Intracellular self‐assembled nanoneedles for efficient lysosomal escape and CRISPR/Cas9 mediated gene editing

Abstract Lysosomal entrapment is a formidable bottleneck for the delivery of clustered regularly interspaced short palindromic repeat/CRISPR‐associated protein (CRISPR/Cas9) gene editing systems. To address this, we developed a nanoneedle platform that are self‐assembled by CRISPR/Cas9 encoding plasmids, triggered by existent cellular metabolic metal ions in tumor cells, leading to lysosomal disruption for effective gene delivery. Based on this, a CRISPR/Cas9 plasmid delivery system termed PlaMnB, was further developed by encapsulation of CRISPR/Cas9‐expressing Pla smid and Mn O 2 in a B acterial cell membrane system derived from genetically engineered Escherichia coli that are transformed by tumor‐homing‐peptide (THP) genes for tumor targeting and vascular permeation. Once targeted delivered to tumors by the bacterial membrane vehicles decorated by THP, PlaMnB releases manganese ion in the acidic and glutathione‐enriching environment in tumors, leading to the formation of metal plasmid coordination nanoneedles and enhanced lysosomal escape. In addition, a tumor‐specific promoter, telomerase reverse transcriptase was integrated in the CRISPR/Cas9 plasmid, allowing it to exclusively express Cas9 and sgRNA in tumors, but not in normal cells. The integrated rational design of different functional modules in the PlaMnB achieved an efficient and precise intracellular delivery of CRISPR/Cas9 for enhanced vascular permeation, effective lysosomal escape, and minimal off‐target of gene editing.