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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]

Single cell analysis is of great significance to biological and biomedical research. With recent development, lots of in situ amplification methods have been applied for imaging the spatial location of specific biomolecules in situ in single cells. In particular, RCA is a simple and efficient isothermal amplification method, which can locally amplify the fluorescent signal in situ. Due to the ability of detecting single molecules directly in cells and tissues, RCA has been widely used for illustrating many important biological processes. In this review, we summarize recent development of RCA-based method for single cell analysis and discuss their applications. Moreover, we emphasize the utilization of RCA for in situ sequencing, which is a very interesting and emerging research field. • The basic mechanism of rolling circle amplification (RCA) for in situ single cell imaging is summarized. • RCA can be used for analysis of various targets in situ in single cells, including gene expression, single-nucleotide variation, mRNA splicing and protein post-translation modification. • The utilization of RCA for in situ sequencing are discussed. [ABSTRACT FROM AUTHOR]