Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing

• DOI: 10.1021/jacs.8b01551
• Publication Date: 2018-04-18T20:25:31Z
• Authors: Romain Rouet; Benjamin A. Thuma; Marc D. Roy; Nathanael G. Lintner; David M. Rubitski; James E. Finley; Hanna M. Wisniewska; Rima Mendonsa; Ariana Hirsh; Lorena de Oñate; Joan Compte Barrón; Thomas J. McLellan; Justin Bellenger; Xidong Feng; Alison Varghese; Boris A. Chrunyk; Kris Borzilleri; Kevin D. Hesp; Kaihong Zhou; Nannan Ma; Meihua Tu; Robert Dullea; Kim F. McClure; Ross C. Wilson; Spiros Liras; Vincent Mascitti; Jennifer A. Doudna
• Keywords: 1.1 Normal biological development and functioning; 610; Protein Engineering; Cell Line; 03 medical and health sciences; Engineering; Cell Line, Tumor; Genetics; Humans; Cancer; Gene Editing; 0303 health sciences; Tumor; 5.2 Cellular and gene therapies; Molecular Structure; Gene Therapy; General Chemistry; Hep G2 Cells; Endonucleases; Chemical sciences; Chemical Sciences; Generic health relevance; CRISPR-Cas Systems; Digestive Diseases; Biotechnology

CRISPR‐Cas RNA‐guided endonucleases hold great promise for disrupting or correcting genomic sequences through site‐specific DNA cleavage and repair. However, the lack of methods for cell‐ and tissue‐selective delivery currently limits both research and clinical uses of these enzymes. Here we report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9‐ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Live cell imaging studies are consistent with a receptor‐mediated endocytosis mechanism of active uptake of the RNP. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor‐facilitated and cell‐type specific gene editing that did not rely on electroporation or the use of transfection reagents. This approach provides a framework for applications to selective gene editing in the liver and offers a potentially general mechanism for cell‐, tissue‐, or organ‐targeted gene editing by receptor‐mediated delivery of genome‐editing enzymes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.