DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency in brain neurons
DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency in brain neurons
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DOI：
10.1016/j.bbrc.2020.01.132
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发表时间：
2020-04-09
影响因子：
3.1
通讯作者：
Tabuchi, Katsuhiko
中科院分区：
文献类型：
--
作者：
Kurihara, Taiga;Kouyama-Suzuki, Emi;Tabuchi, Katsuhiko
使用CRISPR/Cas9系统的基因敲入可以通过使用子宫内电穿孔将DNA片段引入神经祖细胞来在小鼠脑中的特定神经元群体中实现。使用这种策略，我们以前敲入的EGFP编码序列的β-肌动蛋白基因的N-末端区域，特别是在锥体神经元层2/3的躯体感觉皮层。然而，敲入效率低于转染神经元的2%。在这项研究中，我们试图提高敲入效率使用这个系统。首先，我们改变了β-肌动蛋白供体模板DNA的同源臂的长度，并发现通过将5'和3'同源臂的长度分别延长至1.6kb和2.0kb，敲入效率增加至接近14%。然后，我们测试了DNA修复蛋白RAD 51的作用，当与两种不同的β-肌动蛋白和靶向EGFP敲入模块的camk 2a共转染时，敲入效率增加至2.5倍。与未转染RAD 51的神经元相比，RAD 51过表达并没有改变发育中神经元的迁移、树突棘的密度或形态。RAD 51表达将可用于通过CRISPR/Cas9介导的同源定向修复（HDR）增加体内神经元中的敲入效率。(C)2020爱思唯尔公司All rights reserved.
Gene knock-in using the CRISPR/Cas9 system can be achieved in a specific population of neurons in the mouse brain, by using in utero electroporation to introduce DNA fragments into neural progenitor cells. Using this strategy, we previously knocked-in the EGFP coding sequence into the N-terminal region of the beta-actin gene specifically in the pyramidal neurons in layer 2/3 of the somatosensory cortex. However, the knock-in efficiency was less than 2% of the transfected neurons. In this study, we sought to improve the knock-in efficiency using this system. First, we varied the length of the homology arms of the beta-actin donor template DNA, and found that the knock-in efficiency was increased to similar to 14% by extending the length of the 5' and 3' homology arms to 1.6 kb and 2.0 kb, respectively. We then tested the effect of the DNA repair protein RAD51 and the knock-in efficiency was increased up to 2.5-fold when co-transfecting with two different beta-actin and a camk2a targeting EGFP knock-in modules. The RAD51 overexpression did not alter the migration of developing neurons, density or morphology of the dendritic spines compared to those in neurons not transfected with RAD51. RAD51 expression will be useful for increasing the knock-in efficiency in neurons in vivo by CRISPR/Cas9-mediated homology directed repair (HDR). (C) 2020 Elsevier Inc. All rights reserved.