Recent advances on CRISPR/Cas system-enabled portable detection devices for on-site agri-food safety assay

Agri-food safety has been considered as one of the most important public concerns worldwide. From farm to table, food crops and foods are extremely vulnerable to the contamination by a variety of pollutants from their growth and processing. Moreover, the SARS-CoV-2 detected in the food supply chain during COVID-19 pandemic has posed a greater challenge for rapid and on-site detection of agri-food contaminants in complex and volatile environments. Therefore, the development of rapid, accurate, and on-site detection technologies and portable detection devices is of great importance to ensure the agri-food security. This review comprehensively summarized the recent advances on the construction of CRISPR/Cas systems-based biosensing technologies and their portable detection devices, as well as their promising applications in the field of agri-food safety. First of all, the classification and working principles of CRISPR/Cas systems were introduced. Then, the latest advances on the CRISPR/Cas system-based on-site detection technologies and portable detection devices were also systematically summarized. Most importantly, the state-of-the-art applications of CRISPR/Cas systems-based on-site detection technologies and portable detection devices in the fields of agri-food safety were comprehensively summarized. Impressively, the future opportunities and challenges in this emerging and promising field were proposed. Emerging CRISPR/Cas system-based on-site detection technologies have showed a great potential in the detection of agri-food safety. Impressively, the integration of CRISPR/Cas systems-based biosensing technologies with portable detection devices (e.g., nanopore-based detection devices, lateral flow assay, smartphone-based detection devices, and microfluidic devices) is very promising for the on-site detection of agri-food contaminants. Additionally, CRISPR/Cas system-based biosensing technologies can be further integrated with much more innovative technologies for the development of novel detection platforms to realize the more reliable on-site detection of agri-food safety.

Exploring the entropy-driven amplification reaction and trans-cleavage activity of CRISPR-Cas12a for the development of an electrochemiluminescence biosensor for the detection of the SARS-CoV-2 RdRp gene in real samples and environmental surveillance

In this paper, we present the first idea of using a DNA triple helix structure to inhibit CRISPR-Cas12a activity and apply it to the design of an electrochemiluminescent biosensor for the detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) gene in real samples and environmental surveillance. We employed a segment from the RdRp gene of SARS-CoV-2 by an entropy-driven reaction, which was paired with double-stranded DNA that can activate CRISPR-Cas12a activity by Hoogsteen pairing to form triple-stranded DNA, thereby inhibiting the binding interaction of the double-stranded DNA with CRISPR-Cas12a, which in turn inhibits the trans cleavage activity of CRISPR-Cas12a. The inhibited CRISPR-Cas12a is unable to cut the nucleic acid modified on the electrode surface, resulting in the inability of the ferrocene (Fc) modified on the other end of the nucleic acid to move away from the electrode surface, and thus failing to cause electrochemiluminescence changes in GOAu-Ru modified on the electrode surface. The extent of the electrogenic chemiluminescence change can reflect the concentration of the gene to be tested. Using this system, we achieved the detection of the SARS-CoV-2 RdRp gene with a detection limit of 32.80 aM.

Generation of APN-chimeric gene-edited pigs by CRISPR/Cas9-mediated knock-in strategy

The prevalence of porcine enteric coronaviruses (PECs), including transmissible gastroenteritis virus (TGEV), swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine delta coronavirus (PDCoV), and porcine epidemic diarrhea virus (PEDV), poses a serious threat to animal and public health. Here, we aimed to further optimize the porcine aminopeptidase N (pAPN) gene editing strategy to explore the balance between individual antiviral properties and the biological functions of pAPN in pigs. Finally, APN-chimeric gene-edited pigs were produced through a CRISPR/Cas9-mediated knock-in strategy. Further reproductive tests indicated that these gene-edited pigs exhibited normal pregnancy rates and viability. Notably, in vitro viral challenge assays further demonstrated that porcine kidney epithelial cells isolated from F1-generation gene-edited pigs could effectively inhibit TGEV infection. This study is the first to report the generation of APN-chimeric pigs, which may provide a natural host animal for characterizing PEC infection with APN and help in the development of better antiviral solutions. © 2022 Elsevier B.V.

The Trend of CRISPR-Based Technologies in COVID-19 Disease: Beyond Genome Editing.

Biotechnological approaches have always sought to utilize novel and efficient methods in the prevention, diagnosis, and treatment of diseases. This science has consistently tried to revolutionize medical science by employing state-of-the-art technologies in genomic and proteomic engineering. CRISPR-Cas system is one of the emerging techniques in the field of biotechnology. To date, the CRISPR-Cas system has been extensively applied in gene editing, targeting genomic sequences for diagnosis, treatment of diseases through genomic manipulation, and in creating animal models for preclinical researches. With the emergence of the COVID-19 pandemic in 2019, there is need for the development and modification of novel tools such as the CRISPR-Cas system for use in diagnostic emergencies. This system can compete with other existing biotechnological methods in accuracy, precision, and wide performance that could guarantee its future in these conditions. In this article, we review the various platforms of the CRISPR-Cas system meant for SARS-CoV-2 diagnosis, anti-viral therapeutic procedures, producing animal models for preclinical studies, and genome-wide screening studies toward drug and vaccine development.

