# 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
#### 4.1.2 Cancer Cell Migration and Invasion  
One hallmark of cancer is the ability of tumor cells to migrate, invade adjacent tissues, and metastasize, which increases the probability of tumor recurrence and cancer-related death. In this process, transcriptional and posttranscriptional modifications occur in the cells that may involve classical EMT but may also involve partial EMT [48]. In thyroid cancer, ATC is the most aggressive, highly metastatic, and lethal form of disease [49]; however, in PTC, lymph node metastases are frequently detected [50].  
Platelet-derived growth factor receptor-\(\alpha\) (PDGFRA) is a proangiogenic factor that regulates tumor growth and metastasis and is highly expressed in thyroid cancer, correlating with poor survival [51]. Gene editing of _PDGFRA_ with CRISPR/Cas9 in aggressive thyroid cancer cells blunted PDGFRA expression, reduced cell invasion and reversed EMT genes expression (reduced _VIM, SLUG_, and _N-Cadherin_ and induced _E-cadherin_). Moreover, _PDGFRA_ gene editing reduced spontaneous metastasis to the lung when injected in immunocompromised mice, indicating a beneficial role for PDGFRA inhibitors, such as imatinib to treat aggressive thyroid cancer [51].  
The hematological and neurological expressed 1 (_HN1_) gene is involved in microtubule dynamics and is highly expressed in aggressive thyroid cancer that is associated with poor prognosis characteristics such as age, nodal metastasis and tumor stage [52]. Additionally, _HN1_ gene disruption with CRISPR/Cas9 markedly reduced cell migration and invasiveness in ATC cell lines in vitro and blunted tumor growth in vivo. Moreover, CRISPR/Cas9 mediated knockdown of _HN1_ was accompanied by EMT gene expression reduction and reactivation of epithelial genes. This effect was associated with the loss of HN1 interaction with Stathmin (STMN1), which blocks a-tubulin acetylation [52].  
Mitofusin 2 (MFN2), a mitochondrial dynamin-like GTPase that regulates mitochondrial stability and cell metabolism, is expressed at low levels in aggressive thyroid cancer and correlates with BRAFV600E-like/low thyroid differentiation tumors [53]. Targeting _MFN2_ gene with CRISPR/Cas9 resulted in loss of MFN2 expression in the RAS-mutated ATC cell line Cal62 and induced cell invasion in vitro. Moreover, _MFN2_ knockout induced EMT by activating ZEB1, SLUG and N-cadherin gene expression. On the other hand, overexpression of MFN2 reduced EMT genes in ATC cells, reduced cell invasion and migration in vitro, and reduced tumor formation in vivo [53].  
Another interesting gene is _BAG5_ (BAG cochaperone 5), which is upregulated in PTC and whose expression is associated with fibronectin 1 (FN1) [54]. The depletion of BAG5 protein with CRISPR/Cas9-gene editing reduced cell migration and invasion in thyroid-cancer cells. This effect is associated with FN1 protein downregulation that is negatively targeted by upregulation of miRNA _miR-R-144-3p_, which is repressed indirectly by BAG5 [54].  
Most of the studies cited in this thyroid cancer CRISPR section used the plasmid system to express Cas9 nuclease and the sgRNA sequence (as shown in the Section 3 Dry Lab). However, this method generates a stable overexpression of Cas9 and sgRNA that may not be desired in terms of off-targeting potential. Thus, the strategy of transfecting Cas9 RNPs (Cas9 protein complexed with sgRNA explored in Section 3 Dry-Lab: How do I Use CRISPR In Vitro?) results in a temporary presence of Cas9 complex (24-48 h) to edit the desired target, thus minimizing off-targets. This approach was used to investigate the role of LIMD2 in thyroid cancer metastasis. High levels of LIMD2 are detected in metastatic PTC and CRISPR/Cas9 gene editing of _LIMD2_ in thyroid cancer cell lines BCPAP and TPC1 reduced cell invasion and modified cell polarity [55]. Moreover, LIMD2 knockout led to the dysregulation of phosphoprotein cascade, which included inducers of the EMT process, such as SLUG and TWIST.