Development of new edited cell models to analyze pros and cons of the CRISPR/Cas9 technique

The CRISPR/Cas9 system is a powerful genome-editing tool and its great potential is thoroughly recognized. CRISPR/Cas9 has been applied in several fields, such as plant engineering, development of new cell lines, and animal models. CRISPR/Cas9 exploits a small single guide RNA to direct the Cas9 endonuclease activity against a specific genome sequence, where it introduces a double strand break. The DNA damage is then repaired by the non-homologous and joining or the homology direct repair processes. While the first one allows a gene knock out, the homology direct repair bases its activity on a repair template, thus allowing the introduction of specific modification within a gene target. In the present work, we aimed to analyze the pros and cons of the CRISPR/Cas9 system by developing new edited cell models, that will be useful to study the involvement of specific factors in different human diseases. Particularly, viral infections, cancer diseases, and neurodegenerative disorders represent a major issue for public health. The CRISPR/Cas9 system was fundamental to develop new cell lines useful to study host-retrovirus interaction, the involvement of specific proteins in tumorigenesis and cancer progression, and the importance of particular proteins in the onset of neurodegenerative disorders. Retroviral infection: host-virus interaction HIV-1 and HTLV-1 represent the two major human retroviruses. To assess how specific host proteins modulate HIV-1 infection, different 293T packaging cell lines were developed. Specifically, CRISPR/Cas9 was used to produce 293T ACOT8, HDAC6 ΔBUZ, and HLA-C KO cell lines. To investigate the involvement of TRAF3 in the deregulation of the NF-kB pathway mediated by the HTLV protein Tax1, a TRAF3 KO cell model was developed. Cancer diseases Malignant melanoma and pancreatic cancer are two of the most frequent morbidities. To analyze the oncogenic potential of RUNX2 in the development and progression of malignant melanoma, the Mel-HO RUNX2 KO melanoma cell line was developed through CRISPR/Cas9. To clarify the role of GNA15 in the molecular mechanisms leading to pancreatic adenocarcinoma, CRISPR/Cas9 was employed to generate PT45 GNA15 KO adenocarcinoma cells. Neurodegenerative diseases Neurodegenerative disorders are becoming even more frequent especially due to the increase in the average age of the population. Nowadays no effective cures are available. To understand the molecular mechanism by which GPR3 induces amyloid β deposition in Alzheimer’s disease, CRISPR/Cas9 was used to develop H4 Swe GPR3 KO neuroglioma cells. To fully investigate the involvement of the palmitoyl-protein thioesterase 1 PPT1, in the neuronal ceroid lipofuscinoses neurological disorder, SH-SY5Y PPT1 KO neuroblastoma cell line was developed. The CRISPR/Cas9 pros and cons are analyzed for a full technique understanding, with particular regard to its potential limits, such as off-targets, in vitro genome manipulation, delivery systems, and editing screening. As any technology revolution, CRISPR/Cas9 represents the linchpin of fierce debates involving many scientists and researchers to regulate its employment for gene therapy, germline, and human embryos manipulation.