Search

Technological advances have led to the emergence of lineage tracers, but signal recorders for mammalian systems have remained elusive. Kempton et al. have developed a Cas12a base-editing signal recorder capable of capturing diverse signals and operating in various experimental designs. The recorder enables new opportunities to chronicle cellular history. [ABSTRACT FROM AUTHOR]

Abstract 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.

Copyright of Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao is the property of Chinese Journal of Preventive Veterinary Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Recent advances in the development of CRISPR-Cas genome editing technologies have made it possible to perform targeted mutagenesis and precise gene replacement in crop plants. CRISPR-Cas9 and CRISPR-Cas12a are two main types of widely used genome editing systems. However, when CRISPR-Cas12a editing machinery is expressed from a transgene, some chromosomal targets encountered low editing frequency in important crops like maize and soybean. Here, we report efficient methods to directly generate genome edited lines by delivering Cas12a-gRNA ribonucleoprotein complex (RNP) to immature maize embryos through particle bombardment in an elite maize variety. Genome edited lines were obtained at ~7% frequency without any selection during regeneration via biolistic delivery of Cas12a RNP into immature embryos. Strikingly, the gene editing rate was increased to 60% on average and up to 100% in some experiments when the Cas12a RNP was co-delivered with a PMI selectable marker gene cassette and the induced callus cultures were selected with mannose. We also show that use of higher activity Cas12a mutants resulted in improved editing efficiency in more recalcitrant target sequence. The advances described here provide useful tools for genetic improvement of maize.

Clustered regularly interspaced short palindromic repeats (CRISPR) is a promising technology in the biological world. As one of the CRISPR‐associated (Cas) proteins, Cas12a is an RNA‐guided nuclease in the type V CRISPR‐Cas system, which has been a robust tool for gene editing. In addition, due to the discovery of target‐binding‐induced indiscriminate single‐stranded DNase activity of Cas12a, CRISPR‐Cas12a also exhibits great promise in biosensing. This minireview not only gives a brief introduction to the mechanism of CRISPR‐Cas12a but also highlights the recent developments and applications in biosensing and gene regulation. Finally, future prospects of the CRISPR‐Cas12a system are also discussed. We expect this minireview will inspire innovative work on the CRISPR‐Cas12a system by making full use of its features and advantages. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome engineering and related technologies have revolutionized biotechnology over the last decade by enhancing the efficiency of sophisticated biological systems. Cas12a (Cpf1) is an RNA-guided endonuclease associated to the CRISPR adaptive immune system found in many prokaryotes. Contrary to its more prominent counterpart Cas9, Cas12a recognizes A/T rich DNA sequences and is able to process its corresponding guide RNA directly, rendering it a versatile tool for multiplex genome editing efforts and other applications in biotechnology. While Cas12a has been extensively used in eukaryotic cell systems, microbial applications are still limited. In this review, we highlight the mechanistic and functional differences between Cas12a and Cas9 and focus on recent advances of applications using Cas12a in bacterial hosts. Furthermore, we discuss advantages as well as current challenges and give a future outlook for this promising alternative CRISPR-Cas system for bacterial genome editing and beyond. Key points: • Cas12a is a powerful tool for genome engineering and transcriptional perturbation • Cas12a causes less toxic side effects in bacteria than Cas9 • Self-processing of crRNA arrays facilitates multiplexing approaches [ABSTRACT FROM AUTHOR]

The impact of porcine circovirus (PCV) on the worldwide pig industry is profound, leading to notable economic losses. Early and prompt identification of PCV is essential in managing and controlling this disease effectively. A range of detection techniques for PCV have been developed and primarily divided into two categories focusing on nucleic acid or serum antibody identification. The methodologies encompass conventional polymerase chain reaction (PCR), real-time fluorescence quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), loop-mediated isothermal amplification (LAMP), immunofluorescence assay (IFA), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). Despite their efficacy, these techniques are often impeded by the necessity for substantial investment in equipment, specialized knowledge, and intricate procedural steps, which complicate their application in real-time field detections. To surmount these challenges, a sensitive, rapid, and specific PCV detection method using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a/13a coupled with isothermal amplification, such as enzymatic recombinase amplification (ERA), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP), has been developed. This novel method has undergone meticulous optimization for detecting PCV types 2, 3, and 4, boasting a remarkable sensitivity to identify a single copy per microliter. The specificity of this technique is exemplary, with no observable interaction with other porcine viruses such as PEDV, PRRSV, PRV, and CSFV. Its reliability has been validated with clinical samples, where it produced a perfect alignment with qPCR findings, showcasing a 100% coincidence rate. The elegance of merging CRISPR-Cas technology with isothermal amplification assays lies in its on-site testing without the need for expensive tools or trained personnel, rendering it exceptionally suitable for on-site applications, especially in resource-constrained swine farming environments. This review assesses and compares the process and characteristics inherent in the utilization of ERA/LAMP/RPA-CRISPR-Cas12a/Cas13a methodologies for the detection of PCV, providing critical insights into their practicality and effectiveness.

Strawberry anthracnose, caused by Colletotrichum spp., is a devastating disease that significantly reduces strawberry yield and quality. This study aimed to develop a simple diagnostic method to detect infection by the Colletotrichum gloeosporioides complex (CGSC), the most predominant and virulent Colletotrichum species complex causing strawberry anthracnose in China. In this study, a Cas12aVIP diagnostic method was developed for the rapid detection of CGSC in strawberry seedlings. This method targets the β-tubulin gene and combines recombinase polymerase amplification (RPA), the CRISPR/Cas12a system, and a cationic-conjugated polythiophene derivative [poly(3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT)] mixed with single-stranded DNA (ssDNA). This method shows high sensitivity (ten copies per reaction) and no cross-reactivity against related pathogens. The entire procedure, from sample to result, can be completed within 50 min, including simplified DNA extraction (15 min), RPA reaction (37°C for 20 min), CRISPR/Cas12a detection (37°C for 10 min), and visual detection by the naked eye (1-2 min). Furthermore, the Cas12aVIP assay successfully detected CGSC in naturally infected strawberry seedling samples in field conditions. Asymptomatic infected plants and plant residues have been identified as primary inoculum sources for CGSC. This method enables visible detection without the need for expensive equipment or specialized technical skills, thereby offering an efficient and straightforward approach for detecting CGSC in strawberries. The newly developed detection method can be used to promote healthier strawberry production.

