ZEB1 and ZEB2 gene editing mediated by CRISPR/Cas9 in A549 cell line

Abstract

OBJECTIVES: One of the best approaches for recognition of protein function is the induction of mutations for a gene knockout. In line with this strategy, gene editing tools allow researchers to induce these mutations. Lung cancer is one of the leading causes of death worldwide. ZEB1 and ZEB2 genes are the candidates for this disease. METHODS: The ZEB1 and ZEB2 knockout in the non-small cell lung cancer cell line (A549 cell) was investigated. Purifi cation of recombination plasmids was performed from bacteria and then was transported to the A549 cell line. The deletion of ZEB1 and ZEB2 were examined by PCR. RESULTS: The results demonstrated the mutation and deletion in ZEB1 and ZEB2 genes. Based on the fi ndings of this study, A549 cells were transfected with the vectors carrying the sgRNA/Cas9, simultaneously. The DNA fragment demonstrated the presence of indels in target sites as well as provided the potential of CRISPR/Cas9 system. CONCLUSION: CRISPR/Cas9 offers a great potential as an effi cient technique for editing of ZEB1 and ZEB2 genes in A549 cell line (Tab. 1, Fig. 6, Ref.

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“…This system is a family of DNA sequences found in archae and bacteria, and is composed of two major parts: (i) Cas endonuclease and (ii) guide RNA. The former is responsible to break the target genomic site, whereas the latter is used to identify and lead the Cas endonuclease to the target [277] , [278] , [279] . The CRISPR system is further divided into two main classes and six types.…”

In vivo gene delivery mediated by non-viral vectors for cancer therapy

“…One of the most important obstacles in brain drug delivery is the blood-brain barrier (BBB). Therefore, there are many efforts to overcome this barrier including functionalization and modification of non-viral gene delivery vectors [212,213]. The modification leads to the transcytosis and endocytosis of vectors through cell-penetrating peptides (CPP) mediated transmembrane transport, adsorptive-mediated endocytosis and receptormediated endocytosis [214].…”

“…Only in these systems, Cas9 protein is an essential compartment for DNA interference. Generally, this system contains a nuclease protein (Cas9) and a guide RNA (gRNA) [213]. Since, the gRNA could be replaced by sgRNA (synthetic chimeric single guide RNA), the Cas9 protein could be directed to the target site using sgRNA which consequently leads to the induction of double-stranded DNA breaks (DSBs).…”

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“…[32][33][34] Indeed, EMT-induced cells demonstrate enhanced migration, invasion, and frequently express N-cadherin at the expense of E-cadherin reducing in this way the intercellular adhesion. [35][36][37] Moreover, to intricate an already debated and complex process, cancer cells are often reported to display different degrees of EMT phenotypes. [38][39][40][41] As reported above, SNHGs exert multiple roles being able to act as molecular sponges for several miRNAs (Figure 2) or binding proteins modulating in that way multiple biological processes, including the EMT in cancer.…”

“…Such transition is commonly induced by external microenvironmental factors and it is described as a reversible process, allowing cancer cells to evade regulated cell death, and contributing to immunosuppression in early phases while after dissemination they might revert to an epithelial status 32–34 . Indeed, EMT‐induced cells demonstrate enhanced migration, invasion, and frequently express N‐cadherin at the expense of E‐cadherin reducing in this way the intercellular adhesion 35–37 . Moreover, to intricate an already debated and complex process, cancer cells are often reported to display different degrees of EMT phenotypes 38–41 …”