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: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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