Gene Knockouts in Goats Using CRISPR/Cas9 System and Somatic Cell Nuclear Transfer

The combination of CRISPR/Cas9 and SCNT techniques greatly facilitates the production of gene-edited livestock. Here, we describe the detailed procedure to create gene knockout goats using this strategy starting from the construction of CRISPR/Cas9 targeting vectors to the transfer of cloned embryos...

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Bibliographic Details
Published inMethods in molecular biology (Clifton, N.J.) Vol. 1874; p. 373
Main Authors Fan, Zhiqiang, Yang, Min, Regouski, Misha, Polejaeva, Irina A
Format Journal Article
LanguageEnglish
Published United States 2019
Subjects
Animals
Animals, Genetically Modified
Cells, Cultured
Cloning, Organism - methods
CRISPR-Cas Systems
Embryo Culture Techniques
Female
Fibroblasts - cytology
Fibroblasts - metabolism
Gene Knockout Techniques - methods
Goats
Nuclear Transfer Techniques
RNA, Guide, CRISPR-Cas Systems - genetics
CRISPR/Cas9
Goats
Somatic cell nuclear transfer
Gene knockout
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Summary:The combination of CRISPR/Cas9 and SCNT techniques greatly facilitates the production of gene-edited livestock. Here, we describe the detailed procedure to create gene knockout goats using this strategy starting from the construction of CRISPR/Cas9 targeting vectors to the transfer of cloned embryos into recipient females. In this procedure, the transfection conditions for goat fibroblasts were optimized due to their high sensitivity to electrotransfection, which enabled the isolation of single-cell colonies carrying simultaneous disruption of multiple genes for SCNT with a single co-transfection of pooled CRISPR/Cas9 targeting vectors.
ISSN:1940-6029
DOI:10.1007/978-1-4939-8831-0_22