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Sci Rep. 2015 Dec 1;5:17517. doi: 10.1038/srep17517.

Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos.

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Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
The Key Laboratory of Adolescent Health Assessment and Exercise Intervention of Ministry of Education, East China Normal University, Shanghai 200241, China.
Hainan Provincial Key Laboratory for human reproductive medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Haikou 570102, China.
The Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas 77030, USA.


The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously. Generating novel genetically modified mouse and rat models is one valuable application of this system. Through the injection of Cas9 protein instead of mRNA into embryos, we observed fewer off-target effects of Cas9 and increased point mutation knock-in efficiency. Large genomic DNA fragment (up to 95 kb) deletion mice were generated for in vivo study of lncRNAs and gene clusters. Site-specific insertion of a 2.7 kb CreERT2 cassette into the mouse Nfatc1 locus allowed labeling and tracing of hair follicle stem cells. In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis.

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