posted on 2023-11-03, 13:03authored byCarmen Escalona-Noguero, Hernán Alarcón-Iniesta, María López-Valls, Luis Paul del Carpio, Josep M. Piulats, Álvaro Somoza, Begoña Sot
Uveal melanoma (UM) is a rare ocular tumor characterized
by high
metastasis risk and poor prognosis. The in-depth 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.