CRISPR/Cas12a has been widely used in molecular diagnostics due to its excellent trans-cleavage activity. However, conventional reporters, such as F/Q-labeled single-stranded DNA (ssDNA) reporters, enzyme-labeled reporters, and spherical nucleic acid reporters, require complex modification or labeling processes. In this study, we have developed a rapid, universal, and label-free CRISPR/Cas12a-based biomarker detection platform via designing a G-quadruplex (G4) containing a hairpin structure as the reporter. The hairpin loop design of hairpin G4 improves the cleavage efficiency of Cas12a and the signal strength of the G4 binding ligand. Meanwhile, the incorporation of a G4 binding dye (protoporphyrin IX) eliminates the need for complex modifications. The CRISPR-hairpin G4 detection platform is capable of detecting ssDNA, double-stranded DNA, genetic RNAs, and miRNAs. Moreover, this platform achieves label-free detection in clinical samples, demonstrating its practical applicability and efficiency.

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., Competing Interests: The authors declare no conflict of interest.

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., (© 2024. The Author(s).)

Theileria parvacauses East Coast fever (ECF), one of the most important and lethal tick-borne diseases of cattle in sub-Saharan Africa. ECF is a considerable burden to the livestock industry, causing annual losses exceeding US $300 million. Currently, diagnosis of T. parva infections relies mainly on clinical signs, serology, and microscopic identification of parasites in either blood or lymph fluid samples. However, some of these tests might not indicate ongoing infection and they all lack the sensitivity to detect low-level infections. Molecular tests such as nested and quantitative PCR assays offer high sensitivity for detection of T. parva. However, these tests remain highly complex technologies that are impractical to use in resource-limited settings where economic losses due to the disease have the most significant impact. A field-deployable, point-of-care test will be of significant value in the treatment and control of ECF in endemic areas. For this purpose, we have developed a CRISPR-Cas12a-based pen-side tool that can sensitively and specifically detect T. parva based on the p104 gene. We describe a streamlined, field-applicable diagnostic tool comprising a 20 min recombinase polymerase amplification (RPA) reaction followed by a 60 min CRISPR-Cas12a reaction using a FAM/Biotin lateral flow strip readout. We tested two different RPA primer pairs and four different CRISPR-RNAs (crRNAs). The p104-based assay displayed high sensitivity, detecting as low as one infected lymphocyte per three microliters of blood and universally detecting eight different T. parva strains without detecting DNA from other Theileria spp. such as Theileria mutans and Theileria lestoquardi. This work opens the way for a field-applicable diagnostic tool for the sensitive point-of-care early diagnosis of T. parva infections in cattle., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The Penicillium genus exhibits a broad global distribution and holds substantial economic value in sectors including agriculture, industry, and medicine. Particularly in agriculture, Penicillium species significantly impact plants, causing diseases and contamination that adversely affect crop yields and quality. Timely detection of Penicillium species is crucial for controlling disease and preventing mycotoxins from entering the food chain. To tackle this issue, we implement a novel species identification approach called Analysis of whole GEnome (AGE). Here, we initially applied bioinformatics analysis to construct specific target sequence libraries from the whole genomes of seven Penicillium species with significant economic impact: P. canescens , P. citrinum , P. oxalicum , P. polonicum , P. paneum , P. rubens , and P. roqueforti . We successfully identified seven Penicillium species using the target we screened combined with Sanger sequencing and CRISPR-Cas12a technologies. Notably, based on CRISPR-Cas12a technology, AGE can achieve rapid and accurate identification of genomic DNA samples at a concentration as low as 0.01 ng/µL within 30 min. This method features high sensitivity and portability, making it suitable for on-site detection. This robust molecular approach provides precise fungal species identification with broad implications for agricultural control, industrial production, clinical diagnostics, and food safety.

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

Most viruses that infect plants use RNA to carry their genomic information; timely and robust detection methods are crucial for efficient control of these diverse pathogens. The RNA viruses, potexvirus (Potexvirus, family Alphaflexiviridae), potyvirus (Potyvirus, family Potyviridae), and tobamovirus (Tobamovirus, family Virgaviridae) are among the most economically damaging pathogenic plant viruses, as they are highly infectious and distributed worldwide. Their infection of crop plants, alone or together with other viruses, causes severe yield losses. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and others have been harnessed for the detection of DNA- and RNA-based viruses. However, they have a high rate of non-specific amplification and other drawbacks. The collateral activities of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems such as Cas12 and Cas14 (which act on ssDNA) and Cas13 (which acts on ssRNA) have recently been exploited to develop highly sensitive, specific, and rapid detection platforms. Here, we report the development of a simple, rapid, and efficient RT- RPA method, coupled with a CRISPR/Cas12a-based one-step detection assay, to detect plant RNA viruses. This diagnostic method can be performed at a single temperature in less than 30 min and integrated with an inexpensive commercially available fluorescence visualizer to facilitate rapid, in-field diagnosis of plant RNA viruses. Our developed assay provides an efficient and robust detection platform to accelerate plant pathogen detection and fast-track containment strategies.

