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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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Autores principales: Gutierrez, Karina, Glanzner, Werner G., de Macedo, Mariana P., Rissi, Vitor B., Dicks, Naomi, Bohrer, Rodrigo C., Baldassarre, Hernan, Agellon, Luis B., Bordignon, Vilceu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8876063/
https://www.ncbi.nlm.nih.gov/pubmed/35207459
http://dx.doi.org/10.3390/life12020171
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author Gutierrez, Karina
Glanzner, Werner G.
de Macedo, Mariana P.
Rissi, Vitor B.
Dicks, Naomi
Bohrer, Rodrigo C.
Baldassarre, Hernan
Agellon, Luis B.
Bordignon, Vilceu
author_facet Gutierrez, Karina
Glanzner, Werner G.
de Macedo, Mariana P.
Rissi, Vitor B.
Dicks, Naomi
Bohrer, Rodrigo C.
Baldassarre, Hernan
Agellon, Luis B.
Bordignon, Vilceu
author_sort Gutierrez, Karina
collection PubMed
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.
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spelling pubmed-88760632022-02-26 Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos Gutierrez, Karina Glanzner, Werner G. de Macedo, Mariana P. Rissi, Vitor B. Dicks, Naomi Bohrer, Rodrigo C. Baldassarre, Hernan Agellon, Luis B. Bordignon, Vilceu Life (Basel) Article 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. MDPI 2022-01-25 /pmc/articles/PMC8876063/ /pubmed/35207459 http://dx.doi.org/10.3390/life12020171 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Gutierrez, Karina
Glanzner, Werner G.
de Macedo, Mariana P.
Rissi, Vitor B.
Dicks, Naomi
Bohrer, Rodrigo C.
Baldassarre, Hernan
Agellon, Luis B.
Bordignon, Vilceu
Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos
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 Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8876063/
https://www.ncbi.nlm.nih.gov/pubmed/35207459
http://dx.doi.org/10.3390/life12020171
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