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

Abstract

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.

Amplification-Free COVID-19 Detection by Digital Droplet REVEALR

“…When coupled to sequence-specific detection modalities, such as those found in CRISPR-Cas enzymes, these systems offer sensitive and highly accurate platforms for viral RNA detection. 21 Similar detection routes can also be achieved using DNAzyme constructs that have been engineered into point-of-care diagnostics by introducing an analyte recognition domain into the scaffold. 22 However, despite their many benefits, amplification-based approaches are cumbersome due to the need for a sample preamplification step that places an additional burden of time and cost on the assay.…”

“…Finally, the catalytic mechanism of the ddREVEALR system allows for greater targetability of the viral genome, as the targetability of CRISPR-based system is restricted by the protospacer adjacent motif (PAM) for Cas9 and Cas12 or protospacer flanking site (PFS) for Cas13. 21 …”

“…In the field of nucleic acid recognition, specifically, isothermal amplification strategies have emerged as rapid and inexpensive alternatives to quantitative RT-PCR, which remains the gold standard for SARS-CoV-2 detection. When coupled to sequence-specific detection modalities, such as those found in CRISPR-Cas enzymes, these systems offer sensitive and highly accurate platforms for viral RNA detection . Similar detection routes can also be achieved using DNAzyme constructs that have been engineered into point-of-care diagnostics by introducing an analyte recognition domain into the scaffold .…”

“…This property allows for higher purity and greater scalability of the reagents, as cellular processes are difficult to scale and can be prone to unwanted viral or bacterial contamination. Finally, the catalytic mechanism of the ddREVEALR system allows for greater targetability of the viral genome, as the targetability of CRISPR-based system is restricted by the protospacer adjacent motif (PAM) for Cas9 and Cas12 or protospacer flanking site (PFS) for Cas13 …”

“…16 ddREVEALR compares favorably to related CRISPR-based approaches for amplification-free viral RNA detection. Unlike CRISPR, which relies on RNA and protein-based reagents that are temperature-sensitive and must be stored and transported at low temperatures, 21 ddREVEALR is a DNA-based proteinfree system (Figure S11) that is stable at room temperature and, as such, avoids the costly expense of the cold storage. The DNA components of ddREVEALR are also easier and cheaper to produce as they can be made by chemical synthesis rather than cellular protein expression and purification.…”