Takayama Kazuo, Igai Keisuke, Hagihara Yasuko, Hashimoto Rina, Hanawa Morifumi, Sakuma Tetsushi, Tachibana Masashi, Sakurai Fuminori, Yamamoto Takashi, Mizuguchi Hiroyuki
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
PRESTO, Japan Science and Technology Agency, Saitama 332-0012, Japan.
Nucleic Acids Res. 2017 May 19;45(9):5198-5207. doi: 10.1093/nar/gkx130.
Genome editing research of human ES/iPS cells has been accelerated by clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALEN) technologies. However, the efficiency of biallelic genetic engineering in transcriptionally inactive genes is still low, unlike that in transcriptionally active genes. To enhance the biallelic homologous recombination efficiency in human ES/iPS cells, we performed screenings of accessorial genes and compounds. We found that RAD51 overexpression and valproic acid treatment enhanced biallelic-targeting efficiency in human ES/iPS cells regardless of the transcriptional activity of the targeted locus. Importantly, RAD51 overexpression and valproic acid treatment synergistically increased the biallelic homologous recombination efficiency. Our findings would facilitate genome editing study using human ES/iPS cells.
成簇规律间隔短回文重复序列/CRISPR相关蛋白9（CRISPR/Cas9）和转录激活样效应核酸酶（TALEN）技术加速了人类胚胎干细胞/诱导多能干细胞的基因组编辑研究。然而，与转录活跃基因不同，转录不活跃基因的双等位基因遗传工程效率仍然很低。为提高人类胚胎干细胞/诱导多能干细胞中的双等位基因同源重组效率，我们对辅助基因和化合物进行了筛选。我们发现，无论靶向位点的转录活性如何，RAD51过表达和丙戊酸处理均可提高人类胚胎干细胞/诱导多能干细胞中的双等位基因靶向效率。重要的是，RAD51过表达和丙戊酸处理可协同提高双等位基因同源重组效率。我们的研究结果将促进利用人类胚胎干细胞/诱导多能干细胞进行的基因组编辑研究。
