# Gene Editing with CRISPR/Cas Methodology and Thyroid Cancer: Where Are We?
## 4 Gene Editing with CRISPR/Cas in Thyroid Cancer Research
### Targeting Noncoding Genes in Thyroid Research  
In the context of gene-editing noncoding RNAs, the literature on thyroid research is still scarce, with few papers about small RNAs/microRNAs, and long noncoding RNAs. The strategy of gene editing is particularly interesting to blunt the expression of oncogenic miRNAs in thyroid cancer. MiRNAs are small non-coding RNAs (18-22 nt) that regulate target mRNA expression post-transcriptionally by interacting with the 3\({}^{\prime}\)-unstranslatedregion (UTR); furthermore, miRNAs may exert oncogenic or tumor suppressor roles according to the target mRNAs [34]. Among the deregulated miRNAs, _miR-146b-5p_ is the most overexpressed in papillary thyroid cancer and is associated with aggressive clinical-pathological characteristics such as extrathyroidal extension [68]. Moreover, a cluster of miRNAs, _miR-17-92_ that transcribes seven mature miRNAs, is also linked to aggressive tumors [69] and is overexpressed in anaplastic thyroid cancer [69; 70]. Our group used the CRISPR/Cas9n system with mutant nickase Cas9n, which requires a double-sgRNA strategy to target DNA, to disturb the structure of a miRNA gene in two different studies, one targeting _miR-17-92_ and the other targeting _miR-146b-5p_[71; 72]. First, to disrupt the expression of the _miR-17-92_ cluster, we edited a particular region in _MIR17 HG_ (the host gene for _miR-17-92_) by targeting a double-sgRNA+Cas9n to the proximal region of _miR-17_ that contains a splicing site [73]. As a result, we observed a strong reduction in the expression of all cluster miRNAs in ATC cell line KTC2, especially _miR-17_ which was closer to the gene-editing site [71]. As a functional effect, ATC cells showed reduction in cell viability, cell migration and colony-formation ability, but restored the expression of thyroid transcription factors, PAX8 and NKX2-1, and responsiveness to TGF\(\theta\) antimitogenic cytokine [71]. In a further study, we showed that CRISPR/Cas9n can be directed to target and edit the _MIR146B_ gene, which transcribes the most overexpressed miRNA in thyroid cancer, _miR-146b-5p_. We also used the double-sgRNA strategy to target the _miR-146b-5p_ sequence and disrupt the precursor hairpin structure. We observed strong reduction in _miR-146b-5p_ mature level that was sufficient to result in important functional effects in the ATC cell line KTC2, such as impairment of cell proliferation, cell migration and cell growth in immunocompromised mice [72]. These results indicate the importance of miRNAs for thyroid cancer biology, and that targeting miRNAs with CRISPR/Cas9n may become a promising approach for aggressive thyroid cancer. In the context of small noncoding RNAs (<200 nt), _nc886_ (officially called vault RNA 2-1) is a small 101-nt RNA transcribed by RNA polymerase III with intriguing similarities to vault RNA and pre-miRNA that are still under investigation [74]. Nevertheless, _nc886_ acts as a direct inhibitor of protein kinase R (EIF2 AK2: eukaryotic translation initiation factor 2-alpha kinase 2), thus regulating cell growth [75]. High levels of _nc886_ are associated with lymph-node metastasis and tumor aggressiveness in thyroid cancer [76]. The temporary knock-down of _nc886_ with siRNA results in cell death and activation of PKR (phosphor-PKR) in thyroid cells. To study the effect of permanent _nc886_ deletion, a segment of approximately 280 bp that contained the _nc886_ transcript was removed from the genome by using a double-sgRNA strategy with wild-type Cas9. Because of _nc886_ knockout, thyroid cells exhibited an impairment of the tumoral phenotype such as a reduction in cell proliferation, migration, and invasion associated with blockage of transcriptional programs (target gene expression) that support an oncogenic role [76]. Long-noncoding RNAs (>200 nt) are also an important class of noncoding RNAs that exert functional roles in thyroid cancer, with a diverse range of transcriptional and posttranscriptional effects [77].