A one-pot isothermal Cas12-based assay for the sensitive detection of microRNAs

Abstract

amplification-free nucleic acid detection. However, these approaches are not truly comparable to RT-qPCR, due to either low sensitivity without pre-amplification 42 or the limitations arising from highly specialized devices and instruments needed. Lastly, it is worth noting that most, if not all, of the existing CRISPR-based methods leverage on the trans-cleavage activity of Cas proteins, while the exploration of the specific cis-cleavage activity-the main mechanism of CRISPR-Cas systems for gene editing-for biosensing remains largely untapped.In this Article, we report a one-step, one-pot isothermal CRISPR-Cas12a assay, which we named 'Endonucleolytically Exponentiated Rolling Circle Amplification with CRISPR-Cas12a' (EXTRA-CRISPR) for the rapid and specific detection of miRNAs with sensitivity comparable to RT-PCR (Fig. 1). The EXTRA-CRISPR assay offers three major distinctions from the existing CRISPR-based biosensing methods. First, it represents a strategy to simultaneously harness both cis-cleavage and trans-cleavage activities of the CRISPR-Cas12a system. It exploits the specific cis-cleavage activity to transform conventional linear RCA to enable exponential amplification of target sequences, in addition to the trans-cleavage reaction for amplicon detection and signal amplification. Second, by engineering a modular padlock probe design and the reaction kinetics, we incorporate multiple reactions for target-mediated ligation, RCA, Cas12a binding and nucleolytic cleavage into one collaboratively coupled reaction network, creating a robust one-step, single-tube isothermal assay for miRNA analysis. Third, this one-pot isothermal miRNA assay affords comparable analytical performance with standard RT-qPCR, including high sensitivity with a single-digit femtomolar detection limit, single-nucleotide specificity and rapid and flexible turnaround (from 20 min to 3 h for the entire analysis depending on targets and samples). Lastly, one-pot EXTRA-CRISPR technology vastly simplifies the assay workflow and negates the need for specialized instruments, which provides an adaptable modality for POC diagnostics.As proof of concept of potential applications, we adapted the EXTRA-CRISPR assay to quantifying miRNA biomarkers in extracellular vesicles (EVs) for the liquid-biopsy-based diagnosis of pancreatic ductal adenocarcinoma (PDAC). EVs, including exosomes of 50-150 nm in size, are emerging as a promising candidate for liquid biopsy because nature portfolio | reporting summary

Extraction-free, one-pot CRISPR/Cas12a detection of microRNAs directly from extracellular vesicles

“…To maximize the assay performance, we first optimized the EXTRA-CRISPR assays 12 that we initially developed to target four miRNAs associated with human pancreatic ductal adenocarcinoma (PDAC), namely miR-1246, 13 miR-21, 14 miR-451a, 14 miR-196a 13 (Table S1, ESI†). Systematic optimizations of several key parameters for the EXTRA-CRISPR assays were conducted, including the concentrations of reporter, RNP, SplintR ligase, and Phi29 polymerase, as reported in Fig.…”

“…Therefore, we first sought to mitigate these potential interfering effects by the simple dilution method. To this end, we targeted miR-451a which was found to present in PC1 sEVs at a very low endogenous level, 12 so that we can better assess the effects of the buffer and exogenous impurities in EV preparations via spiking in and measuring synthetic miR-451. In this experiment, PC1 sEV samples were first diluted with water to different levels and then spiked with synthetic miR-451a at the final concentration of 50 fM.…”

“…We recently developed a CRISPR-based one-pot assay termed ''EXTRA-CRISPR'' (Endonucleolytically eXponenTiated Rolling circle Amplification with CRISPR-Cas12a) for the robust detection of miRNA. 12 EXTRA-CRISPR is a novel strategy that simultaneously leverages the cis-cleavage and trans-cleavage activities of Cas12a, where the cis-cleavage transforms the conventional linear RCA to the exponential amplification, and the trans-cleavage enables amplicon detection and signal generation. In this work, we sought to combine the EXTRA-CRISPR assay with a thermolysis-based sample-pretreatment method to enable the extraction-free, one-pot detection of miRNAs in small EVs (sEVs, 50-150 nm, also termed ''exosomes'') with a vastly streamlined workflow.…”

“…S2 (ESI †) display the typical real-time curves obtained for the individual assays, suggesting a flexible reaction time from 20 min to 3 h depending on the target concentration. 12 Using the calibration curves (Fig. 2F), the limits of detection (LoDs) defined by the 3s rule were determined to be 1.64 fM (5-500 fM linear range), 1.35 fM (5-500 fM linear range), 4.14 fM (5-500 fM linear range), and 7.96 fM (20-500 fM linear range) for miR-21, miR-196a, miR-451a, and miR-1246, respectively.…”

“…3E, a weak signal was detected even without sEV pre-treatment by thermolysis (i.e., 0 min lysis time). This is because we employed a denaturing (80 1C, 5 min) and annealing procedure 12 to confer efficient hybridization of the padlock probe with its target, which leads to partial lysis of sEVs. The highest signal for miR-1246 was observed with incubating PC1 sEV samples at 90 1C for 10 min.…”