通过增强微同源末端连接实现基因盒在哺乳动物细胞和受精卵中的敲入。
Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ.
作者信息
Aida Tomomi, Nakade Shota, Sakuma Tetsushi, Izu Yayoi, Oishi Ayu, Mochida Keiji, Ishikubo Harumi, Usami Takako, Aizawa Hidenori, Yamamoto Takashi, Tanaka Kohichi
机构信息
Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), 1-5-45, Yushima, Bunkyo, Tokyo, 113-8510, Japan.
Laboratory of Recombinant Animals, MRI, TMDU, 2-3-10, Surugadai, Kanda, Chiyoda, Tokyo, 101-0062, Japan.
出版信息
BMC Genomics. 2016 Nov 28;17(1):979. doi: 10.1186/s12864-016-3331-9.
BACKGROUND
Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining.
RESULTS
Here, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes.
CONCLUSIONS
Our results provide a technical platform for high-throughput knock-in.
背景
尽管CRISPR/Cas技术能够实现一步法基因盒敲入，但构建包含长同源臂的靶向载体对于高通量敲入而言是一个费力的过程。我们最近开发了基于CRISPR/Cas的精确整合到目标染色体（PITCh）系统，用于在无微同源性介导的末端连接介导的情况下进行无长同源臂的基因盒敲入。
结果
在这里，我们确定核酸外切酶1（Exo1）是人类细胞中PITCh的增强子。通过将Exo1与PITCh定向供体载体相结合，我们在人类细胞和小鼠受精卵中都实现了基因盒和floxed等位基因的便捷一步敲入。
结论
我们的结果为高通量敲入提供了一个技术平台。
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