**2.1. Bacterial adaptive immunity: acquisition, biogenesis and interference**

The defense mechanism of CRISPR/Cas9 system can be divided into three stages, which are spacer acquisition or adaptation, crRNA biogenesis and interference (**Figure 4**).

Conceptually, the spacer acquisition process can be further divided into two steps, which are protospacer selection and integration of the spacer into the CRISPR array to synthesize new repeat [44]. The protospacer selection step of the spacer acquisition stage is guided by a conserved DNA sequence element, namely protospacer adjacent motif (PAM), that is located downstream of the DNA target, which has the sequence of 5'-NGG-3 [44]. Next, the selected protospacer will be integrated into the leader-repeat boundary of CRISPR array as new spacer and is duplicated in order to synthesize new repeats. Each repeat is a 29 nucleotide sequence, and repeats are interspaced by five intervening 32-nucleotide nonrepetitive sequences [30].

Then, the CRISPR will be transcribed into a long precursor crRNA (pre-crRNA) with the help of Cas proteins (Cas1, Cas2, Cas9 and Cas4/casn2) or simply known as biogenesis. During this process, the tracrRNA will be transcribed from the bacterial genome as it is needed for processing of the pre-crRNA into a mature guide crRNA. Next, the antirepeat sequence of tracrRNA will enable the complementary base pairing with each pre-crRNA repeats, resulting in the formation of a crRNA-tracrRNA duplex [45].

During the target interference stage, the crRNA-tracrRNA duplex recognizes the PAM sequence located downstream of the protospacer in the foreign DNA and triggers the 'non– self-activation,' which prevents self-targeting of CRISPR array. After that, the duplex guides the Cas9 endonuclease to bind and cleave the DNA target, resulting in the formation of DSBs. Additionally, the DNA target specificity duplex is provided by what is called the 'seed sequence,' which is located at approximately 12 bases upstream of the PAM sequence that matches the RNA with the DNA target [29] (**Figure 4**).
