization chain reaction (CHA) dual signal amplification sensing strategies. The products of both reactions can efficiently and separately activate CRISPR/Cas12a which greatly amplifies the fluorescent signal. The method has good linearity in p53 detection with the concentration ranged from 0.1 fM to 0.5 pM with ultra-low detection limit of 0.096 fM. It also showed good performance in serum, offering potentials for early disease detection. SIGNIFICANCE: The designed dual amplification dynamic DNA network system exhibits an ultra-sensitive fluorescence biosensing for p53 gene identification. The method is simple to operate and requires only one buffer for the experiment, and meanwhile shows smart designability which could be used for a wide range of markers. Thus, we believe the present work will provide a potential tool for the construction and development of sensitive fluorescent biosensors for diseases.

Versatile plant genome engineering using anti-CRISPR-Cas12a systems.

CRISPR-Cas12a genome engineering systems have been widely used in plant research and crop breeding. To date, the performance and use of anti-CRISPR-Cas12a systems have not been fully established in plants. Here, we conduct in silico analysis to identify putative anti-CRISPR systems for Cas12a. These putative anti-CRISPR proteins, along with known anti-CRISPR proteins, are assessed for their ability to inhibit Cas12a cleavage activity in vivo and in planta. Among all anti-CRISPR proteins tested, AcrVA1 shows robust inhibition of Mb2Cas12a and LbCas12a in E. coli. Further tests show that AcrVA1 inhibits LbCas12a mediated genome editing in rice protoplasts and stable transgenic lines. Impressively, co-expression of AcrVA1 mitigates off-target effects by CRISPR-LbCas12a, as revealed by whole genome sequencing. In addition, transgenic plants expressing AcrVA1 exhibit different levels of inhibition to LbCas12a mediated genome editing, representing a novel way of fine-tuning genome editing efficiency. By controlling temporal and spatial expression of AcrVA1, we show that inducible and tissue specific genome editing can be achieved in plants. Furthermore, we demonstrate that AcrVA1 also inhibits LbCas12a-based CRISPR activation (CRISPRa) and based on this principle we build logic gates to turn on and off target genes in plant cells. Together, we have established an efficient anti-CRISPR-Cas12a system in plants and demonstrate its versatile applications in mitigating off-target effects, fine-tuning genome editing efficiency, achieving spatial-temporal control of genome editing, and generating synthetic logic gates for controlling target gene expression in plant cells.

A multi-AS-PCR-coupled CRISPR/Cas12a assay for the detection of ten single-base mutations.

Single-nucleotide polymorphism (SNP) detection is critical for diagnosing diseases, and the development of rapid and accurate diagnostic tools is essential for treatment and prevention. Allele-specific polymerase chain reaction (AS-PCR) is widely used for detecting SNPs with multiplexing capabilities, while CRISPR-based technologies provide high sensitivity and specificity in targeting mutation sites through specific guide RNAs (gRNAs). In this study, we have integrated the high sensitivity and specificity of CRISPR technology with the multiplexing capabilities of AS-PCR, achieving the simultaneous detection of ten single-base mutations. As for Multi-AS-PCR, our research identified that competitive inhibition of primers targeting the same loci, coupled with divergent amplification efficiencies of these primers, could result in diminished amplification efficiency. Consequently, we adjusted and optimized primer combinations and ratios to enhance the amplification efficacy of Multi-AS-PCR. Finally, we successfully developed a novel nested Multi-AS-PCR-Cas12a method for multiplex SNPs detection. To evaluate the clinical utility of this method in a real-world setting, we applied it to diagnose rifampicin-resistant tuberculosis (TB). The limit of detection (LoD) for the nested Multi-AS-PCR-Cas12a was 102 aM, achieving sensitivity, specificity, positive predictive value, and negative predictive value of 100 %, 93.33 %, 90.00 %, and 100 %, respectively, compared to sequencing. In summary, by employing an innovative design that incorporates a universal reverse primer alongside ten distinct forward allele-specific primers, the nested Multi-AS-PCR-Cas12a technique facilitates the parallel detection of ten rpoB gene SNPs. This method also holds broad potential for the detection of drug-resistant gene mutations in infectious diseases and tumors, as well as for the screening of specific genetic disorders.

Capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for highly sensitive visual detection of HPV DNA.

The rapid and specific identification and sensitive detection of human papillomavirus (HPV) infection is critical for preventing cervical cancer, particularly in resource-limited regions. In this work, we hope to propose a capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for point-of-care (POC) DNA testing. Using the thermal reversibility of DNA hydrogel and capillarity, the novel DNA hydrogel distance sensor can be rapidly and simply constructed by loading an ultra-thin CRISPR/Cas12a-responsive DNA-crosslinked hydrogel film at the end of the capillary tube. The target DNA-specific recombinase polymerase reaction (RPA) amplicons activate the trans-cleavage activity of the Cas12a enzyme, cleaving the crosslinked DNA in hydrogel film, and causing an increase of hydrogel's permeability. As a result, a sample solution containing target DNA travels into the capillary tube at a longer distance compared to the negative samples. Reading the solution traveling distance in capillary tubes, the novel sensor realizes target DNA detection without any special equipment. Benefiting from the exponential target amplification of RPA and multiple turnover response of trans-cleavage of CRISPR/Cas12a, the developed sensor can visually and specifically detect as low as 1 aM HPV 16 DNA within 30 min. These outstanding features, including exceptional sensitivity and specificity, simple and portable design, mild measurement conditions, quick turnaround time, and user-friendly read-out, make the novel distance sensor a promising option for POC diagnostic applications.

Dumbbell probe initiated multi-rolling circle amplification assisted CRISPR/Cas12a for highly sensitive detection of clinical microRNA.

A novel miRNA detection technique named Dumbbell probe initiated multi-Rolling Circle Amplification assisted CRISPR/Cas12a (DBmRCA) was developed relying on the ligation-free dumbbell probe and the high-sensitivity CRISPR/Cas12a signal out strategy. This DBmRCA assay streamlines miRNA quantification within a mere 30-min timeframe and with exceptional analytical precision. The efficacy of this method was validated by assessing miRNA levels in clinical samples, revealing distinct expression panel of miR-200a and miR-126 in lung cancer/adjacent/normal tissue specimens. Moreover, a predictive model was established to classify benign and malignant tumor. Due to its time efficiency, enhanced sensitivity, and streamlined workflow, this assay would be a reliable tool for miRNA analysis in clinical settings, offering potential guidance for early diagnosis and treatment of lung cancer.

Cas12a RNP-mediated co-transformation enables transgene-free multiplex genome editing, long deletions, and inversions in citrus chromosome.

Introduction: Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a devastating disease worldwide. Previously, we successfully generated canker-resistant Citrus sinensis cv. Hamlin lines in the T0 generation. This was achieved through the transformation of embryogenic protoplasts using the ribonucleoprotein (RNP) containing Cas12a and one crRNA to edit the canker susceptibility gene, CsLOB1, which led to small indels. Methods: Here, we transformed embryogenic protoplasts of Hamlin with RNP containing Cas12a and three crRNAs. Results: Among the 10 transgene-free genome-edited lines, long deletions were obtained in five lines. Additionally, inversions were observed in three of the five edited lines with long deletions, but not in any edited lines with short indel mutations, suggesting long deletions maybe required for inversions. Biallelic mutations were observed for each of the three target sites in four of the 10 edited lines when three crRNAs were used, demonstrating that transformation of embryogenic citrus protoplasts with Cas12a and three crRNAs RNP can be very efficient for multiplex editing. Our analysis revealed the absence of off-target mutations in the edited lines. These cslob1 mutant lines were canker- resistant and no canker symptoms were observed after inoculation with Xcc and Xcc growth was significantly reduced in the cslob1 mutant lines compared to the wild type plants. Discussion: Taken together, RNP (Cas12a and three crRNAs) transformation of embryogenic protoplasts of citrus provides a promising solution for transgene-free multiplex genome editing with high efficiency and for deletion of long fragments.

Optimization of CRISPR/Cas12a detection assay and its application in the detection of Echinococcus granulosus.

