A Multi-functional Vector for delivery of Genome Editing Plasmid Targeting β-Catenin to Re-modulate Cancer Cell Properties.

Accurate and efficient delivery of genome editing plasmids to targeted cells is of critical importance in genome editing. Herein, we prepared a multifunctional delivery vector with combination of ligand-mediated selectivity and peptide mediated transmembrane function to effectively deliver plasmids to targeted cancerous cells. In the delivery system, the CRISPR-Cas9 plasmid is combined with protamine with membrane and nuclear translocating activities and co-precipitated with CaCO3, which is further decorated by AS1411 functionalized carboxymethyl chitosan (ACMC) and TAT peptide functionalized carboxymethyl chitosan (TCMC). The AS1411 mediated tumor cell/nuclear targeting and TAT induced enhanced endocytosis result in obviously increased cellular uptake and nuclear transport. As a result, the CRISPR-Cas9 plasmid can be efficiently delivered to cancer cell nuclei to mediate genome editing, resulting in an efficacious knockout of CTNNB1 gene coding β-catenin. More importantly, down-regulation of β-catenin could effectively prevent its enrichment in nucleus and then significantly down-regulate the expression of proteins, such as vimentin, Snail, MMP-2, MMP-9, CD44, Nanog, and Oct4 to prevent tumor progression and metastasis. The edited cancerous cells exhibit favorable re-modulated properties including inhibited growth, suppressed migration and invasion, and reduced cancer stemness.