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The CRISPR technology, which does not require complex instruments, expensive reagents or professional operators, has attracted a lot of attention. When utilizing the CRISPR-Cas system for detection, the pre-amplification step is often necessary to enhance sensitivity. However, this approach tends to introduce complexity and prolong the time required. To address this issue, we employed Pd@PCN-222 nanozyme to label single-stranded DNA, referred to as Pd@PCN-222 CRISPR nanozyme, which serves as the reporter of the CRISPR system. Pd@PCN-222 nanozyme possess exceptional catalytic activity for the reduction of H 2 O 2. Compared with traditional electrochemical probe ferrocene and methylene blue without catalytic activity, there is a significant amplification of the electrochemical signal. So the need for pre-amplification was eliminated. In this study, we constructed a CRISPR-Cas system for ochratoxin A, utilizing the Pd@PCN-222 CRISPR nanozyme to amplified signal avoiding pre-amplification with outstanding detection of 1.21 pg/mL. Furthermore, we developed a microfluidic electrochemical chip for the on-site detection of ochratoxin A. This achievement holds significant promise in establishing a practical on-site detection platform for identifying food safety hazards. Schematic illustration of the electrochemical biosensor based on CRISPR-Cas12a integrated Pd@PCN-222 for OTA detection. [Display omitted] • The enzyme formed by the combination of Pd@PCN-222 and CRISPR successfully avoids the pre-amplification step and reduces experimental time. • Combined with the OTA aptamer, a CRISPR-Cas system for detecting OTA was constructed with a detection range of 0.005-50ng/mL and a minimum detection limit of 1.21pg/mL. [ABSTRACT FROM AUTHOR]

The detection of low-abundance mutation genes of the epidermal growth factor receptor (EGFR) exon 21 (EGFR L858R) plays a crucial role in the diagnosis of non-small cell lung cancer (NSCLC), as it enables early cancer detection and facilitates the development of treatment strategies. A detection platform was developed by combining the M scI restriction enzyme with the recombinase-aided isothermal amplification (RAA) technique (MRE-RAA). During the RAA process, "TGG^CCA" site of the wild-type genes was cleaved by the MscI restriction enzyme, while only the low-abundance mutation genes underwent amplification. Notably, when the RAA product was combined with CRISPR-Cas system, the sensitivity of detecting the EGFR L858R mutation increased by up to 1000-fold for addition of the MscI restriction enzyme. This achievement marked the first instance of attaining an analytical sensitivity of 0.001%. Furthermore, a disk-shaped microfluidic chip was developed to automate pretreatment while concurrently analyzing four blood samples. The microfluidic features of the chip include DNA extraction, MRE-RAA, and CRISPR-based detection. The fluorescence signal is employed for detection in the microfluidic chip, which is visible to the naked eye upon exposure to blue light irradiation. Furthermore, this platform has the capability to facilitate early diagnosis for various types of cancer by enabling high-sensitivity detection of low-abundance mutation genes. [Display omitted] [ABSTRACT FROM AUTHOR]

There is a broad diversity among Cas12a endonucleases that possess nucleic acid detection and gene-editing capabilities, but few are studied extensively. Here, we present an exhaustive investigation of 23 Cas12a orthologs, with a focus on their cis - and trans -cleavage activities in combination with noncanonical crRNAs. Through biochemical assays, we observe that some noncanonical crRNA:Cas12a effector complexes outperform their corresponding wild-type crRNA:Cas12a. Cas12a can recruit crRNA with modifications such as loop extensions and split scaffolds. Moreover, the tolerance of Cas12a to noncanonical crRNA is also observed in mammalian cells through the formation of indels. We apply the adaptability of Cas12a:crRNA complexes to detect SARS-CoV-2 in clinical nasopharyngeal swabs, saliva samples, and tracheal aspirates. Our findings further expand the toolbox for next-generation CRISPR-based diagnostics and gene editing. [Display omitted] • Engineered crRNAs with variable stem-loop designs demonstrate adaptability with Cas12a • cCRISPR broadens the gene editing toolbox by screening 207 combinations of Cas12a and crRNAs • cCRISPR detects SARS-CoV-2 in saliva, nasopharyngeal swabs, and tracheal aspirates Nguyen et al. explore the diversity and adaptability of 23 Cas12a orthologs in combination with their canonical and 8 noncanonical crRNAs. A total of 207 combinations of Cas12a and crRNAs were applied for clinical nucleic acid detection and gene editing in cells. The data expand the toolbox for next-generation CRISPR-based diagnostics and genome engineering. [ABSTRACT FROM AUTHOR]

Monkeypox virus (MPXV) poses a global health emergency, necessitating rapid, simple, and accurate detection to manage its spread effectively. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique has emerged as a promising next-generation molecular diagnostic approach. Here, we developed a highly sensitive and specific CRISPR-Cas12a assisted nanopore (SCAN) with isothermal recombinase polymerase amplification (RPA) for MPXV detection. The RPA-SCAN method offers a sensitivity unachievable with unamplified SCAN while also addressing the obstacles of PCR-SCAN for point-of-care applications. We demonstrated that size-counting of single molecules enables analysis of reaction-time dependent distribution of the cleaved reporter. Our MPXV-specific RPA assay achieved a limit of detection (LoD) of 19 copies in a 50 μL reaction system. By integrating 2 μL of RPA amplifications into a 20 μL CRISPR reaction, we attained an overall LoD of 16 copies/μL (26.56 aM) of MPXV at a 95% confidence level using the SCAN sensor. We also verified the specificity of RPA-SCAN in distinguishing MPXV from cowpox virus with 100% accuracy. These findings suggest that the isothermal RPA-SCAN device is well-suited for highly sensitive and specific Monkeypox detection. Given its electronic nature and miniaturization potential, the RPA-SCAN system paves the way for diagnosing a wide array of other infectious pathogens at the point of care. [Display omitted] [ABSTRACT FROM AUTHOR]

Efficient extracellular vesicle (EV) enrichment and timely internal RNA detection for cancer diagnostics are highly desirable and remain a challenge. Here, we report a rapid EV aggregation induced in-situ microRNA detection technology based on cationic lipid-polymer hybrid nanoparticles encapsulating cascade system of catalytic hairpin assembly and CRISPR-Cas12a (CLHN-CCC), allowing for EV enrichment in three-dimensional space and in-situ detection of internal microRNAs in one step within 30 min. The enrichment efficiency (>90%) of CLHN-CCC is demonstrated in artificial EVs, cell-secreted EVs and serum EVs, which is 5-fold higher than that of traditional ultracentrifugation. The sensitive detection of artificial EVs and internal miR-1290 was achieved with the limit of detection of 10 particles/μL and 0.07 amol, respectively. After lyophilization, CLHN-CCC shows no obvious loss of performance within 6 months, making it much more robust and user friendly. This technique could sensitively (sensitivity = 92.9%) and selectively (selectivity = 85.7%) identify low amount miR-1290 in serum EVs, distinguishing early-stage pancreatic cancer patients from healthy subjects, showing high potential for clinical applications. [ABSTRACT FROM AUTHOR]