Cystic echinococcosis, resulting from infection with Echinococcus granulosus, poses a significant challenge as a neglected tropical disease owing to the lack of any known effective treatment. Primarily affecting under-resourced, remote, and conflict-ridden regions, the disease is compounded by the limitations of current detection techniques, such as microscopy, physical imaging, ELISA, and qPCR, which are unsuitable for application in these areas. The emergence of CRISPR/Cas12a as a promising tool for nucleic acid detection, characterized by its unparalleled specificity, heightened sensitivity, and rapid detection time, offers a potential solution. In this study, we present a one-pot CRISPR/Cas12a detection method for E. granulosus (genotype G1, sheep strain) integrating recombinase polymerase amplification (RPA) with suboptimal protospacer adjacent motif (PAM) and structured CRISPR RNA (crRNA) to enhance reaction efficiency. The evaluation of the assay's performance using hydatid cyst spiked dog feces and the examination of 62 dog fecal samples collected from various regions of Western China demonstrate its efficacy. The assay permits visual observation of test results about 15 minutes under blue light and displays superior portability and reaction speed relative to qPCR, achieving a sensitivity level of 10 copies of standard plasmids of the target gene. Analytic specificity was verified against four tapeworm species (E. multilocularis, H. taeniaeformis, M. benedeni, and D. caninum) and two other helminths (T. canis and F. hepatica), with negative results also noted for Mesocestoides sp. This study presents a rapid, sensitive, and time-efficient DNA detection method for E. granulosus of hydatid cyst spiked and clinical dog feces, potential serving as an alternative tool for field detection. This novel assay is primarily used to diagnose the definitive host of E. granulosus. Further validation using a larger set of clinical fecal samples is warranted, along with additional exploration of more effective approaches for nucleic acid release.

Visual detection of fungicide resistance by combining RPA and CRISPR/Cas12a in peach Brown rot fungus Monilinia fructicola.

BACKGROUND: Peach brown rot caused by Monilinia fructicola severely affects the quality and yield of peach, resulting in large economic losses worldwide. Methyl benzimidazole carbamate (MBC) fungicides and sterol demethylation inhibitor (DMI) fungicides are among the most applied chemical classes used to control the disease but resistance in the target pathogen has made them risky choices. Timely monitoring of resistance to these fungicides in orchards could prevent control failure in practice. RESULTS: In the current study, we developed methods based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a systems to detect MBC and DMI resistance based on the E198A mutation in the ß-tubulin (MfTub2) gene and the presence of the Mona element in the upstream region of the MfCYP51, respectively. For MBC resistance, RPA primers were designed that artificially incorporated PAM sites to facilitate the CRISPR/Cas12a reaction. Subsequently, specific tcrRNAs were designed based on the E198A mutation site. For the detection of the Mona element, we designed RPA primers M-DMI-F2/M-DMI-R1 that in combination with crRNA1 detected 'Mona' and distinguished resistant from sensitive strains. CONCLUSION: Both methods exhibited high sensitivity and specificity, requiring only a simple isothermal device to obtain results within 1 h at 37 °C. The FQ-reporter enabled visualization with a handheld UV or white light flashlight. This method was successfully used with purified DNA from lab cultures and crude DNA from symptomatic fruit tissue, highlighting its potential for on-site detection of resistant strains in orchards. © 2024 Society of Chemical Industry.

Establishment of a rapid detection method for Mycoplasma pneumoniae based on RPA-CRISPR-Cas12a technology.

Mycoplasma pneumoniae can cause respiratory infections and pneumonia, posing a serious threat to the health of children and adolescents. Early diagnosis of Mycoplasma pneumoniae infection is crucial for clinical treatment. Currently, diagnostic methods for Mycoplasma pneumoniae infection include pathogen detection, molecular biology techniques, and bacterial culture, all of which have certain limitations. Here, we developed a rapid, simple, and accurate detection method for Mycoplasma pneumoniae that does not rely on large equipment or complex operations. This technology combines the CRISPR-Cas12a system with recombinase polymerase amplification (RPA), allowing the detection results to be observed through fluorescence curves and immunochromatographic lateral flow strips.It has been validated that RPA-CRISPR/Cas12a fluorescence analysis and RPA-CRISPR/Cas12-immunochromatographic exhibit no cross-reactivity with other common pathogens, and The established detection limit was ascertained to be as low as 102 copies/µL.Additionally, 49 clinical samples were tested and compared with fluorescence quantitative polymerase chain reaction, demonstrating a sensitivity and specificity of 100%. This platform exhibits promising clinical performance and holds significant potential for clinical application, particularly in settings with limited resources, such as clinical care points or resource-constrained areas.