A novel strain of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) disease (COVID-19) has been recently identified as an infectious disease affecting the respiratory system of humans. This disease is caused by SARS-CoV-2 that was identified in Chinese patients having severe pneumonia and flu-like symptoms. COVID-19 is a contagious disease that spreads rapidly via droplet particles arising through sneezing and coughing action of an infected person. The reports of asymptomatic carriers changed the scenario of symptom based-diagnosis in COVID-19 and intensified the need for proper diagnosis of the majority of the population to combat the rapid transmission of virus. The diagnosis of positive cases is necessary to ensure prompt care to affected people and also to curb further spread of infection in the population. Collecting samples at the right time and from the exact anatomical site is crucial for proper molecular diagnosis. After the complete genome sequence was available, China formulated RT-PCR as a primary diagnostic procedure for detecting SARS-CoV-2. Many in-house and commercial diagnostic kits have been developed or are under development that have a potential to lower the burden of diagnosis on the primary diagnostic techniques like RT-PCR. Serological based diagnosis is another broad category of testing that can detect different serum antibodies like IgG, IgM, and IgA in an infected patient. PCR-based diagnostic procedures that are commonly used for pathogen detection need sophisticated machines and assistance of a technical expert. Despite their reliable accuracy, they are not cost-effective tests, which a common man can afford, so it becomes imperative to look for other diagnostic approaches, which could be cost effective, rapid, and sensitive with consistent accuracy. To make such diagnostics available to the common man, many techniques can be exploited among, which are Point of Care (POC), also known as bed side testing, which is developing as a portable and promising tool in pathogen diagnosis. Other lateral flow assay (LFA)-based techniques like SHERLOCK, CRISPR-Cas12a (AIOD-CRISPR), and FNCAS9 editor-limited uniform detection assay (FELUDA), etc. have shown promising results in rapid detection of pathogens. Diagnosis holds a critical importance in the pandemic situation when there is no potential drug for the pathogen available in the market. This review sums up the different diagnostic approaches designed or proposed to combat the crisis of widespread diagnosis due to the sudden outbreak of a novel pathogen, SARS-CoV-2 in 2019.

Enterocytozoon hepatopenaei (EHP) is a parasite that infects pacific whiteleg shrimp, Penaeus vannamei, causing growth retardation and uneven size distributions that lead to severe losses in shrimp productivity. Routine monitoring is crucial to timely prevention and management of EHP, but field-deployable diagnostic kits for EHP are still scarce. Here, we proposed the use of recombinase polymerase amplification (RPA) and CRISPR-Cas12a fluorescence assay, henceforth RPA-Cas12a, for detection of EHP. Targeting ptp2 gene, RPA-Cas12a could detect as few as 50 copies of DNA and showed no reactivity with closely related microsporidia. The entire procedure could be performed at a temperature close to 37 °C within 1 h. Naked eye visualization was possible with UV/blue-light excitation or lateral flow detection. Thus, RPA-Cas12a is a rapid, sensitive and specific detection platform that requires no sophisticated equipment and shows promise for on-site surveillance of EHP.

Amycolatopsis mediterranei U32 is an industrial producer of rifamycin SV, whose derivatives have long been the first-line antimycobacterial drugs. In order to perform genetic modification in this important industrial strain, a lot of efforts have been made in the past decades and a homologous recombination-based method was successfully developed in our laboratory, which, however, requires the employment of an antibiotic resistance gene for positive selection and did not support convenient markerless gene deletion. Here in this study, the clustered regularly interspaced short palindromic repeat (CRISPR) system was employed to establish a genome editing system in A. mediterranei U32. Specifically, the Francisella tularensis subsp. novicida Cas12a (FnCas12a) gene was first integrated into the U32 genome to generate target-specific double-stranded DNA (dsDNA) breaks (DSBs) under the guidance of CRISPR RNAs (crRNAs). Then, the DSBs could be repaired by either the non-homologous DNA end-joining (NHEJ) system or the homology-directed repair (HDR) pathway, generating inaccurate or accurate mutations in target genes, respectively. Besides of A. mediterranei, the present work may also shed light on the development of CRISPR-assisted genome editing systems in other species of the Amycolatopsis genus.

Abstract 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.

The efficient signal amplification capacity of several class 2 CRISPR-Cas systems with trans-cleavage activity has exhibited great value in molecular diagnostics, but its potential application for non-nucleic-acid targets is yet underdeveloped. Here, we deploy CRISPR-Cas system for the ultrasensitive detection of protease biomarkers by the coupling of proteolysis-triggered transcription. In this strategy, a protease-activatable RNA polymerase is adopted for the conversion of each protease-catalyzed proteolysis event into the output of multiple programable RNA sequences by in vitro transcription, and the transcribed RNA subsequently serves as the guide RNA of Cas12a proteins with trans-cleavage activity. The rational design of the transcribed RNA efficiently couples the signal conversion and amplification of proteolysis-triggered transcription and the self-signal amplification of CRISPR-Cas12a, resulting in a two-stage amplified detection of target protease. The versatility of this strategy has been demonstrated in the detection of protease biomarkers including MMP-2 and thrombin with femtomolar sensitivity, which is 5–6 orders of magnitude lower than that of the standard peptide-based methods. Moreover, the proposed method has been further applied in the analysis of MMP-2 secreted by different cancer cell lines as well the assessment of MMP-2 activity in clinical serum samples, providing a generic method for the ultrasensitive detection of protease biomarkers in biochemical research and clinical diagnosis. [ABSTRACT FROM AUTHOR]

Rapid and sensitive detection of various analytes is in high demand. Apart from its application in genome editing, CRISPR–Cas also shows promises in nucleic acid detection applications. To further exploit the potential of CRISPR–Cas for detection of diverse analytes, we present a versatile biosensing platform that couples the excellent affinity of aptamers for broad-range analytes with the collateral single-strand DNA cleavage activity of CRISPR–Cas12a. We demonstrated that the biosensors developed by this platform can be used to detect protein and small molecule in human serum with a complicated background, i.e., the tumor marker alpha fetoprotein and cocaine with the detection limits of 0.07 fmol/L and 0.34 μmol/L, respectively, highlighting the advantages of simplicity, sensitivity, short detection time, and low cost compared with the state-of-the-art biosensing approaches. Altogether, this biosensing platform with plug-and-play design show great potential in the detection of diverse analytes. [ABSTRACT FROM AUTHOR]

