1 Department of Medical Ultrasound, The Second Affiliated Hospital, School of Medicine, South China University of Technology, Guangzhou, China;
 2 Department of Gastrointestinal Surgery, The Second Clinical Medical College, Jinan University, Shenzhen China; 
 3 Department of Gastrointestinal Surgery, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, China
 4 Department of Orthopedics, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China;
 5 SXUltrasonic (Shenzhen) Ltd., Shenzhen 518000, China; 
 6 Center for Cell and Gene Circuit Design, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China *
The epithelial-mesenchymal transition (EMT), a process in which epithelial cells undergo a series of biochemical changes to acquire a mesenchymal phenotype, has been linked to tumor metastasis. Here, we present a novel strategy for knocking out the EMT-related Cdh2 gene, which encodes N-cadherin through CRISPR/Cas9-mediated gene editing by an ultrasound combined with biosynthetic nanobubbles (Gas Vesicles, GVs). Polyethyleneimine were employed as a gene delivery vector to deliver sgRNA into 4T1 cells that stably express the Cas9 protein, resulting in the stable Cdh2 gene- knockout cell lines. TheWestern blotting assay confirmed the absence of an N-cadherin protein in these Cdh2 gene-knockout 4T1 cell lines. Significantly reduced tumor cell migration was observed in the Cdh2 gene-knockout 4T1 cells in comparison with the wild-type cells. Our study demonstrated that an ultrasound combined with GVs could effectively mediate CRISPR/Cas9 gene editing of a Cdh2 gene to inhibit tumor invasion and metastasis.