Efficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos.
Somatic cell nuclear transfer or cytoplasm microinjection have been used to generate genome-edited farm animals; however, these methods have several drawbacks that reduce their efficiency. This study aimed to develop electroporation conditions that allow delivery of CRISPR/Cas9 system to bovine zygotes for efficient gene knockout. We optimized electroporation conditions to deliver Cas9:sgRNA ribonucleoproteins to bovine zygotes without compromising embryo development. Higher electroporation pulse voltage resulted in increased membrane permeability; however, voltages above 15 V/mm decreased embryo developmental potential. The zona pellucida of bovine embryos was not a barrier to efficient RNP electroporation. Using parameters optimized for maximal membrane permeability while maintaining developmental competence we achieved high rates of gene editing when targeting bovine OCT4, which resulted in absence of OCT4 protein in 100% of the evaluated embryos and the expected arrest of embryonic development at the morula stage. In conclusion, Cas9:sgRNA ribonucleoproteins can be delivered efficiently by electroporation to zona-intact bovine zygotes, resulting in efficient gene knockouts.
Main Authors: | , , , |
---|---|
Other Authors: | |
Format: | Artigo de periódico biblioteca |
Language: | Ingles English |
Published: |
2021-08-12
|
Subjects: | CRISPR, Genome editing, Bovino, Reprodução Animal, Embrião Animal, Genoma, Embryo (animal), |
Online Access: | http://www.alice.cnptia.embrapa.br/alice/handle/doc/1133487 https://doi.org/10.3389/fgene.2020.570069 |
Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
id |
dig-alice-doc-1133487 |
---|---|
record_format |
koha |
spelling |
dig-alice-doc-11334872021-08-12T13:00:54Z Efficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos. CAMARGO, L. S. de A. OWEN, J. R. VAN EENENNAAM, A. L. ROSS, P. J. LUIZ SERGIO DE ALMEIDA CAMARGO, CNPGL; JOSEPH R. OWEN, University of California; ALISON L. VAN EENENNAAM, University of California; PABLO JUAN ROSS, University of California. CRISPR Genome editing Bovino Reprodução Animal Embrião Animal Genoma Embryo (animal) Somatic cell nuclear transfer or cytoplasm microinjection have been used to generate genome-edited farm animals; however, these methods have several drawbacks that reduce their efficiency. This study aimed to develop electroporation conditions that allow delivery of CRISPR/Cas9 system to bovine zygotes for efficient gene knockout. We optimized electroporation conditions to deliver Cas9:sgRNA ribonucleoproteins to bovine zygotes without compromising embryo development. Higher electroporation pulse voltage resulted in increased membrane permeability; however, voltages above 15 V/mm decreased embryo developmental potential. The zona pellucida of bovine embryos was not a barrier to efficient RNP electroporation. Using parameters optimized for maximal membrane permeability while maintaining developmental competence we achieved high rates of gene editing when targeting bovine OCT4, which resulted in absence of OCT4 protein in 100% of the evaluated embryos and the expected arrest of embryonic development at the morula stage. In conclusion, Cas9:sgRNA ribonucleoproteins can be delivered efficiently by electroporation to zona-intact bovine zygotes, resulting in efficient gene knockouts. 2021-08-12T13:00:45Z 2021-08-12T13:00:45Z 2021-08-12 2020 Artigo de periódico Frontiers in Genetics, v. 11, article 570069, 2020. http://www.alice.cnptia.embrapa.br/alice/handle/doc/1133487 https://doi.org/10.3389/fgene.2020.570069 Ingles en openAccess |
institution |
EMBRAPA |
collection |
DSpace |
country |
Brasil |
countrycode |
BR |
component |
Bibliográfico |
access |
En linea |
databasecode |
dig-alice |
tag |
biblioteca |
region |
America del Sur |
libraryname |
Sistema de bibliotecas de EMBRAPA |
language |
Ingles English |
topic |
CRISPR Genome editing Bovino Reprodução Animal Embrião Animal Genoma Embryo (animal) CRISPR Genome editing Bovino Reprodução Animal Embrião Animal Genoma Embryo (animal) |
spellingShingle |
CRISPR Genome editing Bovino Reprodução Animal Embrião Animal Genoma Embryo (animal) CRISPR Genome editing Bovino Reprodução Animal Embrião Animal Genoma Embryo (animal) CAMARGO, L. S. de A. OWEN, J. R. VAN EENENNAAM, A. L. ROSS, P. J. Efficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos. |
description |
Somatic cell nuclear transfer or cytoplasm microinjection have been used to generate genome-edited farm animals; however, these methods have several drawbacks that reduce their efficiency. This study aimed to develop electroporation conditions that allow delivery of CRISPR/Cas9 system to bovine zygotes for efficient gene knockout. We optimized electroporation conditions to deliver Cas9:sgRNA ribonucleoproteins to bovine zygotes without compromising embryo development. Higher electroporation pulse voltage resulted in increased membrane permeability; however, voltages above 15 V/mm decreased embryo developmental potential. The zona pellucida of bovine embryos was not a barrier to efficient RNP electroporation. Using parameters optimized for maximal membrane permeability while maintaining developmental competence we achieved high rates of gene editing when targeting bovine OCT4, which resulted in absence of OCT4 protein in 100% of the evaluated embryos and the expected arrest of embryonic development at the morula stage. In conclusion, Cas9:sgRNA ribonucleoproteins can be delivered efficiently by electroporation to zona-intact bovine zygotes, resulting in efficient gene knockouts. |
author2 |
LUIZ SERGIO DE ALMEIDA CAMARGO, CNPGL; JOSEPH R. OWEN, University of California; ALISON L. VAN EENENNAAM, University of California; PABLO JUAN ROSS, University of California. |
author_facet |
LUIZ SERGIO DE ALMEIDA CAMARGO, CNPGL; JOSEPH R. OWEN, University of California; ALISON L. VAN EENENNAAM, University of California; PABLO JUAN ROSS, University of California. CAMARGO, L. S. de A. OWEN, J. R. VAN EENENNAAM, A. L. ROSS, P. J. |
format |
Artigo de periódico |
topic_facet |
CRISPR Genome editing Bovino Reprodução Animal Embrião Animal Genoma Embryo (animal) |
author |
CAMARGO, L. S. de A. OWEN, J. R. VAN EENENNAAM, A. L. ROSS, P. J. |
author_sort |
CAMARGO, L. S. de A. |
title |
Efficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos. |
title_short |
Efficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos. |
title_full |
Efficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos. |
title_fullStr |
Efficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos. |
title_full_unstemmed |
Efficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos. |
title_sort |
efficient one-step knockout by electroporation of ribonucleoproteins into zona-intact bovine embryos. |
publishDate |
2021-08-12 |
url |
http://www.alice.cnptia.embrapa.br/alice/handle/doc/1133487 https://doi.org/10.3389/fgene.2020.570069 |
work_keys_str_mv |
AT camargolsdea efficientonestepknockoutbyelectroporationofribonucleoproteinsintozonaintactbovineembryos AT owenjr efficientonestepknockoutbyelectroporationofribonucleoproteinsintozonaintactbovineembryos AT vaneenennaamal efficientonestepknockoutbyelectroporationofribonucleoproteinsintozonaintactbovineembryos AT rosspj efficientonestepknockoutbyelectroporationofribonucleoproteinsintozonaintactbovineembryos |
_version_ |
1756027645761421312 |