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Effects of electroporation treatment using different concentrations of Cas9 protein with gRNA targeting Myostatin ( MSTN ) genes on the development and gene editing of porcine zygotes
Author(s) -
Le Quynh A.,
Hirata Maki,
Nguyen Nhien T.,
Takebayashi Koki,
Wittayarat Manita,
Sato Yoko,
Namula Zhao,
Nii Masahiro,
Tanihara Fuminori,
Otoi Takeshige
Publication year - 2020
Publication title -
animal science journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.606
H-Index - 38
eISSN - 1740-0929
pISSN - 1344-3941
DOI - 10.1111/asj.13386
Subject(s) - zygote , biology , electroporation , crispr , genome editing , cas9 , blastocyst , gene , embryo , microbiology and biotechnology , genetics , embryogenesis
This study was conducted to investigate the effect of seven concentrations of Cas9 protein (0, 25, 50, 100, 200, 500, and 1,000 ng/µl) on the development and gene editing of porcine embryos. This included the target editing and off‐target effect of embryos developed from zygotes that were edited via electroporation of the Cas9 protein with guide RNA targeting Myostatin genes. We found that the development to blastocysts of electroporated zygotes was not affected by the concentration of Cas9 protein. Although the editing rate, which was defined as the ratio of edited blastocysts to total examined blastocysts, did not differ with Cas9 protein concentration, the editing efficiency, which was defined as the frequency of indel mutations in each edited blastocyst, was significantly decreased in the edited blastocysts from zygotes electroporated with 25 ng/µl of Cas9 protein compared with that of blastocysts from zygotes electroporated with higher Cas9 protein concentrations. Moreover the frequency of indel events at the two possible off‐target sites was not significantly different with different concentrations of Cas9 protein. These results indicate that the concentration of Cas9 protein affects gene editing efficiency in embryos but not the embryonic development, gene editing rate, and non‐specific cleavage of off‐target sites.