# Gene Editing with CRISPR/Cas Methodology and Thyroid Cancer: Where Are We?
## 3 Dry-Lab: How Do I Use CRISPR In Vitro? To start a gene editing experiment with CRISPR/Cas9, two essential components are mandatory: (1) Cas9 (or other Cas endonuclease) and (2) sgRNA. A third optional component is a selection marker that facilitates the screening. The delivery of CRISPR/Cas9 (containing sgRNA) frequently uses a plasmid system that can be easily transfected into cells, which is explored below. It is necessary to express Cas endonuclease (Cas9, for example) and sgRNA simultaneously to achieve DNA double-strand break, single-strand break or gene modulation (in this case, dCas9 + sgRNA expression should be kept constant). One of the easiest methods is to clone the sgRNA sequence under the control of the U6 promoter in a plasmid that also expresses Cas9 endonuclease (or other Cas variants, discussed in the upper sections) and a selection marker, such as puromycin resistance, or a fluorescent reporter such as GFP. The Zhang Lab [18] constructed these plasmids, which are available in Addgene as PX459 or PX458, respectively, for Cas9 + puromycin resistance, or Cas9 + GFP. We use these plasmids as examples. The cloning methodology is user-friendly and uses the restriction site created by the digestion of the plasmid PX459 or PX458 with Bbsl endonuclease for sgRNA cloning as shown in Figure 5. The resulting open linear plasmid has cohesive ends with a 5\({}^{\prime}\)-CACC overhang and a 3\({}^{\prime}\)-CAAA overhang for ligation with sgRNA (DNA sequence). We recommend testing at least three different sgRNAs sequences in order to select the most efficient. The sgRNA sequence can be efficiently designed using available online tools, such as ChopChop [23] or Gene Perturbation Platform (GPP)/CRISPRk [24] that have algorithms to predict the efficiency of double-strand break and score sgRNAs according to the off-target prediction. These tools are especially efficient for coding-genes. After choosing the sgRNAs, the sequence is ordered as a pair of DNA oligonucleotides that contain the 20-nt target region (PAM sequence should be removed from the sgRNA sequence) and the cohesive ends to ligate into the Bbsl digested plasmid (PX458 or PX459, for example) when these oligonucleotides are annealed to form a double-strand DNA (Figure 5). In the coding-gene context (Figure 6), the sgRNA design should preferentially favor the targeting of the coding-gene's first exons, closer to the start codon region (ATG), if possible. This strategy aims to disrupt the coding sequence from the start codon and, therefore, truncate the protein or create a premature stop codon. Figure 5: Plasmid system to express CRISPR/Cas9 system. (**A**) CRISPR/Cas9 plasmid contains a site for Bbsl digestion that creates the cloning site for the 20-nt sequence sgRNA; (**B**) The sgRNA sequence is ligated into Bbsl-digested plasmids PX458 (GFP) or PX459 (puromycin resistance) as annealed DNA oligonucleotides with cohesive ends as shown in the figure CACC in the 5\({}^{\prime}\) and CAAA in the 3\({}^{\prime}\). In terms of targeting noncoding RNAs, the main objective is to disrupt structural or regulatory regions. For example, when targeting a microRNA(miRNA) gene (explored in Section 4.2 targeting noncoding genes in thyroid research), the sgRNA may be directed to the desired strand of miRNA (either 5p or 3p) to remove its DNA sequence. Alternatively, sgRNAs may target the structural regions of primary or precursor miRNA processing, for example DROSHA/DICER binding sites or stem loop region (Figure 7). After plasmid sequence confirmation, cells are transfected and selected for the marker (GFP or puromycin) to generate a mixed population. This mixed population can be initially screened for the target gene or protein expression (dependent upon if it is a coding or noncoding gene). Moreover, the genomic DNA can be used to assess the extent of gene editing by a surveyor assay or T7 endonuclease assay (both assays use nucleases that recognize DNA annealing mismatch).