Convergent Arrangement of sgRNA and Cas9 in CRISPRsome for Transcellular Trafficking

The emergence of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing systems has led to the appreciation of CRISPR as a versatile and powerful gene-editing tool in the molecular biology laboratory and a potentially potent therapeutic for the treatment of a variety of intractable diseases. Nonetheless, one of the most onerous obstacles that restricts the wide availability of CRISPR technology from wide practical applications is the lack of appropriate delivery vehicles for the simultaneous transportation of multicomponent CRISPR systems to the intracellular targets. Herein, we attempted to create an intriguing nanometer-scaled vesicle platform (CRISPRsome) that, with the aid of a block copolymer of poly(ethylene glycol)-polylysine, simultaneously combined the fundamental components of the CRISPR system into the CRISPRsome, particularly the ribonucleic sgRNA and proteinic Cas9 endonuclease precisely located at the intermediate membrane and the internal reservoir regions of the nanometer-scaled vesicles, respectively. Furthermore, biofunctionalization strategies, including using redox-responsive disulfide linkages between lysine units to cross-link the well-defined CRISPRsome structures and surface functionalization with the cyclic Arg-Gly-Asp ligand to stimulate receptor-mediated endocytosis, were proposed in the well-defined CRISPRsome. The subsequent results validated its appreciable gene knockout efficacies toward cancerous cells with the green fluorescent protein (GFP) reporter gene, thereby highlighting the prosperous potential of CRISPRsome to facilitate the use of CRISPR technology in a variety of practical applications.