A pH-engineering regenerative DNA tetrahedron ECL biosensor for the assay of SARS-CoV-2 RdRp gene based on CRISPR/Cas12a trans-activity.
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ABSTRACT:
SUBMITTER: Zhang K
PROVIDER: S-EPMC8440004 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Development of CRISPR/Cas-based in vitro diagnostic devices, or CRISPR/Cas-Dx, has become an intensely researched area. Among the different classes of CRISPR/Cas-Dx, the class based on the Cas12a enzyme (i.e., CRISPR/Cas12a-Dx or simply Cas12a-Dx), is predominantly employed for detecting DNA targets. Current research in Cas12a-Dx has focused on appending Cas12a-Dx to preamplification techniques or coupling Cas12a-Dx to different detection modalities, which has inevitably overshadowed the detection performance of Cas12a-Dx and overlooked its intrinsic detection capability without preamplification. We recognize that Cas12a-Dx, which relies on DNA-activated Cas12a to cleave single-stranded DNA, shares significant similarity with other nuclease-based DNA biosensors, whose performances can be influenced by parameters ranging from the reaction buffer to the reaction temperature. We are thus inspired to probe the limits of preamplification-free Cas12a-Dx by exploring and systematically evaluating several potential parameters that may impact its detection sensitivity and time. Using a previously reported fluorescence-based Cas12a-Dx as the test bed, we have identified that the Cas12a enzyme, the reaction buffer, the substrate label, the substrate concentration, and the reaction temperature can be optimized to significantly improve the signal-to-background ratio and the reaction rate of Cas12a-Dx. Based on these findings, we have improved the limit of detection (LOD) of the Cas12a-Dx to 100 fM, while reduced the time-to-positive to <46 min, representing the most sensitive LOD without preamplification and the fastest time-to-positive for this LOD to date. More broadly, our work provides a roadmap for further advancing Cas12a-Dx and perhaps other classes of CRISPR/Cas-Dx.
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Project description:Early diagnosis and timely management of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are the keys to preventing the spread of the epidemic and controlling new infection clues. Therefore, strengthening the surveillance of the epidemic and timely screening and confirming SARS-CoV-2 infection is the primary task. In this work, we first proposed the idea of activating CRISPR-Cas12a activity using double-stranded DNA amplified by a three-dimensional (3D) DNA walker. We applied it to the design of an electrochemiluminescent (ECL) biosensor to detect the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene. We first activated the cleavage activity of CRISPR-Cas12a by amplifying the target DNA into a segment of double-stranded DNA through the amplification effect of a 3D DNA walker. At the same time, we designed an MXene based ECL material: PEI-Ru@Ti3C2@AuNPs, and constructed an ECL biosensor to detect the RdRp gene based on this ECL material as a framework. Activated CRISPR-Cas12a cleaves the single-stranded DNA on the surface of this sensor and causes the ferrocene modified at one end of the DNA to move away from the electrode surface, increasing the ECL signal. The extent of the change in electrochemiluminescence reflects the concentration of the gene to be measured. Using this system, we detected the SARS-CoV-2 RdRp gene with a detection limit of 12.8 aM. This strategy contributes to the rapid and convenient detection of SARS-CoV-2-associated nucleic acids and promotes the clinical application of ECL biosensors based on CRISPR-Cas12a and novel composite materials.
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Project description:In addition to cis-cleavage activity that recognizes and cleaves nucleic acid sequences, a trans-cleavage activity that indiscriminately and non-specifically cleaves single-stranded DNA or RNA has been discovered in some Cas proteins, including Cas12a and Cas13a. Various detection methods using this activity have been widely reported. Herein, we describe a new highly efficient DNA reporter (5'-TTATT-CCCCC-3'; TTATT-5C) that outperformed the existing AT-rich DNA reporter (5'-TTATT-3') used in most Cas12a-based target nucleic detection assays. By systematically investigating the effect of DNA reporter length and sequence on the trans-cleavage activity of Cas12a, we achieved up to a 100-fold increase in fluorescence signal intensity derived from the trans-cleavage activity of Cas12a compared to that achieved using the existing AT-rich DNA reporter. The new DNA reporter was also applied, along with the existing AT-rich DNA reporter, for the detection of the Salmonella enterotoxin (stn) gene. Importantly, both detection speed and limit were significantly enhanced with the new DNA reporter. In addition, polymerase chain reaction (PCR) was adopted to the CRISR/Cas-Based system of the new DNA reporter, thereby confirming its practical applicability. The high-efficiency DNA reporter described herein can pave the way for further improving the trans-cleavage activity of other Cas proteins, as well as the sensitivity of CRISPR/Cas-Based systems.Supplementary informationThe online version contains supplementary material available at 10.1007/s13206-022-00081-0.
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Project description:Clostridium species have gained attention in industrial and medical applications, and the development of genetic tools has enabled the advancement of the CRISPR-Cas systems for these bacteria. Compared to the primarily used Cas9 from Streptococcus pyogenes, the utilization of Cas12a (previously known as Cpf1) proteins remains incomplete in clostridia, although they exhibit potential advantages, including T-rich recognition for Clostridium genomes and lower toxicity. In this study, we expanded the CRISPR-Cas tools in clostridia by establishing a CRISPR-Cas12a system with two different cas12a genes (Ascas12a from Acidaminococcus and Fncas12a from Francisella novicida). The optimized tetracycline-inducible systems were initially determined by the glucuronidase reporter and were used to drive expression of the cas12a genes and crRNAs. Our results demonstrate that the CRISPR-FnCas12a system offers flexible target selection in clostridia, and a specific folding pattern of the precursor crRNA is important to enable high mutation generation efficiency. By using sacB (encoding levansucrase) as a negative marker for plasmid curing and determining the optimal size of the donor DNA template for gene integration in the CRISPR-FnCas12a system, we achieved highly efficient and rapid genome modification, exemplified by the successful engineering of clostridia (Clostridium butyricum and Clostridium sporogenes) to produce near-infrared fluorescence from biliverdin and hemin.IMPORTANCEContinued efforts in developing the CRISPR-Cas systems will further enhance our understanding and utilization of Clostridium species. This study demonstrates the development and application of a genome-engineering tool in two Clostridium strains, Clostridium butyricum and Clostridium sporogenes, which have promising potential as probiotics and oncolytic agents. Particular attention was given to the folding of precursor crRNA and the role of this process in off-target DNA cleavage by Cas12a. The results provide the guidelines necessary for efficient genome engineering using this system in clostridia. Our findings not only expand our fundamental understanding of genome-engineering tools in clostridia but also improve this technology to allow use of its full potential in a plethora of biotechnological applications.
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