Most viruses that infect plants use RNA to carry their genomic information; timely and robust detection methods are crucial for efficient control of these diverse pathogens. The RNA viruses, potexvirus (Potexvirus , family Alphaflexiviridae), potyvirus (Potyvirus , family Potyviridae), and tobamovirus (Tobamovirus , family Virgaviridae) are among the most economically damaging pathogenic plant viruses, as they are highly infectious and distributed worldwide. Their infection of crop plants, alone or together with other viruses, causes severe yield losses. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and others have been harnessed for the detection of DNA- and RNA-based viruses. However, they have a high rate of non-specific amplification and other drawbacks. The collateral activities of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems such as Cas12 and Cas14 (which act on ssDNA) and Cas13 (which acts on ssRNA) have recently been exploited to develop highly sensitive, specific, and rapid detection platforms. Here, we report the development of a simple, rapid, and efficient RT- RPA method, coupled with a CRISPR/Cas12a-based one-step detection assay, to detect plant RNA viruses. This diagnostic method can be performed at a single temperature in less than 30 min and integrated with an inexpensive commercially available fluorescence visualizer to facilitate rapid, in-field diagnosis of plant RNA viruses. Our developed assay provides an efficient and robust detection platform to accelerate plant pathogen detection and fast-track containment strategies. [ABSTRACT FROM AUTHOR]

A novel strain of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) disease (COVID-19) has been recently identified as an infectious disease affecting the respiratory system of humans. This disease is caused by SARS-CoV-2 that was identified in Chinese patients having severe pneumonia and flu-like symptoms. COVID-19 is a contagious disease that spreads rapidly via droplet particles arising through sneezing and coughing action of an infected person. The reports of asymptomatic carriers changed the scenario of symptom based-diagnosis in COVID-19 and intensified the need for proper diagnosis of the majority of the population to combat the rapid transmission of virus. The diagnosis of positive cases is necessary to ensure prompt care to affected people and also to curb further spread of infection in the population. Collecting samples at the right time and from the exact anatomical site is crucial for proper molecular diagnosis. After the complete genome sequence was available, China formulated RT-PCR as a primary diagnostic procedure for detecting SARS-CoV-2. Many in-house and commercial diagnostic kits have been developed or are under development that have a potential to lower the burden of diagnosis on the primary diagnostic techniques like RT-PCR. Serological based diagnosis is another broad category of testing that can detect different serum antibodies like IgG, IgM, and IgA in an infected patient. PCR-based diagnostic procedures that are commonly used for pathogen detection need sophisticated machines and assistance of a technical expert. Despite their reliable accuracy, they are not cost-effective tests, which a common man can afford, so it becomes imperative to look for other diagnostic approaches, which could be cost effective, rapid, and sensitive with consistent accuracy. To make such diagnostics available to the common man, many techniques can be exploited among, which are Point of Care (POC), also known as bed side testing, which is developing as a portable and promising tool in pathogen diagnosis. Other lateral flow assay (LFA)-based techniques like SHERLOCK, CRISPR-Cas12a (AIOD-CRISPR), and FNCAS9 editor-limited uniform detection assay (FELUDA), etc. have shown promising results in rapid detection of pathogens. Diagnosis holds a critical importance in the pandemic situation when there is no potential drug for the pathogen available in the market. This review sums up the different diagnostic approaches designed or proposed to combat the crisis of widespread diagnosis due to the sudden outbreak of a novel pathogen, SARS-CoV-2 in 2019. [ABSTRACT FROM AUTHOR]

For the first time, three 2-input elementary AND, OR, INHIBIT logic gates have been constructed by using CRISPR-Cas12a system. These logic gates utilised the intrinsic advantages of programmability, sequence specificity and high base resolution of CRISPR-Cas12a system. Among them, the AND gate owned the potentials as a built-in biosensor that responded rapidly to external pathogenic bacteria such as Staphylococcus aureus with high sensitivity and specificity. We applied the CRISPR-Cas12a based bacterial detection after a target-amplification using PCR. The total sample-to-answer time was appropriately 2.0 h, the limit of detection (LOD) was 103 CFU/mL, and the dynamic range was 103–107 CFU/mL. Also, the sequence addressability enabled this AND logic gate to accurately trace back and distinguish input genes. These above-mentioned features were highly ideal to incur a rapid response to pathogenic bacteria for decision making. Our results not only validated the possibility of using CRISPR-Cas systems for constructing bio-computing devices but also provided a prototype of biosensor for rapid and intelligent pathogenic bacteria detection. Image 1 • For the first time, three 2-input elementary AND, OR, INHIBIT logic gates have been constructed by CRISPR-Cas12a system. • The AND gate responded rapidly to Staphylococcus aureus with high sensitivity and specificity. • The total time was 2.0 h, the limit of detection (LOD) was 103 CFU/mL, and the dynamic range was 103–107 CFU/mL. • The sequence addressability enabled this AND logic gate to accurately trace back and distinguish input genes. • These above-mentioned features were highly ideal to incur a rapid response to pathogenic bacteria for decision making. [ABSTRACT FROM AUTHOR]