Dual electrochemical signal "signal-on-off" sensor based on CHA-Td-HCR and CRISPR-Cas12a for MUC1 detection.

Mucin 1 (MUC1) is frequently overexpressed in various cancers and is essential for early cancer detection. Current methods to detect MUC1 are expensive, time-consuming, and require skilled personnel. Therefore, developing a simple, sensitive, highly selective MUC1 detection sensor is necessary. In this study, we proposed a novel "signal-on-off" strategy that, in the presence of MUC1, synergistically integrates catalytic hairpin assembly (CHA) with DNA tetrahedron (Td)-based nonlinear hybridization chain reaction (HCR) to enhance the immobilization of electrochemically active methylene blue (MB) on magnetic nanoparticles (MNP), marking the MB signal "on". Concurrently, the activation of CRISPR-Cas12a by isothermal amplification products triggers the cleavage of single-stranded DNA (ssDNA) at the electrode surface, resulting in a reduction of MgAl-LDH@Fc-AuFe-MIL-101 (containing ferrocene, Fc) on the electrode, presenting the "signal-off" state. Both MB and MgAl-LDH@Fc-AuFe-MIL-101 electrochemical signals were measured and analyzed. Assay parameters were optimized, and sensitivity, stability, and linear range were assessed. Across a concentration spectrum of MUC1 spanning from 10 fg/mL to 100 ng/mL, the MB and MgAl-LDH@Fc-AuFe-MIL-101 signals were calibrated with each other, demonstrating a "signal-on-off" dual electrochemical signaling pattern. This allows for the precise and quantitative detection of MUC1 in clinical samples, offering significant potential for medical diagnosis.

High-Throughput and Integrated CRISPR/Cas12a-Based Molecular Diagnosis Using a Deep Learning Enabled Microfluidic System.

CRISPR/Cas-based molecular diagnosis demonstrates potent potential for sensitive and rapid pathogen detection, notably in SARS-CoV-2 diagnosis and mutation tracking. Yet, a major hurdle hindering widespread practical use is its restricted throughput, limited integration, and complex reagent preparation. Here, a system, microfluidic multiplate-based ultrahigh throughput analysis of SARS-CoV-2 variants of concern using CRISPR/Cas12a and nonextraction RT-LAMP (mutaSCAN), is proposed for rapid detection of SARS-CoV-2 and its variants with limited resource requirements. With the aid of the self-developed reagents and deep-learning enabled prototype device, our mutaSCAN system can detect SARS-CoV-2 in mock swab samples below 30 min as low as 250 copies/mL with the throughput up to 96 per round. Clinical specimens were tested with this system, the accuracy for routine and mutation testing (22 wildtype samples, 26 mutational samples) was 98% and 100%, respectively. No false-positive results were found for negative (n = 24) samples.

A novel system with robust compatibility and stability for detecting Sugarcane yellow leaf virus based on CRISPR-Cas12a.

Sugarcane yellow leaf virus (SCYLV) can reduce sugarcane productivity. A novel detection system based on reverse transcription-multienzyme isothermal rapid amplification (RT-MIRA) combined with CRISPR-Cas12a, named RT-MIRA-CRISPR-Cas12a, was developed. This innovative approach employs crude leaf extract directly as the reaction template, streamlining the extraction process for simplicity and speed. Combining RT-MIRA and CRISPR-Cas12a in one reaction tube increases the ease of operation while reducing the risk of aerosol contamination. In addition, it exhibits sensitivity equivalent to qPCR, boasting a lower detection limit of 25 copies. Remarkably, the entire process, from sample extraction to reaction completion, requires only 52-57 minutes, just a thermostat water bath. The result can be observed and judged by the naked eye.IMPORTANCESugarcane yellow leaf disease (SCYLD) is an important viral disease that affects sugarcane yield. There is an urgent need for rapid, sensitive, and stable detection methods. The reverse transcription-multienzyme isothermal rapid amplification combined with CRISPR-Cas12a (RT-MIRA-CRISPR-Cas12a) method established in this study has good specificity and high sensitivity. In addition, the system showed good compatibility and stability with the crude leaf extract, as shown by the fact that the crude extract of the positive sample could still be stably detected after 1 week when placed at 4°C. RT-MIRA-CRISPR-Cas12a, reverse transcription polymerase chain reaction (RT-PCR), and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect SCYLV on 33 sugarcane leaf samples collected from the field, and it was found that the three methods reached consistent conclusions. This Cas12a-based detection method proves highly suitable for the rapid on-site detection of the SCYLV.

