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The persistence of the embryonic (optic) fissure into adulthood and development of the falciform process in the eye varied among three fish species: guppy Poecilia reticulata, Mozambique tilapia Tilapia mossambica, and brown trout Sal mo trutta. The falciform process is a ridge of pigmenied and vascular tissue associated with the embryonic fissure in teleosts. In guppies, the embryonic fissure closed during embryonic development, and no falciform process developed. In Mozambique tilapias, the embryonic fissure persisted into adulthood but was lined only with retinal pigment epithelium and lacked photoreceptive layers; it was associated with a partially formed falciform process. In brown trout, the embryonic fissure remained well defined with both retinal pigment epithelium and photoreceptor layers, and the falciform process was well developed. The temporal zone of the fissure in adult brown trout exhibited active growth and had a ventral area of high cone density. The well-developed fissure in brown trout and the oblique orientation of its ventrotemporal cones relative to incident light may be correlated with the species* ability to locate prey above and in front of its body. This system also may allow brown trout to detect polarized light, which could assist in navigation during migration.

Targeted knock-in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)-assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining.The objective of this study was to explore whether CRISPR-assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS-1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a singlestranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS-1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS-1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS-1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos.

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