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Foodborne pathogenic infection has brought multifaceted issues to human life, leading to an urgent demand for advanced detection technologies. CRISPR/Cas-based biosensors have the potential to address various challenges that exist in conventional assays such as insensitivity, long turnaround time and complex pretreatments. In this perspective, we review the relevant strategies of CRISPR/Cas-assisted diagnostics on foodborne pathogens, focusing on biosensing platforms for foodborne pathogens based on fluorescence, colorimetric, (electro)chemiluminescence, electrochemical, and surface-enhanced Raman scattering detection. It summarizes their detection principles by the clarification of foodborne pathogenic bacteria, fungi, and viruses. Finally, we discuss the current challenges or technical barriers of these methods against broad application, and put forward alternative solutions to improve CRISPR/Cas potential for food safety. [ABSTRACT FROM AUTHOR]

Food fraud threats the sustainable development of the food industry. Accurate and rapid techniques for food authentication can protect consumers' rights and interests, safeguard public health, and maintain the food market. Applying quantitative polymerase chain reaction (qPCR) or sequencing to detect nucleic acid components has been a specific and sensitive strategy for food authentication, yet requires trained personnel and costly equipment. The rapidly evolving clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR/Cas) system has become an efficient toolbox to address the need for on-site and time-effective detection. In this review, we first outline the basic principles of the CRISPR/Cas system. Next, we summarize the features of pre-amplification and preamplification-free nucleic acid assays using CRISPR-based techniques. We highlight on-site nucleic acid tests for food authentication enabled by Cas effectors. Particularly, we exemplify approaches employing CRISPR-based tools for authentication analysis in food, including milk, halal food, seafood products, plant ingredients, and genetically modified organisms. Finally, we discuss the challenges of transforming CRISPR-based tools for practical use for food authentication. • CRISPR-based tools promise sensitive on-site food authentication. • CRISPR-Cas system has become an efficient toolbox to address the need for time-effective and cost-effective detection. • The ongoing advances, significance, and challenges of CRISPR-based methods for food authentication were reviewed. • On-site nucleic acid test for food authentication enabled by Cas effectors was highlighted. [ABSTRACT FROM AUTHOR]

Pathogenic biosafety is a worldwide concern. Tools for analyzing pathogenic biosafety, that are precise, rapid and field-deployable, are highly demanded. Recently developed biotechnological tools, especially those utilizing CRISPR/Cas systems which can couple with nanotechnologies, have enormous potential to achieve point-of-care (POC) testing for pathogen infection. In this review, we first introduce the working principle of class II CRISPR/Cas system for detecting nucleic acid and non-nucleic acid biomarkers, and highlight the molecular assays that leverage CRISPR technologies for POC detection. We summarize the application of CRISPR tools in detecting pathogens, including pathogenic bacteria, viruses, fungi and parasites and their variants, and highlight the profiling of pathogens' genotypes or phenotypes, such as the viability, and drug-resistance. In addition, we discuss the challenges and opportunities of CRISPR-based biosensors in pathogenic biosafety analysis. • CRISPR-leveraged biosensors for POC testing of pathogenic biosafety. • Integration of bioaffinities broadens target scope of CRISPR-based biosensing. • CRISPR-enabled pathogenic biosafety analysis was highlighted. • Challenges of CRISPR-based biosensing in pathogenic biosafety were discussed. [ABSTRACT FROM AUTHOR]

Pathogenic infection remains the primary threat to human health, such as the global COVID-19 pandemic. It is important to develop rapid, sensitive and multiplexed tools for detecting pathogens and their mutated variants, particularly the tailor-made strategies for point-of-care diagnosis allowing for use in resource-constrained settings. The rapidly evolving CRISPR/Cas systems have provided a powerful toolbox for pathogenic diagnostics via nucleic acid tests. In this review, we firstly describe the resultant promising class 2 (single, multidomain effector) and recently explored class 1 (multisubunit effector complexes) CRISPR tools. We present diverse engineering nucleic acid diagnostics based on CRISPR/Cas systems for pathogenic viruses, bacteria and fungi, and highlight the application for detecting viral variants and drug-resistant bacteria enabled by CRISPR-based mutation profiling. Finally, we discuss the challenges involved in on-site diagnostic assays and present emerging CRISPR systems and CRISPR cascade that potentially enable multiplexed and preamplification-free pathogenic diagnostics. • Class 2 and recently explored class 1 CRISPR tools are presented. • POCT strategies for detecting pathogenic viruses, bacteria and fungi are introduced. • CRISPR-based profiling of viral variants and drug-resistant bacteria is highlighted. • Challenges for multiplexing and preamplification-free diagnostic assay are discussed. [ABSTRACT FROM AUTHOR]