How to realize fM-level detection of viral DNA without target amplification is a challenge. Traditionally, preamplification of low-abundant DNA targets is a prerequisite, which not only increases the risk of infectious material leakage, enlarges the chance of false positive detection, but also prolongs the overall detection time. Herein, we developed a combinatory CRISPR-Cas12a detection system from three aspects to enhance its limit of detection (LOD) to the femtomolar level which is three orders of magnitude lower than a conventional protocol. Specifically, the detection sensitivity of CRISPR-Cas12a system was enhanced by utilizing multiplex crRNAs for simultaneous recognition of multiple distinct sites of the same viral DNA target, by employing a novel molecular reporter with G-triplex structure that exhibits a significantly enhanced cleaving tendency by Cas12a, by exploring the optimal molecular coexistence reaction environment for maintaining enzyme activity which was the key for sensing. It is worth noting that we have for the first time discovered an environment where sensitive G-triplex reporter forms at a low K+ concentration without damaging CRISPR-Cas12a activity. Using this system, we demonstrated ultrasensitive detection of plasmids containing monkeypox (Mpox) viral DNA sequences in a mimicking physiological scenario. More importantly, we realized sensitive and specific detection and classification of human papillomavirus (HPV) subtypes from clinical samples. Moreover, this design successfully circumvented intricate procedures and the signal readout was not contingent upon the use of unconventional equipment, suggesting its great promise of expanding into a portable, field-deployable test kit for rapid, sensitive, and specific detection of various pathogens. • From 3 aspects, a CRISPR-Cas12a system was developed with LOD to fM level, which is superior to the conventional protocol. • A low K+ buffer that can stabilize the Cas12a nucleases-sensitive G-triplex reporters was constructed for the first time. • One-step preamplification-free accurate detection and classification of HPV infection in patient samples was achieved. [ABSTRACT FROM AUTHOR]

The establishment of sensitive and facile colorimetric platform based on the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is of great significance for in vitro diagnosis. Herein, we develop a dual-enzyme cascade amplification strategy based on CRISPR-Cas12a and glucose oxidase (GOx) for instrument-free and sensitive detection of target analytes. HPV-16 DNA as the model nucleic acid target directly initiated CRISPR-Cas12a-based signal transduction, resulting in the enzymatic cleavage of ssDNA linker and the release of GOx from magnetic nanoparticles 1 (MNPs1). Following simple magnetic separation, the supernatant containing GOx was taken out and used to catalyze the substrate, resulting in a visually detectable color change. The detection limit (LOD) of HPV-16 DNA was as low as 1 pM, and the entire process could be completed within 70 min without the need for expensive equipment. Notably, the dual-enzyme cascade amplification strategy was successfully applied to the detection of non-nucleic acid targets, such as ATP, via a simple signal transduction process. The visual LOD for ATP detection reaches 2.5 μM. The approach provides a robust, sensitive and reliable point-of-care biosensing platform for the detection of target analytes. [Display omitted] • CRISPR-Cas12a-based dual-enzyme cascade amplification strategy has been designed. • HPV-16 and ATP can be visually analyzed with satisfactory detection performance. • The operation procedure is simple without expensive instruments. [ABSTRACT FROM AUTHOR]

A simple and low-cost paper-based microfluidic biosensor has been developed for on-site detection of Pb(II) by using CRISPR-Cas12a assisted smartphone device. Pb(II) induced G-quadruplex can inhibit the activation of the CRISPR-Cas12a system and subsequently prevent the cleavage of single-stranded DNA around gold nanoparticles. Thus, the resultant dispersed gold nanoparticles will flow into the detection zone and be subsequently captured by DNA nano-flowers in the detection zone of the paper-based microfluidic chip, resulting in the corresponding color change. The R/G values analyzed through our established SmartIons APP, correlate well with Pb(II) concentrations with a linear detection range of 0.1 μM - 10 μM and a lowest detection limit of 18.3 nM. The paper-based microfluidic biosensor exhibits high specificity, good anti-interference ability, stability and applicability. In view of low cost, easy customization and multiple channel, paper-based microfluidic biosensor has great potential for the on-site detection of Pb(II). [Display omitted] • For the first time, the inhibition of CRISPR-Cas12a system by Pb(II) has been studied. • Smartphone detection of paper-based microfluidic biosensor has been developed. • The biosensor can sensitively detect Pb(II) with a lowest detection limit of 18.3 nM. • The biosensor can be applied to on-site detect Pb(II) in environmental samples. [ABSTRACT FROM AUTHOR]

Background: Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a severe nosocomial threat, prompting a need for efficient detection methods. Traditional approaches, such as bacterial culture and PCR, are time-consuming and cumbersome. The CRISPR-based gene editing system offered a potential approach for point-of-care testing of CRAB., Methods: We integrated recombinase polymerase amplification (RPA) and CRISPR-Cas12a system to swiftly diagnose CRAB-associated genes, OXA-51 and OXA-23 . This multiplex RPA-CRISPR-Cas12a system eliminates bulky instruments, ensuring a simplified UV lamp-based outcome interpretation., Results: Operating at 37°C to 40°C, the entire process achieves CRAB diagnosis within 90 minutes. Detection limits for OXA-51 and OXA-23 genes are 1.3 × 10 -6 ng/μL, exhibiting exclusive CRAB detection without cross-reactivity to common pathogens. Notably, the platform shows 100% concordance with PCR when testing 30 clinical Acinetobacter baumannii strains., Conclusion: In conclusion, our multiplex RPA coupled with the CRISPR-Cas12a system provides a fast and sensitive CRAB detection method, overcoming limitations of traditional approaches and holding promise for efficient point-of-care testing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhou, Liang, Liao, Pan, Wang, Ma, Yi, Tan, Li and Wei.)

CRISPR-Cas technology has widely been applied to detect single-nucleotide mutation and is considered as the next generation of molecular diagnostics. We previously reported the combination of nucleic acid amplification (NAA) and CRISPR-Cas12a system to distinguish major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. However, the mixture of NAA and CRISPR-Cas12a reagents in one tube could interfere with the efficiency of NAA and CRISPR-Cas12a cleavage, which in turn affects the detection sensitivity. In the current study, we employed a novel photoactivated CRISPR-Cas12a strategy integrated with recombinase polymerase amplification (RPA) to develop one-pot RPA/CRISPR-Cas12a genotyping assay for detecting SARS-CoV-2 Omicron sub-lineages. The new system overcomes the potential inhibition of RPA due to early CRISPR-Cas12a activation and cleavage of the target template in traditional one-pot assay using photocleavable p-RNA, a complementary single-stranded RNA to specifically bind crRNA and precisely block Cas12a activation. The detection can be finished in one tube at 39℃ within 1 h and exhibits a low limit of detection of 30 copies per reaction. Our results demonstrated that the photocontrolled one-pot RPA/CRISPR-Cas12a assay could effectively identify three signature mutations in the spike gene of SARS-CoV-2 Omicron variant, namely, R346T, F486V, and 49X, and distinguish Omicron BA.1, BA.5.2, and BF.7 sub-lineages. Furthermore, the assay achieved a sensitivity of 97.3% and a specificity of 100.0% and showed a concordance of 98.3% with Sanger sequencing results.IMPORTANCEWe successfully developed one-pot recombinase polymerase amplification/CRISPR-Cas12a genotyping assay by adapting photocontrolled CRISPR-Cas technology to optimize the conditions of nucleic acid amplification and CRISPR-Cas12a-mediated detection. This innovative approach was able to quickly distinguish severe acute respiratory syndrome coronavirus 2 Omicron variants and can be readily modified for detecting any nucleic acid mutations. The assay system demonstrates excellent clinical performance, including rapid detection, user-friendly operations, and minimized risk of contamination, which highlights its promising potential as a point-of-care testing for wide applications in resource-limiting settings., Competing Interests: The authors declare no conflict of interest.

