CRISPR Assays for Disease Diagnosis: Progress to and Barriers Remaining for Clinical Applications

Abstract

Numerous groups have employed the special properties of CRISPR/Cas systems to develop platforms that have broad potential applications for sensitive and specific detection of nucleic acid (NA) targets. However, few of these approaches have progressed to commercial or clinical applications. This review summarizes the properties of known CRISPR/Cas systems and their applications, challenges associated with the development of such assays, and opportunities to improve their performance or address unmet assay needs using nano-/micro-technology platforms. These include rapid and efficient sample preparation, integrated single-tube, amplification-free, quantifiable, multiplex, and non-NA assays. Finally, this review discusses the current outlook for such assays, including remaining barriers for clinical or point-of-care applications and their commercial development.

Detection of the Uveal Melanoma-Associated Mutation GNAQ Q209P from Liquid Biopsy Using CRISPR/Cas12a Technology

“…This sensitivity is insufficient for the direct detection of uveal melanoma mutations from either tumor tissue or liquid biopsy. Cas12a sensing must then be coupled to prior nucleic acid amplification . Among the different amplification techniques available, we chose PCR because it is a cost-effective, specific, and semiquantitative method.…”

“…Cas12a sensing must then be coupled to prior nucleic acid amplification. 22 Among the different amplification techniques available, we chose PCR because it is a costeffective, specific, and semiquantitative method. To assess the potential suitability of our strategy for detecting GNAQ Q209P from physiological samples, we isolated genomic DNA (gDNA) from two cell lines: OMM 1.3, a uveal melanoma cell line containing the GNAQ Q209P mutation; and HEK293, a cell line with wild-type GNAQ.…”

Leveraging Cas13a's trans-cleavage on RNA G-quadruplexes for amplification-free RNA detection

“…CRISPR-Cas systems are extensively employed in nucleic acid testing (NAT), leveraging the trans-cleavage activity of Cas proteins activated by CRISPR RNA (crRNA) targeting. 1 Previous studies have primarily focused on harnessing Cas13's trans -cleavage activity towards ssRNA, and relatively complex strategies are required to ultimately achieve essential sensitivity for target amplification-free detection (Table S1, ESI†). 2–4 We previously uncovered that Cas12a can trans -cleave DNA G-quadruplex (G4) and G-triplex (G3) structures, and demonstrated that the high-order DNA structure-based reporter improved the detection sensitivity by up to 20 fold for NAT (albeit without a fully disclosed mechanism).…”