猪受精卵 CRISPR/Cas9 基因组编辑前透明带处理。
Zona pellucida treatment before CRISPR/Cas9-mediated genome editing of porcine zygotes.
机构信息
Faculty of Veterinary Science, Guangdong Ocean University, Zhanjiang, China.
Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan.
出版信息
Vet Med Sci. 2022 Jan;8(1):164-169. doi: 10.1002/vms3.659. Epub 2021 Oct 21.
BACKGROUND
Increasing the permeability of the zona pellucida (ZP) of oocytes before CRISPR/Cas9 electroporation may improve the efficiency of gene editing; however, the effects of this approach on subsequent developmental processes are unclear. In this study, the effects of ZP treatment before electroporation on embryonic development and gene editing in porcine embryos were evaluated.
METHODS
The ZP of zygotes was weakened or removed by exposure to 0.5% actinase E, followed by electroporation of the Cas9 protein with guide RNA targeting GGTA1.
RESULTS
The blastocyst formation rate of ZP-free zygotes after electroporation was significantly lower (p < 0.05) than that of ZP-intact zygotes. The mutation rate in blastocysts from ZP-weakened zygotes was similar to that in ZP-intact zygotes, whereas ZP removal increased the mutation rate. The mutation efficiency in blastocysts from electroporated zygotes did not differ among ZP treatment groups.
CONCLUSIONS
Our results indicate that weakening the ZP does not affect the developmental competence, mutation rate, or mutation efficiency of electroporated zygotes, whereas ZP removal has a detrimental effect on embryonic development but may increase the mutation rate.
背景
在 CRISPR/Cas9 电穿孔前增加卵透明带（ZP）的通透性可能会提高基因编辑的效率；然而，这种方法对随后的发育过程的影响尚不清楚。在这项研究中，评估了电穿孔前ZP 处理对猪胚胎发育和基因编辑的影响。
方法
通过暴露于 0.5%的纤维蛋白溶酶，使受精卵的 ZP 减弱或去除，然后用靶向 GGTA1 的 Cas9 蛋白和向导 RNA 进行电穿孔。
结果
电穿孔后 ZP 缺失的受精卵的囊胚形成率明显低于 ZP 完整的受精卵（p<0.05）。ZP 减弱的受精卵囊胚中的突变率与 ZP 完整的受精卵相似，而 ZP 去除则增加了突变率。电穿孔受精卵囊胚中的突变效率在 ZP 处理组之间没有差异。
结论
我们的结果表明，减弱 ZP 不会影响电穿孔受精卵的发育能力、突变率或突变效率，而去除 ZP 对胚胎发育有不利影响，但可能会增加突变率。
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