Feline parvovirus (FPV) is highly infectious for cats and other Felidae and often causes severe damage to young kittens. In this study, we incorporated recombinase polymerase amplification (RPA) and Cas12a-mediated detection and developed an RPA-Cas12a-based real-time or end-point fluorescence detection method to identify the NS1 gene of FPV. The total time of RPA-Cas12a-based fluorescence assay is approximately 25 min. The assay presented a limit of detection (LOD) of 1 copies/μl (25 copies/per reaction), with no cross-reactivity with several feline pathogens. The clinical performance of the assay was examined using total genomic DNA purified from 60 clinical specimens and then compared to results obtained with qPCR detection of FPV with 93.3% positive predictive agreement and 100% negative predictive agreement. Together, the rapid reaction, cost-effectiveness, and high sensitivity make the RPA-Cas12a-based fluorescence assay a fascinating diagnostic tool that will help minimize infection spread through instant detection of FPV., (© 2024 Ting Wang et al., published by Sciendo.)

Norovirus is highly infectious and rapidly transmissible and represents a major pathogen of sporadic cases and outbreaks of acute gastroenteritis worldwide, causing a substantial disease burden. Recent years have witnessed a dramatic increase in norovirus outbreaks in China, significantly higher than in previous years, among which GII norovirus is the predominant prevalent strain. Therefore, rapid norovirus diagnosis is critical for clinical treatment and transmission control. Hence, we developed a molecular assay based on RPA combined with the CRISPER-CAS12a technique targeting the conserved region of the GII norovirus genome, the results of which could be displayed by fluorescence curves and immunochromatographic lateral-flow test strips. The reaction only required approximately 50 min, and the results were visible by the naked eye with a sensitivity reaching 10 2 copies/μl. Also, our method does not cross-react with other common pathogens that cause intestinal diarrhea. Furthermore, this assay was easy to perform and inexpensive, which could be widely applied for detecting norovirus in settings including medical institutions at all levels, particularly township health centers in low-resource areas., (© 2024 Xinyi Hu et al., published by Sciendo.)

Survival crises stalk many animals, especially endangered and rare animals. Accurate species identification plays a pivotal role in animal resource conservation. In this study, we developed an animal species identification method called Analysis of whole-GEnome (AGE), which identifies species by finding species-specific sequences through bioinformatics analysis of the whole genome and subsequently recognizing these sequences using experimental technologies. To clearly demonstrate the AGE method, Cervus nippon , a well-known endangered species, and a closely related species, Cervus elaphus , were set as model species, without and with published genomes, respectively. By analyzing the whole genomes of C. nippon and C. elaphus , which were obtained through next-generation sequencing and online databases, we built specific sequence databases containing 7,670,140 and 570,981 sequences, respectively. Then, the species specificities of the sequences were confirmed experimentally using Sanger sequencing and the CRISPR-Cas12a system. Moreover, for 11 fresh animal samples and 35 commercially available products, our results were in complete agreement with those of other authoritative identification methods, demonstrating AGE's precision and potential application. Notably, AGE found a mixture in the 35 commercially available products and successfully identified it. This study broadens the horizons of species identification using the whole genome and sheds light on the potential of AGE for conserving animal resources.

Background: Diaporthe aspalathi and Diaporthe caulivora are two of the fungal pathogens causing soybean stem canker (SSC) in soybean, which is one of the most widespread diseases in soybean growing regions and can cause 100% loss of yield. Current methods for the detection of fungal pathogens, including morphological identification and molecular detection, are mostly limited by the need for professional laboratories and staff. To develop a detection method for potential on-site diagnosis for two of the fungal pathogens causing SSC, we designed a rapid assay combining recombinase polymerase amplification (RPA) and CRISPR-Cas12a-based diagnostics to specifically detect D. aspalathi and D. caulivora., Results: The translation elongation factor 1-alpha gene was employed as the target gene to evaluate the specificity and sensitivity of this assay. The RPA/CRISPR-Cas12a system has excellent specificity to distinguish D. aspalathi and D. caulivora from closely related species. The sensitivities of RPA/CRISPR-Cas12a-based fluorescence detection and lateral flow assay for D. aspalathi and D. caulivora are 14.5 copies and 24.6 copies, respectively. This assay can detect hyphae in inoculated soybean stems at 12 days after inoculation and has a recovery as high as 86% for hyphae-spiked soybean seed powder. The total time from DNA extraction to detection was not more than 60 min., Conclusion: The method developed for rapid detection of plant pathogens includes DNA extraction with magnetic beads or rapid DNA extraction, isothermal nucleic acid amplification at 39 °C, CRISPR-Cas12a cleavage reaction at 37 °C, and lateral flow assay or endpoint fluorescence visualization at room temperature. The RPA and CRISPR-Cas12a reagents can be preloaded in the microcentrifuge tube to simplify the procedures in the field. Both RPA and CRISPR-Cas12a reaction can be realized on a portable incubator, and the results are visualized using lateral flow strips or portable flashlight. This method requires minimal equipment and operator training, and has promising applications for rapid on-site disease screening, port inspection, or controlling fungal pathogen transmission in crop. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Fusobacterium nucleatum (Fn), as a conditional pathogen, can cause a range of oral and gastrointestinal diseases. However, existing clinical detection methods require expensive equipment and complex procedures, which are inconvenient for large-scale screening in epidemiological research. The purpose of this study was to establish a reliable, rapid, and inexpensive detection method based on CRISPR/Cas12a technology for the detection of Fn. Specific recombinase polymerase amplification (RPA) primer sequences and crRNA sequences were designed based on the nusG gene of Fn. Subsequently, a fluorescence assay and a lateral flow immunoassay were established using the RPA and CRISPR-Cas12a system (RPA-CRISPR-Cas12a). Sensitivity validation revealed a limit of detection of 5 copies/µL. This method could distinguish Fn from other pathogens with excellent specificity. Furthermore, the RPA-CRISPR-Cas12a assay was highly consistent with the classical quantitative real-time PCR method when testing periodontal pocket samples. This makes it a promising method for the detection of Fn and has the potential to play an increasingly important role in infectious disease testing.IMPORTANCE Fusobacterium nucleatum (Fn) naturally exists in the microbial communities of the oral and gastrointestinal tracts of healthy individuals and can cause inflammatory diseases in the oral and gastrointestinal tracts. Recent studies have shown that Fn is closely associated with the occurrence and development of gastrointestinal cancer. Therefore, the detection of Fn is very important. Unlike the existing clinical detection methods, this study established a fluorescence-based assay and lateral flow immunoassay based on the RPA and CRISPR-Cas12a system (RPA-CRISPR-Cas12a), which is fast, reliable, and inexpensive and can complete the detection within 30-40 minutes. This makes it a promising method for the detection of Fn and has the potential to play an increasingly important role in infectious disease testing., Competing Interests: The authors declare no conflict of interest.

Rapid, accurate, and noninvasive detection of biomarkers in saliva, urine, or nasal fluid is essential for the identification, early diagnosis, and monitoring of cancer, organ failure, transplant rejection, vascular diseases, autoimmune disorders, and infectious diseases. We report the development of an Immuno-CRISPR-based lateral flow assay (LFA) using antibody-DNA barcode complexes with magnetic enrichment of the target urinary biomarkers CXCL9 and CXCL10 for naked eye detection (ImmunoMag-CRISPR LFA). An intermediate approach involving a magnetic bead-based Immuno-CRISPR assay (ImmunoMag-CRISPR) resulted in a limit of detection (LOD) of 0.6 pg/mL for CXCL9. This value surpasses the detection limits achieved by previously reported assays. The highly sensitive detection method was then re-engineered into an LFA format with an LOD of 18 pg/mL for CXCL9, thereby enabling noninvasive early detection of acute kidney transplant rejection. The ImmunoMag-CRISPR LFA was tested on 42 clinical urine samples from kidney transplant recipients, and the assay could determine 11 positive and 31 negative urinary samples through a simple visual comparison of the test line and the control line of the LFA strip. The LFA system was then expanded to quantify the CXCL9 and CXCL10 levels in clinical urine samples from images. This approach has the potential to be extended to a wide range of point-of-care tests for highly sensitive biomarker detection.

