Generation of Knockout Cell Lines for the Invasion-regulating Scaffold Gene AFAPILI in Sarcoma Cells by CRISPR/Cas9 Technology
Shapiro, Rebecca L.
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There are over fifty types of sarcomas known in the world today. However, in most cases, the molecular mechanisms which cause their poor prognoses are still a mystery. Cancer metastasis is one of the most unifying characteristics of late-stage cancers. It is therefore important to try to further understanding of the molecular mechanisms underlying metastasis and the progression of tumors throughout the body. Structures known as invadopodia have been found to cause metastasis of tumors by infiltrating the basement membrane and extracellular membrane of cells, allowing tumor cells to spread throughout the body. AFAPILI, an identified oncogene, is characterized in many different components of invadopodia, indicating a direct link to metastasis. AFAPILI is known to directly correlate to the amount of cancer metastasis in cancer patients, making it an ideal target for gene therapies. The Clustered Regulatory Interspaced Short Palindromic Repeat (CRISPR) gene engineering system was used in this study to target a specific 20 nucleotide sequence of AFAPILI and remove it from osteosarcoma cells (U20S), generating a knockout cell line. Our study successfully engineered a CRISPR/Cas9 system through the confirmed removal of AFAPILI. This was shown via a red fluorescent tag which replaced AFAPILI in the U20S cell line. This successful knockout of AFAPILI will aid in our understanding of cancer metastasis, helpfully leading to potential therapies for sarcoma cancer patients.