CRISPR-Based Diagnostics: Challenges and Potential Solutions toward Point-of-Care Applications.

The COVID-19 pandemic has challenged the conventional diagnostic field and revealed the need for decentralized Point of Care (POC) solutions. Although nucleic acid testing is considered to be the most sensitive and specific disease detection method, conventional testing platforms are expensive, confined to central laboratories, and are not deployable in low-resource settings. CRISPR-based diagnostics have emerged as promising tools capable of revolutionizing the field of molecular diagnostics. These platforms are inexpensive, simple, and do not require the use of special instrumentation, suggesting they could democratize access to disease diagnostics. However, there are several obstacles to the use of the current platforms for POC applications, including difficulties in sample processing and stability. In this review, we discuss key advancements in the field, with an emphasis on the challenges of sample processing, stability, multiplexing, amplification-free detection, signal interpretation, and process automation. We also discuss potential solutions for revolutionizing CRISPR-based diagnostics toward sample-to-answer diagnostic solutions for POC and home use.

Recent advances on CRISPR/Cas system-enabled portable detection devices for on-site agri-food safety assay

Background Agri-food safety has been considered as one of the most important public concerns worldwide. From farm to table, food crops and foods are extremely vulnerable to the contamination by a variety of pollutants from their growth and processing. Moreover, the SARS-CoV-2 detected in the food supply chain during COVID-19 pandemic has posed a greater challenge for rapid and on-site detection of agri-food contaminants in complex and volatile environments. Therefore, the development of rapid, accurate, and on-site detection technologies and portable detection devices is of great importance to ensure the agri-food security. Scope and approach This review comprehensively summarized the recent advances on the construction of CRISPR/Cas systems-based biosensing technologies and their portable detection devices, as well as their promising applications in the field of agri-food safety. First of all, the classification and working principles of CRISPR/Cas systems were introduced. Then, the latest advances on the CRISPR/Cas system-based on-site detection technologies and portable detection devices were also systematically summarized. Most importantly, the state-of-the-art applications of CRISPR/Cas systems-based on-site detection technologies and portable detection devices in the fields of agri-food safety were comprehensively summarized. Impressively, the future opportunities and challenges in this emerging and promising field were proposed. Key findings and conclusions Emerging CRISPR/Cas system-based on-site detection technologies have showed a great potential in the detection of agri-food safety. Impressively, the integration of CRISPR/Cas systems-based biosensing technologies with portable detection devices (e.g., nanopore-based detection devices, lateral flow assay, smartphone-based detection devices, and microfluidic devices) is very promising for the on-site detection of agri-food contaminants. Additionally, CRISPR/Cas system-based biosensing technologies can be further integrated with much more innovative technologies for the development of novel detection platforms to realize the more reliable on-site detection of agri-food safety.

Diagnostic accuracy of CRISPR technology for detecting SARS-CoV-2: a systematic review and meta-analysis.

OBJECTIVE: To evaluate the diagnostic accuracy of CRISPR-Cas technology for SARS-CoV-2. METHODS: RT-qPCR is defined as the reference standard. Data was collected and assessed by Quality Assessment of Diagnostic Accuracy Studies (QUADAS)-2 tool. A bivariate model for pooling was employed and subgroups analysis was used to explore heterogeneity. RESULTS: 2264 samples from 28 articles were extracted for evaluating the accuracy of CRISPR technology for diagnosing SARS-CoV-2. The pooled sensitivity and specificity of CRISPR technology were 0.98 (95% CI: 0.95-0.99) and 1.0 (95% CI: 0.98-1.00), respectively. High risks in patient selection bias and unclear risk of index test bias may affect accuracy. Subgroup analysis showed that CRISPR-Cas12 is applicable for molecular diagnostics for its active editing characteristics. RT-LAMP and RT-RPA are usually used for pre-amplification and fluorescence detection to output results quantitatively. Nasopharyngeal swabs and dual-genes perform greatly in our study. CONCLUSION: The results concluded from all studies showed that CRISPR technology is a promising molecular method for detecting SARS-CoV-2. Standard methods including comparable sample material, patient selection, operating procedure and operators should be established.

Conventional and Microfluidic Methods for the Detection of Nucleic Acid of SARS-CoV-2.