Aberrant DNA methylation is closely associated with various diseases, particularly cancer, and its precise detection plays an essential role in disease diagnosis and monitoring. In this study, we present a novel DNA methylation detection method (namely meHOLMES), which integrates both the TET2/APOBEC-mediated cytosine deamination step and the CRISPR-Cas12a-based signal readout step. TET2/APOBEC efficiently converts unmethylated cytosine to uracil, which is subsequently changed to thymine after PCR amplification. Utilizing a rationally designed crRNA, Cas12a specifically identifies unconverted methylated cytosines and generates detectable signals using either fluorescent reporters or lateral flow test strips. meHOLMES quantitatively detects methylated CpG sites with or without Protospacer Adjacent Motif (PAM) sequences in both artificial and real biological samples. In addition, meHOLMES can complete the whole detection process within 6 h, which is much faster than traditional bisulfite-based sample pre-treatment method. Above all, meHOLMES provides a simpler, faster, more accurate, and cost-effective approach for quantitation of DNA methylation levels in a sequence-independent manner., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Importance: Pseudorabies virus (PRV) causes high mortality and miscarriage rates in the infected swine, and the eradication policy coupled with large-scale vaccination of live attenuated vaccines has been adopted globally against PRV. Differential diagnosis of the vaccinated and infected swine is highly demanded. Our multienzyme isothermal rapid amplification (MIRA)-Cas12a detection method described in this study can diagnose PRV with a superior sensitivity comparable to the quantitative PCR (qPCR) and a competitive detection speed (only half the time as qPCR needs). The portable feature and the simple procedure of MIRA-Cas12a make it easier to deploy for clinical diagnosis, even in resource-limited settings. The MIRA-Cas12a method would provide immediate and accurate diagnostic information for policymakers to respond promptly., Competing Interests: The authors declare no conflict of interest.

Class II Type V endonucleases have increasingly been adapted to develop sophisticated and easily accessible synthetic biology tools for genome editing, transcriptional regulation, and functional genomic screening in a wide range of organisms. One such endonuclease, Cas12a, presents itself as an attractive alternative to Cas9-based systems. The ability to mature its own guide RNAs (gRNAs) from a single transcript has been leveraged for easy multiplexing, and its lack of requirement of a tracrRNA element, also allows for short gRNA expression cassettes. To extend these functionalities into the industrially relevant oleaginous yeast Yarrowia lipolytica, we developed a set of CRISPR-Cas12a vectors for easy multiplexed gene knockout, repression, and activation. We further extended the utility of this CRISPR-Cas12a system to functional genomic screening by constructing a genome-wide guide library targeting every gene with an eightfold coverage. Pooled CRISPR screens conducted with this library were used to profile Cas12a guide activities and develop a machine learning algorithm that could accurately predict highly efficient Cas12a gRNA. In this protocols chapter, we first present a method by which protein coding genes may be functionally disrupted via indel formation with CRISPR-Cas12a systems. Further, we describe how Cas12a fused to a transcriptional regulator can be used in conjunction with shortened gRNA to achieve transcriptional repression or activation. Finally, we describe the design, cloning, and validation of a genome-wide library as well as a protocol for the execution of a pooled CRISPR screen, to determine guide activity profiles in a genome-wide context in Y. lipolytica. The tools and strategies discussed here expand the list of available synthetic biology tools for facile genome engineering in this industrially important host., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Efficient and sensitive detection of targets is one of the motivations for constant development and innovation of various biosensors. CRISPR-Cas12a, a new generation of gene editing tools, has shown excellent application potential in biosensor design and construction. By combining with the specific recognition element-aptamer, a single-stranded oligonucleotide obtained by systematic evolution of ligands by exponential enrichment (SELEX) in vitro screening, CRISPR-Cas12a also shows superior performance non-nucleic acid targets detection, such as small molecules, proteins, virus and pathogenic bacteria. However, aptamer and CRISPR-Cas12a (CRISPR-Cas12a/Apt) still face some problems in non-nucleic acid target detection, such as single signal response mode and narrow linear range. The development of diverse CRISPR-Cas12a/Apt biosensors is necessary to meet the needs of various detection environments. In this review, the working principle of CRISPR-Cas12a/Apt was introduced and recent progress in CRISPR-Cas12a/Apt in the application of non-nucleic acid target detection was summarized. Moreover, the requirements of critical parameters such as crRNA sequence, activator sequence, and reaction system in the design of CRISPR-Cas12a/Apt biosensors were discussed, which could provide the reference for the design of efficient and sensitive novel non-nucleic acid target biosensors. In addition, the challenges and prospects of CRISPR-Cas12a/Apt-based biosensor were further presented.

Three worldwide historical plague pandemics resulted in millions of deaths. Yersinia pestis, the etiologic agent of plague, is also a potential bioterrorist weapon. Simple, rapid, and specific detection of Y. pestis is important to prevent and control plague. However, the high similarity between Y. pestis and its sister species within the same genus makes detection work problematic. Here, the genome sequence from the Y. pestis CO92 strain was electronically separated into millions of fragments. These fragments were analyzed and compared with the genome sequences of 539 Y. pestis strains and 572 strains of 20 species within the Yersinia genus. Altogether, 97 Y. pestis-specific tags containing two or more single nucleotide polymorphism sites were screened out. These 97 tags efficiently distinguished Y. pestis from all other closely related species. We chose four of these tags to design a Cas12a-based detection system. PCR–fluorescence methodology was used to test the specificity of these tags, and the results showed that the fluorescence intensity produced by Y. pestis was significantly higher than that of non-Y. pestis (p < 0.0001). We then employed recombinase polymerase amplification and lateral flow dipsticks to visualize the results. Our newly developed plasmid-independent, species-specific library of tags completely and effectively screened chromosomal sequences. The detection limit of our four-tag Cas12a system reached picogram levels.

