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Suppression of mosaic mutation by co-delivery of CRISPR associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation
Gene-modified animals, including pigs, can be generated efficiently by introducing CRISPR associated protein 9 (CRISPR/Cas9) into zygotes. However, in many cases, these zygotes tend to become mosaic mutants with various different mutant cell types, making it difficult to analyze the phenotype of gen...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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The Society for Reproduction and Development
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040215/ https://www.ncbi.nlm.nih.gov/pubmed/31761839 http://dx.doi.org/10.1262/jrd.2019-088 |
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author | YAMASHITA, Shiro KOGASAKA, Yuhei HIRADATE, Yuuki TANEMURA, Kentaro SENDAI, Yutaka |
author_facet | YAMASHITA, Shiro KOGASAKA, Yuhei HIRADATE, Yuuki TANEMURA, Kentaro SENDAI, Yutaka |
author_sort | YAMASHITA, Shiro |
collection | PubMed |
description | Gene-modified animals, including pigs, can be generated efficiently by introducing CRISPR associated protein 9 (CRISPR/Cas9) into zygotes. However, in many cases, these zygotes tend to become mosaic mutants with various different mutant cell types, making it difficult to analyze the phenotype of gene-modified founder animals. To reduce the mosaic mutations, we introduced three-prime repair exonuclease 2 (Trex2), an exonuclease that improves gene editing efficiency, into porcine zygotes along with CRISPR/Cas9 via electroporation. Although the rate of porcine blastocyst formation decreased due to electroporation (25.9 ± 4.6% vs. 41.2 ± 2.0%), co-delivery of murine Trex2 (mTrex2) mRNA with CRISPR/Cas9 did not affect it any further (25.9 ± 4.6% vs. 31.0 ± 4.6%). In addition, there was no significant difference in the diameter of blastocysts carrying CRISPR/Cas9 (164.7 ± 10.2 μm), and those with CRISPR/Cas9 + mTrex2 (151.9 ± 5.1 μm) as compared to those from the control group (178.9 ± 9.0 μm). These results revealed that mTrex2 did not affect the development of pre-implantation embryo. We also found bi-allelic, as well as mono-allelic, non-mosaic homozygous mutations in the blastocysts. Most importantly, co-delivery of mTrex2 mRNA with CRISPR/Cas9 increased non-mosaic mutant blastocysts (29.3 ± 4.5%) and reduced mosaic mutant blastocysts (70.7 ± 4.5%) as compared to CRISPR/Cas9 alone (5.6 ± 6.4% and 92.6 ± 8.6%, respectively). These data suggest that the co-delivery of CRISPR/Cas9 and mTrex2 is a useful method to suppress mosaic mutation. |
format | Online Article Text |
id | pubmed-7040215 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | The Society for Reproduction and Development |
record_format | MEDLINE/PubMed |
spelling | pubmed-70402152020-03-02 Suppression of mosaic mutation by co-delivery of CRISPR associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation YAMASHITA, Shiro KOGASAKA, Yuhei HIRADATE, Yuuki TANEMURA, Kentaro SENDAI, Yutaka J Reprod Dev Original Article Gene-modified animals, including pigs, can be generated efficiently by introducing CRISPR associated protein 9 (CRISPR/Cas9) into zygotes. However, in many cases, these zygotes tend to become mosaic mutants with various different mutant cell types, making it difficult to analyze the phenotype of gene-modified founder animals. To reduce the mosaic mutations, we introduced three-prime repair exonuclease 2 (Trex2), an exonuclease that improves gene editing efficiency, into porcine zygotes along with CRISPR/Cas9 via electroporation. Although the rate of porcine blastocyst formation decreased due to electroporation (25.9 ± 4.6% vs. 41.2 ± 2.0%), co-delivery of murine Trex2 (mTrex2) mRNA with CRISPR/Cas9 did not affect it any further (25.9 ± 4.6% vs. 31.0 ± 4.6%). In addition, there was no significant difference in the diameter of blastocysts carrying CRISPR/Cas9 (164.7 ± 10.2 μm), and those with CRISPR/Cas9 + mTrex2 (151.9 ± 5.1 μm) as compared to those from the control group (178.9 ± 9.0 μm). These results revealed that mTrex2 did not affect the development of pre-implantation embryo. We also found bi-allelic, as well as mono-allelic, non-mosaic homozygous mutations in the blastocysts. Most importantly, co-delivery of mTrex2 mRNA with CRISPR/Cas9 increased non-mosaic mutant blastocysts (29.3 ± 4.5%) and reduced mosaic mutant blastocysts (70.7 ± 4.5%) as compared to CRISPR/Cas9 alone (5.6 ± 6.4% and 92.6 ± 8.6%, respectively). These data suggest that the co-delivery of CRISPR/Cas9 and mTrex2 is a useful method to suppress mosaic mutation. The Society for Reproduction and Development 2019-11-24 2020-02 /pmc/articles/PMC7040215/ /pubmed/31761839 http://dx.doi.org/10.1262/jrd.2019-088 Text en ©2020 Society for Reproduction and Development This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. (CC-BY-NC-ND 4.0: https://creativecommons.org/licenses/by-nc-nd/4.0/) |
spellingShingle | Original Article YAMASHITA, Shiro KOGASAKA, Yuhei HIRADATE, Yuuki TANEMURA, Kentaro SENDAI, Yutaka Suppression of mosaic mutation by co-delivery of CRISPR associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation |
title | Suppression of mosaic mutation by co-delivery of CRISPR associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation |
title_full | Suppression of mosaic mutation by co-delivery of CRISPR associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation |
title_fullStr | Suppression of mosaic mutation by co-delivery of CRISPR associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation |
title_full_unstemmed | Suppression of mosaic mutation by co-delivery of CRISPR associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation |
title_short | Suppression of mosaic mutation by co-delivery of CRISPR associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation |
title_sort | suppression of mosaic mutation by co-delivery of crispr associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040215/ https://www.ncbi.nlm.nih.gov/pubmed/31761839 http://dx.doi.org/10.1262/jrd.2019-088 |
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