Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos
Cytoplasmic microinjection (CI) of the CRISPR/Cas9 system enabled the induction of site-specific mutations in porcine zygotes and resulting pigs. However, mosaicism is a serious problem for genetically modified pigs. In the present study, we investigated suitable timing and concentration of CRISPR/C...
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| Format: | Article |
| Language: | English |
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The Society for Reproduction and Development
2019-02-01
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| Series: | The Journal of Reproduction and Development |
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| Online Access: | https://www.jstage.jst.go.jp/article/jrd/65/3/65_2018-116/_pdf/-char/en |
| _version_ | 1827752385257144320 |
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| author | Fuminori TANIHARA Maki HIRATA Nhien Thi NGUYEN Quynh Anh LE Takayuki HIRANO Takeshige OTOI |
| author_facet | Fuminori TANIHARA Maki HIRATA Nhien Thi NGUYEN Quynh Anh LE Takayuki HIRANO Takeshige OTOI |
| author_sort | Fuminori TANIHARA |
| collection | DOAJ |
| description | Cytoplasmic microinjection (CI) of the CRISPR/Cas9 system enabled the induction of site-specific mutations in porcine zygotes and resulting pigs. However, mosaicism is a serious problem for genetically modified pigs. In the present study, we investigated suitable timing and concentration of CRISPR/Cas9 components for introduction into oocytes/zygotes by CI, to reduce mosaicism in the resulting blastocysts. First, we introduced 20 ng/μl of Cas9 protein and guide RNA (gRNA), targeting the α-1,3-galactosyltransferase (GalT) gene in oocytes before in vitro fertilization (IVF), in zygotes after IVF, or in oocytes/zygotes before and after IVF, twice. CI treatment had no detrimental effects on blastocyst formation rates. The highest value of the rate of mutant blastocysts was observed in zygotes injected after IVF. Next, we injected Cas9 protein and gRNA into zygotes after IVF at a concentration of 20 ng/μl each (20 ng/μl group) or 100 ng/μl each (100 ng/μl group). The ratio of the number of blastocysts that carried mutations to the total number of blastocysts examined in the 100 ng/μl group was significantly higher (P < 0.05) than that in the 20 ng/μl group. Although no blastocysts from the 20 ng/μl group carried a biallelic mutation, 16.7% of blastocysts from the 100 ng/μl group carried a biallelic mutation. In conclusion, increasing the concentration of Cas9 protein and gRNA is effective in generating biallelic mutant blastocysts. To reduce mosaicism, however, further optimization of the timing of CI, and the concentration of CRISPR/Cas9 components, is needed. |
| first_indexed | 2024-03-11T07:16:37Z |
| format | Article |
| id | doaj.art-76d1562c480a4902a3acd9befe4f324d |
| institution | Directory Open Access Journal |
| issn | 0916-8818 1348-4400 |
| language | English |
| last_indexed | 2024-03-11T07:16:37Z |
| publishDate | 2019-02-01 |
| publisher | The Society for Reproduction and Development |
| record_format | Article |
| series | The Journal of Reproduction and Development |
| spelling | doaj.art-76d1562c480a4902a3acd9befe4f324d2023-11-17T08:16:14ZengThe Society for Reproduction and DevelopmentThe Journal of Reproduction and Development0916-88181348-44002019-02-0165320921410.1262/jrd.2018-116jrdEffects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryosFuminori TANIHARA0Maki HIRATA1Nhien Thi NGUYEN2Quynh Anh LE3Takayuki HIRANO4Takeshige OTOI5Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8513, JapanFaculty of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8513, JapanFaculty of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8513, JapanFaculty of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8513, JapanFaculty of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8513, JapanFaculty of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8513, JapanCytoplasmic microinjection (CI) of the CRISPR/Cas9 system enabled the induction of site-specific mutations in porcine zygotes and resulting pigs. However, mosaicism is a serious problem for genetically modified pigs. In the present study, we investigated suitable timing and concentration of CRISPR/Cas9 components for introduction into oocytes/zygotes by CI, to reduce mosaicism in the resulting blastocysts. First, we introduced 20 ng/μl of Cas9 protein and guide RNA (gRNA), targeting the α-1,3-galactosyltransferase (GalT) gene in oocytes before in vitro fertilization (IVF), in zygotes after IVF, or in oocytes/zygotes before and after IVF, twice. CI treatment had no detrimental effects on blastocyst formation rates. The highest value of the rate of mutant blastocysts was observed in zygotes injected after IVF. Next, we injected Cas9 protein and gRNA into zygotes after IVF at a concentration of 20 ng/μl each (20 ng/μl group) or 100 ng/μl each (100 ng/μl group). The ratio of the number of blastocysts that carried mutations to the total number of blastocysts examined in the 100 ng/μl group was significantly higher (P < 0.05) than that in the 20 ng/μl group. Although no blastocysts from the 20 ng/μl group carried a biallelic mutation, 16.7% of blastocysts from the 100 ng/μl group carried a biallelic mutation. In conclusion, increasing the concentration of Cas9 protein and gRNA is effective in generating biallelic mutant blastocysts. To reduce mosaicism, however, further optimization of the timing of CI, and the concentration of CRISPR/Cas9 components, is needed.https://www.jstage.jst.go.jp/article/jrd/65/3/65_2018-116/_pdf/-char/encrispr/cas9cytoplasmic microinjectiongene editingmosaicismporcine zygote |
| spellingShingle | Fuminori TANIHARA Maki HIRATA Nhien Thi NGUYEN Quynh Anh LE Takayuki HIRANO Takeshige OTOI Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos The Journal of Reproduction and Development crispr/cas9 cytoplasmic microinjection gene editing mosaicism porcine zygote |
| title | Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos |
| title_full | Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos |
| title_fullStr | Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos |
| title_full_unstemmed | Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos |
| title_short | Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos |
| title_sort | effects of concentration of crispr cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos |
| topic | crispr/cas9 cytoplasmic microinjection gene editing mosaicism porcine zygote |
| url | https://www.jstage.jst.go.jp/article/jrd/65/3/65_2018-116/_pdf/-char/en |
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