Background: Thermophilic filamentous fungus Myceliophthora thermophila has great capacity for biomass degradation and is an attractive system for direct production of enzymes and chemicals from plant biomass. Its industrial importance inspired us to develop genome editing tools to speed up the genetic engineering of this fungus. First-generation CRISPR–Cas9 technology was developed in 2017 and, since then, some progress has been made in thermophilic fungi genetic engineering, but a number of limitations remain. They include the need for complex independent expression cassettes for targeting multiplex genomic loci and the limited number of available selectable marker genes. Results: In this study, we developed an Acidaminococcus sp. Cas12a-based CRISPR system for efficient multiplex genome editing, using a single-array approach in M. thermophila. These CRISPR–Cas12a cassettes worked well for simultaneous multiple gene deletions/insertions. We also developed a new simple approach for marker recycling that relied on the novel cleavage activity of the CRISPR–Cas12a system to make DNA breaks in selected markers. We demonstrated its performance by targeting nine genes involved in the cellulase production pathway in M. thermophila via three transformation rounds, using two selectable markers neo and bar. We obtained the nonuple mutant M9 in which protein productivity and lignocellulase activity were 9.0- and 18.5-fold higher than in the wild type. We conducted a parallel investigation using our transient CRISPR–Cas9 system and found the two technologies were complementary. Together we called them CRISPR–Cas-assisted marker recycling technology (Camr technology). Conclusions: Our study described new approaches (Camr technology) that allow easy and efficient marker recycling and iterative stacking of traits in the same thermophilic fungus strain either, using the newly established CRISPR–Cas12a system or the established CRISPR–Cas9 system. This Camr technology will be a versatile and efficient tool for engineering, theoretically, an unlimited number of genes in fungi. We expect this advance to accelerate biotechnology-oriented engineering processes in fungi. [ABSTRACT FROM AUTHOR]

Summary: CRISPR‐Cas9 and Cas12a are two powerful genome editing systems. Expression of CRISPR in plants is typically achieved with a mixed dual promoter system, in which Cas protein is expressed by a Pol II promoter and a guide RNA is expressed by a species‐specific Pol III promoter such as U6 or U3. To achieve coordinated expression and compact vector packaging, it is desirable to express both CRISPR components under a single Pol II promoter. Previously, we demonstrated a first‐generation single transcript unit (STU)‐Cas9 system, STU‐Cas9‐RZ, which is based on hammerhead ribozyme for processing single guide RNAs (sgRNAs). In this study, we developed two new STU‐Cas9 systems and one STU‐Cas12a system for applications in plants, collectively called the STU CRISPR 2.0 systems. We demonstrated these systems for genome editing in rice with both transient expression and stable transgenesis. The two STU‐Cas9 2.0 systems process the sgRNAs with Csy4 ribonuclease and endogenous tRNA processing system respectively. Both STU‐Cas9‐Csy4 and STU‐Cas9‐tRNA systems showed more robust genome editing efficiencies than our first‐generation STU‐Cas9‐RZ system and the conventional mixed dual promoter system. We further applied the STU‐Cas9‐tRNA system to compare two C to T base editing systems based on rAPOBEC1 and PmCDA1 cytidine deaminases. The results suggest STU‐based PmCDA1 base editor system is highly efficient in rice. The STU‐Cas12a system, based on Cas12a' self‐processing of a CRISPR RNA (crRNA) array, was also developed and demonstrated for expression of a single crRNA and four crRNAs. Altogether, our STU CRISPR 2.0 systems further expanded the CRISPR toolbox for plant genome editing and other applications. [ABSTRACT FROM AUTHOR]

Background: CRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities. Cas12a-mediated genome editing is temperature sensitive in plants, but a lack of a comprehensive understanding on Cas12a temperature sensitivity in plant cells has hampered effective application of Cas12a nucleases in plant genome editing. Results: We compared AsCas12a, FnCas12a, and LbCas12a for their editing efficiencies and non-homologous end joining (NHEJ) repair profiles at four different temperatures in rice. We found that AsCas12a is more sensitive to temperature and that it requires a temperature of over 28 °C for high activity. Each Cas12a nuclease exhibited distinct indel mutation profiles which were not affected by temperatures. For the first time, we successfully applied AsCas12a for generating rice mutants with high frequencies up to 93% among T0 lines. We next pursued editing in the dicot model plant Arabidopsis, for which Cas12a-based genome editing has not been previously demonstrated. While LbCas12a barely showed any editing activity at 22 °C, its editing activity was rescued by growing the transgenic plants at 29 °C. With an early high-temperature treatment regime, we successfully achieved germline editing at the two target genes, GL2 and TT4, in Arabidopsis transgenic lines. We then used high-temperature treatment to improve Cas12a-mediated genome editing in maize. By growing LbCas12a T0 maize lines at 28 °C, we obtained Cas12a-edited mutants at frequencies up to 100% in the T1 generation. Finally, we demonstrated DNA binding of Cas12a was not abolished at lower temperatures by using a dCas12a-SRDX-based transcriptional repression system in Arabidopsis. Conclusion: Our study demonstrates the use of high-temperature regimes to achieve high editing efficiencies with Cas12a systems in rice, Arabidopsis, and maize and sheds light on the mechanism of temperature sensitivity for Cas12a in plants. [ABSTRACT FROM AUTHOR]

A rapid and accurate on-site diagnostic test for pathogens including influenza viruses is critical for preventing the spread of infectious diseases. Two types of influenza virus, A and B cause seasonal flu epidemics, whereas type A can cause influenza pandemics. To specifically detect influenza A (IAV) and B (IBV) viruses, we developed a clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system-based assay. By coupling reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), a CRISPR-Cas12a DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) detected IAV and IBV titers as low as 1 × 100 plaque forming units (PFUs) per reaction without exhibiting cross-reactivity. Only 75 to 85 min were required to detect IAV and IBV, depending on isothermal nucleic acid amplification methods, and results were verified using a lateral flow strip assay that does not require additional analytic equipment. Taken together, our findings establish RT-RPA and RT-LAMP-coupled DETECTR-based diagnostic tests for rapid, specific and high-sensitivity detection of IAV and IBV using fluorescence and lateral flow assays. The diagnostic test developed in this study can be used to distinguish IAV and IBV infections, a capability that is necessary for monitoring and preventing the spread of influenza epidemics and pandemics.

Rapid, efficient, specific and sensitive diagnostic techniques are critical for selecting appropriate treatments for drug-resistant bacterial infections. To address this challenge, we have developed a novel diagnostic method, called the D ual-Cas T andem D iagnostic P latform (DTDP), which combines the use of Cas9 nickase (Cas9n) and Cas12a. DTDP works by utilizing the Cas9n-sgRNA complex to create a nick in the target strand's double-stranded DNA (dsDNA). This prompts DNA polymerase to displace the single-stranded DNA (ssDNA) and leads to cycles of DNA replication through nicking, displacement, and extension. The ssDNA is then detected by the Cas12a-crRNA complex (which is PAM-free), activating trans -cleavage and generating a fluorescent signal from the fluorescent reporter. DTDP exhibits a high sensitivity (1 CFU/mL or 100 ag/μL), high specificity (specifically to MRSA in nine pathogenic species), and excellent accuracy (100%). The dual RNA recognition process in our method improves diagnostic specificity by decreasing the limitations of Cas12a in detecting dsDNA protospacer adjacent motifs (PAMs) and leverages multiple advantages of multi-Cas enzymes in diagnostics. This novel approach to pathogenic microorganism detection has also great potential for clinical diagnosis. Schematic of the DTDP strategy for bacterial diagnostics. [Display omitted] • A PAM-free CRISPR/Cas12a diagnostic platform based on Cas9 nickase-mediated strand displacement. • The platform has high specificity due to the double RNA recognition system. • The stability of the platform is better than qPCR in serum sample. [ABSTRACT FROM AUTHOR]