In this study, an ultrasensitive electrochemiluminescence (ECL) aptasensing method was developed for lipopolysaccharide (LPS) determination based on CRISPR-Cas12a accessory cleavage activity. Tris (2,2'-bipyridine) dichlororuthenium (II) (Ru(bpy) 3 2+) was adsorbed on the surface of a glassy carbon electrode (GCE) coated with a mixture of gold nanoparticles (AuNPs) and Nafion film via electrostatic interaction. The obtained ECL platform (Ru(bpy) 3 2+/AuNP/Nafion/GCE) exhibited strong ECL emission. Thiol-functionalized single-stranded DNA (ssDNA) was modified with a ferrocenyl (Fc) group and autonomously assembled on the ECL platform of Ru(bpy) 3 2+/AuNP/Nafion/GCE via thiol-gold bonding, resulting in the quenching of ECL emission. After hybridization of the LPS aptamer strand (AS) with its partial complementary strand (CS), the formed double-stranded DNA (dsDNA) could activate CRISPR-Cas12a to indiscriminately cleave ssDNA-Fc on the surface of Ru(bpy) 3 2+/AuNP/Nafion/GCE, resulting in recovery of the ECL intensity of Ru(bpy) 3 2+ due to the increasing distance between Fc and the electrode surface. The combination of LPS and AS suppressed the formation of dsDNA, inhibited the activation of CRISPR-Cas12a, and prevented further cleavage of ssDNA-Fc. This mechanism aided in upholding the integrity of ssDNA-Fc on the surface of the electrode and was combined with ECL quenching induced by the target. The ECL intensity decreased linearly as the concentration of LPS increased from 1 to 50,000 pg/mL and followed a logarithmic relationship. This method exhibited a remarkably low detection limit of 0.24 pg/mL, which meets the requirement for low-concentration detection of LPS in the human body. The proposed method demonstrates the capacity of CRISPR-Cas12a to perform non-specific cutting of single-stranded DNA and transform the resultant cutting substances into changes in the ECL signal. By amalgamating this approach with the distinct identification abilities of LPS and its aptamers, a simple, responsive, and discriminatory LPS assay was established that holds immense significance for clinical diagnosis. [Display omitted] • An ECL aptasensing method for the determination of LPS was proposed. • The method was based on CRISPR-Cas12a accessory cleavage activity. • The method achieved a lower DL of 0.24 pg/mL for detection of LPS. • The method can be applied to determine LPS in human serum samples. [ABSTRACT FROM AUTHOR]

Given the highly mutagenic and carcinogenic nature of Aflatoxin M1 (AFM1), the quantity assessment of AFM1 residues in milk and dairy products is necessary to maintain consumer health and food safety. Herein, CRISPR-Cas12a-based colorimetric aptasensor was developed using the catalytic activity of flower-like nanozymes of MnO 2 and trans -cleavage property of CRISPR-Cas12a system to quantitatively detect AFM1. The basis of the developed colorimetric aptasensor relies on whether or not the CRISPR-Cas12a system is activated, as well as the contrast in oxidase-mimicking capability exhibited by flower-like MnO 2 nanozymes when AFM1 is absent or present. When AFM1 is not present in the sample, single-stranded DNA (ssDNA) is degraded by the activated CRISPR-Cas12a, and the solution turns into yellow due to the catalytic activity of the nanozymes. While, in the attendance of AFM1, ssDNA degradation does not occur due to the inactivation of the CRISPR-Cas12a. Therefore, with the adsorption of the ssDNA on the MnO 2 nanozymes, their catalytic activity decreases, and the solution color becomes pale yellow due to less oxidation of the chromogenic substrate. In this aptasensor, the relative absorbance changes increased linearly from 6 to 160 ng L−1, and the detection limit was 2.1 ng L−1. The developed aptasensor displays a selective detection performance and a practical application for quantitative analysis of AFM1 in milk samples. The results of the introduced aptasensor open up the way to design other selective and sensitive aptasensors for the detection of other mycotoxins by substitution of the used sequences. [Display omitted] • CRISPR-Cas12a-based colorimetric aptasensor was developed for detection of AFM1. • The biosensor used cleavage property of the CRISPR-Cas12a, and its combination with aptamer and MnO 2 as nanozyme. • The oxidase-like activity of the flower-like nanozyme was contingent upon the activation and deactivation of CRISPR-Cas12a. • The aptasensor showed a LOD of 2.1 ng L−1. • The aptasensor displayed a practical application for quantitative analysis of AFM1 in milk sample. [ABSTRACT FROM AUTHOR]

The significant role of cell-free DNA (cfDNA) for disease diagnosis, including cancer, has garnered a lot of attention. The challenges of creating target-specific primers and the possibility of false-positive signals make amplification-based detection methods problematic. Fluorescent biosensors based on CRISPR-Cas have been widely established, however they still require an amplification step before they can be used for detection. To detect cfDNA, researchers have created a CRISPR-Cas12a-based nucleic acid amplification-free fluorescent biosensor that uses a combination of fluorescence and colorimetric signaling improved by duplex-specific nuclease (DSN). DSN-assisted signal recycling is initiated in H1@MBs when the target cfDNA activates the CRISPR-Cas12a complex, leading to the degradation of single-strand DNA (ssDNA) sequences. This method has an extremely high detection limit for the BRCA-1 breast cancer gene. In addition to measuring viral DNA in a field-deployable and point-of-care testing (POCT) platform, this fast and highly selective sensor can be used to evaluate additional nucleic acid biomarkers. [Display omitted] • The approach output both colorimetry and fluorescence signals. • The approach is highly-possible to greatly reduce outside noise and increase the precision of the analysis. • The approach displays remarkable selectivity thanks to CRISPR-Cas12a system based accurate target recognition. [ABSTRACT FROM AUTHOR]

