期刊
MOLECULAR THERAPY-NUCLEIC ACIDS
卷 5, 期 -, 页码 -出版社
CELL PRESS
DOI: 10.1038/mtna.2016.100
关键词
AAV; CRISPR/Cas9; homologous recombination; human embryonic stem cells; next generation sequencing; NRL
资金
- Chinese National Program on Key Basic Research Project (973 Program) [2013CB967502]
- Natural Science Foundation of China [81201181/H1818]
- Natural Science Foundation of Zhejiang Province, China [LY13H040013]
- Ministry of Health [WKJ2013-2-023, 2016KYA145, 2016KYA146]
- Wenzhou city grant [Y20140633, Y20150071]
- Wenzhou Medical University [QTJ12011]
- Eye Hospital at WenZhou Medical University [YNZD201602]
- Zhejiang provincial
Clustered interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low. Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases. However, whether CRISPR/Cas9mediated double strand breaks and AAV based donor DNA mediated homologous recombination approaches could be combined to create a novel CRISPR/Cas9-AAV genetic tool for highly specific gene editing is not clear. Here we demonstrate that using CRISPR/Cas9-AAV, we could successfully knock-in a DsRed reporter gene at the basic motifleucine zipper transcription factor (NRL) locus in human embryonic stem cells. For the first time, this study provides the proof of principle that these two technologies can be used together. CRISPR/Cas9-AAV, a new genome editing tool, offers a platform for the manipulation of human genome.
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