Simple knockout by electroporation of engineered endonucleases into intact rat embryos

Abstract

Engineered endonucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, provide a powerful approach for genome editing in animals. However, the microinjection of endonucleases into embryos requires a high skill level, is time consuming, and may cause damage to embryos. Here, we demonstrate that the electroporation of endonuclease mRNAs into intact embryos can induce editing at targeted loci and efficiently produce knockout rats. It is noteworthy that the electroporation of ZFNs resulted in an embryonic survival rate (91%) and a genome-editing rate (73%) that were more than 2-fold higher than the corresponding rates from conventional microinjection. Electroporation technology provides a simple and effective method to produce knockout animals.

Figure 1. Introducing mRNA into embryos by electroporation.

(a) Super Electroporator NEPA 21 (left) and glass chamber with metal plates (right). Embryos were placed in a line between the metal plates (arrow). (b) Illustration of electric pulses delivered by the electroporator. (c) The first poring pulse with high voltage and short duration makes micro-holes in the zona pellucida and oolemma (left), and mRNA in PBS was then moved into the cytoplasm with a few first transfer pulses with a low voltage and long duration after the poring pulse (middle). The mRNA was then transferred into oocytes by the polarity-changed second transfer pulse (right). (d) Fluorescence analysis of tetramethylrhodamine-labelled dextran that was introduced into embryos by electroporation with the pulse width adjusted to 0, 0.5, 1.5, and 2.5 ms.