In 1987, the presence of CRISPR, in the Escherichia coli genome was reported by Nakata et al. from the Osaka University, Japan while Cas was discovered in 2002 by Jansen et al. Further discovery of in both Gram-positive and Gram-negative bacteria, along with archaea, led to the obvious questions regarding the relevance of CRISPR to these organisms. Later on, in the mid-2000s, the functionality and importance of CRISPR was first realized in prokaryotes wherein the CRISPR system is a key component of their adaptive immunity, which protects prokaryotes from attack by viruses, plasmids, and other invasive genetic elements. The CRISPR defense mechanism protects bacteria via three basic stages: spacer acquisition (adaptation), CRISPR RNA (crRNA) biogenesis (expression or maturation), and target interference (Figure ). CRISPR/Cas systems are composed of a Cas operon and a CRISPR array. The CRISPR array contains the unique sequences that are nestled in between the palindromic repeats (spacers) which are bits of foreign DNA, originated from mobile genetic elements such as bacteriophages, transposons, or plasmids that have previously infected the prokaryote. Cas gene (CRISPR-associated nuclease protein) is usually found adjacent to CRISPR which codes the nuclease protein (Cas protein) responsible for destroying or cleaving viral nucleic acid. In the first stage (spacer acquisition), upon phage infection, a sequence of the invading DNA (protospacer) is incorporated into the CRISPR array by the Cas1-Cas2 complex to form immune memory. In the second stage referred to as the CRISPR RNA (crRNA) biogenesis, when foreign nucleic acids invade again, the CRISPR array is transcribed to generate crRNA and a complementary trans-activating crRNA (tracrRNA), which form a double-stranded RNA structure. At the interference stage, crRNA-tracrRNA complex recruit Cas proteins to specifically destroy the invading nucleic acid. Adjacent to the crRNA-targeted sequence on the invading DNA, a short sequence named as the protospacer adjacent motif (PAM) plays an essential role in the adaptation and interference stages, which the CRISPR/Cas complex recognizes target DNA binding. The absence of a PAM sequence can alter the affinity between Cas and the target DNA, since specific PAM sequence recognition serves to discriminate nonself-target sequences from non-target sequences. 49