Microfluidics: the propellant of CRISPR-based nucleic acid detection

Microfluidic platforms can integrate clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection procedures, reduce the need for manual operation, and shorten turnaround times. Microfluidics enables multiplex CRISPR detection in a low-cost way. For example, droplet fusion-based combinatorial arrayed reactions can detect 169 human-associated viruses simultaneously. The confinement effect in small-volume partitions (microchambers or water-in-oil droplets) promotes the concentration of nucleic acids and improves the reaction efficiency, thus realizing amplification-free digital CRISPR detection. Microfluidic-assisted CRISPR detection platforms combine the advantages of CRISPR (high specificity) and microfluidics (miniaturization/integration/easy-to-automation), which is promising for point-of-care applications. Since the discovery of collateral cleavage activity, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems have become the new generation of nucleic acid detection tools. However, their widespread application remains limited. A pre-amplification step is required to improve the sensitivity of CRISPR systems, complicating the operating procedure and limiting quantitative precision. In addition, nonspecific collateral cleavage activity makes it difficult to realize multiplex detection in a one-pot CRISPR reaction with a single Cas protein. Microfluidics, which can transfer nucleic acid analysis process to a chip, has the advantages of miniaturization, integration, and automation. Microfluidics coupled with CRISPR systems improves the detection ability of CRISPR, enabling fast, high-throughput, integrated, multiplex, and digital detection, which results in the further popularization of CRISPR for a range of scenarios. Since the discovery of collateral cleavage activity, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems have become the new generation of nucleic acid detection tools. However, their widespread application remains limited. A pre-amplification step is required to improve the sensitivity of CRISPR systems, complicating the operating procedure and limiting quantitative precision. In addition, nonspecific collateral cleavage activity makes it difficult to realize multiplex detection in a one-pot CRISPR reaction with a single Cas protein. Microfluidics, which can transfer nucleic acid analysis process to a chip, has the advantages of miniaturization, integration, and automation. Microfluidics coupled with CRISPR systems improves the detection ability of CRISPR, enabling fast, high-throughput, integrated, multiplex, and digital detection, which results in the further popularization of CRISPR for a range of scenarios. movement of a liquid solution along the surface of a solid caused by the greater attraction of the molecules in the liquid to the surface of the solid than the interaction between liquid molecules. Capillary forces can have a critical impact at the microscale. when Cas12, Cas13, or Cas14 protein binds its target under the guidance of guide RNA, the Cas protein cleaves not only the target, but also other nucleic acids nearby in the solution. Cas12 and Cas14 cleave nearby ssDNA molecules, and Cas13 cleaves nearby RNA molecules. CRISPR-based nucleic acid detection method that combines RPA and the CRISPR/Cas12a system. versatile electrophoretic technique that uses a discontinuous electrical field to create sharp boundaries between the sample constituents, realizing sample preconcentration, separation, mixing, and chemical reaction acceleration. system that can integrate sample processing, separation, and analysis on a miniaturized chip. Liquid solutions can be manipulated in the microchambers or microchannels of the chip. small single-stranded noncoding RNA molecules that function in RNA silencing and post-transcriptional regulation of gene expression; they are ~22-nucleotides long. CRISPR-based nucleic acid detection method that combines PCR and the CRISPR/Cas12a system. CRISPR-based nucleic acid detection method that combines RPA and the CRISPR/Cas13a system. transcription activator-like effector (TALEs) are proteins that are secreted by Xanthomonas bacteria. TALEs can be engineered to bind a desired DNA sequence. By combining such an engineered TALE with a DNA cleavage domain, the DNA sequence can be cut at specific positions. when a fluid flows through a constricted section of a pipe, the pressure of fluid reduces and the velocity increases. comprises a DNA-binding zinc-finger protein (ZFP) domain at the N terminus and the FokI nuclease cleavage domain at the C terminus. The ZFN system is capable of making site-specific double-stranded DNA breaks to realize gene edits.