Late-Breaking Abstract: Production of a Gene Knock-In Bull Calf by Embryo-Mediated Genome Editing

Genome editing offers an opportunity to introduce targeted gene insertions into livestock breeding programs. Molecular geneticists have typically employed a donor repair template and the homologous recombination (HR) pathway in somatic cells to introduce gene knockins into livestock genomes, followe...

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Bibliographic Details
Published in:Journal of animal science 2020-11, Vol.98, p.358-358
Main Authors: Owen, Joseph R, Henning, Sadie L, McNabb, Bret R, Lin, Jason C, Young, Amy E, Murray, James D, Ross, Pablo J, Van Eenennaam, Alison L
Format: Article
Language:English
Subjects:
Animal husbandry
Blastocysts
Chromosome 17
Chromosomes
Cloning
CRISPR
Deoxyribonucleic acid
DNA
Editing
Embryos
Fluorescence
Gene sequencing
Genes
Genome editing
Genomes
Genotypes
Green fluorescent protein
gRNA
Homologous recombination
Homology
Insertion
Livestock
Livestock breeding
Microinjection
Nucleotide sequence
Offspring
Placenta
Reagents
Reporter gene
Sex
Somatic cells
Studies
Ultrasound
Zygotes
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Summary:Genome editing offers an opportunity to introduce targeted gene insertions into livestock breeding programs. Molecular geneticists have typically employed a donor repair template and the homologous recombination (HR) pathway in somatic cells to introduce gene knockins into livestock genomes, followed by cloning. Editing embryos directly to achieve targeted gene knock-ins is inefficient, especially for introducing large DNA sequences. Here we report using a one-step method to produce a gene knock-in bull calf by cytoplasmic microinjection of CRISPR/Cas9 reagents into a bovine embryo. In vitro fertilized one-cell bovine zygotes were injected with a gRNA/Cas9 ribonucleoprotein complex and homology mediated end joining donor template containing the sex determining region Y (SRY) gene, the green fluorescent protein (gfp) reporter gene driven by the SV40 promoter, and one kilobase homology arms targeting the H11 safe harbor locus on bovine chromosome 17. Seven-day blastocysts were evaluated using fluorescent microscopy, and nine green fluorescent embryos were transferred to synchronized recipients. Ultrasound evaluation at 35 days revealed one pregnancy. In April 2020, a healthy 50 kg male calf was born. DNA was extracted from placenta, blood and a fibroblast line derived from the calf and analyzed for SRY-GFP knock-in, as well as genotypic sex. PCR and Sanger sequencing revealed the biallelic edit of the target location on chromosome 17, with the insertion of three or seven copies of the SRY-GFP construct in addition to donor plasmid backbone, or a 26 base pair insertion, and an XY genotype. Future analysis of the XX offspring inheriting the SRY gene on chromosome 17 from this knock-in bull will reveal whether inheritance of the bovine SRY gene is sufficient to trigger the male developmental pathway in cattle.
ISSN:0021-8812
1525-3163