Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos
CRISPR/Cas9 technology is a powerful tool used for genome manipulation in different cell types and species. However, as with all new technologies, it still requires improvements. Different factors can affect CRISPR/Cas efficiency in zygotes, which influence the total cost and complexity for creating...
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MDPI AG
2022-01-01
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| author | Karina Gutierrez Werner G. Glanzner Mariana P. de Macedo Vitor B. Rissi Naomi Dicks Rodrigo C. Bohrer Hernan Baldassarre Luis B. Agellon Vilceu Bordignon |
| author_facet | Karina Gutierrez Werner G. Glanzner Mariana P. de Macedo Vitor B. Rissi Naomi Dicks Rodrigo C. Bohrer Hernan Baldassarre Luis B. Agellon Vilceu Bordignon |
| author_sort | Karina Gutierrez |
| collection | DOAJ |
| description | CRISPR/Cas9 technology is a powerful tool used for genome manipulation in different cell types and species. However, as with all new technologies, it still requires improvements. Different factors can affect CRISPR/Cas efficiency in zygotes, which influence the total cost and complexity for creating large-animal models for research. This study evaluated the importance of zygote cell cycle stage between early-injection (within 6 h post activation/fertilization) versus late-injection (14–16 h post activation/fertilization) when the CRISPR/Cas9 components were injected and the inhibition of the homologous recombination (HR) pathway of DNA repair on gene editing, embryo survival and development on embryos produced by fertilization, sperm injection, somatic cell nuclear transfer, and parthenogenetic activation technologies. Injections at the late cell cycle stage decreased embryo survival (measured as the proportion of unlysed embryos) and blastocyst formation (68.2%; 19.3%) compared to early-stage injection (86.3%; 28.8%). However, gene editing was higher in blastocysts from late-(73.8%) vs. early-(63.8%) injected zygotes. Inhibition of the HR repair pathway increased gene editing efficiency by 15.6% in blastocysts from early-injected zygotes without compromising embryo development. Our finding shows that injection at the early cell cycle stage along with HR inhibition improves both zygote viability and gene editing rate in pig blastocysts. |
| first_indexed | 2024-03-09T21:35:12Z |
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| institution | Directory Open Access Journal |
| issn | 2075-1729 |
| language | English |
| last_indexed | 2024-03-09T21:35:12Z |
| publishDate | 2022-01-01 |
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| series | Life |
| spelling | doaj.art-49dce14483fc4f0e9dcbc4586060022e2023-11-23T20:45:12ZengMDPI AGLife2075-17292022-01-0112217110.3390/life12020171Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine EmbryosKarina Gutierrez0Werner G. Glanzner1Mariana P. de Macedo2Vitor B. Rissi3Naomi Dicks4Rodrigo C. Bohrer5Hernan Baldassarre6Luis B. Agellon7Vilceu Bordignon8Department of Animal Science, McGill University, Sainte-Anne-de-Bellevue, QC H9X 3V9, CanadaDepartment of Animal Science, McGill University, Sainte-Anne-de-Bellevue, QC H9X 3V9, CanadaDepartment of Animal Science, McGill University, Sainte-Anne-de-Bellevue, QC H9X 3V9, CanadaDepartment of Agriculture, Biodiversity and Forests, Federal University of Santa Catarina, Curitibanos 89520-000, BrazilDepartment of Animal Science, McGill University, Sainte-Anne-de-Bellevue, QC H9X 3V9, CanadaDepartment of Animal Science, McGill University, Sainte-Anne-de-Bellevue, QC H9X 3V9, CanadaDepartment of Animal Science, McGill University, Sainte-Anne-de-Bellevue, QC H9X 3V9, CanadaSchool of Human Nutrition, McGill University, Sainte-Anne-de-Bellevue, QC H9X 3V9, CanadaDepartment of Animal Science, McGill University, Sainte-Anne-de-Bellevue, QC H9X 3V9, CanadaCRISPR/Cas9 technology is a powerful tool used for genome manipulation in different cell types and species. However, as with all new technologies, it still requires improvements. Different factors can affect CRISPR/Cas efficiency in zygotes, which influence the total cost and complexity for creating large-animal models for research. This study evaluated the importance of zygote cell cycle stage between early-injection (within 6 h post activation/fertilization) versus late-injection (14–16 h post activation/fertilization) when the CRISPR/Cas9 components were injected and the inhibition of the homologous recombination (HR) pathway of DNA repair on gene editing, embryo survival and development on embryos produced by fertilization, sperm injection, somatic cell nuclear transfer, and parthenogenetic activation technologies. Injections at the late cell cycle stage decreased embryo survival (measured as the proportion of unlysed embryos) and blastocyst formation (68.2%; 19.3%) compared to early-stage injection (86.3%; 28.8%). However, gene editing was higher in blastocysts from late-(73.8%) vs. early-(63.8%) injected zygotes. Inhibition of the HR repair pathway increased gene editing efficiency by 15.6% in blastocysts from early-injected zygotes without compromising embryo development. Our finding shows that injection at the early cell cycle stage along with HR inhibition improves both zygote viability and gene editing rate in pig blastocysts.https://www.mdpi.com/2075-1729/12/2/171cell cycleDNA repairgene editingpigszygotes |
| spellingShingle | Karina Gutierrez Werner G. Glanzner Mariana P. de Macedo Vitor B. Rissi Naomi Dicks Rodrigo C. Bohrer Hernan Baldassarre Luis B. Agellon Vilceu Bordignon Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos Life cell cycle DNA repair gene editing pigs zygotes |
| title | Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos |
| title_full | Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos |
| title_fullStr | Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos |
| title_full_unstemmed | Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos |
| title_short | Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos |
| title_sort | cell cycle stage and dna repair pathway influence crispr cas9 gene editing efficiency in porcine embryos |
| topic | cell cycle DNA repair gene editing pigs zygotes |
| url | https://www.mdpi.com/2075-1729/12/2/171 |
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