Refinements in embryo manipulation applied to CRISPR technology in livestock.

The implementation of CRISPR technology in large animals requires further improvements in embryo manipulation and transfer to be applied with commercial purposes. In this study we report (a) developmental competence of CRISPR/Cas microinjected zygotes subjected to in vitro culture in large scale programs in sheep; (b) pregnancy outcomes after early-stage (2-8-cell) embryo transfer into the oviduct or the uterine horn; and (c) embryo survival and birth rate after vitrification/warming of CRISPR/Cas microinjected zygotes. Experiment 1 consisted of a retrospective analysis to evaluate embryo developmental rate of in vitro produced zygotes subjected to CRISPR/Cas microinjection (n = 7,819) compared with a subset of non-microinjected zygotes (n = 701). Development rates to blastocyst on Day 6 were 20.0% for microinjected zygotes and 44.9% for non-injected zygotes (P < 0.05). In Experiment 2, CRISPR/Cas microinjected zygotes were transferred on Day 2 after in vitro fertilization (2-8 cell embryos) into the oviductal ampulla (n = 262) or into the uterine horn (n = 276) in synchronized recipient ewes at prefixed time (i.e., approximately two days after ovulation). Pregnant/transferred recipients (24.0% vs. 25.0%), embryo survival/transferred embryos (6.9% vs. 6.2%), and born lambs/pregnant embryos (72.2% vs. 100.0%) did not differ significantly in the two groups. In Experiment 3, CRISPR/Cas microinjected zygotes were maintained under in vitro culture until blastocyst stage (Day 6), and subjected to vitrification/warming via the Cryotop method (n = 474), while a subset of embryos were left fresh as control group (n = 75). Embryos were transferred into the uterine horn of recipient females at prefixed time 8.5 days after the estrous synchronization treatment (i.e., approximately six days after ovulation). Pregnancy rate (30.8% vs. 48.0%), embryo survival rate (14.8% vs. 21.3%), and birth rate (85.7% vs. 75.0%) were not different (PNS) between vitrified and fresh embryos, respectively. In conclusion, the current study in sheep embryos reports (a) suitable developmental rate after CRISPR/Cas microinjection (i.e., 20%), even though it was lower than non-microinjected zygotes; (b) similar outcomes when Day 2-embryos were placed into the uterine horn instead of the oviduct, avoiding both time-consuming and invasive oviduct manipulation, and extended in vitro culture during one week; (c) promising pregnancy and birth rates obtained with vitrification of CRISPR/Cas microinjected embryos. This knowledge on in vitro embryo development, timing of embryo transfer, and cryopreservation of CRISPR/Cas microinjected zygotes have practical implications for the implementation of genome editing technology in large animals.