The CRISPR-Cas13a Gemini System for noncontiguous target RNA activation.

Simultaneous multi-target detection and multi-site gene editing are two key factors restricting the development of disease diagnostic and treatment technologies. Despite numerous explorations on the source, classification, functional features, crystal structure, applications and engineering of CRISPR-Cas13a, all reports use the contiguous target RNA activation paradigm that only enables single-target detection in vitro and one-site gene editing in vivo. Here we propose a noncontiguous target RNA activation paradigm of Cas13a and establish a CRISPR-Cas13a Gemini System composed of two Cas13a:crRNA binary complexes, which can provide rapid, simultaneous, highly specific and sensitive detection of two RNAs in a single readout, as well as parallel dual transgene knockdown. CRISPR-Cas13a Gemini System are demonstrated in the detection of two miRNAs (miR-155 and miR-375) for breast cancer diagnosis and two small RNAs (EBER-1 and EBER-2) for Epstein-Barr virus diagnosis using multiple diagnostic platforms, including fluorescence and colorimetric-based lateral flow systems. We also show that CRISPR-Cas13a Gemini System can knockdown two foreign genes (EGFP and mCherry transcripts) in mammalian cells simultaneously. These findings suggest the potential of highly effective and simultaneous detection of multiple biomarkers and gene editing of multiple sites.

Polydopamine-assisted aptamer-carrying tetrahedral DNA microelectrode sensor for ultrasensitive electrochemical detection of exosomes.

BACKGROUND: Exosomes are nanoscale extracellular vesicles (30-160 nm) with endosome origin secreted by almost all types of cells, which are considered to be messengers of intercellular communication. Cancerous exosomes serve as a rich source of biomarkers for monitoring changes in cancer-related physiological status, because they carry a large number of biological macromolecules derived from parental tumors. The ultrasensitive quantification of trace amounts of cancerous exosomes is highly valuable for non-invasive early cancer diagnosis, yet it remains challenging. Herein, we developed an aptamer-carrying tetrahedral DNA (Apt-TDNA) microelectrode sensor, assisted by a polydopamine (PDA) coating with semiconducting properties, for the ultrasensitive electrochemical detection of cancer-derived exosomes. RESULTS: The stable rigid structure and orientation of Apt-TDNA ensured efficient capture of suspended exosomes. Without PDA coating signal amplification strategy, the sensor has a linear working range of 102-107 particles mL-1, with LOD of ~ 69 exosomes and ~ 42 exosomes for EIS and DPV, respectively. With PDA coating, the electrochemical signal of the microelectrode is further amplified, achieving single particle level sensitivity (~ 14 exosomes by EIS and ~ 6 exosomes by DPV). CONCLUSIONS: The proposed PDA-assisted Apt-TDNA microelectrode sensor, which integrates efficient exosome capture, sensitive electrochemical signal feedback with PDA coating signal amplification, provides a new avenue for the development of simple and sensitive electrochemical sensing techniques in non-invasive cancer diagnosis and monitoring treatment response.

A dual amplification-based CRISPR/Cas12a biosensor for sensitive detection of miRNA in prostate cancer.

MicroRNA (miRNA) has gained significant attention as a potential biomarker for cancer clinics, and there is an urgent need for developing sensing strategies with high selectivity, sensitivity, and low background. In vitro diagnosis based on Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated protein (CRISPR/Cas) technology could simplify the detection procedure, improve sensitivity and selectivity, and has broad application prospects as the next-generation molecular diagnosis technology. We propose a novel dual signal amplification strategy, called CENTER, which integrates the CRISPR/Cas12a system, an entropy-driven DNA signaling network, and strand displacement amplification to achieve ultrasensitive detection of miR-141, a potential marker for prostate cancer. The experimental results demonstrate that CENTER can distinguish single nucleotide mutations, and the strategy exhibits a good linear calibration curve ranging from 100 aM to 1 pM. Due to dual signal amplification, the detection limit is as low as 34 aM. We proposed a method for identifying miR-141 expressed in human serum and successfully distinguished between prostate cancer patients (n = 20) and healthy individuals (n = 15) with an impressive accuracy of 94%. Overall, CENTER shows great promise for the detection of miRNA.

Analysis and Biomedical Applications of Functional Cargo in Extracellular Vesicles.

Extracellular vesicles (EVs) can facilitate essential communication among cells in a range of pathophysiological conditions including cancer metastasis and progression, immune regulation, and neuronal communication. EVs are membrane-enclosed vesicles generated through endocytic origin and contain many cellular components, including proteins, lipids, nucleic acids, and metabolites. Over the past few years, the intravesicular content of EVs has proven to be a valuable biomarker for disease diagnostics, involving cancer, cardiovascular diseases, and central nervous system diseases. This review aims to provide insight into EV biogenesis, composition, function, and isolation, present a comprehensive overview of emerging techniques for EV cargo analysis, highlighting their major technical features and limitations, and summarize the potential role of EV cargos as biomarkers in disease diagnostics. Further, progress and remaining challenges will be discussed for clinical diagnostic outlooks.

Living Cell Nanoporation and Exosomal RNA Analysis Platform for Real-Time Assessment of Cellular Therapies.

Efficient transfection of therapeutic agents and timely potency testing are two key factors hindering the development of cellular therapy. Here we present a cellular-nanoporation and exosome assessment device, a quantitative platform for nanochannel-based cell electroporation and exosome-based in situ RNA expression analysis. In its application to transfection of anti-miRNAs and/or chemotherapeutics into cells, we have systematically described the differences in RNA expression in secreted exosomes and assessed cellular therapies in real time.

A CRISPR/Cas13a-powered catalytic electrochemical biosensor for successive and highly sensitive RNA diagnostics.

Rapid and specific quantitation of a variety of RNAs with low expression levels in early-stage cancer is highly desirable but remains a challenge. Here, we present a dual signal amplification strategy consisting of the CRISPR/Cas13a system and a catalytic hairpin DNA circuit (CHDC), integrated on a reusable electrochemical biosensor for rapid and accurate detection of RNAs. Signal amplification is accomplished through the unique combination of the CRISPR/Cas13a system with CHDC, achieving a limit of detection of 50 aM within a readout time of 6 min and an overall process time of 36 min, using a measuring volume of 10 µL. Enzymatic regeneration of the sensor surface and ratiometric correction of background signal allow up to 37 sequential RNA quantifications by square-wave voltammetry on a single biosensor chip without loss of sensitivity. The reusable biosensor platform could selectively (specificity = 0.952) and sensitively (sensitivity = 0.900) identify low expression RNA targets in human serum, distinguishing early-stage patients (n = 20) suffering from non-small-cell lung carcinoma (NSCLC) from healthy subjects (n = 30) and patients with benign lung disease (n = 12). Measurement of six NSCLC-related RNAs (miR-17, miR-155, TTF-1 mRNA, miR-19b, miR-210 and EGFR mRNA) shows the ability of the electrochemical CRISPR/CHDC system to be a fast, low-cost and highly accurate tool for early cancer diagnostics.