Solid-Phase Extraction and Enhanced Amplification-Free Detection of Pathogens Integrated by Multifunctional CRISPR-Cas12a.

Public healthcare demands effective and pragmatic diagnostic tools to address the escalating challenges in infection management in resource-limited areas. Recent advances in clustered regularly interspaced short palindromic repeat (CRISPR)-based biosensing promise the development of next-generation tools for disease diagnostics, including point-of-care (POC) testing for infectious diseases. The currently prevailing strategy of developing CRISPR/Cas-based diagnostics exploits only the target identification and trans-cleavage activity of a CRISPR-Cas12a/Cas13a system to provide diagnostic results, and they need to be combined with an additional preamplification reaction to enhance sensitivity. In contrast to this dual-function strategy, here, we present a new approach that collaboratively integrates the triple functions of CRISPR-Cas12a: target identification, sequence-specific enrichment, and signal generation. With this approach, we develop a nucleic acid assay termed Solid-Phase Extraction and Enhanced Detection Assay integrated by CRISPR-Cas12a (SPEEDi-CRISPR) that negates the need for preamplification but significantly improves the detection of limit (LOD) from the pM to fM level. Specifically, using Cas12a-coated magnetic beads, this assay combines efficient solid-phase extraction and enrichment of DNA targets enabled by the sequence-specific affinity of CRISPR-Cas12a with fluorogenic detection by activated Cas12a on beads. SPEEDi-CRISPR, for the first time, leverages the possibility of employing CRISPR/Cas12a in nucleic acid extraction and integrates the ability of both enrichment and detection of CRISPR/Cas into a single platform. Our proof-of-concept studies revealed that the SPEEDi-CRISPR assay has great specificity to distinguish HPV-18 from HPV-16, and Parvovirus B19, in addition to being able to detect HPV-18 at a concentration as low as 2.3 fM in 100 min and 4.7 fM in 60 min. Furthermore, we proved that this assay can be coupled with two point-of-care testing strategies: the smartphone-based fluorescence detector and the lateral flow assay. Overall, these results suggested that our assay could pave a new way for developing CRISPR diagnostics.

Solid-Phase Extraction and Enhanced Amplification-Free Detection of Pathogens Integrated by Dual-Functional CRISPR-Cas12a.

Public healthcare demands effective and pragmatic diagnostic tools to address the escalating challenges in infection management in resource-limited areas. Recent advance in CRISPR-based biosensing promises the development of next-generation tools for disease diagnostics, including point-of-care (POC) testing for infectious diseases. Currently prevailing strategy of developing CRISPR assays exploits only the non-specific trans-cleavage function of a CRISPR-Cas12a/Cas13a system for detection and combines it with an additional pre-amplification reaction to enhance the sensitivity. In contrast to this single-function strategy, here we present a new approach that collaboratively integrates the dual functions of CRISPR-Cas12a: sequence-specific binding and trans-cleavage activity. With this approach, we developed a POC nucleic acid assay termed Solid-Phase Extraction and Enhanced Detection assay Integrated by CRISPR-Cas12a (SPEEDi-CRISPR) that negates the need for preamplification but significantly improves the detection of limit (LOD) from the pM to fM level. Specifically, using Cas12a-coated magnetic beads, this assay combines efficient solid-phase extraction and enrichment of DNA targets enabled by the sequence-specific affinity of CRISPR-Cas12a with the fluorogenic detection by the activated Cas12a on beads. Our proof-of-concept study demonstrated that the SPEEDi-CRISPR assay affords an improved detection sensitivity for human papillomavirus (HPV)-18 with a LOD of 2.3 fM and excellent specificity to discriminate HPV-18 from HPV-16, Parvovirus B19, and scramble HPV-18. Furthermore, this robust assay was readily coupled with a portable smartphone-based fluorescence detector and a lateral flow assay for quantitative detection and visualized readout, respectively. Overall, these results should suggest that our dual-function strategy could pave a new way for developing the next-generation CRISPR diagnostics and that the SPEEDi-CRISPR assay provides a potentially useful tool for point-of-care testing.

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

The use of microRNAs as clinical cancer biomarkers is hindered by the absence of accurate, fast and inexpensive assays for their detection in biofluids. Here we report a one-step and one-pot isothermal assay that leverages rolling-circle amplification and the endonuclease Cas12a for the accurate detection of specific miRNAs. The assay exploits the cis-cleavage activity of Cas12a to enable exponential rolling-circle amplification of target sequences and its trans-cleavage activity for their detection and for signal amplification. In plasma from patients with pancreatic ductal adenocarcinoma, the assay detected the miRNAs miR-21, miR-196a, miR-451a and miR-1246 in extracellular vesicles at single-digit femtomolar concentrations with single-nucleotide specificity. The assay is rapid (sample-to-answer times ranged from 20 min to 3 h), does not require specialized instrumentation and is compatible with a smartphone-based fluorescence detection and with the lateral-flow format for visual readouts. Simple assays for the detection of miRNAs in blood may aid the development of miRNAs as biomarkers for the diagnosis and prognosis of cancers.