Functional analysis of AKT1 knockout in fibrosarcoma cells using CRISPR/Cas9 technology

AKT1 (Protein Kinase B alpha) is a serine/threonine kinase that plays a pivotal role in regulating various cellular processes. To elucidate the role of the AKT1 gene in signaling pathways, this study generated AKT1 knockout (KO) HT-1080 cells using the CRISPR/Cas9 system. Gene-editing efficiency was validated through Sanger DNA sequencing and insertion/deletion (InDel) analysis. Quantitative real-time PCR and Western blot analyses were performed to evaluate the expression levels of AKT1 mRNA and protein, as well as to examine the expression of AKT1 downstream effectors: mTOR, BCL-2, and FOXO1. The AKT1 single-guide RNA sequence was successfully cloned into the CRISPR/Cas9 vector, leading to the establishment of AKT1 KO cells. InDel analysis identified eight editing types, with two dominant populations. The expression levels of AKT1 mRNA and protein were significantly reduced in the KO cells. The expression levels of mTOR, BCL-2, and FOXO1 were significantly altered in the KO cells compared to normal cells. These findings highlight the impact of AKT1 disruption on signaling pathways and provide fundamental insights into the regulatory role of the AKT1 gene.