SAT-111 Rat genome editing by rGONAD (rat Genome-editing via Oviductal Nucleic Acids Delivery) method

Abstract

Recent progress in development of the CRISPR/Cas9 system has been shown to be an efficient gene-editing technology in various organisms. The laboratory rat (Rattus norvegicus) is a common model of human diseases. Although there are several advantages compared with using mice, technology for handling the rat genome has lagged behind. We recently developed a novel method called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) in mice; a novel in vivo genome editing system that does not require ex vivo handling of embryos, and this technology is newly developed and renamed as “improved GONAD (i-GONAD). However, this technology has been limited only to mice. In this study, we attempted to apply GONAD method to “rats”. To test the conditions of electroporation, we prepared 0.5mg/ml Tetramethylrhodamine-labelled dextran and 0.1% trypan blue. To produce genome-edited rats, the annealed crRNA and tracrRNA were mixed with Cas9 protein and/or ssODN so that the final concentrations of components were 30 µM (for crRNA/tracrRNA), 1 mg/ml (for Cas9 protein), 1 µg/µl (for ssODN). Surgical procedures were operated on anesthetized female rats at Day 0.75 of pregnancy (E0.75: corresponding to late 1-cell stage zygotes; at 16:00 of the same day when plugs were confirmed) under observation using a dissecting microscope. Approximately 2.-2.5 µl of electroporation solution was injected into the oviductal lumen from upstream of the ampulla using a micropipette. We determine the most suitable condition for in vivo gene delivery towards rat preimplantation embryos using tetramethylrhodamine-labelled dextran, termed as Rat improved GONAD (rGONAD). Then, to investigate whether this method is feasible to generate genome-edited rats by delivery of CRISPR/Cas9 components, the tyrosinase (Tyr) gene was used as a target. Some pups showed albino-colored coat, indicating disruption of wild-type Tyr gene allele. Furthermore, we confirm that rGONAD method can be used to introduce genetic changes in rat genome by the ssODN-based knock-in. We established the method, namely rGONAD for generating genome editing rats. We confirm that the rGONAD method will facilitate the production of rat genome engineering experiments in many laboratories. Although the rGONAD method has so far been successfully implemented in mice and rats, this concept can be adapted for other animals. Therefore, rGONAD is a powerful method for genome-editing in the research of kidney diseases.