The detection of foodborne pathogens is crucial for ensuring the maintenance of food safety. In the present study, a portable CRISPR-Cas12a triggered photothermal biosensor integrating branch hybrid chain reaction (bHCR) and DNA metallization strategy for sensitive and visual detection of foodborne pathogens was proposed. The sheared probes were utilized to block the locker probes, which enabled preventing the assembly of bHCR in the absence of target bacteria, while target bacteria can activate the cleavage of sheared probes through CRISPR-Cas12a. Therefore, the locker probes functioned as initiating chains, triggering the formation of the branching double-stranded DNA consisting of H1, H2, and H3. The silver particles, which were in situ deposited on the DNA structure, functioned as a signal factor for conducting photothermal detection. Staphylococcus aureus and Listeria monocytogenes were selected as the foodborne pathogens to verify the analytical performance of this CRISPR-Cas12a triggered photothermal sensor platform. The sensor exhibited a sensitive detection with a low detection limit of 1 CFU/mL, while the concentration ranged from 100 to 108 CFU/mL. Furthermore, this method could efficiently detect target bacteria in multiple food samples. The findings demonstrate that this strategy can serve as a valuable reference for the development of a portable platform enabling quantitative analysis, visualization, and highly sensitive detection of foodborne bacteria. [Display omitted] • The introduction of CRISPR-Cas12a and bHCR can significantly enhance the detection accuracy and sensitivity. • Silver nanoparticle-mediated DNA metallization strategy was in the CRISPR-Cas12a system first, providing a photothermal signal for detection. • The photothermal signal can be conveniently captured using a portable infrared camera, eliminating the reliance on large analytical equipment. • Using S. aureus and L. monocytogenes as examples, the proposed approach displayed an exciting analytical ability, where an excellent linear correlation at concentrations ranging from 100 to 108 CFU/mL was obtained, which is fully competent for the food safety test. [ABSTRACT FROM AUTHOR]

Development of new diagnostic methods is essential for disease diagnosis and treatment. In this work, we present a stimuli-responsive incremental DNA machine auto-catalyzed CRISPR-Cas12a (SRI-DNA machine/CRISPR-Cas12a) feedback amplification for ultrasensitive molecular detection of miRNA-21, which is an important biomarker related closely to the initiation and development of cancers, such as esophageal cancer. Strategically, the powerful SRI-DNA machine and efficient trans -cleavage activity of the CRISPR-Cas12a system are ingeniously integrated via a rationally designed probe termed as stem-elongated functional hairpin probe (SEF-HP). The SRI-DNA machine begins with the target miRNA, the trigger of the reaction, binding complementarily to the SEF-HP, followed by autonomously performed mechanical strand replication, cleavage, and displacement circuit at multiple sites. This conversion process led to the amplified generation of numerous DNA activators that are complementary with CRISPR RNA (CrRNA). Once formed the DNA activator/CrRNA heteroduplex, the trans -cleavage activity of the CRISPR-Cas12a was activated to nonspecific cleavage of single-stranded areas of a reporter probe for fluorescence emission. Under optimal conditions, the target miRNA can be detected with a wide linear range and an excellent specificity. As a proof-of-concept, this SRI-DNA machine/CRISPR-Cas12a feedback amplification system is adaptable and scalable to higher-order artificial amplification circuits for biomarkers detection, highlighting its promising potential in early diagnosis and disease treatment. [Display omitted] • Innovative SRI-DNA machine/CRISPR-Cas12a system for ultrasensitive miRNA-21 detection. • Autonomous mechanical processes enable exceptional sensitivity and specificity. • Potential implications for early diagnosis and treatment of esophageal cancer. • Superior performance compared to existing miRNA detection methods. [ABSTRACT FROM AUTHOR]

[Display omitted] • A QD-CRISPR-MNP biosensor was established for Salmonella detection. • Two different cleavage strategies for Salmonella detection were studied. • The biosensor was able to detect Salmonella as low as 86 CFU mL−1 with satisfactory specificity. • The biosensor showed the feasibility for Salmonella detection in spiked chicken breast extracts. Rapid detection methods are urgently needed to help prevent the spread of Salmonella among food products. Herein, a CRISPR-Cas12a-powered, quantum dot-based and magnetic nanoparticle-assisted (QD-CRISPR-MNP) biosensor was proposed for the screening of Salmonella. The QD-CRISPR-MNP biosensor was designed and fabricated relying on the CRISPR-Cas12a system for better guidance of the coupling between magnetic nanoparticles (MNPs) and quantum dots (QDs). In this biosensor, the activity of the CRISPR-Cas12a system was unlocked by Salmonella and displayed collateral cleavage activity towards linker DNAs, which inhibited the coupling of the MNPs and QDs. Then, Salmonella could be quantitatively detected by simply measuring the fluorescence emitted by the QDs on the MNPs surface. This biosensor showed good detection performance for Salmonella with a detection limit of 86 CFU mL−1, and exhibited a satisfactory specificity as well. Moreover, it overcomes the low fluorescence quantum yields and the possible high fluorescence background noise in conventional dye-based CRISPR detection platforms, acting as a powerful alternative for the detection of foodborne pathogens. [ABSTRACT FROM AUTHOR]

Base excision repair (BER) plays an essential role in the repair of DNA damage and maintenance of genomic integrity. As a major BER enzyme, human alkyladenine DNA glycosylase (hAAG) acts on various alkylated bases, and its abnormal activity may lead to elevated risks of different cancers. Herein, we develop a transcription-driven CRISPR RNA (crRNAs) auto-generation-mediated CRISPR-Cas12a system for sensitive and simple detection of hAAG. In this strategy, a hairpin probe containing a deoxyinosine base/T (I/T) pairs and a destroyed T7 promoter sequence serves not only as a probe for hAAG recognition but also as a template for transcription, efficiently avoiding nonspecific transcription. Target hAAG induces the hairpin probe cleavage and subsequent unfolding. The unfolded hairpin probe can initiate the KF polymerase-mediated polymerization reaction and subsequent T7 polymerase-mediated transcription reaction for the production of substantial crRNAs. The resultant crRNAs activate the trans-cleavage activity of CRISPR-Cas12a to cleave signal probes, generating an enhanced fluorescence signal. This strategy can measure hAAG with a limit of detection of 9.25 × 10−11 U/μL and a broad linear range of 1 × 10−10–1 × 10-2 U/μL, and it may accurately measure endogenous hAAG at the single-cell level. Furthermore, the assay can be applied for inhibitor screening, kinetic analysis, and the differentiation of tumor cells from healthy cells, with great potential in disease diagnosis. • We develop a CRISPR RNA auto-generation-mediated CRISPR-Cas12a system. • This strategy can sensitively measure hAAG with a broad linear range. • This strategy can detect endogenous hAAG at the single-cell level. • This strategy can be applied for inhibitor screening and kinetic analysis. • This strategy can differentiate tumor cells from healthy cells. [ABSTRACT FROM AUTHOR]

