CRISPR-Based Diagnostics: Challenges and Potential Solutions toward Point-of-Care Applications.

The COVID-19 pandemic has challenged the conventional diagnostic field and revealed the need for decentralized Point of Care (POC) solutions. Although nucleic acid testing is considered to be the most sensitive and specific disease detection method, conventional testing platforms are expensive, confined to central laboratories, and are not deployable in low-resource settings. CRISPR-based diagnostics have emerged as promising tools capable of revolutionizing the field of molecular diagnostics. These platforms are inexpensive, simple, and do not require the use of special instrumentation, suggesting they could democratize access to disease diagnostics. However, there are several obstacles to the use of the current platforms for POC applications, including difficulties in sample processing and stability. In this review, we discuss key advancements in the field, with an emphasis on the challenges of sample processing, stability, multiplexing, amplification-free detection, signal interpretation, and process automation. We also discuss potential solutions for revolutionizing CRISPR-based diagnostics toward sample-to-answer diagnostic solutions for POC and home use.

Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2.

Type VI CRISPR-Cas systems have been repurposed for various applications such as gene knockdown, viral interference, and diagnostics. However, the identification and characterization of thermophilic orthologs will expand and unlock the potential of diverse biotechnological applications. Herein, we identified and characterized a thermostable ortholog of the Cas13a family from the thermophilic organism Thermoclostridium caenicola (TccCas13a). We show that TccCas13a has a close phylogenetic relation to the HheCas13a ortholog from the thermophilic bacterium Herbinix hemicellulosilytica and shares several properties such as thermostability and inability to process its own pre-CRISPR RNA. We demonstrate that TccCas13a possesses robust cis and trans activities at a broad temperature range of 37 to 70 °C, compared with HheCas13a, which has a more limited range and lower activity. We harnessed TccCas13a thermostability to develop a sensitive, robust, rapid, and one-pot assay, named OPTIMA-dx, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. OPTIMA-dx exhibits no cross-reactivity with other viruses and a limit of detection of 10 copies/µL when using a synthetic SARS-CoV-2 genome. We used OPTIMA-dx for SARS-CoV-2 detection in clinical samples, and our assay showed 95% sensitivity and 100% specificity compared with qRT-PCR. Furthermore, we demonstrated that OPTIMA-dx is suitable for multiplexed detection and is compatible with the quick extraction protocol. OPTIMA-dx exhibits critical features that enable its use at point of care (POC). Therefore, we developed a mobile phone application to facilitate OPTIMA-dx data collection and sharing of patient sample results. This work demonstrates the power of CRISPR-Cas13 thermostable enzymes in enabling key applications in one-pot POC diagnostics and potentially in transcriptome engineering, editing, and therapies.

iSCAN-V2: A One-Pot RT-RPA-CRISPR/Cas12b Assay for Point-of-Care SARS-CoV-2 Detection.

Rapid, specific, and sensitive detection platforms are prerequisites for early pathogen detection to efficiently contain and control the spread of contagious diseases. Robust and portable point-of-care (POC) methods are indispensable for mass screening of SARS-CoV-2. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based nucleic acid detection technologies coupled with isothermal amplification methods provide a straightforward and easy-to-handle platform for detecting SARS-CoV-2 at POC, low-resource settings. Recently, we developed iSCAN, a two-pot system based on coupled loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a reactions. However, in two-pot systems, the tubes must be opened to conduct both reactions; two-pot systems thus have higher inherent risks of cross-contamination and a more cumbersome workflow. In this study, we developed and optimized iSCAN-V2, a one-pot reverse transcription-recombinase polymerase amplification (RT-RPA)-coupled CRISPR/Cas12b-based assay for SARS-CoV-2 detection, at a single temperature in less than an hour. Compared to Cas12a, Cas12b worked more efficiently in the iSCAN-V2 detection platform. We assessed and determined the critical factors, and present detailed guidelines and considerations for developing and establishing a one-pot assay. Clinical validation of our iSCAN-V2 detection module with reverse transcription-quantitative PCR (RT-qPCR) on patient samples showed 93.75% sensitivity and 100% specificity. Furthermore, we coupled our assay with a low-cost, commercially available fluorescence visualizer to enable its in-field deployment and use for SARS-CoV-2 detection. Taken together, our optimized iSCAN-V2 detection platform displays critical features of a POC molecular diagnostic device to enable mass-scale screening of SARS-CoV-2 in low-resource settings.

