# Gene Editing with CRISPR/Cas Methodology and Thyroid Cancer: Where Are We?
## 2 The Principle of CRISPR/Cas
### Mechanism behind Gene Editing  
The Cas endonucleases (i.e., Cas9, Cas12, Cass, etc.) generate a DNA double-strand break at the target region guided by the sgRNA. As this type of DNA break is extremely deleterious because it may lead to loss of DNA content, cells express a DNA damage repair machinery to reunite DNA proficiently. The gene editing per se, meaning changes in the DNA sequence by insertion or deletion of nucleotides, occurs in this process of repairing (ligating) DNA broken strands.  
DNA double-strand breaks can be repaired through the following two main mechanisms: (1) Nonhomologous End Joining Repair (NHE)) or (2) Homologous Directed Repair (HDR) (Figure 3).  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Organism** & \begin{tabular}{c} **CRISPR** \\ **Nuclease** \\ \end{tabular} & **CRISPR Type** & \begin{tabular}{c} **PAM Sequence** \\ **(5’-3’)** \\ \end{tabular} & \begin{tabular}{c} **Size** \\ **(Aminoacids)** \\ \end{tabular} & **Aim** & **Reference** \\ \hline \begin{tabular}{c} _Streptococcus_ \\ _pyogenes_ \\ \end{tabular} & SpCas9 & Class2, Type II & NGG & 1368 aa & Gene editing & [2] \\ \hline \begin{tabular}{c} _Staphylococcus_ \\ _aureus_ \\ \end{tabular} & SaCas9 & Class2, Type II & \begin{tabular}{c} NNGRR or \\ NGRRN \\ \end{tabular} & 1053 aa & Gene editing & [9] \\ \hline \begin{tabular}{c} _Neisseria_ \\ _menitigitidis_ \\ \end{tabular} & NmeCas9 & Class2, Type II & NNNNGATT & 1082 aa & Gene editing & [10] \\ \hline \begin{tabular}{c} _Streptococcus_ \\ _thermophilus_ \\ \end{tabular} & StCas9 & Class2, Type II & NNAGAAW & 1409 aa & Gene editing & [11] \\ \hline \begin{tabular}{c} _Lachospiraceae_ \\ _bacterium_ \\ \end{tabular} & \begin{tabular}{c} LibCas12a \\ (former Cpf1) \\ \end{tabular} & Class2, Type V & TTTV & 1228 aa & Gene editing & [12] \\ \hline \
\begin{tabular}{c} _Delaproteobacteria_ \\ \end{tabular} &
\begin{tabular}{c} DpbCasX \\ (Cas12e) \\ \end{tabular} & Class2, Type II & TTCN & 996 aa & Gene editing & [13] \\ \hline \hline \end{tabular}
\end{table}
Table 1: Class2 Cas nucleases comparison.  
NHEJ is the main mechanism for double-strand break repair because it is proficient at reuniting double-strand break ends. However, it may be unspecific and error-prone, and this property is especially important for gene disruption because it frequently causes microdeletions or insertions that ultimately lead to a loss of protein expression by frame shifting and premature stop-codon insertion.  
On the other hand, HDR occurs less frequently (mainly in the cell cycle's S-G2 phase) and uses endogenous (sequence in the sister chromosome) or exogenous homologous pieces of DNA to ligate double-strand breaks correctly without error. When using an exogenous sequence, a DNA template may contain the gene's correct sequence to correct a mutation through HDR, for example, and must be provided with Cas9 + sgRNA. Nevertheless, a lower frequency of HDR (compared to NHEJ) is a scenario that may be improved by cell synchronization, drug treatment, or molecular biology manipulation to favor gene correction [16].