CRISPR/Cas12a and Hybridization Chain Reaction-Coregulated Magnetic Relaxation Switching Biosensor for Sensitive Detection of Viable Salmonella in Animal-Derived Foods.

We combined a CRISPR/Cas12a system with a hybridization chain reaction (HCR) to develop an ultrasensitive magnetic relaxation switching (MRS) biosensor for detecting viable Salmonella typhimurium (S. typhimurium). Magnetic nanoparticles of two sizes (30 and 1000 nm: MNP30 and MNP1000, respectively) were coupled through HCR. The S. typhimurium gene-activated CRISPR/Cas12a system released MNP30 from the MNP1000-HCR-MNP30 complex through a trans-cleavage reaction. After magnetic separation, released MNP30 was collected from the supernatant and served as a transverse relaxation time (T2) signal probe. Quantitative detection of S. typhimurium is achieved by establishing a linear relationship between the change in T2 and the target gene. The biosensor's limit of detection was 77 CFU/mL (LOD = 3S/M, S = 22.30, M = 0.87), and the linear range was 102-108 CFU/mL. The accuracy for detecting S. typhimurium in real samples is comparable to that of qPCR. Thus, this is a promising method for the rapid and effective detection of foodborne pathogens.

An amplification-free CRISPR-Cas12a assay for titer determination and composition analysis of the rAAV genome.

The viral genome titer is a crucial indicator for the clinical dosing, manufacturing, and analytical testing of recombinant adeno-associated virus (rAAV) gene therapy products. Although quantitative PCR and digital PCR are the common methods used for quantifying the rAAV genome titer, they are limited by inadequate accuracy and robustness. The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a biosensor is being increasingly used in virus detection; however, there is currently no report on its application in the titer determination of gene therapy products. In the present study, an amplification-free CRISPR-Cas12a assay was developed, optimized, and applied for rAAV genome titer determination. The assay demonstrated high precision and accuracy within the detection range of 4 × 109 and 1011 vg/mL. No significant difference was observed between the Cas12a and qPCR assay results (p < 0.05, t test). Moreover, Cas12a exhibited similar activity on both single-stranded and double-stranded DNA substrates. Based on this characteristic, the titers of positive-sense and negative-sense strands were determined separately, which revealed a significant difference between their titers for an in-house reference AAV5-IN. This study presents the inaugural report of a Cas12a assay developed for the titer determination and composition analysis of the rAAV genome.

Development of RPA-Cas12a assay for rapid and sensitive detection of Pneumocystis jirovecii.

Pneumocystis jirovecii is a prevalent opportunistic fungal pathogen that can lead to life-threatening Pneumocystis pneumonia in immunocompromised individuals. Given that timely and accurate diagnosis is essential for initiating prompt treatment and enhancing patient outcomes, it is vital to develop a rapid, simple, and sensitive method for P. jirovecii detection. Herein, we exploited a novel detection method for P. jirovecii by combining recombinase polymerase amplification (RPA) of nucleic acids isothermal amplification and the trans cleavage activity of Cas12a. The factors influencing the efficiency of RPA and Cas12a-mediated trans cleavage reaction, such as RPA primer, crRNA, the ratio of crRNA to Cas12a and ssDNA reporter concentration, were optimized. Our RPA-Cas12a-based fluorescent assay can be completed within  30-40 min, comprising a 25-30 min RPA reaction and a 5-10 min trans cleavage reaction. It can achieve a lower detection threshold of 0.5 copies/µL of target DNA with high specificity. Moreover, our RPA-Cas12a-based fluorescent method was examined using 30 artificial samples and demonstrated high accuracy with a diagnostic accuracy of 93.33%. In conclusion, a novel, rapid, sensitive, and cost-effective RPA-Cas12a-based detection method was developed and demonstrates significant potential for on-site detection of P. jirovecii in resource-limited settings.

