RISPR Cas system
In the CRISPR Cas system, the enzyme Cas9 is an integral part of the construct to which a molecular guide RNA attached targeting it to any possible matching DNA sequence and it is actually used to specify the sites of cleavage that is critical.
The CRISPR landscape, which began around 1987 leading to further discoveries and around 2010, Emmanuelle Charpentier (University of Vienna) identified the role of the Cas9 enzyme in Streptococcus pyogenes which is an efficient enzyme in cutting DNA. Later, Zhang and colleagues at the Broad Institute and the Massachusetts Institute of Technology (MIT) presented several in vitro proof of principle showing that Cas9 may be targeted to genes in bacteria, human cell lines, cultured stem cells and zebrafish (Knut J Egelie et al., 2016). CRISPR/Cas9 can be used to generate knockout (KO) cell lines and in vivo animal models. For example, CRISPR/Cas9 based on lentiviral vectors can generate KO cell lines by depleting one or more genes simultaneously. CRISPR/Cas9-mediated transcriptional regulation, either as activation or repression. Different genome-wide transcriptional library screens of both loss-of-function and gain-of-function mutations are now available, using these screens, different drug-resistant genes were identified. Melanoma whereas, in another study, Gilbert et al. used the dCas9-KRAB system to repress gene expression and concluded that this strategy could be applied to genome-wide genetic screening. Recently, the CRISPR/Cas9 system was also harnessed to address complicated diseases, such as cancer, which result from a perturbation in multiple genes simultaneously and, in some cases, translocation of a chromosome region; for example, the fusion of EML4-ALK in lung cancer (Maddalo, D. et al., 2014).
Cellular models are generated using the CRISPR Cas9 system by introducing the plasmid carrying the Cas9 and sgRNA into the target cells i.e., iPSCs considering these cells have the ability to replace diseased and unhealthy cells with the health once. This system has also been used to generate transgenic mouse using various other approaches, some of which are transplantation-based, exogenous delivery, inducible, translocation-based and many more to get an appropriate model for the study. Applications of CRISPR are not just confined to cells and mice for generating human disease models but also other organisms like rat, zebrafish, sheep, Xenopus, rabbit, pig and monkey (Gulzar Ahmad and Mansoor Amiji, 2018).