Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing

Abstract

Recent use of the CRISPR/Cas9 system has dramatically reduced the time required to produce mutant mice, but the involvement of a time-consuming microinjection step still hampers its application for high-throughput genetic analysis. Here we developed a simple, highly efficient, and large-scale genome editing method, in which the RNAs for the CRISPR/Cas9 system are electroporated into zygotes rather than microinjected. We used this method to perform single-stranded oligodeoxynucleotide (ssODN)-mediated knock-in in mouse embryos. This method facilitates large-scale genetic analysis in the mouse.

Production of genome-edited pluripotent stem cells and mice by CRISPR/Cas [Review]

“…In addition, recent reports showed that gene-edited mice can be generated by direct injection of Cas9 protein, instead of Cas9 mRNA [29,30]. Furthermore, electroporation enables efficient mRNA delivery into mouse and rat zygotes [31,32]. Although microinjection into zygotes requires specialized skills and is difficult for beginners to perform, electroporation is simple and easy for beginners to perform.…”

Delivery of modified mRNA encoding vesicular stomatitis virus matrix protein for colon cancer gene therapy

“…11 These efforts have overcome the aforementioned shortcomings with improved intracellular stability and translational efficiency. 22,23 Nowadays, IVT mRNA has undergone extensive clinical or pre-clinical investigation in the elds of therapeutic cancer vaccination, 24-27 cell programming [28][29][30] and so on, demonstrating great potential.…”

CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists

“…A second round of confirmatory experiments, performed on a gene-by-gene approach, could confirm the gene identification process. The rate-limiting step for such a CRISPR-based genetic screening in mice is oocyte microinjection, which could be replaced by oocyte electroporation (Hashimoto & Takemoto 2015, Qin et al 2015, Takahashi et al 2015.…”