trans Single-Stranded DNA Cleavage via CRISPR/Cas14a1 Activated by Target RNA without Destruction.

As a CRISPR-Cas system (clustered regularly interspaced short palindromic repeats and CRISPR associated proteins), Cas14a1 can cis/trans cleave single-stranded DNA (ssDNA). Here, we describe an unreported capacity of Cas14a1: RNA can trigger the trans ssDNA cleavage. This Cas14a1-based RNA-activated detection platform (Amplification, Transcription, Cas14a1-based RNA-activated trans ssDNA cleavage, ATCas-RNA) has an outstanding specificity for the detection of target RNAs with point mutation resolution, which is better than that of the Cas14a1-based ssDNA-activation. Using ATCas-RNA via a fluorophore quencher-labeled ssDNA reporter (FQ), we were able to detect 1 aM pathogenic nucleic acid within 1 h, and achieve 100 % accuracy with 25 milk samples. This platform can serve as a new tool for high-efficiency nucleic acid diagnostics. Importantly, this work can expand our understanding of Cas14a1 and inspire further mechanisms and applications of Class-2 Cas systems.

Application of Virtual Reality Technology in Post-Stroke Depression.

Post-stroke depression (PSD) is one of the most common complications of emotional disorders after stroke, and the recovery effect of clinical intervention using conventional means is limited. As an emerging technology, virtual reality technology provides a new idea for the clinical rehabilitation of patients with post-stroke depression. Through literature retrieval and review of domestic and foreign studies, this paper summarizes the development of virtual reality technology in psychotherapy. This paper expounds the application value of virtual reality technology in post-stroke depression, summarizes the common types of virtual reality technology combined with conventional intervention to treat post-stroke depression, and analyzes the influence of virtual reality technology on physical and mental rehabilitation of patients with post-stroke depression combined with empirical data. At the same time, the positive significance of virtual reality technology popularization and application is discussed. Finally, it is concluded that virtual reality technology can make up for the shortcomings of traditional treatment in the application of post-stroke depression, and can effectively improve the physical and mental state of patients. The application prospect is broad, but there is still room for development and improvement.

A signal-off Cas14a1-based platform for highly specific detection of methicillin-resistant Staphylococcus aureus.

Antibiotic usage has become very widespread in aquaculture, and the abuse or overuse of antibiotics has led to the evolution of antibiotic-resistance bacteria, which has adverse effects on aquatic products and ecosystems. Moreover, this evolution can potentially cause harm to human health. Thus, there is an urgent need for diagnostic tools for antibiotic-resistant microorganisms. Herein, we proposed a signal-off Cas14a1-based platform (SOCP) for the detection of methicillin-resistant Staphylococcus aureus (MRSA). In this SOCP, we have designed single-stranded DNA (ssDNA) that not only can activate the trans-cleavage ability of dual Cas14a1-sgRNA complex but also can be used as the primers for the amplified methicilin-resistant gene (mecA). When MRSA is present, the primers can be transformed into products with amplification, leading to the signal decrease of trans-cleavage activity of Cas14a1. The SOCP showed high specificity and fair sensitivity for mecA gene and MRSA. In the detection of real samples, this platform also showed consistent results compared with qPCR. The SOCP could provide an alternative tool for the diagnosis of antibiotic-resistant bacteria in aquaculture, food industry and other fields.

Aptamer-based Cas14a1 biosensor for amplification-free live pathogenic detection.

CRISPR-Cas systems have been employed to detect a large variety of pathogenic microorganisms by simply changing the guide RNA sequence. However, these platforms usually rely on nucleic acid extraction and amplification to achieve good sensitivity. Herein, we developed a new platform for the highly specific and sensitive detection of live staphylococcus aureus (S. aureus) based on an Aptamer-based Cas14a1 Biosensor (ACasB), without the need for nucleic acid extraction or amplification. First, the S. aureus specific aptamer was hybrid with a blocker DNA. After the live S. aureus was added, the blocker can be released upon bacteria-aptamer binding. Finally, the released blocker can activate Cas14a1 protein by binding with the sgRNA to generate a change of fluorescent intensity. The ACasB indicates high specificity and sensitivity: it can directly distinguish 400 CFU/ml live S. aureus cells. Comparable to qPCR, the Cas14a1-aptamer biosensor can detect S. aureus with 100% accuracy in complex samples. Therefore, this ACasB for the on-site detection of live S. aureus can broaden its applications in food safety and environmental monitoring.

Cas14a1-mediated nucleic acid detectifon platform for pathogens.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas) based biosensing system provides a novel genomic diagnostic tool for pathogenic detection. However, most of the discovered Cas effectors have poor single strand DNA (ssDNA) target recognition capability with the constraint of protospacer adjacent motif (PAM) sites, which are not suitable for universal pathogenic diagnosis. Herein, we developed a highly sensitive and specific fluorescence tool for bacterial detection by utilizing the unique collateral cleavage activity of a Cas14a1-mediated nucleic acid detection platform (CMP). We combine CMP with molecular amplification to build a CRISPR-Cas based bioanalysis technique, offering fast nucleic acid detection with high sensitivity and specificity. This technique can identify different species of pathogens in milk samples with excellent accuracy. The CMP technique is a promising platform for pathogenic genomic diagnostic in biomedicine and food safety field.