Efficient gene editing of pig embryos by combining electroporation and lipofection
Background and Aim: Mosaicism, which is characterized by the presence of wild-type and more than one mutant allele, poses a serious problem in zygotic gene modification through the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system. Therefore, we use...
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Veterinary World
2024-11-01
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| Series: | Veterinary World |
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| Online Access: | https://www.veterinaryworld.org/Vol.17/November-2024/30.pdf |
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| author | Qingyi Lin Nanaka Torigoe Bin Liu Yuichiro Nakayama Aya Nakai Zhao Namula Megumi Nagahara Fuminori Tanihara Maki Hirata Takeshige Otoi |
| author_facet | Qingyi Lin Nanaka Torigoe Bin Liu Yuichiro Nakayama Aya Nakai Zhao Namula Megumi Nagahara Fuminori Tanihara Maki Hirata Takeshige Otoi |
| author_sort | Qingyi Lin |
| collection | DOAJ |
| description | Background and Aim: Mosaicism, which is characterized by the presence of wild-type and more than one mutant allele, poses a serious problem in zygotic gene modification through the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system. Therefore, we used pig embryos to compare the gene editing efficiencies achieved by combining electroporation and lipofection using different aminopeptidase N (APN)-targeting guide RNA (gRNA) sequences.
Materials and Methods: Six gRNAs (gRNA1–6) with different target sequences were designed to target APN. Zona pellucida (ZP)-intact zygotes collected 10 h after the start of in vitro fertilization (IVF) were electroporated with each gRNA to compare their gene editing efficiency. The gRNA sequences that achieved the lowest and highest mutation rates (gRNA4 and gRNA6, respectively) were selected for additional lipofection to assess gene editing efficiency following combined treatment. As ZP removal is essential for lipofection, ZP-free zygotes were electroporated with gRNA4 or gRNA6 10 h after IVF initiation, followed by lipofection with the same gRNAs 24 or 29 h after IVF initiation. The electroporated ZP-intact and ZP-free zygotes were used as controls.
Results: gRNA4 and gRNA6 exhibited the lowest and highest mutation rates, respectively. gRNA4-targeted ZP-free embryos subjected to additional lipofection 29 h after IVF initiation exhibited significantly higher total and biallelic mutation rates than ZP-intact embryos that received only electroporation. Additional lipofection of gRNA6-targeted embryos had no obvious effect on mutation rates.
Conclusion: Electroporation combined with lipofection using gRNAs with low mutation rates may improve gene editing efficiency in pig embryos. However, the effects may vary based on the timing of gene editing. |
| format | Article |
| id | doaj-art-d63b7b1d0dc04428873395ad1489957d |
| institution | Kabale University |
| issn | 0972-8988 2231-0916 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | Veterinary World |
| record_format | Article |
| series | Veterinary World |
| spelling | doaj-art-d63b7b1d0dc04428873395ad1489957d2024-12-08T06:28:42ZengVeterinary WorldVeterinary World0972-89882231-09162024-11-0117112701270710.14202/vetworld.2024.2701-2707Efficient gene editing of pig embryos by combining electroporation and lipofectionQingyi Lin0https://orcid.org/0000-0002-3558-7812Nanaka Torigoe1https://orcid.org/0009-0001-0272-6142Bin Liu2https://orcid.org/0009-0001-0081-8262Yuichiro Nakayama3https://orcid.org/0009-0000-4046-7260Aya Nakai4https://orcid.org/0009-0008-1675-0494Zhao Namula5https://orcid.org/0000-0002-4048-3161Megumi Nagahara6https://orcid.org/0009-0009-7045-3922Fuminori Tanihara7https://orcid.org/0000-0002-2158-5808Maki Hirata8https://orcid.org/0000-0003-4673-1581Takeshige Otoi9https://orcid.org/0000-0002-0382-959XBio-Innovation Research Center, Tokushima University, Tokushima, Japan; Department of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan.Bio-Innovation Research Center, Tokushima University, Tokushima, Japan; Department of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan.Bio-Innovation Research Center, Tokushima University, Tokushima, Japan; Department of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan.Bio-Innovation Research Center, Tokushima University, Tokushima, Japan; Department of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan.Bio-Innovation Research Center, Tokushima University, Tokushima, Japan; Department of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan.Bio-Innovation Research Center, Tokushima University, Tokushima, Japan; Department of Animal Reproduction, College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang, China.Bio-Innovation Research Center, Tokushima University, Tokushima, Japan; Department of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan.Bio-Innovation Research Center, Tokushima University, Tokushima, Japan; Department of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan.Bio-Innovation Research Center, Tokushima University, Tokushima, Japan; Department of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan.Bio-Innovation Research Center, Tokushima University, Tokushima, Japan; Department of Animal Reproduction, Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan.Background and Aim: Mosaicism, which is characterized by the presence of wild-type and more than one mutant allele, poses a serious problem in zygotic gene modification through the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system. Therefore, we used pig embryos to compare the gene editing efficiencies achieved by combining electroporation and lipofection using different aminopeptidase N (APN)-targeting guide RNA (gRNA) sequences. Materials and Methods: Six gRNAs (gRNA1–6) with different target sequences were designed to target APN. Zona pellucida (ZP)-intact zygotes collected 10 h after the start of in vitro fertilization (IVF) were electroporated with each gRNA to compare their gene editing efficiency. The gRNA sequences that achieved the lowest and highest mutation rates (gRNA4 and gRNA6, respectively) were selected for additional lipofection to assess gene editing efficiency following combined treatment. As ZP removal is essential for lipofection, ZP-free zygotes were electroporated with gRNA4 or gRNA6 10 h after IVF initiation, followed by lipofection with the same gRNAs 24 or 29 h after IVF initiation. The electroporated ZP-intact and ZP-free zygotes were used as controls. Results: gRNA4 and gRNA6 exhibited the lowest and highest mutation rates, respectively. gRNA4-targeted ZP-free embryos subjected to additional lipofection 29 h after IVF initiation exhibited significantly higher total and biallelic mutation rates than ZP-intact embryos that received only electroporation. Additional lipofection of gRNA6-targeted embryos had no obvious effect on mutation rates. Conclusion: Electroporation combined with lipofection using gRNAs with low mutation rates may improve gene editing efficiency in pig embryos. However, the effects may vary based on the timing of gene editing.https://www.veterinaryworld.org/Vol.17/November-2024/30.pdfelectroporationguide rna sequencelipofectionpig embryo |
| spellingShingle | Qingyi Lin Nanaka Torigoe Bin Liu Yuichiro Nakayama Aya Nakai Zhao Namula Megumi Nagahara Fuminori Tanihara Maki Hirata Takeshige Otoi Efficient gene editing of pig embryos by combining electroporation and lipofection Veterinary World electroporation guide rna sequence lipofection pig embryo |
| title | Efficient gene editing of pig embryos by combining electroporation and lipofection |
| title_full | Efficient gene editing of pig embryos by combining electroporation and lipofection |
| title_fullStr | Efficient gene editing of pig embryos by combining electroporation and lipofection |
| title_full_unstemmed | Efficient gene editing of pig embryos by combining electroporation and lipofection |
| title_short | Efficient gene editing of pig embryos by combining electroporation and lipofection |
| title_sort | efficient gene editing of pig embryos by combining electroporation and lipofection |
| topic | electroporation guide rna sequence lipofection pig embryo |
| url | https://www.veterinaryworld.org/Vol.17/November-2024/30.pdf |
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