RESUMO
The CRISPR/Cas9 system enables a versatile set of genomes editing and genetic-based disease modeling tools due to its high specificity, efficiency, and accessible design and implementation. In cancer, the CRISPR/Cas9 system has been used to characterize genes and explore different mechanisms implicated in tumorigenesis. Different experimental strategies have been proposed in recent years, showing dependency on various intrinsic factors such as cancer type, gene function, mutation type, and technical approaches such as cell line, Cas9 expression, and transfection options. However, the successful methodological approaches, genes, and other experimental factors have not been analyzed. We, therefore, initially considered more than 1,300 research articles related to CRISPR/Cas9 in cancer to finally examine more than 400 full-text research publications. We summarize findings regarding target genes, RNA guide designs, cloning, Cas9 delivery systems, cell enrichment, and experimental validations. This analysis provides valuable information and guidance for future cancer gene validation experiments.
Assuntos
Sistemas CRISPR-Cas , Neoplasias , Edição de Genes , Humanos , Mutação , Neoplasias/genética , Oncogenes , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Several genes are significantly mutated in breast cancer but only a small percentage of mutations are well-known to contribute to cancer development. FASN is involved in de novo lipogenesis and the regulation of ERα signaling. However, the effect of genetic mutations affecting FASN in breast cancer has not thoroughly studied. Therefore, we used the CRISPR/Cas9 system to edit the FASN locus in MCF-7 cells and evaluated its biological effect. We obtained four clones carrying mutations and frameshifts in the acyl-transferase domain of FASN. We found that clones had reduced proliferation, migration, viability, and showed alterations in cell cycle profiles. RNA-Seq analysis demonstrates that a lack of fully functional FASN may have a more significant role in proliferation-related genes than in lipid metabolism. We conclude that functional knockouts in FASN contributes to decrease the proliferation and migration of breast cancer cells contrary to point mutations in breast cancer patients.