An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes

Abstract

Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6N mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring.Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.. CC-BY 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

i‐GONAD: A method for generating genome‐edited animals without ex vivo handling of embryos

“…The success of this technology was subsequently reported by Kaneko et al. and other researchers using mice (Chen, Lee, Lee, Modzelewski, & He, ; Hashimoto, Yamashita, & Takemoto, ; Kaneko & Mashimo, ; Qin et al., ; Teixeira et al., ; Tröder et al., ). Although this approach simplified the process of gene delivery into embryos, it still requires ex vivo handling of embryos, such as embryo collection, short periods of embryo culture, and subsequent ET (Figure ; Table ).…”

“… These amounts were determined based on our experiences for zygote microinjection and i ‐GONAD, and on the data from other groups for in vitro EP (Hashimoto et al., ; Tröder et al., ). …”

Gradual centriole maturation associates with the mitotic surveillance pathway in mouse development

“…The Sas-4 −/− mice (Cenpj tm1d(EUCOMM)Wtsi/tm1d(EUCOMM)Wtsi ) (Bazzi & Anderson, 2014a) and the Ift88 −/− null mouse allele generated from the Ift88 fl/fl allele (Ift88 tm1Bky ) (Haycraft et al, 2007) were used in this study. The CRISPR/Cas9 endonuclease-mediated knockouts of 53bp1 −/− and Usp28 −/− were generated by the CECAD in vivo Research Facility using microinjection or electroporation of the corresponding gRNA, Cas9 mRNA and Cas9 protein into fertilized zygotes (Table 1) (Chu et al, 2016;Troder et al, 2018). The gRNA target sequence predictor tool developed by the Broad Institute was used to design gRNAs (Doench et al, 2016).…”

Delivery methods for CRISPR/Cas9 gene editing in crustaceans

“…In rodents, electroporation of Cas9 mRNA with gRNA results in high efficiency of knock-out (67%-88%) and knock-in (33%) in mice and rats (Kaneko and Mashimo 2015). The electroporation method has also been used with Cas9-sgRNA RNP in mice (Troder et al 2018). It should be noted that a special buffer is often used in electroporation experiments to provide appropriate conductivity.…”