Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles

• DOI: 10.1073/pnas.1520244113
• Publication Date: 2016-03-01T04:14:31Z
• Authors: Ming Wang; John A. Zuris; Fantao Meng; Holly Rees; Shuo Sun; Pu Deng; Yong Han; Xue Gao; Dimitra Pouli; Qi Wu; Irene Georgakoudi; David R. Liu; Qiaobing Xu
• Keywords: protein delivery; 570; Green Fluorescent Proteins; Hypothalamus; Cre recombinase; Endosomes; Ceramides; Gene Knockout Techniques; 03 medical and health sciences; Bacterial Proteins; Genes, Reporter; CRISPR-Associated Protein 9; Genes, Synthetic; genome editing; Animals; Humans; CRISPR/Cas9; Drug Carriers; 0303 health sciences; Integrases; lipid nanoparticle; 540; Endonucleases; Lipids; Endocytosis; Cholesterol; CRISPR-Cas Systems; Genetic Engineering; HeLa Cells

Significance The therapeutic potential of protein-based genome editing is dependent on the delivery of proteins to appropriate intracellular targets. Here we report that combining bioreducible lipid nanoparticles and negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the self-assembly of nanoparticles for potent protein delivery and genome editing. The design of bioreducible lipids facilitates the degradation of nanoparticles inside cells in response to the reductive intracellular environment, enhancing the endosome escape of protein. In addition, modulation of protein charge through either genetic fusion of supercharged protein or complexation of Cas9 with its inherently anionic sgRNA allows highly efficient protein delivery and effective genome editing in mammalian cells and functional recombinase delivery in the rodent brain.