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Nucleic Acids Res. 2017 May 19;45(9):5198-5207. doi: 10.1093/nar/gkx130.

Highly efficient biallelic genome editing of human ES/iPS cells using a CRISPR/Cas9 or TALEN system.

Author information

1
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
2
PRESTO, Japan Science and Technology Agency, Saitama 332-0012, Japan.
3
K-CONNEX, Kyoto University, Kyoto 606-8302, Japan.
4
Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085, Japan.
5
Laboratory of Biochemistry and Molecular Biology, School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
6
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan.
7
Laboratory of Regulatory Sciences for Oligonucleotide Therapeutics, Clinical Drug Development Project, Graduate School of Pharmaceutical Sciences, Osaka University Osaka 565-0871, Japan.
8
Global Center for Medical Engineering and Informatics, Osaka University, Osaka 565-0871, Japan.

Abstract

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.

PMID:
28334759
PMCID:
PMC5435997
DOI:
10.1093/nar/gkx130
[Indexed for MEDLINE]
Free PMC Article

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