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.

The aim of the current investigation is to study the embryogenesis of the pseudobranchia (PB) in the Eastern mosquito fish Gambusia holbrooki, Girard, 1859 (Cyprinodontiformes, Poeciliidae). Histological sections from 2-4 mm long embryos revealed the presumable PB as a cell mass surrounded by a row of squamous cells in the operculum, as well as the formationof cartilage and blood vessels, and the beginning of the formation of its lamellae. In 5-7.75 mm long embryos, a prominent rise appeared in the mass of cells and PB lamellae, along withan increase in melanin deposition around the blood vessels. Moreover, a row of squamous cells encircled the rise from the front and back. At this stage, the parallel arrangement of the cartilage-attached lamellae correlated with increased melanin concentration around the blood vessels. In newborns, the differentiated cells increased similar to that appearing in the adult’s stages.