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Cytoplasmic Injection of Zygotes to Genome Edit Naturally Occurring Sequence Variants Into Bovine Embryos
Jingwei Wei1, Brigid Brophy1, Sally-Ann Cole1
1Animal Biotechnology, Ruakura Research Centre, AgResearch Ltd, Hamilton, New Zealand.
Frontiers in Genetics
|July 28, 2022
Summary
Genome editing in cattle embryos can accelerate genetic gain by precisely inserting natural genetic variants. Researchers achieved high conversion rates for specific gene edits, enabling the development of non-mosaic edited animals.
Area of Science:
- Animal genetics
- Genomic engineering
- Biotechnology
Background:
- Genome editing offers a powerful tool for enhancing cattle breeding by introducing targeted genetic variations.
- Homology-directed repair mechanisms are key to precise genome modification using engineered nucleases and repair templates.
- Editing bovine embryos allows for the development of fully edited, non-mosaic animals, aligning with advanced breeding practices.
Purpose of the Study:
- To evaluate the efficiency of genome editing techniques for introducing specific genetic variants into cattle embryos.
- To assess the potential for creating non-mosaic, precisely edited bovine embryos for breeding applications.
- To compare different genome editing tools and delivery methods for optimizing edit efficiency.
Main Methods:
- Utilized CRISPR-Cas9 and TALEN gene editing systems to introduce targeted mutations in bovine zygotes.
- Employed homology-directed repair with specific repair templates for precise sequence variant introduction.
- Developed and applied a biopsy-based screening method to identify non-mosaic edited embryos.
Main Results:
- Achieved up to 11% fully converted embryos for a 3 bp deletion in the PMEL gene using gRNA/Cas9.
- Increased conversion rates to up to 48% for PMEL gene editing using TALEN delivered via plasmid.
- Obtained full conversion rates up to 8% for PRLR gene variants using various gRNA/Cas9 editors and delivery methods.
- Successfully developed a screening strategy to identify non-mosaic edited embryos.
Conclusions:
- Genome editing in bovine embryos is a viable strategy for accelerating genetic gain through precise introduction of natural variants.
- TALEN delivery via plasmid showed higher efficiency for specific edits compared to gRNA/Cas9.
- A biopsy-based screening method is crucial for producing uniformly edited, non-mosaic cattle.
- This approach holds significant potential for precision breeding in cattle.
