Reprogrammable Gel Electrophoresis Detection Assay Using CRISPR-Cas12a and Hybridization Chain Reaction.

Hybridization chain reaction (HCR) is a DNA-based target-induced cascade reaction. Due to its unique enzyme-free amplification feature, HCR is often employed for sensing applications. Much like DNA nanostructures that have been designed to respond to a specific stimulus, HCR employs nucleic acids that reconfigure and assemble in the presence of a specific trigger. Despite its standalone capabilities, HCR is highly modular; therefore, it can be advanced and repurposed when coupled with latest discoveries. To this effect, we have developed a gel electrophoresis-based detection approach which combines the signal amplification feature of HCR with the programmability and sensitivity of the CRISPR-Cas12a system. By incorporating CRISPR-Cas12a, we have achieved greater sensitivity and reversed the signal output from TURN OFF to TURN ON. CRISPR-Cas12a also enabled us to rapidly reprogram the assay for the detection of both ssDNA and dsDNA target sequences by replacing a single reaction component in the detection kit. Detection of conserved, both ssDNA and dsDNA, regions of tobacco curly shoot virus (TCSV) and hepatitis B virus (HepBV) genomes is demonstrated with this methodology. This low-cost gel electrophoresis assay can detect as little as 1.5 fmol of the target without any additional target amplification steps and is about 100-fold more sensitive than HCR-alone approach.

Regulation of the Peroxidase-Like Activity of nGO, MoS2 and WS2 Nanozymes by Using Metal Cations.

Two dimensional nanoparticles (2D-NPs) along with other nanoscale materials have been deemed to be the next generation of artificial enzymes (nanozymes). The low-cost bulk-scale production, ease of storage and modification of such nanomaterials have given nanozymes an advantage over traditional enzymes. Many studies have been aimed at developing methods to increase the performance of these nanozymes, and also identify interfering agents. To investigate the interference of a number of metal cations, we studied the effect of Ti2+ , Fe2+ , Ag+ , Hg2+ , Co2+ , Cu2+ , Ni2+ , Pb2+ , Ca2+ , Zn2+ and Mn2+ in a nanozyme assays of 2D-NPs using ABTS radical formation. Ti2+ , Co2+ , Cu2+ , Ni2+ , Ca2+ , Zn2+ and Mn2+ ions did not display any notable effect on the peroxidase-like activity of nGO, MoS2 and WS2 2D-NPs. However, Fe2+ , Ag+ , Hg2+ and Pb2+ ions' effects on the overall ABTS reaction were significant enough to be visualised by partial least square discriminant analysis (PLSDA). We report that, similar to that of many natural enzymes, the nanozyme activity of 2D-NPs is regulated by a number of metal cations allowing their identification and discrimination by using a statistical analysis tool.

Probing CRISPR-Cas12a Nuclease Activity Using Double-Stranded DNA-Templated Fluorescent Substrates.

The CRISPR-Cas12a nuclease shreds short single-stranded DNA (ssDNA) substrates indiscriminately through trans-cleavage upon activation with a specific target DNA. This shredding activity offered the potential for development of ssDNA-templated probes with fluorescent dye (F) and quencher (Q) labels. However, the formulations of double-stranded DNA (dsDNA)-templated fluorescent probes have not been reported possibly due to unknown (or limited) activity of Cas12a against short dsDNAs. The ssDNA probes have been shown to be powerful for diagnostic applications; however, limiting the probe selections to short ssDNAs could be restrictive from an application and probe diversification standpoint. Here, we report a dsDNA substrate (probe-full) for probing Cas12a trans-cleavage activity upon target detection. A diverse set of Cas12a substrates with alternating dsDNA character were designed and studied using fluorescence spectroscopy. We have observed that probe-full without any nick displayed trans-cleavage performance that was better than that of the form that contains a nick. Different experimental conditions of salt concentration, target concentration, and mismatch tolerance were examined to evaluate the probe performance. The activity of Cas12a was programmed for a dsDNA frame copied from a tobacco curly shoot virus (TCSV) or hepatitis B virus (HepBV) genome by using crRNA against TCSV or HepBV, respectively. While on-target activity offered detection of as little as 10 pM dsDNA target, off-target activity was not observed even at 1 nM control DNAs. This study demonstrates that trans-cleavage of Cas12a is not limited to ssDNA substrates, and Cas12a-based diagnostics can be extended to dsDNA substrates.

