Quencher-free CRISPR-based molecular detection using an amphiphilic DNA fluorescence probe.

Rapid, sensitive, and specific nucleic acid detection methods play crucial roles in clinical diagnostics and healthcare. Here, we report a novel amphiphilic DNA fluorescence probe for CRISPR-based nucleic acid detection. Unlike conventional fluorophore-quencher probe detection system, our amphiphilic DNA fluorescence probe features a hydrophobic Cy5 fluorophore head and a hydrophilic single-stranded DNA (ssDNA) tail.

Programmable Multiplexed Nucleic Acid Detection by Harnessing Specificity Defect of CRISPR-Cas12a.

CRISPR-Cas12a works like a sophisticated algorithm in nucleic acid detection, yet its challenge lies in sometimes failing to distinguish targets with mismatches due to its specificity limitations. Here, the mismatch profiles, including the quantity, location, and type of mismatches in the CRISPR-Cas12a reaction, are investigated and its various tolerances to mismatches are discovered. By harnessing the specificity defect of the CRISPR-Cas12a enzyme, a dual-mode detection strategy is designed, which includes approximate matching and precise querying of target sequences and develop a programmable multiplexed nucleic acid assay.

Interpreting CRISPR-Cas12a enzyme kinetics through free energy change of nucleic acids.

While CRISPR has revolutionized biotechnology, predicting CRISPR-Cas nuclease activity remains a challenge. Herein, through the trans-cleavage feature of CRISPR-Cas12a, we investigate the correlation between CRISPR enzyme kinetics and the free energy change of crRNA and DNA targets from their initial thermodynamic states to a presumed transition state before hybridization. By subjecting computationally designed CRISPR RNAs (crRNAs), we unravel a linear correlation between the trans-cleavage kinetics of Cas12a and the energy barrier for crRNA spacer and single-stranded DNA target unwinding.

Palm-Sized Lab-In-A-Magnetofluidic Tube Platform for Rapid and Sensitive Virus Detection.

Simple, sensitive, and accurate molecular diagnostics are critical for preventing rapid spread of infection and initiating early treatment of diseases. However, current molecular detection methods typically rely on extensive nucleic acid sample preparation and expensive instrumentation. Here, a simple, fully integrated, lab-in-a-magnetofluidic tube (LIAMT) platform is presented for "sample-to-result" molecular detection of virus.

Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR-Cas12a.

The CRISPR-Cas12a system has emerged as a powerful tool for next-generation nucleic acid-based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA-enabled trans-cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by full-size RNA targets, although LbCas12a exhibits weaker trans-cleavage activity than AsCas12a on both single-stranded DNA and RNA substrates.

CRISPR-powered quantitative keyword search engine in DNA data storage.

Despite the growing interest of archiving information in synthetic DNA to confront data explosion, quantitatively querying the data stored in DNA is still a challenge. Herein, we present Search Enabled by Enzymatic Keyword Recognition (SEEKER), which utilizes CRISPR-Cas12a to rapidly generate visible fluorescence when a DNA target corresponding to the keyword of interest is present. SEEKER achieves quantitative text searching since the growth rate of fluorescence intensity is proportional to keyword frequency.

CRISPR-Powered DNA Computing and Digital Display.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has unique specificity for recognizing and cleaving target DNA complementary to the CRISPR guide sequence. Here, we report on a CRISPR-powered DNA computing and digital display system in which programmed DNA targets serve as the input and an ON/OFF fluorescence signal indicates a TRUE/FALSE output. This system allows the establishment of a one-to-one relationship between input and output, enabling multilevel DNA logic computing.

Electrochemical biotoxicity detection on a microfluidic paper-based analytical device via cellular respiratory inhibition.

A novel microfluidic paper-based analytical device (μPAD) was developed with benzoquinone (BQ)-mediated E. coli respiration method to measure the biotoxicities of pollutants. Functional units including sample injection, fluid-cell separation, all-carbon electrode-enabled electrochemical detection, were integrated on a piece of chromatography paper.