1. Efficient generation of FVII gene knockout mice using CRISPR/Cas9 nuclease and truncated guided RNAs
- Author
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Liyou An, Xiumei Zhu, Pingping Ling, Lan Yang, Jiao Liu, Shiwei Chang, Yanhong Liu, Yeshu Hu, Giorgio Antonio Presicce, Fenli Zhang, Fuliang Du, and Yexiang Chen
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0301 basic medicine ,Article ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Genome editing ,Gene expression ,Animals ,CRISPR ,Gene ,Gene knockout ,Gene Editing ,Nuclease ,Multidisciplinary ,biology ,Cas9 ,RNA ,Factor VII ,Endonucleases ,Molecular biology ,030104 developmental biology ,NIH 3T3 Cells ,biology.protein ,Prothrombin ,CRISPR-Cas Systems ,030217 neurology & neurosurgery ,RNA, Guide, Kinetoplastida - Abstract
We investigated the effects of 5′-end truncated CRISPR RNA-guided Cas9 nuclease (tru-RGN, 17/18 nucleotides) on genome editing capability in NIH/3T3 cells, and its efficiencies on generating Factor VII (FVII) gene-knockout (KO) mice. In cultured cells, RGNs on-target editing activity had been varied when gRNAs was truncated, higher at Site Two (tF7–2 vs. F7–2, 49.5 vs. 30.1%) while lower in other two sites (Site One, tF7–1 vs.F7–1, 12.1 vs. 23.6%; Site Three, tF7–3 vs.F7–3, 7.7 vs 10.9%) (P FVII KO mice were generated with higher efficiency at Site Two (80.1 vs. 35.8%) and Site One (55.0 vs 3.7%) (P 0.05) when compared with standard RGN controls. Knockout FVII mice demonstrated a delayed prothrombin time and decreased plasma FVII expression. Our study first demonstrates that truncated gRNAs to 18 complementary nucleotides and Cas9 nucleases, can effectively generate FVII gene KO mice with a significantly higher efficiency in a site-dependent manner. In addition, the off-target frequency was much lower in KO mice than in cell lines via RGN expression vector-mediated genome editing.
- Published
- 2016
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