Preliminary evaluation of CRISPR-Cas9-edited cells targeting MCTP2, a candidate gene for familial non-medullary thyroid cancer

Thyroid cancer is increasing in incidence worldwide, with its most dominant form arising from follicular cells. A familial variant of this cancer, known as familial non-medullary thyroid cancer (FNMTC), shows a hereditary component but its genetic predisposition remains poorly understood. Several candidate genes have been identified but none have achieved consistent validation at either the genetic or functional level. Through a previous Whole Genome Sequencing analysis in FNMTC families, several mutations cosegregating with the disease were identified in the MCTP2 (multiple C2 and transmembrane domain containing 2) which was subsequently identified as a promising candidate gene. To explore its function, thyroid immortalized cells were edited using CRISPR-Cas9 technology with an aim of generating MCTP2 knockout model. In this thesis, the validity and efficacy of the CRISPR-Cas9 approach were assessed using PCR and gel electrophoresis, together with subsequent techniques like Sanger sequencing and Real-time qPCR to validate the proper gene knockout. The analyses confirmed successful editing and revealed markedly reduced MCTP2 expression in the majority of tested clones, establishing a preliminary model for investigating the possible role of MCTP2 in FNMTC predisposition.