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.

The collateral cleavage function is only a corollary of programmable nuclease activity of certain Cas effectors, such as Cas12 and Cas13, but it can be utilised to amplify fluorescence signals in various CRISPR/Cas-based biosensing systems. In this work, this special signal amplification capability of CRISPR/Cas12a ribonucleoproteins has been employed to increase the sensitivity of a broad class of commercial ELISA systems with undisclosed chemistry except for the use of horseradish peroxidase (HRP), a common signal reporting molecule. We demonstrated that such ELISA systems with HRP on the detection antibody, can be amplified by 2 orders of magnitude using an example commercial IFN-γ ELISA kit where the detection limit was decreased from 312.5 pg/mL to 1.2 pg/mL. The detection range was simultaneously increased from 2 orders to 3 orders of magnitude. Our CRISPR/Cas12a-based ELISA Sensitivity Amplifier (CES-Amplifier) approach is based on a hybrid single strand DNA oligo and antibody conjugate targeting the HRP enzyme. The CES-Amplifier can be directly integrated into commercial ELISA kits to replace their original last step, without any additional changes of the ELISA kit reagents or setup. In this way, our CES-Amplifier provides a versatile and affordable approach for expanding CRISPR/Cas-based biosensing to a wide range of non-nucleic acid analytes.

A versatile CRISPR/Cas12a-based sensitivity amplifier suitable for commercial HRP-based ELISA kits

A simple and versatile CRISPR/Cas12a-based immunosensing platform: Towards attomolar level sensitivity for small protein diagnostics.

The exceptional programable trans -cleavage ability of type V and VI CRISPR/Cas nucleases paved the way for ultrasensitive CRISPR/Cas based sensing of nucleic acid and alternative targets. However, the enhancement of the trans -cleavage activity of Cas effector with organic chemical agents has not been explored thus far. We report here chemically enhanced trans -cleavage activity of Cas12a and Cas13a nucleases which improves sensor performance in CRISPR/Cas biosensing. Improved trans -ssDNA cleavage of Cas12a and trans -ssRNA cleavage of Cas13a were demonstrated by using sulfhydryl reductants and non-ionic surfactants. DTT and PVA were demonstrated to be the most effective chemical enhancers in both cases. By using a fluorescence resonance energy transfer (FRET)-based intramolecular distance measurements, we identified the mechanism of this enhancement to be the conformation change of the ribonucleoprotein and quantified it to be major (about 50% increase of a relevant intramolecular distance). These chemical enhancers have been integrated into the established CRISPR/Cas biosensing protocols without additional modifications. For the detection of Helicobacter Pylori DNA and SARS-CoV-2 RNA, we found a decreased reaction time by 75% to 83% and 4 to 6-fold increased sensitivity. These results indicate that chemical enhancers provide a versatile and broadly applicable approach to break through the barriers of long reaction time and sensitivity in CRISPR/Cas sensors. • Enhanced trans -cleavage activity of Cas12a and Cas13a by chemical enhancers • Sulfhydryl reductants and non-ionic surfactants are two types of chemical enhancers • The mechanism of this enhancement is the conformation change of Cas RNP • The reaction time of chemical enhanced CRISPR/Cas system reduced 75% to 83% • The sensitivity of chemical enhanced CRISPR/Cas system increased 4 to 6 folds 

Increasing trans-cleavage Catalytic Efficiency of Cas12a and Cas13a With Chemical Enhancers: Application to Amplified Nucleic Acid Detection

Oncolytic viruses (OVs), including oncolytic herpes simplex viruses (oHSVs), are promising therapeutics against cancer. Here, we report two ICP6‐mutated HSVs (type I) generated by CRISPR/Cas9, rHSV1/∆RR (with ICP6 ribonucleotide reductase [RR] domain deleted) and rHSV1/∆ICP6 (with a complete deletion of ICP6), exhibiting potent antitumor efficacy against lung adenocarcinoma. Both the mutants showed strong cytotoxicity in vitro, comparable with the control viruses expressing intact ICP6, but in relatively lower titers. Moreover, these mutant viruses exhibited preferential killing ability against lung tumor cells rather than normal lung fibroblast cells. Further, unlike the control HSV‐1 causing severe illness or death in the mouse model, the ICP6‐mutated viruses did not induce significant pathogenicity but instead effectively reduced tumor burden in vivo and led to 100% survival of the animals, indicating notable antitumor activity and attenuated virulence. In addition, rHSV1/∆RR seemed to have even better antitumor efficacy than rHSV1/∆ICP6, albeit no statistical significance in inhibition of tumor volume. Histopathologically, rHSV1/∆RR induced massive neutrophil infiltration to the tumor microenvironment and consistently, triggered more antitumor immune and neutrophil chemotactic cytokines or higher expression levels of them (indicated by quantitative polymerase chain reaction and transcriptome analyses). These results demonstrate the anti‐adenocarcinoma potential of the CRISPR/Cas9‐engineered ICP6 mutant HSV1, especially the rHSV1/∆RR, which likely induces stronger innate antitumor immune response. Together, these findings may provide new valuable clues for further development of OV‐based therapeutics against lung adenocarcinoma or other types of tumors.

Antitumor efficacy of CRISPR/Cas9–engineered ICP6 mutant herpes simplex viruses in a mouse xenograft model for lung adenocarcinoma

Cytokines are typical mediators of the immune response. Single cytokine measurement is unable to reflect the true complexity of these physiological processes and multiplex detection is required. In this work, we developed an optical fibre based biosensor for multiplex detection of cytokines. This multiplex biosensor has been successfully applied for the measurement of proinflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the linear range of 12.5–200 pg mL−1 with the limit of detection (LOD) of 12.5 pg mL−1. Additionally, it has compatible performance with ELISA kit for the monitoring of cytokines from rat PBMC cell culture mediums stimulated by LPS or UV radiation. Moreover, this universal platform could be applied for multiplex detection of other proteins by simply changing the capture/detection antibody pairs. This fibre based multiplex biosensor is designed for in-situ measurement of target cytokines, which is significant for the investigation of inflammatory processes or disease prognosis and treatment monitoring. 

Yi Li

Papers

A versatile CRISPR/Cas12a-based sensitivity amplifier suitable for commercial HRP-based ELISA kits

A simple and versatile CRISPR/Cas12a-based immunosensing platform: Towards attomolar level sensitivity for small protein diagnostics.

Increasing trans-cleavage Catalytic Efficiency of Cas12a and Cas13a With Chemical Enhancers: Application to Amplified Nucleic Acid Detection

Antitumor efficacy of CRISPR/Cas9–engineered ICP6 mutant herpes simplex viruses in a mouse xenograft model for lung adenocarcinoma

A fluorescent immunosensor on optical fibre for the multiplex detection of proinflammatory cytokines