Efficient CRISPR/Cas9-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease.
Efficient CRISPR/Cas9-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease.
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通过核酸酶 Zygote 电穿孔进行高效 CRISPR/Cas9 介导的小鼠基因组编辑。
DOI：
10.1534/genetics.115.176594
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发表时间：
2015-06
期刊：
影响因子：
3.3
通讯作者：
Wang H
中科院分区：
文献类型：
--
作者：
Qin W;Dion SL;Kutny PM;Zhang Y;Cheng AW;Jillette NL;Malhotra A;Geurts AM;Chen YG;Wang H
成簇规则间隔短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）系统是细菌和古细菌中的适应性免疫系统，最近已被用于基因组工程。突变小鼠可以通过将CRISPR/Cas9组分直接递送到小鼠受精卵中在一个步骤中产生。尽管该技术是稳健的，但递送仍然是一个瓶颈，因为它涉及将组分手动注射到小鼠受精卵的原核或细胞质中，这在技术上要求很高并且固有地低通量。为了克服这一限制，我们采用电穿孔作为一种手段，将CRISPR/Cas9组分（包括Cas9信使RNA、单向导RNA和供体寡核苷酸）高效递送到小鼠受精卵中，并回收具有靶向非同源末端连接和同源定向修复突变的活小鼠。我们的结果表明，通过受精卵电穿孔可以高效地获得携带CRISPR/Cas9介导的靶向突变的小鼠。
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is an adaptive immune system in bacteria and archaea that has recently been exploited for genome engineering. Mutant mice can be generated in one step through direct delivery of the CRISPR/Cas9 components into a mouse zygote. Although the technology is robust, delivery remains a bottleneck, as it involves manual injection of the components into the pronuclei or the cytoplasm of mouse zygotes, which is technically demanding and inherently low throughput. To overcome this limitation, we employed electroporation as a means to deliver the CRISPR/Cas9 components, including Cas9 messenger RNA, single-guide RNA, and donor oligonucleotide, into mouse zygotes and recovered live mice with targeted nonhomologous end joining and homology-directed repair mutations with high efficiency. Our results demonstrate that mice carrying CRISPR/Cas9-mediated targeted mutations can be obtained with high efficiency by zygote electroporation.