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Efficient generation of FVII gene knockout mice using CRISPR/Cas9 nuclease and truncated guided RNAs.


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??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.

SUBMITTER: An L 

PROVIDER: S-EPMC4853708 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Efficient generation of FVII gene knockout mice using CRISPR/Cas9 nuclease and truncated guided RNAs.

An Liyou L   Hu Yeshu Y   Chang Shiwei S   Zhu Xiumei X   Ling Pingping P   Zhang Fenli F   Liu Jiao J   Liu Yanhong Y   Chen Yexiang Y   Yang Lan L   Presicce Giorgio Antonio GA   Du Fuliang F  

Scientific reports 20160503


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 < 0.  ...[more]

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