TL;DR
This investigation aimed to device rapid and cost effective screening system for identification of new variants of Cas9 proteins in environmental bacterial species and developed and optimized a cost effective screening tool C9CSS to preliminary identify CIRISPR type-II locus in bacteria.
This protocol minimizes off-target editing, shortens experimental timelines, reduces screening workload, and provides a reliable platform for investigating resistance mechanisms, validating candidate genes, and supporting functional genomics studies in clinically relevant bacterial pathogens.
This chapter outlines a comprehensive methodology for the design, assembly, and functional assessment of CRISPR/dCas9 systems optimized for tomato to investigate pathogen-associated responses.
Examination of the predicted secondary structure of the tracrRNA–crRNA duplex suggests that the features required for Cas9-catalyzed DNA cleavage at specific sites can be captured within a single chimeric RNA.
Molecular diagnostic technologies play an indispensable role in modern medicine and public health. However, traditional diagnostic platforms frequently face an inherent trade-off between laboratory-grade analytical precision and the speed and operational simplicity required for point-of-care testing. In recent years, the emergence of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein system has precipitated disruptive technological changes to this field. CRISPR-Cas system possesses high-fidelity target recognition capability and exhibits a distinctive trans-cleavage activity upon activation, which functions as signal amplification. This technology alleviates the inherent trade-off between sensitivity and portability. This review systematically summarizes the core molecular mechanisms of CRISPR-Cas detection platforms, addressing the differences in substrate preference and cleavage behavior among mainstream effector proteins (e.g., Cas9, Cas12, Cas13, and Cas14) and prokaryotic Argonaute (pAgo) proteins. Furthermore,this review sorts out the technological iteration path of detection platforms and presents the applications of this technology in fields such as infectious disease surveillance, cancer liquid biopsy, preliminary screening of genetic diseases, food and environmental safety, and veterinary port quarantine. Despite the challenges in quantitative accuracy and anti-interference ability, CRISPR biosensors are powerfully driving precision medicine towards decentralized, on-site, and accessible Point-of-Care Testing (POCT).
Examination of the predicted secondary structure of the tracrRNA–crRNA duplex suggests that the features required for Cas9-catalyzed DNA cleavage at specific sites can be captured within a single chimeric RNA.
Examination of the predicted secondary structure of the tracrRNA–crRNA duplex suggests that the features required for Cas9-catalyzed DNA cleavage at specific sites can be captured within a single chimeric RNA.