Quantification of extracellular tumor markers has shown great promise for non-invasive cancer diagnosis. Combined detection of multiple tumor markers instead of a single one is valuable for accurate diagnosis. Here, we integrate CRISPR-Cas12a with DNA catalytic hairpin assembly (CHA) to doubly amplify the output signal for detecting microRNA-182 (miR-182), which is overexpressed by gastric cancer patients. Additionally, we develop a CHA system with self-replicating capacity (SRCHA) to realize dual-signal amplification for the detection of carcinoembryonic antigen (CEA), a broad-spectrum tumor marker. The proposed cascade amplification strategies enable ultrasensitive detection of miR-182 and CEA with low LODs of 0.063 fM and 4.8 pg mL−1, respectively. Moreover, we design a ternary "AND" logic gate using different concentrations of miR-182 and CEA as inputs, which demonstrates intelligent diagnosis of gastric cancer staging with a high accuracy of 93.3% in a clinical cohort of 30 individuals. Overall, our study expands the application of CRISPR-Cas12a in biosensing and provides a new diagnostic strategy for non-invasive liquid biopsy of gastric cancer before resorting to a traumatic tissue biopsy. A dual-signal amplification platform enables ultrasensitive detection of miR-182 and CEA, and is coupled with a ternary "AND" logic gate for intelligent diagnosis of gastric cancer staging with high accuracy. [Display omitted] • A CRISPR-Cas12a-assisted CHA platform enables ultrasensitive detection of miR-182 with dual-signal amplification. • A self-replicating CHA platform enables ultrasensitive detection of CEA with dual-signal amplification. • Accurate staging diagnosis of gastric cancer is realized using a ternary "AND" logic gate. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (MRSA) strains are prevalent foodborne superbugs and pose significant pathogens for both humans and animals. The sensitive and specific diagnosis of MRSA holds paramount importance for its prevention and treatment. Herein, we develop a T 7 exonuclease-assisted PAM-free D ual CRISPR- C as12a B iosensor (TDCB) targeting the antibiotic resistance gene MecA for ultrasensitive and highly specific MRSA detection. After generation of single-stranded DNA (ssDNA) obtained with T7 exonuclease digestion of amplified MRSA genome, the trans -cleavage activity of dual-Cas12a-crRNA complexes was initiated without the requirement of protospacer adjacent motifs (PAM). The fluorescent signal obtained with TDCB biosensor was stronger, compared with the single-Cas12a biosensor. TDCB detects the MRSA genome and cells with a high sensitivity of 100 ag/mL and 1 cfu/mL, respectively. Moreover, TDCB can distinguish MRSA from tested 6 non-MRSA bacterial species from each other. We further validated TDCB using MRSA-infected tilapia fishes and obtained results consistent with the qPCR assay. This TDCB technology, a novel Cas12a-based biosensor that detects genomic regions without reliance on PAM motifs, can have very broad utilization in hospitals, food and feed industry, and fish and dairy farming. [Display omitted] • A dual-CRISPR-Cas12a biosensor was prepared. • The biosensor is PAM motifs-free for detection of MecA gene. • The diagnostic result of MRSA-infected tilapia is consistent with qPCR. • The biosensor has high sensitivity with 1 cfu/mL. [ABSTRACT FROM AUTHOR]

8-oxoguanine (8-oxoG) based DNA damage is the most common type of DNA damage which greatly affect gene expression. Therefore, accurate quantification of 8-oxoG based DNA damage is of high clinical significance. However, current methods for 8-oxoG detection struggle to balance convenience, low cost, and sensitivity. Herein, we have proposed and investigated the shortened crRNA mode of CRISPR-Cas12a system and greatly enhanced its signal-to-noise ratio. Taking advantages of the shortened crRNA mode, we further developed a CRISPR-enhanced structure-switching aptamer assay (CESA) for 8-oxoG. The analytical performance of CESA was thoroughly investigated via detecting free 8-oxoG and 8-oxoG on gDNA. The CESA displayed impressive sensitivity for free 8-oxoG, with detection and quantification limits of 32.3 pM and 0.107 nM. These limits modestly rose to 64.5 pM and 0.215 nM when examining 8-oxoG on gDNA. To demonstrate the clinical practicability and significance of the CESA system, we further applied it to measuring 8-oxoG levels in 7 plasma samples (Cervical carcinoma, 11.87 ± 0.69 nM VS. Healthy control, 2.66 ± 0.42 nM), 24 seminal plasma samples (Asthenospermia, 22.29 ± 7.48 nM VS. Normal sperm, 9.75 ± 3.59 nM), 10 breast-tissue gDNA samples (Breast cancer, 2.77 ± 0.63 nM/μg VS. Healthy control, 0.41 ± 0.09 nM/μg), and 24 sperm gDNA samples (Asthenospermia, 28.62 ± 4.84 VS. Normal sperm, 16.67 ± 3.31). This work not only proposes a novel design paradigm of shortened crRNA for developing CRISPR-Cas12a based biosensors but also offers a powerful tool for detecting 8-oxoG based DNA damage. [ABSTRACT FROM AUTHOR]

Mild traumatic brain injury (mTBI) may cause increasing risk of long-term sequelae during brain development among children and adolescents. Recent investigations have declared that salivary microRNAs (miRNAs) in mTBI were reliable diagnostic markers. Herein, a versatile biosensor, namely, self-primer exponential amplified Cas12a tandem (SP-ExACT) reaction for quantifying mTBI-related miRNAs was established by integrating CRISPR-Cas12a with copper(I) 1,2,4-triazolate coordination polymer ([Cu(tz)]) nanosheets. Unlike conventional strand displacement amplification, we designed DNA hairpins with integrated primer and template sequences to generate multiple single-stranded DNA (ssDNA) identical to targets. They could trigger the following rolling circle amplification together and then activate Cas12a to indiscriminately cleave the intact fluorophores-labeled ssDNA that absorbed on [Cu(tz)] nanosheets. The corresponding fluorescence signal would recover for the desorption of cleaved short ssDNA from nanosheets. Owing to the remarkable performance of SP-ExACT-[Cu(tz)] reaction, this biosensor could detect let-7a, miR-30e and miR-21 with a LOD of 11.6, 5.4, 7.4 fM, respectively. This study explored a novel fluorescence biosensor based on [Cu(tz)] nanosheets combined with CRISPR-Cas12a to reduce initial background signal, and combined multiple signal amplification strategies to achieve ultra-sensitive detection. Moreover, multiple mTBI-related miRNAs detection could avoid false positive/negative results, and a universal detection platform was constructed through changing corresponding sequence. • A SP-ExACT-[Cu(tz)] system to detect mTBI-related miRNAs in human saliva was explored. • Target-induced hairpin intramolecular directed strand displacement amplification. • Ingenious integration of dual signal amplification strategies and CRISPR system. • [Cu(tz)] nanosheets for DNA adsorption and fluorescence quenching was applied to reducing background signals. [ABSTRACT FROM AUTHOR]

Given the targeted binding ability and cleavage activity of the emerging CRISPR/Cas12a assay which transduces the target into its cleavage activity exhibited broadly prospective applications in integrated sensing and actuating system. Here, we elaborated a universal approach to quickly activate CRISPR/Cas12a for low-abundance biomarker detection based on the amplification strategy of a target-induced spherical nucleic acid enzyme (SNAzyme) network that could accelerate the output of activators. Specifically, multifunctional Y-shaped probes and hairpin probes (HPs, which contained the specific sequence of the activators of CRISPR/Cas12a and the substrate chain of DNAzyme) were rationally designed to construct SNAzyme. Target recognition induced disassembly of the Y-shaped probes, which released DNAzyme strands to active DNAzyme and accompanied by SNAzyme self-assembly into SNAzyme network. Interestingly, compared with randomly dispersed SNAzyme, the reaction kinetics of the SNAzyme network enhanced 1.6 times in response to Α-methyl acyl-CoA racemase (AMACR, a biomarker for prostate cancer), which was attributed to the promoted catalytic efficiency of DNAzyme by the confined SNAzyme network. Benefiting from these, the prepared biosensor based on electrochemiluminescence (ECL) platform by loading AuAg nanoclusters (AuAgNCs) into metal-organic framework-5 (MOF-5) exhibited satisfying detection performance for AMACR with a wide linear range (0.001 μg/mL to 100 μg/mL) and a low detection limit (1.0 × 10−4 μg/mL, which exhibited significant potential in clinical diagnoses. [ABSTRACT FROM AUTHOR]