Bio-SCAN: A CRISPR/dCas9-Based Lateral Flow Assay for Rapid, Specific, and Sensitive Detection of SARS-CoV-2.

Simple, rapid, specific, and sensitive point-of-care detection methods are needed to contain the spread of SARS-CoV-2. CRISPR/Cas9-based lateral flow assays are emerging as a powerful alternative for COVID-19 diagnostics. Here, we developed Bio-SCAN (biotin-coupled specific CRISPR-based assay for nucleic acid detection) as an accurate pathogen detection platform that requires no sophisticated equipment or technical expertise. Bio-SCAN detects the SARS-CoV-2 genome in less than 1 h from sample collection to result. In the first step, the target nucleic acid sequence is isothermally amplified in 15 min via recombinase polymerase amplification before being precisely detected by biotin-labeled nuclease-dead SpCas9 (dCas9) on commercially available lateral flow strips. The resulting readout is visible to the naked eye. Compared to other CRISPR-Cas-based pathogen detection assays, Bio-SCAN requires no additional reporters, probes, enhancers, reagents, or sophisticated devices to interpret the results. Bio-SCAN is highly sensitive and successfully detected a clinically relevant level (4 copies/µL) of synthetic SARS-CoV-2 RNA genome. Similarly, Bio-SCAN showed 100% negative and 96% positive predictive agreement with RT-qPCR results when using clinical samples (86 nasopharyngeal swab samples). Furthermore, incorporating variant-specific sgRNAs in the detection reaction allowed Bio-SCAN to efficiently distinguish between the α, ß, and δ SARS-CoV-2 variants. Also, our results confirmed that the Bio-SCAN reagents have a long shelf life and can be assembled locally in nonlaboratory and limited-resource settings. Furthermore, the Bio-SCAN platform is compatible with the nucleic acid quick extraction protocol. Our results highlight the potential of Bio-SCAN as a promising point-of-care diagnostic platform that can facilitate low-cost mass screening for SARS-CoV-2.

A Novel Miniature CRISPR-Cas13 System for SARS-CoV-2 Diagnostics.

Rapid, point-of-care (POC) diagnostics are essential to mitigate the impacts of current (and future) epidemics; however, current methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require complicated laboratory tests that are generally conducted off-site and require substantial time. CRISPR-Cas systems have been harnessed to develop sensitive and specific platforms for nucleic acid detection. These detection platforms take advantage of CRISPR enzymes' RNA-guided specificity for RNA and DNA targets and collateral trans activities on single-stranded RNA and DNA reporters. Microbial genomes possess an extensive range of CRISPR enzymes with different specificities and levels of collateral activity; identifying new enzymes may improve CRISPR-based diagnostics. Here, we identified a new Cas13 variant, which we named as miniature Cas13 (mCas13), and characterized its catalytic activity. We then employed this system to design, build, and test a SARS-CoV-2 detection module coupling reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the mCas13 system to detect SARS-CoV-2 in synthetic and clinical samples. Our system exhibits sensitivity and specificity comparable to other CRISPR systems. This work expands the repertoire and application of Cas13 enzymes in diagnostics and for potential in vivo applications, including RNA knockdown and editing. Importantly, our system can be potentially adapted and used in large-scale testing for diverse pathogens, including RNA and DNA viruses, and bacteria.

Vigilant: An Engineered VirD2-Cas9 Complex for Lateral Flow Assay-Based Detection of SARS-CoV2.

Rapid, sensitive, and specific point-of-care testing for pathogens is crucial for disease control. Lateral flow assays (LFAs) have been employed for nucleic acid detection, but they have limited sensitivity and specificity. Here, we used a fusion of catalytically inactive SpCas9 endonuclease and VirD2 relaxase for sensitive, specific nucleic acid detection by LFA. In this assay, the target nucleic acid is amplified with biotinylated oligos. VirD2-dCas9 specifically binds the target sequence via dCas9 and covalently binds to a FAM-tagged oligonucleotide via VirD2. The biotin label and FAM tag are detected by a commercially available LFA. We coupled this system, named Vigilant (VirD2-dCas9 guided and LFA-coupled nucleic acid test), to reverse transcription-recombinase polymerase amplification to detect SARS-CoV2 in clinical samples. Vigilant exhibited a limit of detection of 2.5 copies/µL, comparable to CRISPR-based systems, and showed no cross-reactivity with SARS-CoV1 or MERS. Vigilant offers an easy-to-use, rapid, cost-effective, and robust detection platform for SARS-CoV2.

iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2.

The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.