Establishment of a technological system via zygote injection of CRISPR/Cas9 system for the generation of gene modified ziwuling black goats

Zygote injection of CRISPR/Cas9 system is an efficient method for generation of gene modified animals. Efficiency of zygote collection, embryo survival rate after micro-injection and pregnancy rate of surrogate are the key influence factors for the generation of gene modified animals using this strategy. Here, we investigated the influence of CIDR treatment at different time during the estrus cycle to the superovulation, and analyzed the influence of the estrus terminal time between embryo donor and embryo receptor to the pregnancy rate in Ziwuling black goats. We designed three sgRNAs targeting myostatin ( MSTN ) and also optimized the micro-injected parameter of the CRISPR/Cas9 system. The results showed that embryo donor goats effectively increase the corpus luteum amount (15.25±3.69) and zygote amount (13.875±4.015) when they were treated with CIDR at the fourth day of estrus cycle. The mixture including Cas9 mRNA (60 ng/μL) and sgRNAs (60 ng/μL) targeting MSTN was injected into zygote cytoplasm using a micromanipulator at the optimized injection parameters (Pi, Ti and Pc was 300, 0.5 and 60, respectively). Embryo cleavage rate was 76.79 % after 24 h of embryo injection. Pregnancy rate of embryo receptors whose terminal estrus time was delayed 12–16 h compared with that of embryo donor was the highest (38.46%). A total of 19 living lambs were obtained, and indels were detected in all the lambs using TA cloning sequencing. Overall, we established the technological system based on the zygote injection of CRISPR/Cas9 in Ziwuling black goats. The established technological system is the basic for generation of gene modified goats.