生物
清脆的
转基因
Cas9
同源重组
基因靶向
基因组编辑
基因
引导RNA
转基因小鼠
计算生物学
核糖核酸
胚胎
同源(生物学)
合子
基因敲除
同源定向修复
细胞生物学
遗传学
胚胎发生
DNA修复
DNA错配修复
作者
Xuan Yao,Meiling Zhang,Xing Wang,Weiwen Ying,Xin Hu,Peng Dai,Fei-Long Meng,Linqi Shi,Yun Sun,Ning Yao,Wanxia Zhong,Yun Li,Keliang Wu,Weiping Li,Zi‐Jiang Chen,Hui Yang
标识
DOI:10.1016/j.devcel.2018.04.021
摘要
The targeting efficiency of knockin sequences via homologous recombination (HR) is generally low. Here we describe a method we call Tild-CRISPR (targeted integration with linearized dsDNA-CRISPR), a targeting strategy in which a PCR-amplified or precisely enzyme-cut transgene donor with 800-bp homology arms is injected with Cas9 mRNA and single guide RNA into mouse zygotes. Compared with existing targeting strategies, this method achieved much higher knockin efficiency in mouse embryos, as well as brain tissue. Importantly, the Tild-CRISPR method also yielded up to 12-fold higher knockin efficiency than HR-based methods in human embryos, making it suitable for studying gene functions in vivo and developing potential gene therapies.
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