Probiotic bacterial adsorption coupled with CRISPR/Cas12a system for mercury (II) ions detection.

The complex sample matrix poses significant challenges in accurately detecting heavy metals. In view of its superior performance for the biological adsorption of heavy metals, probiotic bacteria can be explored for functional unit to eliminate matrix interference. Herein, Lactobacillus rhamnosus (LGG) demonstrates a remarkable tolerance and can adsorb up to 300 µM of Hg2+, following the Freundlich isotherm model with the correlation coefficient (R2) value of 0.9881. Subsequently, by integrating the CRISPR/Cas12a system, a sensitive and specific fluorescent biosensor, "Cas12a-MB," has been developed for Hg2+ detection. Specifically, Hg2+ adsorbed onto LGG can specifically bind to the nucleic acid probe, thereby inhibiting the binding of the probe to LGG and the subsequent activation of the CRISPR/Cas12a system. Under optimal experimental conditions, with the detection time of 90 min and the detection limit of 0.44 nM, the "Cas12a-MB" biosensor offers a novel, eco-friendly approach for Hg2+ detection, showcasing the innovative application of probiotics in biosensor.

Hydrophilic/hydrophobic modified microchip for detecting multiple gene doping candidates using CRISPR-Cas12a and RPA.

With significant advancements in understanding gene functions and therapy, the potential misuse of gene technologies, particularly in the context of sports through gene doping (GD), has come to the forefront. This raises concerns regarding the need for point-of-care testing of various GD candidates to counter illicit practices in sports. However, current GD detection techniques, such as PCR, lack the portability required for on-site multiplexed detection. In this study, we introduce an integrated microfluidics-based chip for multiplexed gene doping detection, termed MGD-Chip. Through the strategic design of hydrophilic and hydrophobic channels, MGD-Chip enables the RPA and CRISPR-Cas12a assays to be sequentially performed on the device, ensuring minimal interference and cross-contamination. Six potential GD candidates were selected and successfully tested simultaneously on the platform within 1 h. Demonstrating exceptional specificity, the platform achieved a detection sensitivity of 0.1 nM for unamplified target plasmids and 1 aM for amplified ones. Validation using mouse models established by injecting IGFI and EPO transgenes confirmed the platform's efficacy in detecting gene doping in real samples. This technology, capable of detecting multiple targets using portable elements, holds promise for real-time GD detection at sports events, offering a rapid, highly sensitive, and user-friendly solution to uphold the integrity of sports competitions.

CRISPR/Cas genome editing in soybean: Challenges and new insights to overcome existing bottlenecks.

BACKGROUND: Soybean is a worldwide-cultivated crop due to its applications in the food, feed, and biodiesel industries. Genome editing in soybean began with ZFN and TALEN technologies; however, CRISPR/Cas has emerged and shortly became the preferable approach for soybean genome manipulation since it is more precise, easy to handle, and cost-effective. Recent reports have focused on the conventional Cas9 nuclease, Cas9 nickase (nCas9) derived base editors, and Cas12a (formally Cpf1) as the most commonly used genome editors in soybean. Nonetheless, several challenges in the complex plant genetic engineering pipeline need to be overcome to effectively edit the genome of an elite soybean cultivar. These challenges include (1) optimizing CRISPR cassette design (i.e., gRNA and Cas promoters, gRNA design and testing, number of gRNAs, and binary vector), (2) improving transformation frequency, (3) increasing the editing efficiency ratio of targeted plant cells, and (4) improving soybean crop production. AIM OF REVIEW: This review provides an overview of soybean genome editing using CRISPR/Cas technology, discusses current challenges, and highlights theoretical (insights) and practical suggestions to overcome the existing bottlenecks. KEY SCIENTIFIC CONCEPTS OF REVIEW: The CRISPR/Cas system was discovered as part of the bacterial innate immune system. It has been used as a biotechnological tool for genome editing and efficiently applied in soybean to unveil gene function, improve agronomic traits such as yield and nutritional grain quality, and enhance biotic and abiotic stress tolerance. To date, the editing efficiency has been validated using protoplasts and hairy root assays, while stable plant transformation relies on Agrobacterium-mediated and particle bombardment methods. Nevertheless, most steps of the CRISPR/Cas workflow require optimizations to achieve a more effective genome editing in soybean plants.