Tild-CRISPR Allows for Efficient and Precise Gene Knockin in Mouse and Human Cells

Publication date: 21 May 2018
Source:Developmental Cell, Volume 45, Issue 4
Author(s): Xuan Yao, Meiling Zhang, Xing Wang, Wenqin Ying, Xinde Hu, Pengfei Dai, Feilong Meng, Linyu Shi, Yun Sun, Ning Yao, Wanxia Zhong, Yun Li, Keliang Wu, Weiping Li, Zi-jiang Chen, Hui Yang
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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Yao et al. describe Tild-CRISPR, a targeting strategy using PCR-amplified or precisely enzyme-cut transgene donor sequence. Tild-CRISPR yields robust knockin efficiency in mouse and human embryos, as well as mouse brain in vivo, suitable for studying gene functions in vivo and developing potential gene therapies.


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