CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) is a natural adaptive immune system found in about one-third of bacteria and almost all archaea. Proteins of its effector module have shown great efficiency to be used for targeted genome editing, especially CRISPR associated protein 9 (Cas9). Owing to the relatively easy and affordable application of CRISPR/Cas9 system, its usage spans almost all areas of biological research including gene therapy, synthetic biology, bioimaging, drug discovery and personalized medicine, among other applications. Cas9 is a multidomain protein that cleaves the target DNA via the action of 2 different nuclease domains, namely RuvC and HNH nucleases. Naturally, for Cas9 to functionalize properly, it requires 2 types of RNA; the target-specific CRISPR RNA (crRNA) and the structural transactivating CRISPR RNA (tracrRNA). However, engineered CRISPR systems utilize single-guide RNA (sgRNA), which contains a 20-nucleotide variable region used to specify targeted DNA sequence. In addition, Cas9 contains a unique protein fold that is called PAM (Protospacer Adjacent Motif)-interacting domain, which recognizes a short specific DNA sequence of the non-target DNA strand4. After recognizing PAM sequence, DNA duplex gets unwound, then the guide RNA anneals to the target DNA strand, then RuvC and HNH nuclease domains cause a double strand break (3~4 nucleotides upstream of the PAM sequence). The specificity of CRISPR-associated proteins still a matter that needs more effort to be fully understood. Notwithstanding its versatile potential usage, the problem of off-target effect still hinders CRISPR/Cas application with full confidence.
employing evolutionary analysis methods to investigate the specificity of Cas9
training in evolutionary analysis; research experiences in CRISPR/Cas9 system