Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.

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SHERLOCK: nucleic acid detection with CRISPR nucleases.

The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world. Rapid and early detection of SARS-CoV-2 facilitates early intervention and prevents the disease spread. Here, we present an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay for one-pot, ultrasensitive, and visual SARS-CoV-2 detection. By targeting SARS-CoV-2's nucleoprotein gene, two CRISPR RNAs without protospacer adjacent motif (PAM) site limitation are introduced to develop the AIOD-CRISPR assay and detect the nucleic acids with a sensitivity of few copies. We validate the assay by using COVID-19 clinical swab samples and obtain consistent results with RT-PCR assay. Furthermore, a low-cost hand warmer (~$0.3) is used as an incubator of the AIOD-CRISPR assay to detect clinical samples within 20 min, enabling an instrument-free, visual SARS-CoV-2 detection at the point of care. Thus, our method has the significant potential to provide a rapid, sensitive, one-pot point-of-care assay for SARS-CoV-2.

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Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread to nearly every corner of the globe, causing societal instability. The resultant coronavirus disease 2019 (COVID-19) leads to fever, sore throat, cough, chest and muscle pain, dyspnoea, confusion, anosmia, ageusia and headache. These can progress to life-threatening respiratory insufficiency, also affecting the heart, kidney, liver and nervous systems. The diagnosis of SARS-CoV-2 infection is often confused with that of influenza and seasonal upper respiratory tract viral infections. Due to available treatment strategies and required containments, rapid diagnosis is mandated. This Review brings clarity to the rapidly growing body of available and in-development diagnostic tests, including nanomaterial-based tools. It serves as a resource guide for scientists, physicians, students and the public at large.

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Diagnostics for SARS-CoV-2 infections.

An accurate, rapid, and cost-effective biosensor for the quantification of disease biomarkers is vital for the development of early-diagnostic point-of-care systems. The recent discovery of the trans-cleavage property of CRISPR type V effectors makes CRISPR a potential high-accuracy bio-recognition tool. Herein, a CRISPR-Cas12a (cpf1) based electrochemical biosensor (E-CRISPR) is reported, which is more cost-effective and portable than optical-transduction-based biosensors. Through optimizing the in vitro trans-cleavage activity of Cas12a, E-CRIPSR was used to detect viral nucleic acids, including human papillomavirus 16 (HPV-16) and parvovirus B19 (PB-19), with a picomolar sensitivity. An aptamer-based E-CRISPR cascade was further designed for the detection of transforming growth factor β1 (TGF-β1) protein in clinical samples. As demonstrated, E-CRISPR could enable the development of portable, accurate, and cost-effective point-of-care diagnostic systems.

Exploring the Trans-Cleavage Activity of CRISPR-Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor.

Ultra-sensitive and high-throughput CRISPR-p owered COVID-19 diagnosis.

CRISPR/Cas Systems towards Next-Generation Biosensing.

Monitoring of human serum albumin (HSA) in a point-of-care fashion is urgently needed in particular for elderly or chronically ill patients. Herein, a dual-state emissive chalcone probe having the feature of aggregation-induced emission was designed and synthesized. The concentration of HSA can be evaluated by the ratios of emission from probes in aggregated and monomeric state, which gives a visually discernible red-to-green color change. A simple, portable paper-based analytical device have been fabricated by integration of the recognition probe in the detection pad and employed for HSA test using the whole blood samples. This paper-based assay shows the analytical capability comparable to the standard testing methods but is in a point-of-care fashion, providing a promising tool for at-home HSA detection and HSA-related disease diagnosis.

Paper‐Based Ratiometric Fluorescence Analytical Devices towards Point‐of‐Care Testing of Human Serum Albumin

CRISPR/Cas Multiplexed Biosensing: A Challenge or an Insurmountable Obstacle?

CRISPR/Cas12a-powered immunosensor suitable for ultra-sensitive whole Cryptosporidium oocyst detection from water samples using a plate reader.

A sandwich immunosensor was successfully developed for monitoring of interleukin-1β (IL-1β) in rat whole blood. The substrate stainless steel (SS) was first coated with a polydopamine layer and subsequently grafted with poly(ethylene glycol) methacrylate brushes, onto which a sandwich immunosensor was modified for detection of IL-1β. The device has been successfully applied for monitoring of IL-1β with a limit of detection of 4.7 pg mL−1, and a linear detection range of 12.5–200 pg mL−1. Good specificity and selectivity for monitoring of IL-1β in rat macrophage secretion were achieved. Furthermore, this device was validated by detection of IL-1β in rat whole blood samples with greater concentrations observed in obese rats compared to control, and strong positive correlation between concentrations of IL-1β and blood glucose. These results suggest this device is feasible for direct detection of target analytes in biological samples.

Yi Li

Papers

CRISPR/Cas Systems towards Next-Generation Biosensing.

Paper‐Based Ratiometric Fluorescence Analytical Devices towards Point‐of‐Care Testing of Human Serum Albumin

CRISPR/Cas Multiplexed Biosensing: A Challenge or an Insurmountable Obstacle?

CRISPR/Cas12a-powered immunosensor suitable for ultra-sensitive whole Cryptosporidium oocyst detection from water samples using a plate reader.

Polymer brush based fluorescent immunosensor for direct monitoring of interleukin-1β in rat blood