In vivo selective gene knockout through in situ delivery of RNA-guided Cas9 nuclease using lipidoid

Abstract

Efficient methods for creating targeted genetic modifications have long been sought for the investigation of gene function and the development of therapeutic modalities for various diseases, including genetic disorders. Although such modifications are possible using custom-designed artificial nucleases are currently in use ie. Zinc finger nucleases (ZFNs), TAL effector nucleases (TALEN) and CRISPR/Cas9-based RNA guided RNA endonucleases (RGEN). Among them, RGEN based genome editing was found to be more efficient and easy to clone. RNA-guided endonucleases (RGENs), which are based on the clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR-associated (Cas) system, have recently emerged as a simple and efficient tool for genome editing. However, in vitro genome editing using CRISPR system is successfully achieved in mouse, drosophila, c eligans and human cells, it’s in vivo usage in multi-cellular organism is not yet been iv tested. In this study, we delivered RGEN plasmid DNA that express eGFP targeting guided RNA to eGFP expressing mouse by intradermal injection using Lipidoid. Our results showed significant decrease in eGFP signals at the site of injection indicating efficient silencing of eGFP gene in vivo. Thus, the RGEN system can be used to knock out specific genes at specified site in vivo, providing a potential tool for in vivo gene therapy using CRISPR.