Fumonisin B 1 (FB 1) is a common mycotoxin found in agricultural products. Therefore, establishing rapid detection methods is an important means to prevent the harm of FB 1. This study was based on the clustered, regularly spaced short palindromic repeats (CRISPR)-Cas12a system with the introduction of aptamer F10. This study aimed to design CRISPR RNA (crRNA) to specifically recognize the aptamer F10, which formed the competitive relationship between the target molecule and crRNA for nucleic acid aptamer. Finally, a new type of biosensor was established using the trans-cleavage activity of Cas12a protein and amplifying the signal with a fluorescent probe. This study observed a linear relationship of FB 1 concentrations in the range of 25–500 nM. The standard curve was y = −0.2586 × x + 775.3, R 2 = 0.9922, and the limit of detection (LOD) was 16.84 nM. The recovery of the maize sample was 92.2–101.93% and 92.24–103.96%. The total assay time was 40 min. The biosensor showed high specificity and sensitivity for FB 1 after the introduction of the aptamer F10, and the workflow was simple, convenient, and fast, which was suitable for point-of-care (POC) detection in the field. Our designed biosensor validated the principle of detecting small molecules using CRISPR-Cas12a systems, further facilitating its application in the diagnostic field. [Display omitted] • A small molecule fluorescent biosensor based on CRISPR-Cas12a. • The introduced aptamer F10 has high affinity and specificity for FB 1. • The crRNA was designed to specifically recognize the aptamer F10. • The double-blind method was used to detect corn flour and wheat flour. [ABSTRACT FROM AUTHOR]

Conventional pathogen detection strategies based on the molecular structure or chemical characteristics of biomarkers can only provide the "physical abundance" of microorganisms, but cannot reflect the "biological effect abundance" in the true sense. To address this issue, we report an erythrocyte membrane-encapsulated biomimetic sensor cascaded with CRISPR-Cas12a (EMSCC). Taking hemolytic pathogens as the target model, we first constructed an erythrocyte membrane-encapsulated biomimetic sensor (EMS). Only hemolytic pathogens with biological effects can disrupt the erythrocyte membrane (EM), resulting in signal generation. Then the signal was amplified by cascading CRISPR-Cas12a, and more than 6.67 × 104-fold improvement in detection sensitivity compared to traditional erythrocyte hemolysis assay was achieved. Notably, compared with polymerase chain reaction (PCR) or enzyme linked immunosorbent assay (ELISA)-based quantification methods, EMSCC can sensitively respond to the pathogenicity change of pathogens. For the detection of simulated clinical samples based on EMSCC, we obtained an accuracy of 95% in 40 samples, demonstrating its potential clinical value. [ABSTRACT FROM AUTHOR]

Cross-border pathogens such as the African swine fever virus (ASFV) still pose a socio-economic threat. Cheaper, faster, and accurate diagnostics are imperative for healthcare and food safety applications. Currently, the discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has paved the way for the diagnostics based on Cas13 and Cas12/14 that exhibit collateral cleavage of target and single-stranded DNA (ssDNA) reporter. The reporter is fluorescently labeled to report the presence of a target. These methods are powerful; however, fluorescence-based approaches require expensive apparatuses, complicate results readout, and exhibit high-fluorescence background. Here, we present a new CRISPR–Cas-based approach that combines polymerase chain reaction (PCR) amplification, Cas12a, and a probe-based lateral flow biosensor (LFB) for the simultaneous detection of seven types of ASFV. In the presence of ASFVs, the LFB responded to reporter trans-cleavage by naked eyes and achieved a sensitivity of 2.5 × 10−15 M within 2 h, and unambiguously identified ASFV from swine blood. This system uses less time for PCR pre-amplification and requires cheaper devices; thus, it can be applied to virus monitoring and food samples detection.

Various methods for editing specific sites in the Escherichia coli chromosome are available, and gene-size (∼1 kb) integration into a single site or to introduce deletions, short insertions or point mutations into multiple sites can be conducted in a short period of time. However, a method for rapidly integrating multiple gene-size sequences into different sites has not been developed yet. Here, we describe a method and plasmid system that makes it possible to simultaneously insert genes into multiple specific loci of the E. coli genome without the need for chromosomal markers. The method uses a CRISPR-Cas12a system to eliminate unmodified cells by double-stranded DNA cleavage in conjunction with the phage-derived λ-Red recombinases to facilitate recombination between the chromosome and the donor DNA. We achieved the insertion of up to 3 heterologous genes in one round of recombination and selection. To demonstrate the practical application of this gene-insertion method, we constructed a recombinant E. coli producing an industrially useful chemical, 5-aminolevulinic acid (ALA), with high-yield. Moreover, a similar two-plasmid system was built to edit the genome of the extremophile Halomonas bluephagenesis.

Importance: A method for Neisseria gonorrhoeae (NG)/ Chlamydia trachomatis (CT) detection is developed using multiplex-recombinase polymerase amplification and Cas12a/Cas13a. This method can detect NG and CT simultaneously with high sensitivity and specificity. This method has great potential to be further developed into larger-scale screening and point-of-care testing (POCT)., Competing Interests: The authors declare no conflict of interest.

Duck Tembusu virus (DTMUV) is an emerging pathogen that poses a serious threat to the duck industry in China. Currently, polymerase chain reaction (PCR), quantitative PCR (qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) are commonly used for DTMUV detection. However, these methods require complex steps and special equipment and easily cause false-positive results. Therefore, we urgently need to establish a simple, sensitive and specific method for the clinical field detection of DTMUV. In this study, we developed an RT-LAMP-based CRISPR-Cas12a assay targeting the C gene to detect DTMUV with a limited detection of 3 copies/μL. This assay was specific for DTMUV without cross-reaction with other common avian viruses and only required some simple pieces of equipment, such as a thermostat water bath and blue/UV light transilluminator. Furthermore, this assay showed 100% positive predictive agreement (PPA) and negative predictive agreement (NPA) relative to SYBR Green qPCR for DTMUV detection in 32 cloacal swabs and 22 tissue samples, supporting its application for clinical field detection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Ding, Huang, Li, Tang, Li, Feng, Zhao, Zhang, Yuan, Shan and Jiao.)

Human rhinovirus B (HRV-B) is a major human viral pathogen that can be responsible for various kinds of infections. Due to the health risks associated with HRV-B, it is therefore crucial to explore a rapid, specific, and sensitive method for surveillance. Herein, we exploited a novel detection method for HRV-B by combining reverse-transcription recombinase polymerase amplification (RT-RPA) of nucleic acids isothermal amplification and the trans-cleavage activity of Cas12a. Our RT-RPA-Cas12a-based fluorescent assay can be completed within 35-45 min and obtain a lower detection threshold to 0.5 copies/µL of target RNA. Meanwhile, crRNA sequences without a specific protospacer adjacent motif can effectively activate the trans-cleavage activity of Cas12a. Moreover, our RT-RPA-Cas12a-based fluorescent method was examined using 30 clinical samples, and exhibited high accuracy with positive and negative predictive agreement of 90% and 100%, respectively. Taken together, a novel promising, rapid and effective RT-RPA-Cas12a-based detection method was explored and shows promising potential for on-site HRV-B infection in resource-limited settings., (© 2023. The Author(s).)

