One-step genome editing of porcine zygotes through the electroporation of a CRISPR/Cas9 system with two guide RNAs
- PDF / 1,726,307 Bytes
- 8 Pages / 595.276 x 790.866 pts Page_size
- 80 Downloads / 173 Views
One-step genome editing of porcine zygotes through the electroporation of a CRISPR/Cas9 system with two guide RNAs Maki Hirata 1 & Manita Wittayarat 2 & Fuminori Tanihara 1 Koki Takebayashi 1 & Takeshige Otoi 1
3
4
1
1
& Yoko Sato & Zhao Namula & Quynh Anh Le & Qingyi Lin &
Received: 2 June 2020 / Accepted: 8 September 2020 / Published online: 25 September 2020 / Editor: Tetsuji Okamoto # The Society for In Vitro Biology 2020
Abstract In the present study, we investigated whether electroporation could be used for one-step multiplex CRISPR/Cas9-based genome editing, targeting IL2RG and GHR in porcine embryos. First, we evaluated and selected guide RNAs (gRNAs) by analyzing blastocyst formation rates and genome editing efficiency. This was performed in embryos electroporated with one of three different gRNAs targeting IL2RG or one of two gRNAs targeting GHR. No significant differences in embryo development rates were found between control embryos and those subjected to electroporation, irrespective of the target gene. Two gRNAs targeting IL2RG (nos. 2 and 3) contributed to an increased biallelic mutation rate in porcine blastocysts compared with gRNA no. 1. There were no significant differences in the mutation rates between the two gRNAs targeting GHR. In our next experiment, the mutation efficiency and the development of embryos simultaneously electroporated with gRNAs targeting IL2RG and GHR were investigated. Similar embryo development rates were observed between embryos electroporated with two gRNAs and control embryos. When IL2RG-targeting gRNA no. 2 was used with GHR-targeting gRNAs no. 1 or no. 2, a significantly higher double biallelic mutation rate was observed than with IL2RG-targeting gRNA no. 3. In conclusion, we demonstrate the feasibility of using electroporation to transfer multiple gRNAs and Cas9 into porcine zygotes, enabling the double biallelic mutation of multiple genes with favorable embryo survival. Keywords IL2RG . GHR . CRISPR/Cas9 . Electroporation . Pig
Introduction Domestic pigs have been shown to be an effective model organism for biomedical research, as their background Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11626-020-00507-9) contains supplementary material, which is available to authorized users. * Fuminori Tanihara [email protected] 1
Laboratory of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, 2272-1 Ishii, Myozai-gun, Tokushima 779-3233, Japan
2
Faculty of Veterinary Science, Prince of Songkla University, Songkhla, Thailand
3
School of Biological Science, Tokai University, Sapporo, Japan
4
College of Agricultural Science, Guangdong Ocean University, Guangdong, China
physiology and genetic heterogeneity are closely related to those of humans (Abkowitz et al. 1995; Meurens et al. 2012). Pigs are considered to have a high reproductive performance, as heat cycles start as early as 5 mo old. They also have a short generation interval and a high number of offspring per litter (
Data Loading...
