# Gene Editing with CRISPR/Cas Methodology and Thyroid Cancer: Where Are We?
## 2 The Principle of CRISPR/Cas
### New Applications for CRISPR/Cas: Tailoring Cas Activity  
Since the groundbreaking report of CRISPR/Cas9-mediated gene editing [2], intense investigations of Cas9 protein crystallography and biochemistry have resulted in important advances in the understanding of endonuclease function. It also permitted repurposing Cas9 variants into new applications such as CRISPR or CRISPRa (explored later in this section) that perturb gene expression without changing the DNA sequence (epigenetic modulation) by inactivating or activating gene transcription. The nuclease activity of Cas9 generates a double-strand DNA break and can be modulated through specific mutations in the nuclease domain. The RuvC (D10A) or HNH (H840A) mutations individually render Cas9 enzyme single-strand break activity (called nickase: Cas9n), meaning it cuts only one strand of DNA. An endogenous repair system easily repairs this type of DNA damage; however, the Cas9n system can be used for gene editing in a double-sgRNA strategy (Table 2), where a double-strand break occurs only in the DNA region targeted by a pair of sgRNA complementary to a different strand of DNA, and requires an optimal distance (minus 4 to 40-nt). This strategy reduces off-targets because only on-targeting results in double-strand breaks [17], and is better exemplified in Section 4.2. Targeting Noncoding Genes in Thyroid Research. The double-mutant (RuvC D10A and HNH H840A) is considered a \"dead\" Cas9 (dCas9) that does not cleave or nick the DNA strand, but it still interacts with target DNA region guided by the sgRNA. This property is especially noteworthy in disturbing gene expression when dCas9 impairs the recruitment of transcriptional complexes or blocks RNA polymerase [21]. In this context, dCas9 may be fused to different protein domains that can result either in gene expression silencing or gene induction [22], which we explore below in detail (Figure 4). Figure 3: DNA repair mechanism. Mechanisms of gene editing mediated by NHEJ and HDR after CRISPR/Cas-induced double-strand break. CRISPRR interference (CRISPRRi): the presence of dCas9 complex (dCas9 + sgRNA) in a coding region interferes with transcriptional elongation, RNA polymerase binding, or transcription factor binding leading to reduction of gene transcription [21]. In this study, the authors directed dCas9 + sgRNAs to the coding region of _mRFP_ (mouse red fluorescent protein) and observed that the sgRNA targeting of the coding region's proximal sequences (in the non-template strand) resulted in a 10 to 300-fold inhibition of gene expression. Moreover, targeting the proximal promoter (minus 35-nt box region) resulted in a strong knockdown of _mRFP_ gene expression [21]. Further study of this mechanism led to the development of the CRISPR methodology that relies on the use of dCas9 fused to the repressive chromatin-modifier domain in the N-terminal region, such as Kruppel-associated box (KRAB) of Kox1, to improve CRISPR silencing efficiency [22]. Indeed, targeting the dCas9-KRAB complex (+sgRNA) to the proximal promoter or the first exons of _EGFP_ (enhanced green fluorescent protein) resulted in robust inhibition of gene transcription (\(\sim\)15-fold) because KRAB recruited chromatin modifiers to proximal regulatory elements [22]. CRISPR activation (CRISPRRa): In the CRISPR system, dCas9 protein fused transcriptional activators, such as VP64 (tetrameric repeat of VP16 from Herpes virus) or the p65 activation domain, induces target gene expression [22] (Figure 4B). Indeed, targeting sgRNA to the upstream activation sequence of the _Gal4_ gene, for example, resulted in a 25-fold and 12-fold activation for dCas9-VP64 and dCas9-p65, respectively. Further optimization of this system led to the discovery that the tandem fusion of VP64, p65, and Rta, in this order, generates a hybrid VP64-p65-Rta activator (called as VPR), which improves the activation efficiency in the CRISPR system [20].