Nucleic acid testing (NAT) played a crucial role in containing the spread of SARS-CoV-2 during the epidemic. The gold standard technique, the quantitative real-time polymerase chain reaction (qRT-PCR) technique, is currently used by the government and medical boards to detect SARS-CoV-2. Due to the limitations of this technology, it is not capable of meeting the needs of large-scale rapid detection. To solve this problem, many new techniques for detecting nucleic acids of SARS-CoV-2 have been reported. Therefore, a review that systematically and comprehensively introduces and compares various detection technologies is needed. In this paper, we not only review the traditional NAT but also provide an overview of microfluidic-based NAT technologies and summarize and discuss the characteristics and development prospects of these techniques.

iSCAN-V2: A One-Pot RT-RPA-CRISPR/Cas12b Assay for Point-of-Care SARS-CoV-2 Detection.

Rapid, specific, and sensitive detection platforms are prerequisites for early pathogen detection to efficiently contain and control the spread of contagious diseases. Robust and portable point-of-care (POC) methods are indispensable for mass screening of SARS-CoV-2. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based nucleic acid detection technologies coupled with isothermal amplification methods provide a straightforward and easy-to-handle platform for detecting SARS-CoV-2 at POC, low-resource settings. Recently, we developed iSCAN, a two-pot system based on coupled loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a reactions. However, in two-pot systems, the tubes must be opened to conduct both reactions; two-pot systems thus have higher inherent risks of cross-contamination and a more cumbersome workflow. In this study, we developed and optimized iSCAN-V2, a one-pot reverse transcription-recombinase polymerase amplification (RT-RPA)-coupled CRISPR/Cas12b-based assay for SARS-CoV-2 detection, at a single temperature in less than an hour. Compared to Cas12a, Cas12b worked more efficiently in the iSCAN-V2 detection platform. We assessed and determined the critical factors, and present detailed guidelines and considerations for developing and establishing a one-pot assay. Clinical validation of our iSCAN-V2 detection module with reverse transcription-quantitative PCR (RT-qPCR) on patient samples showed 93.75% sensitivity and 100% specificity. Furthermore, we coupled our assay with a low-cost, commercially available fluorescence visualizer to enable its in-field deployment and use for SARS-CoV-2 detection. Taken together, our optimized iSCAN-V2 detection platform displays critical features of a POC molecular diagnostic device to enable mass-scale screening of SARS-CoV-2 in low-resource settings.

CRISPR-Cas System: A Promising Diagnostic Tool for Covid-19.

More than a year has passed since the beginning of the 2019 novel coronavirus diseases (COVID-19) pandemic which has created massive problems globally affecting all aspects of people's life. Due to the emergence of new strains of the SARS-CoV-2, pandemic risk still remains, despite the start of vaccination. Therefore, rapid diagnostic tests are essential to control infection, improve clinical care and stop the spread of the disease. Recently CRISPR-based diagnostic tools have facilitated rapid diagnostic. Here, we review the diagnostic applications of CRISPR-Cas system in COVID-19.

The potential use of Drosophila as an in vivo model organism for COVID-19-related research: a review.

The world urgently needs effective antiviral approaches against emerging viruses, as shown by the coronavirus disease 2019 (COVID-19) pandemic, which has become an exponentially growing health crisis. Scientists from diverse backgrounds have directed their efforts towards identifying key features of SARS-CoV-2 and clinical manifestations of COVID-19 infection. Reports of more transmissible variants of SARS-CoV-2 also raise concerns over the possibility of an explosive trajectory of the pandemic, so scientific attention should focus on developing new weapons to help win the fight against coronaviruses that may undergo further mutations in the future. Drosophila melanogaster offers a powerful and potential in vivo model that can significantly increase the efficiency of drug screening for viral and bacterial infections. Thanks to its genes with functional human homologs, Drosophila could play a significant role in such gene-editing studies geared towards designing vaccines and antiviral drugs for COVID-19. It can also help rectify current drawbacks of CRISPR-based therapeutics like off-target effects and delivery issues, representing another momentous step forward in healthcare. Here I present an overview of recent literature and the current state of knowledge, explaining how it can open up new avenues for Drosophila in our battle against infectious diseases.

Applications of the CRISPR-Cas system for infectious disease diagnostics.

INTRODUCTION: Rapid and accurate diagnostic approaches are essential for impeding the spread of infectious diseases. This review aims to summarize current progress of clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems in the applications for diagnostics of infectious diseases including the ongoing COVID-19 epidemic. AREAS COVERED: In this review, we discuss class 2 CRISPR-Cas biosensing systems-based diagnostics in various emerging and reemerging infectious diseases, CRISPR-Cas systems have created a new era for early diagnostics of infectious diseases, especially with the discovery of the collateral cleavage activity of Cas12 and Cas13. We mainly focus on different CRISPR-Cas effectors for the detection of pathogenic microorganisms as well as provide a detailed explanation of the pros and cons of CRISPR-Cas biosensing systems. In addition, we also introduce future research perspectives. EXPERT COMMENTARY: However, further improvement of newly discovered systems and engineering existing ones should be developed to increase the specificity, sensitivity or stability of the diagnostic tools. It may be a long journey to finish the clinical transition from research use. CRISPR-Cas approaches will emerge as more promising and robust tools for infectious disease diagnosis in the future.