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

Abstract

Uveal melanoma (UM) is a rare ocular tumor characterized by high metastasis risk and poor prognosis. The indepth characterization of UM's molecular profile is critical for better disease classification and prognosis. Furthermore, the development of detection tools to monitor UM evolution upon treatment is of great interest for designing optimal therapeutic strategies. However, commonly used techniques, such as ddPCR or NGS, are costly, and they involve sophisticated equipment and complex experimental design. The development of alternative sensing methods that are fast, simple, and inexpensive would be of great benefit to improve UM's diagnosis and management, especially when combined with liquid biopsy. Samples from liquid biopsy can be obtained with minimal invasiveness, and the detection of circulating tumor DNA (ctDNA) in UM patients' plasma has proven useful for the diagnosis of metastasis, prognosis prediction, and disease monitoring. In this context, CRISPR/Cas12a-derived molecular sensors, thanks to their high specificity and sensitivity and their potential for point of care diagnosis, are particularly interesting. Here, we developed a CRISPR/Cas12a-based approach for the specific detection of the UM-related mutation GNAQ Q209P that relies on the design of highly specific crRNAs. Coupled with allele-specific PCR, it constitutes a sensitive platform for liquid biopsy detection, capable of sensing GNAQ Q209P in plasma samples with a low ctDNA concentration and fractional abundance. Finally, our method was validated using plasma samples from metastatic UM patients.

Recent Advances in Molecular and Genetic Research on Uveal Melanoma

“…This inconsistency makes the use of ctDNA in diagnosing primary UM still a subject of debate and underscores the need for better detection methods. For example, a recent study using a CRISPR/Cas12a-based fluorescent sensor was able to detect the GNAQ Q209P mutation in patients’ plasma with a minimum of 68 ctDNA copies/mL and 3% of fractional abundance of mutant GNAQ [ 102 ]. Although this technology is highly sensitive, the plasma from only four patients was tested using this technique.…”