# Gene Editing with CRISPR/Cas Methodology and Thyroid Cancer: Where Are We?
## 2 The Principle of CRISPR/Cas  
### What Is CRISPR/Cas?  
The CRISPR/Cas system is an endogenous RNA-guided prototype of immune defense against viral infection (DNA or RNA) in bacteria and archaea, initially described as a peculiar region of repetitive sequences detected downstream of the _lap_ gene's translation termination codon in _E. coli_[3]. The CRISPR locus is composed of palindromic sequences of 14 bp (repeats) that are interspaced by a 32-nucleotide (32-nt) sequence and repeated several times in the genome. The Cluster of Regularly Interspaced Palindromic Region (CRISPR), as it was named in 2002 [4], contains sequences of foreign DNA incorporated between the palindromic repeats (CRISPR array region). This system constitutes a prototype of adaptive immune responses by storing information of previous infections in the DNA, which is transcribed into CRISPR RNAs (crRNAs) [5].  
As an essential part of the system, CRISPR-associated genes (Cas) are located adjacent to the CRISPR region and participate in several steps of adaptive immunity, in a process defined by the following three phases: adaptation, expression, and interference [6]. In the adaptation phase, specific Cas genes (i.e., Cas1 and Cas2) recognize the invading DNA, which is fragmented and incorporated into the CRISPR locus spacer region. In the expression phase, the CRISPR array is transcribed into crRNA, a small RNA that contains a 20-nt specific sequence (targeting the foreign DNA) and a segment of repeats that interacts with trans-activating crRNA (tracrRNA). This RNA structure is loaded into the Interference Cas protein, Cas9. In the interference phase, Cas9 endonuclease is complexed with crRNA + tracrRNA and targets foreign DNA based on a perfect complementarity match to the 20-nt of crRNA, which leads to the cleavage of the double-strand DNA due to the nuclease activity of Cas9.  
The existence of an array of differences in the CRISPR/Cas loci structure in bacteria and archaea led to the classification of CRISPR systems based on the interference phase's effector proteins. The current classification divided CRISPR into two classes and six types with particular Cas proteins [7]. The main difference between Class 1 and Class 2 is the composition of the Cas protein that participates in the interference Phase. In Class1, the CRISPR system recruits a large complex composed of several Cas proteins in the interference phase (Types I, III, and IV). On the other hand, in Class2 CRISPR, a single protein exerts multiple functions required for the interference phase (Types II, V, and VI). The focus of this review is the Cas9 protein, the most used Cas endonuclease for gene editing purposes. Additionally, Cas9 is a member of Class2 Type II CRISPR, in which a single protein is responsible for the interaction with guide RNA (crRNA + tracrRNA), DNA scanning, and double-strand break due to nuclease activity of the RuvC and HNH nuclease domains (Figure 1).  
Figure 1: The basic components CRISPR/Cas9 system. (**A**) The crRNA associated with tracrRNA enters Cas9 endonuclease and guides the protein throughout the foreign DNA in search of a full-complementarity region adjacent to a PAM sequence in order to produce the double-strand break. (**B**) Detailed view of interaction of crRNA with the target DNA sequence (complementary sequence) and PAM sequence in the non-complementary strand of DNA.  
Understanding the principle of RNA-guided DNA cleavage by the CRISPR/Cas system in bacteria led to the repurposing of Cas9 to target DNA in eukaryotes, thus envisioning gene editing applications for functional investigation. In this new context, the single multifunctional protein Cas9 emerged as a catalyzer of gene editing with CRISPR/Cas due to the relative simplicity of expressing a single protein allied to the sgRNA (and other requirements, which we explore next).