Respiratory tract infections are associated with the most common diseases transmitted among people and remain a huge threat to global public health. Rapid and sensitive diagnosis of causative agents is critical for timely treatment and disease control. Here, we developed a novel method based on recombinase polymerase amplification (RPA) combined with CRISPR-Cas12a to detect three viral pathogens, including SARS-CoV-2, influenza A, and influenza B, which cause similar symptom complexes of flu cold in the respiratory tract. The detection method can be completed within 1 h, which is faster than other standard detection methods, and the limit of detection is approximately 10 2 copies/μL. Additionally, this detection system is highly specific and there is no cross-reactivity with other common respiratory tract pathogens. Based on this assay, we further developed a more simplified RPA/CRISPR-Cas12a system combined with lateral flow assay on a manual microfluidic chip, which can simultaneously detect these three viruses. This low-cost detection system is rapid and sensitive, which could be applied in the field and resource-limited areas without bulky and expensive instruments, providing powerful tools for the point-of-care diagnostic., (© 2023 Wiley Periodicals LLC.)

Helicobacter pylori (H. pylori) is a zoonotic gastric microorganism capable of efficient interspecies transmission. Domesticated companion animals, particularly dogs and cats, serve as natural reservoirs for H. pylori. This phenomenon facilitates the extensive dissemination of H. pylori among households with pets. Hence, the prompt and precise identification of H. pylori in companion animals holds paramount importance for the well-being of both animals and their owners. With the assistance of Multienzyme Isothermal Rapid Amplification (MIRA) and CRISPR-Cas12a system, we successfully crafted a highly adaptable optical detection platform for H. pylori. Three sensor systems with corresponding visual interpretations were proposed. This study demonstrated a rapid turnaround time of approximately 45 min from DNA extraction to the result display. Moreover, this platform topped germiculture and real-time PCR in terms of sensitivity or efficiency in clinical diagnoses of 66 samples. This platform possesses significant potential as a versatile approach and represents the premiere application of CRISPR for the non-invasive detection of H. pylori in companion animals, thereby mitigating the dissemination of H. pylori among household members., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Background: Brucellosis is a common zoonotic disease caused by Brucella, which causes enormous economic losses and public burden to epidemic areas. Early and precise diagnosis and timely culling of infected animals are crucial to prevent the infection and spread of Brucella. In recent years, RNA-guided CRISPR/Cas12a(Clustered Regularly Interspaced Short Palindromic Repeats and its associated protein 12a) nucleases have shown great promise in nucleic acid detection. This research aims to develop a CRISPR/CAST (CRISPR/Cas12a Test strip) package that can rapidly detect Brucella nucleic acid during on-site screening, especially on remote family pastures. The CRISPR/Cas12a system combined with recombinase polymerase amplification (RPA), and lateral flow read-out., Results: We selected the conserved gene bp26, which commonly used in Brucella infection detection and compared on Genbank with other Brucella species. The genomes of Brucella abortus 2308, Brucella suis S2, Brucella melitansis 16 M, and Brucella suis 1330, et al. were aligned, and the sequences were found to be consistent. Therefore, the experiments were only performed on B. melitensis. With the CRISPR/CAST package, the assay of Brucella nucleic acid can be completed within 30 min under isothermal temperature conditions, with a sensitivity of 10 copies/μl. Additionally, no antigen cross-reaction was observed against Yersinia enterocolitica O:9, Escherichia coli O157, Salmonella enterica serovar Urbana O:30, and Francisella tularensis. The serum samples of 398 sheep and 100 cattle were tested by the CRISPR/CAST package, of which 31 sheep and 8 cattle were Brucella DNA positive. The detection rate was consistent with the qPCR results and higher than that of the Rose Bengal Test (RBT, 19 sheep and 5 cattle were serum positive)., Conclusions: The CRISPR/CAST package can accurately detect Brucella DNA in infected livestock within 30 min and exhibits several advantages, including simplicity, speed, high sensitivity, and strong specificity with no window period. In addition, no expensive equipment, standard laboratory, or professional operators are needed for the package. It is an effective tool for screening in the field and obtaining early, rapid diagnoses of Brucella infection. The package is an efficient tool for preventing and controlling epidemics., (© 2023. BioMed Central Ltd., part of Springer Nature.)

With the increasing acreage of genetically modified crops worldwide, rapid and efficient detection technologies have become very important for the regulation and screening of GM organisms. We constructed a method based on loop-mediated isothermal amplification (LAMP), CRISPR-Cas12a and lateral flow assay (LAMP-CRISPR-Cas12a-LFA). It is an intuitive, sensitive and specific fluorescence detection and test strip system to detect CP4-EPSPS and Cry1Ab/Ac genes in field screening. The LAMP-CRISPR-Cas12a-LFA method has a limit of detection (LOD) of 100 copies based on lateral flow test strips after optimization of the conditions with screened specific primers, and the entire detection process can be completed within 1 h at 61 °C. The system was used to evaluate field test samples and showed high reproducibility after testing products containing CP4-EPSPS and Cry1Ab/Ac genes, and both were detectable. The LAMP-CRISPR-Cas12a-LFA method established in this paper functions as a rapid field detection method. It requires only one portable thermostatic instrument, which renders it compatible with the rapid detection of field samples and useable at experimental workstations, in law enforcement field work, and in local inspection and quarantine departments., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Deformed wing virus (DWV) is one of the important pathogens of the honey bee ( Apis mellifera) , which consists of three master variants: types A, B, and C. Among them, DWV types A (DWV-A) and B (DWV-B) are the most prevalent variants in honey bee colonies and have been linked to colony decline. DWV-A and DWV-B have different virulence, but it is difficult to distinguish them via traditional methods. In this study, we established a visual detection assay for DWV-A and DWV-B using recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) 12a fluorescence system (RPA-CRISPR-Cas12a-LFD). The limit of detection of this system was ~6.5 × 10 0 and 6.2 × 10 1 copies/μL for DWV-A and DWV-B, respectively. The assays were specific and non-cross-reactive against other bee viruses, and the results could be visualized within 1 h. The assays were validated by extracting cDNA from 36 clinical samples of bees that were suspected to be infected with DWV. The findings were consistent with those of traditional reverse transcription-quantitative polymerase chain reaction, and the RPA-CRISPR-Cas12a assay showed the specific, sensitive, simple, and appropriate detection of DWV-A and DWV-B. This method can facilitate the visual and qualitative detection of DWV-A and DWV-B as well as the monitoring of different subtypes, thereby providing potentially better control and preventing current and future DWV outbreaks.

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., (© 2023 Wiley-VCH GmbH.)

The persistent infection of high-risk-human papillomavirus type 16 (HPV16) is considered an essential element for suffering cervical cancer. Despite polymerase chain reaction, loop-mediated amplification, and microfluidic chips are used to detect the HPV16, these methods still exist some drawbacks including time-consuming and false positive results. The CRISPR-Cas system is widely used in the region of biological detection due to its precise targeted recognition capability. In this contribution, the novel solution-gated graphene transistor sensor is designed to realize the unamplified and label-free detection of HPV16 DNA. Using the precise recognition of the CRISPR-Cas12a system and the gate functionalization, HPV16 DNA can be precisely identified without need the amplification and labeling. The limit of detection of the sensor can be up to 8.3 × 10 -18  m and the detection can be within 20 min. Additionally, the heat-Inactivated clinical samples can be clearly distinguished by the sensor the diagnosis results have a high degree of agreement with q-PCR detection., (© 2023 Wiley-VCH GmbH.)