Knockout Mice Embryo By CRISPR/Cas9 And Cryopreservation Of Spermatozoa From Cynomolgus Monkeys (Macaca Fascicularis)

(CRISPR)/CRISPR-associated proteins (Cas) systems, found in bacteria and archaea, carry out the adaptive immune responses against invading genetic elements (virus or plasmid) by using RNAs to guide site-specific cleavage of genetic material. Type II of these systems(also called CRISPR/Cas9), Currently, have already modified the genome of many species with this system including mouse.This system could site-specifically cut eukaryote DNA in mouse embryos by co-injection of Cas9 mRNA and sgRNA.However, there is little verification of microinjection methods for generating knockout mice using this approach.In this study, the verification of microinjection methods of the CRISPR/Cas system, using ICR embryos by co-injection of Cas9 mRNA and sgRNA into the pronucleus or the cytoplasm.was reported.On the base of experiments above, the best microinjection concentration of Cas9 mRNA and sgRNA was talked about. The results were follows:1.Transgenic embryos collected at 23-24 hours after hCG-treated reach the highest blastocyst rate2. The developmental rates between embryos with microinjecting and embryos without microinjection when considering number of 2-cell embryos as the parameters has a insignificant difference3. Injection of Cas9 mRNA and sgRNA into the cytoplasm was the most efficient method in terms of the numbers of viable blastocyst stage embryos4. The knockout efficiency rates between embryos with microinjecting into the cytoplasm and microinjection into the pronucleus has a insignificant difference5. The Best way of microinjection concentration for knockout efficiency:20 ng/ul Cas9mRNA and 10 ng/ul sgRNA.In order to established a novel method for sperm cryopreservation of cynomolgus macaque by using the egg yolk free cryoprotective medium SpermCryoTM All-round, we evaluate the effects of the holding time (10 and 30 min, respectively) in liquid nitrogen and different cooling rates (-69,-183 and-435 ℃/min, respectively) on sperm cryosurvival of cynomolgus macaques. The optimized cropreserving protocol for cynomolgus macaque sperm by using the egg yolk free medium SpermCryo in our study can achieve preferable cryosurvial rate, and the optimized protocol is easy to use and can save time and labors. Our results will benefit the study for understanding the mechanism of cell injury during cryopreservation and will improve the assistant reproductive technique of nonhuman primate. The result showed:1. Sperm cryopreserved for 10 min in liquid nitrogen acheived higher post-thaw survival rate than that of 30 min.2.Sperm cryopreserved at fast and medium cooling rate (-183 and-435℃/min) showed higher post-Thaw survival rate than sperm cryopreserved at slow freezing rate (-69℃/min).