• A tool allows for direct visualization of single-cell non-repetitive genes. • In situ activation of trans-cleavage via CRISPR/Cas12a inside cells was explored. • Combining multiple Cas12a binding improves the signal-to-background ratio. • Single-cell investigation of S. Typhimurium under salt stress was proceeded. The spread of drug-resistance bacteria is a serious issue of environment. Tools allowing to image single-cell genes can provide key information about the spatial pattern and heterogeneity of cell population. Herein, we explored the possibility of in situ activation of collateral trans-cleavage activity of CRISPR/Cas12a inside cells, to achieve a direct detection of single-cell non-repetitive genes. CRISPR/Cas12a allows to recognize target genes without the need for denaturation or digestion process. Particularly, the target gene-activated trans-cleavage by CRISPR/Cas12a inside cells outputs an amplified signal for the gene recognition, allowing to visualize non-repetitive genes. The signal-to-background ratio for imaging drug-resistance gene, oqxB in the Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) was further improved by combining multiple binding of Cas12a, enabled imaging of drug-resistance S. Typhimurium isolated from poultry farm and in the intestinal tract sections. Single-cell investigation of S. Typhimurium under salt stress indicated that drug-sensitive strain owned a survival advantage over drug-resistance strain at high-content salt environment. This gene imaging methods holds potential for detecting the spread of drug resistance in the environment and serves as a means to investigate the relationship between genotype and phenotype at single-cell level. [ABSTRACT FROM AUTHOR]

Animal bacterial infection is increasingly threatening human health. Here we report a nucleic acid amplification-free CRISPR genetic assay that allows to rapidly screen potential food-origin antimicrobial probiotics. The assay (termed CRISPRzyme assay) is based on a CRISPR-DNAzyme cascade, where the target gene sequentially activated Cas12a protein and DNAzyme, yielding a limit of detection of 62 CFU Vibrio parahaemolyticus , 86 CFU Salmonella Typhimurium , and 82 CFU Listeria monocytogenes. The elimination of nucleic acid amplification shortens processing time and operational complexity. The assay was used to rapidly screen antimicrobial probiotics by end-measurement of fluorescence of pathogenic bacteria. Particularly, it can estimate the in vivo antimicrobial effect due to its capacity for pathogen quantification in complex samples. We found that isolates of Bacillus and lactic acid bacteria separated from fermented food exhibited strong antimicrobial activity for fish pathogen, Vibrio parahaemolyticus , and identified surfactin as the key antimicrobial component. The CRISPRzyme assay could ease antimicrobial probiotics screening, and constitutes a new tool for combatting pathogenic bacterial contamination and infection. [ABSTRACT FROM AUTHOR]

Food authenticity is a critical issue owing economic or ethnic considerations. Herein, we introduce CRISPR-Cas system to food authentication via detecting DNA barcodes of livestock and poultry in both unprocessed and processed food samples, allowing for a specific molecular traceability of meat species. We integrated the specific sequence recognition of CRISPR/Cas12 system and the efficient amplification of PCR method to identify DNA barcodes to improve the accuracy of pork component identification. This method, termed CRISPR- Ca s-based P CR DNA bar cod ing method (CAPCOD), can detect pork contamination diluted into 0.1% (w/w) in raw meat mixture, which ranks one of the most sensitive assays for food authenticity. And it has been applied to identify pork contents in complex processed halal food including beef luncheon meat, spiced beef and non-halal food including sausage, dried pork slices, pork balls and ham sausage. CRISPR-Cas-based DNA barcoding method is promising to serve as sensitive and reliable analytical tools for food authenticity. [Display omitted] • Introduction of CRISPR-Cas into food authenticity. • The sensor can identify pork contents in processed food. • Down to 0.1%(w/w) pork contamination can be detected. [ABSTRACT FROM AUTHOR]