Novel nucleic acid detection strategies based on CRISPR-Cas systems: From construction to application.

Beyond their widespread application as genome-editing and regulatory tools, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems also play a critical role in nucleic acid detection due to their high sensitivity and specificity. Recently developed Cas family effectors have opened the door to the development of new strategies for detecting different types of nucleic acids for a variety of purposes. Precise and efficient nucleic acid detection using CRISPR-Cas systems has the potential to advance both basic and applied biological research. In this review, we summarize the CRISPR-Cas systems used for the recognition and detection of specific nucleic acids for different purposes, including the detection of genomic DNA, nongenomic DNA, RNA, and pathogenic microbe genomes. Current challenges and further applications of CRISPR-based detection methods will be discussed according to the most recent developments.

Avenues Toward microRNA Detection In Vitro: A Review of Technical Advances and Challenges.

Over the decades, the biological role of microRNAs (miRNAs) in the post-transcriptional regulation of gene expression has been discovered in many cancer types, thus initiating the tremendous expectation of their application as biomarkers in the diagnosis, prognosis, and treatment of cancer. Hence, the development of efficient miRNA detection methods in vitro is in high demand. Extensive efforts have been made based on the intrinsic properties of miRNAs, such as low expression levels, high sequence homology, and short length, to develop novel in vitro miRNA detection methods with high accuracy, low cost, practicality, and multiplexity at point-of-care settings. In this review, we mainly summarized the newly developed in vitro miRNA detection methods classified by three key elements, including biological recognition elements, additional micro-/nano-materials and signal transduction/readout elements, their current challenges and further applications are also discussed.

Assembly of TALE-based DNA scaffold for the enhancement of exogenous multi-enzymatic pathway.

Synthetic scaffold systems, which exhibit enzyme clustering effect, have been considered as an important parallel approach for metabolic flux control and pathway enhancement. Here, we described an improved DNA-based scaffold system for synthetic tri-enzymatic pathway in Escherichia coli. With plasmid DNA serving as scaffold and exogenous enzymes fused with rationally designed transcription activator-like effectors (TALEs), our approach successfully clustered three TALE-fused enzymes and significantly increased the production of a mevalonate-producing tri-enzymatic pathway with the optimized scaffold structure and plasmid copy number. These results further suggested the scalability and robustness of the TALE-based scaffold system, and we can assume that it can be used on numerous multi-enzyme metabolic pathways due to its programmable features.

Highly Effective and Low-Cost MicroRNA Detection with CRISPR-Cas9.

MicroRNAs have been reported as related to multiple diseases and have potential applications in diagnosis and therapeutics. However, detection of miRNAs remains improvable, given their complexity, high cost, and low sensitivity as of currently. In this study, we attempt to build a novel platform that detects miRNAs at low cost and high efficacy. This detection system contains isothermal amplification, detecting and reporting process based on rolling circle amplification, CRISPR-Cas9, and split-horseradish peroxidase techniques. It is able to detect trace amount of miRNAs from samples with mere single-base specificity. Moreover, we demonstrated that such scheme can effectively detect target miRNAs in clinical serum samples and significantly distinguish patients of non-small cell lung cancer from healthy volunteers by detecting the previously reported biomarker: circulating let-7a. As the first to use CRISPR-Cas9 in miRNA detection, this method is a promising approach capable of being applied in screening, diagnosing, and prognosticating of multiple diseases.

Spatial organization of heterologous metabolic system in vivo based on TALE.

For years, prokaryotic hosts have been widely applied in bio-engineering. However, the confined in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. We developed a new method to accelerate a heterologous metabolic system by integrating a transcription activator-like effector (TALE)-based scaffold system into an Escherichia coli chassis. The binding abilities of the TALEs to the artificial DNA scaffold were measured through ChIP-PCR. The effect of the system was determined through a split GFP study and validated through the heterologous production of indole-3-acetic acid (IAA) by incorporating TALE-fused IAA biosynthetic enzymes in E. coli. To the best of our knowledge, we are the first to use the TALE system as a scaffold for the spatial organization of bacterial metabolism. This technique might be used to establish multi-enzymatic reaction programs in a prokaryotic chassis for various applications.