Highly efficient biallelic genome editing of human ES/iPS cells using a CRISPR/Cas9 or TALEN system.
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.
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.|K-CONNEX, Kyoto University, Kyoto 606-8302, Japan.|Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085, Japan.
Kazuo
K
Takayama
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
Keisuke
K
Igai
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
Yasuko
Y
Hagihara
Laboratory of Biochemistry and Molecular Biology, School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
Rina
R
Hashimoto
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
Morifumi
M
Hanawa
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan.
Tetsushi
T
Sakuma
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
Masashi
M
Tachibana
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.|Laboratory of Biochemistry and Molecular Biology, School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.|Laboratory of Regulatory Sciences for Oligonucleotide Therapeutics, Clinical Drug Development Project, Graduate School of Pharmaceutical Sciences, Osaka University Osaka 565-0871, Japan.
Fuminori
F
Sakurai
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima 739-8526, Japan.
Takashi
T
Yamamoto
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.|Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085, Japan.|Laboratory of Biochemistry and Molecular Biology, School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.|Global Center for Medical Engineering and Informatics, Osaka University, Osaka 565-0871, Japan.
Hiroyuki
H
Mizuguchi
D000483:Alleles; D064113:CRISPR-Cas Systems / Q000235:genetics*; D000072669:Gene Editing; D015894:Genome, Human; D059765:Homologous Recombination / Q000187:drug effects; D006720:Homozygote; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051135:Rad51 Recombinase / Q000235:genetics; D000069896:Transcription Activator-Like Effector Nucleases / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014635:Valproic Acid / Q000494:pharmacology
D014635:Valproic Acid; D051135:Rad51 Recombinase; D000069896:Transcription Activator-Like Effector Nucleases