Long-lasting infection of high-risk HPV infection has long been recognized as the leading cause of cervical cancer and has garnered widespread attention. However, in resource-limited areas, effective HPV screening is often not easily accessible, resulting in a continuous growth in the incidence and mortality rates of cervical cancer in these areas. In this study, we present a rapid, efficient, and accurate method for HPV detection based on the CRISPR/Cas12a dual-chamber "one-pot" test (DROPT) system. Clinical samples are able to be rapidly tested in the DROPT system to get quantitative fluorescence readout with a simple one-time press. The entire reaction process, including RPA and CRISPR assays, happens within 30 min in a portable device composed of an isothermal heating chamber, laser diode source, and silicon photodiode. The results demonstrate that this approach achieves a remarkable limit of detection of 10−18 M. In the testing of HPV16/18 in 60 cervical swab samples, the system reached a sensitivity and specificity of 100% and 95.8%, respectively. Consequently, the DROPT system offers ease of use, requires no large-scale equipment or laboratory personnel, and effectively avoids aerosol contamination, which potentially fills the gap for HPV self-testing and on-site nucleic acid testing. • A integrated "use-and-throw" tube is designed to accommodate both RPA and CRISPR. • The DROPT tube achieve HPV self-testing and POCT with compatible portable device. • The DROPT system eliminates the need of complex operations and minimizes aerosol contamination. • The LOD of the DROPT system achieved 10−18 M in 30 min for HPV16/18 plasmids. • A specificity and sensitivity of 100% and 95.8% was achieved in the detection of HPV patient samples. [ABSTRACT FROM AUTHOR]

Detection of a pathogen is crucial prior to all prophylaxis and post exposure treatment, as it can prevent further disease manifestation. In this study, we have developed a nucleic acid pre-amplification based CRISPR diagnostic for detection and surveillance of Bacillus anthracis Sterne. Strand Invasion Based isothermal Amplification (SIBA) platform and Cas12a (CRISPR endo -nuclease) was used to develop CRISPR-SIBA, a multifaceted diagnostic platform. SIBA was employed as the isothermal pre-amplification platform. CRISPR-Cas12a based collateral trans-cleavage reaction was used to ensure and enhance the specificity of the system. Efficiency of the detection system was evaluated by detecting Bacillus anthracis Sterne in complex wastewater sample backgrounds. Previously reported, Prophage 3, Cya and Pag gene s of Bacillus anthracis were used as targets for this assay. The amplification system provided reliable and specific detection readout, with a sensitivity limit of 100 colony forming units in 40 min. The endpoint fluorescence from CRISPR collateral cleavage reactions gave a detection limit of 105 to 106 CFUs. The experiments conducted in this study provide the evidence for SIBA's applicability and compatibility with CRISPR-Cas system and its efficiency to specifically detect Bacillus anthracis Sterne. CRISPR-SIBA can be translated into developing cost-effective diagnostics for pathogens in resource constrained settings. [Display omitted] [ABSTRACT FROM AUTHOR]

Purpose: Accurate detection and identification of pathogens and their associated resistance mechanisms are essential prerequisites for implementing precision medicine in the management of Carbapenem-resistant Enterobacterales (CRE). Among the various resistance mechanisms, the production of KPC carbapenemase is the most prevalent worldwide. Consequently, this study aims to develop a convenient and precise nucleic acid detection platform specifically for the bla KPC gene., Methods: The initial phase of our research methodology involved developing a CRISPR/Cas12a detection framework, which was achieved by designing highly specific single-guide RNAs (sgRNAs) targeting the bla KPC gene. To enhance the sensitivity of this system, we incorporated three distinct amplification techniques-polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA)-into the CRISPR/Cas12a framework. Subsequently, we conducted a comparative analysis of the sensitivity and specificity of these three amplification methods when used in combination with the CRISPR/Cas12a system. Additionally, we assessed the clinical applicability of the methodologies by evaluating fluorescence readouts from 80 different clinical isolates. Furthermore, we employed lateral flow assay technology to provide a visual representation of the results, facilitating point-of-care testing., Results: Following a comparative analysis of the sensitivity and specificity of the three methods, we identified the RPA-Cas12a approach as the optimal detection technique. Our findings demonstrated that the limit of detection (LoD) of the RPA-Cas12a platform was 1 aM (~1 copy/µL) for plasmid DNA and 5 × 10³ fg/µL for genomic DNA. Furthermore, both the sensitivity and specificity of the platform achieved 100% upon validation with 80 clinical isolates., Conclusion: These findings suggest that the developed RPA-Cas12a platform represents a promising tool for the cost-effective, convenient, and accurate detection of the bla KPC gene., Competing Interests: The authors report no conflicts of interest in this work, including employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding., (© 2024 Li et al.)