Universal CRISPR-Cas12a and Toehold RNA Cascade Reaction on Paper Substrate for Visual Salmonella Genome Detection.

Salmonella, the most prevalent food-borne pathogen, poses significant medical and economic threats. Swift and accurate on-site identification and serotyping of Salmonella is crucial to curb its spread and contamination. Here, a synthetic biology cascade reaction is presented on a paper substrate using CRISPR-Cas12a and recombinase polymerase amplification (RPA), enabling the programming of a standard toehold RNA switch for a genome of choice. This approach employs just one toehold RNA switch design to differentiate between two different Salmonella serotypes, i.e., S. Typhimurium and S. Enteritidis, without the need for reengineering the toehold RNA switch. The sensor exhibits high sensitivity, capable of visually detecting as few as 100 copies of the whole genome from a model Salmonella pathogen on a paper substrate. Furthermore, this robust assay is successfully applied to detect whole genomes in contaminated milk and lettuce samples, demonstrating its potential in real sample analysis. Due to its versatility and practical features, genomes from different organisms can be detected by merely changing a single RNA element in this universal cell-free cascade reaction.

Recombinase amplified CRISPR enhanced chain reaction (RACECAR) for viral genome detection.

We have developed a 'recombinase amplified CRISPR enhanced chain reaction' (RACECAR) assay that can detect as little as 40 copies of hepatitis B virus (HBV) genome using a benchtop spectrofluorometer. The limit of detection was determined to be 3 copies of HBV genome. The specificity of RACECAR was confirmed against hepatitis A virus (HAV). This assay can detect the genomic targets directly in serum samples without an extraction step. The 4 h-long fluorometric assay was developed by combining three tiers of isothermal amplification processes and can be repurposed for any target of choice. This highly modular reaction setup is an untapped resource that can be incorporated into the front-runners of molecular diagnostics.

Recognition of DNA Target Formulations by CRISPR-Cas12a Using a dsDNA Reporter.

CRISPR-Cas12a is a powerful platform for DNA-based diagnostics. The detection scheme relies on unselective shredding of a fluorescent ssDNA reporter upon target DNA recognition. To extend the reporter library beyond ssDNAs, we discovered a fluorescent reporter type using a dsDNA template. In this design, the fluorescence of the dsDNA reporter is quenched via contact-quenching mechanism. Upon detection, the quenched fluorescence recovers with the activation Cas12a complex. Here, we compared the probing performance of two dsDNA reporters with two ssDNA reporters. The rate of the Cas12a trans-cleavage reaction was studied using one of the dsDNA reporters under different settings. The detection of different sizes of dsDNA or ssDNA targets was studied systematically under three different temperatures. Lower thresholds for ssDNA and dsDNA target size were identified. The mismatch tolerance and target specificity were examined for both ssDNA and dsDNA targets, separately. The probing performance of the dsDNA reporter was evaluated in a random DNA pool with and without target strands. We report that dsDNA can serve as a tunable fluorescence reporter template expanding the toolbox for Cas12a-based diagnostics.

Machine-Learning Single-Stranded DNA Nanoparticles for Bacterial Analysis.

A two-dimensional nanoparticle-single-stranded DNA (ssDNA) array has been assembled for the detection of bacterial species using machine-learning (ML) algorithms. Out of 60 unknowns prepared from bacterial lysates, 54 unknowns were predicted correctly. Furthermore, the nanosensor array, supported by ML algorithms, was able to distinguish wild-type Escherichia coli from its mutant by a single gene difference. In addition, the nanosensor array was able to distinguish untreated wild-type E. coli from those treated with antimicrobial drugs. This work demonstrates the potential of nanoparticle-ssDNA arrays and ML algorithms for the discrimination and identification of complex biological matrixes.