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Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo
npg 201 5 Nature America, Inc. All rights reserved.
John A Zuris1,2, David B Thompson1,2, Yilai Shu36, John P Guilinger1,2, Jeffrey L Bessen1,2, Johnny H Hu1,2, Morgan L Maeder710, J Keith Joung710, Zheng-Yi Chen3,4 & David R Liu1,2
Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.
Therapeutic proteins including peptide hormones1, cytokines2 and monoclonal antibodies3 have achieved widespread success as research tools and are among the fastest growing classes of drugs4. Many powerful and potentially therapeutic proteins have been discovered or engineered over the past two decades, including enzymes capable of metabolic complementation5, neutralizing antibodies against intra-cellular targets6, engineered transcription factors7 and programmable genome-editing enzymes8. Although protein biologics have proven effective for extracellular targets, their use to address intracellular targets is comparatively undeveloped because most proteins are unable to spontaneously enter mammalian cells. Enabling exogenous proteins to access intracellular targets is most commonly achieved by delivery of their encoding DNA sequences through chemical transfection9, electro-poration10 or viral delivery11. The introduction of exogenous DNA into cells, however, raises the possibility of permanent recombination into the genome, potential disruption of endogenous genes and long-term exposure to the encoded agent. The recent development of methods to deliver in vitrotranscribed mRNAs or mRNA analogs has offered an alternative to DNA delivery without requiring nuclear...