Journal
IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL
Volume 56, Issue 8, Pages 614-621Publisher
SPRINGER
DOI: 10.1007/s11626-020-00507-9
Keywords
IL2RG; GHR; CRISPR; Cas9; Electroporation; Pig
Categories
Funding
- Program of Open Innovation Platform with Enterprises, Research Institute and Academia (OPERA) from the Japan Science and Technology Agency (JST) [JPMJOP1613]
- KAKENHI grants from the Japan Society for the Promotion of Science (JSPS) [JP17H03938, JP18K12062, JP19K16014]
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In the present study, we investigated whether electroporation could be used for one-step multiplex CRISPR/Cas9-based genome editing, targetingIL2RGandGHRin 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 targetingIL2RGor one of two gRNAs targetingGHR. No significant differences in embryo development rates were found between control embryos and those subjected to electroporation, irrespective of the target gene. Two gRNAs targetingIL2RG(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 targetingGHR. In our next experiment, the mutation efficiency and the development of embryos simultaneously electroporated with gRNAs targetingIL2RGandGHRwere investigated. Similar embryo development rates were observed between embryos electroporated with two gRNAs and control embryos. WhenIL2RG-targeting gRNA no. 2 was used withGHR-targeting gRNAs no. 1 or no. 2, a significantly higher double biallelic mutation rate was observed than withIL2RG-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.
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