PDF(Pubmed)
Abstract：
Microinjection of CRISPR/Cas9 requires the availability of zygotes that implies animal breeding, superovulation schemes, and embryo collection. Vitrification of zygotes may allow having ready-to-use embryos and to temporally dissociate the workload of embryo production from microinjection. In this study, fresh (F group) or vitrified (V group) zygotes were microinjected with CRISPR/Cas9 system to test the hypothesis that vitrified zygotes could be a suitable source of embryos for microinjection. In Experiment 1 (in vitro evaluation), B6D2F1/J zygotes were microinjected and cultured until blastocyst stage. Embryo survival and cleavage rates after microinjection were similar between groups (~50% and ~80% respectively; P = NS). Development rate was significantly higher for F than V group (55.0% vs. 32.6%, respectively; P<0.05). Mutation rate did not show statistical differences among groups (P = NS). In Experiment 2 (in vivo evaluation), C57BL/6J zygotes were microinjected and transferred to recipient females. Embryo survival was significantly lower in fresh than in vitrified zygotes (49.2% vs. 62.7%, respectively; P<0.05). Cleavage rate did not show statistical differences (~70%; P = NS). Pregnancy rate (70.0% vs. 58.3%) and birth rate (11.9% vs. 11.2%) were not different between groups (F vs. V group; P = NS). Offspring mutation rate was higher for F than V group, in both heterodimer analysis (73.7% vs. 33.3%, respectively; P = 0.015) and Sanger sequencing (89.5% vs. 41.7%, respectively; P = 0.006). In conclusion, vitrified-warmed zygotes present a viable alternative source for CRISPR/Cas9 microinjection when the production of fresh embryos is impeded by limited technical support. The possibility of zygote cryobanking to perform microinjection sessions on demand seems to be a suitable alternative to avoid the breeding and maintenance of animals all over the year, enhancing the implementation of CRISPR technology.
摘要：
CRISPR/Cas9的显微注射需要受精卵的可用性，这意味着动物育种，超数排卵计划,和胚胎收集。受精卵的玻璃化可以允许具有即用型胚胎，并在时间上将胚胎生产的工作量与显微注射分离。在这项研究中,用CRISPR/Cas9系统显微注射新鲜（F组）或玻璃化（V组）受精卵，以检验玻璃化受精卵可能是显微注射胚胎的合适来源的假设。在实验1（体外评估）中，将B6D2F1/J受精卵显微注射并培养直至胚泡期。显微注射后的胚胎存活率和卵裂率在组间相似(分别为~50%和~80%;P=NS)。F组的发育率明显高于V组（55.0％vs.32.6%,分别；P<0.05）。组间突变率无统计学差异(P=NS)。在实验2（体内评估）中，将C57BL/6J受精卵显微注射并转移至受体雌性。新鲜受精卵的胚胎存活率显着低于玻璃化受精卵（49.2％vs.62.7%,分别；P<0.05）。卵裂率无统计学差异(~70%;P=NS)。妊娠率（70.0%vs.58.3%）和出生率（11.9%vs.11.2%)组间没有差异(Fvs.V组；P=NS）。F组后代突变率高于V组,在两种异源二聚体分析中(73.7%vs.33.3%,分别为；P=0.015）和Sanger测序（89.5%vs.41.7%,分别为；P=0.006)。总之,当有限的技术支持阻碍了新鲜胚胎的产生时,玻璃化加热的受精卵为CRISPR/Cas9显微注射提供了可行的替代来源.合子冷冻保存按需进行显微注射的可能性似乎是避免一年四季动物繁殖和维持的合适选择，加强CRISPR技术的实施。
