# Gene Editing with CRISPR/Cas Methodology and Thyroid Cancer: Where Are We?
## 4 Gene Editing with CRISPR/Cas in Thyroid Cancer Research
### Targeting Coding Genes in Thyroid Cancer  
#### 4.1.1 MAPK Pathway  
Thyroid cancer is the most frequent endocrine cancer [25], and arises from two distinct main parenchymal cells, the thyroid-hormone producing follicular cells and the calcitonin-secreting C-cells (also called parafollicular cells). The majority of thyroid cancer is derived from follicular cells, and classified as well-differentiated (papillary and follicular) and undifferentiated (anplastic) types, which are the focus on the present review, whereas the C-cells derived cancer, the medullary thyroid cancer, is less prevalent [26]. The most frequent histotype of thyroid cancer is the papillary thyroid cancer (PTC, approximately 80-85%) which in general exhibits a low mortality. On the other hand, the less common histotype, the anaplastic thyroid cancer (ATC, 1%) presents an extremely aggressive clinical behavior. The mitogen-activated protein kinase (MAPK) pathway contains the majority of driver oncogenes in both of the subtypes [27], and additional alterations, such as in _TERT_ promoter and _TP53_ genes in ATC [28; 29]. In PTC, the MAPK oncogenes are the highly mutated _BRAF_ (60-70%) gene, and less frequently the mutations in the isoforms of _RAS_, and the _RET_ gene translocations (_RET/PTC_ rearrangement), alterations that lead to overactivation of this signaling [30; 31; 32]. Besides the proliferative effect, MAPK signaling is involved in the control of cell differentiation, motility, invasion, apoptosis, and noncoding signaling [33; 34; 35; 36]. BRAF\({}^{\rm V600E}\) point mutations are the most common event in PTC (~60%) and are detected in poorly differentiated thyroid cancer (PDTC) and ATC, approximately 33% and 45%, respectively [32; 37]. Classically, mouse models BRAFF\({}^{\rm V600E}\) targeted to thyroid cells develop PTC with high penetrance and facilitated important discoveries in thyroid-cancer development and progression [38; 39; 40]. However, even in the CRISPR era, mouse genetic manipulating is still a laborious and time-consuming process. In this context, zebrafish constitute an alternative model that can be easily manipulated in vitro and can facilitate rapid evaluation of the effects of genetic manipulation, as the development of major organs occurs within 5 days. Indeed, a zebrafish BRAFF\({}^{\rm V600E}\) model (named Tg-BRAF-TOM) showed that BRAF activation in thyroid cells induced severe thyroid gland disorganization and impairment of thyroid hormone production as soon as 5 days postfertilization [41]. Moreover, adult Tg-BRAF-TOM animals displayed thyroid cancer that resembled human histopathological features. The RNAseq analysis revealed the activation of the epithelial-mesenchymal transition (EMT) program (as observed in larvae), and gene-set-enrichmentanalysis identified the transcription factor _twist3_ (orthologue of _TWIST2_) when Tg-BRAF-TOM profile was compared to human PTC in TCGA database [32]. Targeting the _twist3_ gene with CRISPR/Cas9-mediated gene editing resulted in a loss of function of the twist3 in zebrafish larvae and led to the partial restoration of thyroid follicles in Tg-BRAF-TOM animals revealing the potentiality of the technique in vivo [41]. Despite the classical MAPK genetic alteration, the gene expression of key MAPK genes were also detected in thyroid cancer. For example, the overexpression of epidermal growth factor receptor (EGFR) is reported in the thyroid cancer database from The Cancer Genome Atlas (TCGA) [32] and in aggressive ATC [42]. Indeed, CRISPR/Cas9-mediated _EGFR_ gene editing in ATC cell line _SW579_ resulted in EGFR knockout at the protein level that blocked ERK and AKT phosphorylation [42]. Moreover, EGFR loss induced cell growth arrest in the S-phase, and reduced colony formation ability. In the context of negative regulators of MAPK signaling, the NF2 protein (also known as Merlin) is a scaffold protein that regulates the receptor-mediated signaling pathway, such as MAPK and Hippo signaling [43].