1. Single-Step Genome Editing of Small Ruminant Embryos by Electroporation
- Author
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Mahdi, Ahmed K, Medrano, Juan F, and Ross, Pablo J
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,Medicinal and Biomolecular Chemistry ,Chemical Sciences ,Microbiology ,Genetics ,Animals ,CRISPR-Cas Systems ,Electroporation ,Gene Editing ,Goats ,Ruminants ,Sheep ,Zygote ,RNA ,Small Untranslated ,genome editing ,CRISPR-Cas9 ,electroporation ,single step ,small ruminants ,Other Chemical Sciences ,Other Biological Sciences ,Chemical Physics ,Biochemistry and cell biology ,Medicinal and biomolecular chemistry - Abstract
We investigated the possibility of single-step genome editing in small ruminants by CRISPR-Cas9 zygote electroporation. We targeted SOCS2 and PDX1 in sheep embryos and OTX2 in goat embryos, utilizing a dual sgRNA approach. Gene editing efficiency was compared between microinjection and three different electroporation settings performed at four different times of embryo development. Electroporation of sheep zygotes 6 h after fertilization with settings that included short high-voltage (poring) and long low-voltage (transfer) pulses was efficient at producing SOCS2 knock-out blastocysts. The mutation rate after CRISPR/Cas9 electroporation was 95.6% ± 8%, including 95.4% ± 9% biallelic mutations; which compared favorably to 82.3% ± 8% and 25% ± 10%, respectively, when using microinjection. We also successfully disrupted the PDX1 gene in sheep and the OTX2 gene in goat embryos. The biallelic mutation rate was 81 ± 5% for PDX1 and 85% ± 6% for OTX2. In conclusion, using single-step CRISPR-Cas9 zygote electroporation, we successfully introduced biallelic deletions in the genome of small ruminant embryos.
- Published
- 2022