CRISPR-Cas12a with robust trans-cleavage activity were employed to mitigate background fluorescence signal, achieving sensitive detection of miRNA-21. The activation of trans-cleavage activity of Cas12a was achieved by utilizing cDNA as a trigger. Upon the presence of target miRNA-21, cDNA hybridizes with it forming a DNA/RNA double-stranded structure. Exonuclease III (ExoIII) facilitates the degradation of cDNA, releasing the target for subsequent cycles. Due to cDNA degradation, the trans-cleavage activity of Cas12a remains unactivated and does not disrupt the synthesis template of copper nanoparticles. Addition of Cu 2+ and AA leads to the formation of highly fluorescent copper nanoparticles. Conversely, in absence of miRNA-21, intact cDNA activates trans-cleavage activity of Cas12a, resulting in degradation of the synthesis template and failure in synthesizing fluorescent copper nanoparticles. This method exhibits excellent selectivity with a low limit of detection (LOD) at 5 pM. Furthermore, we successfully applied this approach to determine miRNA-21 in cell lysates and human serum samples, providing a new approach for sensitive determination of biomarkers in biochemical research and disease diagnosis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The detection of carcinoembryonic antigen (CEA) holds significant importance in the early diagnosis of cancer. However, current methods are hindered by limited accessibility and specificity. This study proposes a rapid and convenient Cas12a-based assay for the direct detection of CEA in clinical serum samples, aiming to address these limitations. The protocol involves a rolling machine operation, followed by a 5-min Cas12a-mediated cleavage process. The assay demonstrates the capability to detect human serum with high anti-interference performance and a detection limit as low as 0.2 ng/mL. The entire testing procedure can be accomplished in 75 min without centrifugation steps, and successfully reduced the limit of detection of traditional DNA walking machine by 50 folds. Overall, the testing procedure can be easily implemented in clinical settings., (© 2024. The Author(s).)

The CRISPR-Cas12a system has emerged as a powerful tool for next-generation nucleic acid-based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA-enabled trans-cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by full-size RNA targets, although LbCas12a exhibits weaker trans-cleavage activity than AsCas12a on both single-stranded DNA and RNA substrates. Remarkably, we find that the RNA-activated Cas12a possesses higher specificity in recognizing mutated target sequences compared to DNA activation. Based on these findings, we develop the "Universal Nuclease for Identification of Virus Empowered by RNA-Sensing" (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM-less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay based on double-stranded DNA activation, demonstrating unrestricted target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a-based nucleic acid detection and further enhance the understanding of CRISPR-Cas biochemistry., (© 2024 Wiley-VCH GmbH.)

Global food safety stands out as a prominent public concern, affecting populations worldwide. The recurrent challenge of food safety incidents reveals the need for a robust inspection framework. In recent years, the integration of isothermal nucleic acid amplification with CRISPR-Cas12a techniques has emerged as a promising tool for molecular detection of food hazards, presenting next generation of biosensing for food safety detection. This paper provides a comprehensive review of the current state of research on the synergistic application of isothermal nucleic acid amplification and CRISPR-Cas12a technology in the field of food safety. This innovative combination not only enriches the analytical tools, but also improving assay performance such as sensitivity and specificity, addressing the limitations of traditional methods. The review summarized various detection methodologies by the integration of isothermal nucleic acid amplification and CRISPR-Cas12a technology for diverse food safety concerns, including pathogenic bacterium, viruses, mycotoxins, food adulteration, and genetically modified foods. Each section elucidates the specific strategies employed and highlights the advantages conferred. Furthermore, the paper discussed the challenges faced by this technology in the context of food safety, offering insightful discussions on potential solutions and future prospects.

Human bocavirus (HBoV) 1 is considered an important pathogen that mainly affects infants aged 6-24 months, but preventing viral transmission in resource-limited regions through rapid and affordable on-site diagnosis of individuals with early infection of HBoV1 remains somewhat challenging. Herein, we present a novel faster, lower cost, reliable method for the detection of HBoV1, which integrates a recombinase polymerase amplification (RPA) assay with the CRISPR/Cas12a system, designated the RPA-Cas12a-fluorescence assay. The RPA-Cas12a-fluorescence system can specifically detect target gene levels as low as 0.5 copies of HBoV1 plasmid DNA per microliter within 40 min at 37°C without the need for sophisticated instruments. The method also demonstrates excellent specificity without cross-reactivity to non-target pathogens. Furthermore, the method was appraised using 28 clinical samples, and displayed high accuracy with positive and negative predictive agreement of 90.9% and 100%, respectively. Therefore, our proposed rapid and sensitive HBoV1 detection method, the RPA-Cas12a-fluorescence assay, shows promising potential for early on-site diagnosis of HBoV1 infection in the fields of public health and health care. The established RPA-Cas12a-fluorescence assay is rapid and reliable method for human bocavirus 1 detection. The RPA-Cas12a-fluorescence assay can be completed within 40 min with robust specificity and sensitivity of 0.5 copies/μl., (© 2023 The Authors. Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.)

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]

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.

[Display omitted] • A novel fluorescent sensor for Hg2+ detection is developed based on T–Hg2+–T structure and CRISPR-Cas12a. • The fluorescent sensor exhibits high sensitivity for Hg2+ detection with a detection limit down to 23 pM. • The developed sensor can be applied in the detection of Hg2+ in real samples. • This work broadens the applications of CRISPR/Cas systems in environmental and food monitoring. In this work, we developed a novel label-free fluorescent sensor for the highly sensitive detection of mercury ions (Hg2+) based on the coordination chemistry of thymine-Hg2+-thymine (T-Hg2+-T) structures and the properties of CRISPR-Cas12a systems. Most notably, two T-rich sequences (a blocker and an activator) were designed to form stable double-stranded structures in the presence of Hg2+ via the T-Hg2+-T base pairing. The formation of T-T mismatched double-stranded DNA between the blocker and the activator prevented the cleavage of G-rich sequences by Cas12a, allowing them to fold into G-quadruplex-thioflavin T complexes, resulting in significantly enhanced fluorescence. Under the optimized conditions, the developed sensor showed an excellent response for Hg2+ detection in the linear range of 0.05 to 200 nM with a detection limit of 23 pM. Moreover, this fluorescent sensor exhibited excellent selectivity and was successfully used for the detection of Hg2+ in real samples of Zhujiang river water and tangerine peel , demonstrating its potential in environmental monitoring and food safety applications. [ABSTRACT FROM AUTHOR]

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]