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Construction of VPS34-Knockout A549/293T Cell Line by CRISPR/Cas9 System
Liu Yulan1, Li Yahui1, Zhang Yina2, Liu Fei1, Hu Boli1*, Zhou Jiyong1,2
1College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China; 2College of Animal Science, Zhejiang University, Hangzhou 310058, China
Abstract: The aim of this study is to generate VPS34 gene knockout A549/293T cell lines by CRISPR/ Cas9 system and preliminarily used them in autophagy research. Three VPS34-targeting sgRNAs were designed by online software and then cloned into pX459 vector. The recombinant vectors were transfected into A549/293T cells and screened by puromycin. The results of PCR sequencing and Western blot showed that sgRNA-1 had the best knockout effect. Object to subclone the two kinds of cells which were transfected with sgRNA-1， the deletion of bases were found after PCR sequencing. At the same time， the VPS34 protein expression was not detected in the corresponding monoclonal cells by Western blot. Finally， the VPS34 gene deletions in A549 cells and 293T cells were successfully obtained. As VPS34 plays an important role in the initiation of autophagy， in VPS34 knockout A549/293T cells， we found that the transformation of LC3-I to LC3-II was significantly inhibited， and the P62 was accumulated in large quantities. The results showed that the autophagy in VPS34 deleted cell lines were significantly inhibited. At the same time， we found that VPS34 gene knockout could reduce the growth rate of A549 cell. Our results showed that VPS34 in A549/293T cells was successfully deleted using CRISPR/Cas9 technology， which provide a powerful tool for autophagy studies in the future.
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