B Shen

D Li

ED Rosen

FA Ran

H Wang

H Yang

J Zhou

JA Doudna

JG Doench

JJ Emeis

JP Guilinger

L An

L Chong

L Cong

M Jiang

MA Moreno-Mateos

N Wyvekens

S Mizuno

SQ Tsai

SW Cho

T Gaj

T Horii

T Sakurai

V Iyer

W Fujii

X Wu

Y Fu

Y Kimura

Y Ma

Y Wu

Scientific Reports

AbstractWe 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 &lt; 0.05). Out of 15 predicated off–target sites, tru-RGNs showed significantly decreased frequencies at 5 sites. By microinjecting tru-RGN RNAs into zygotes, 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 &lt; 0.05), but not at Site three (39.4 vs 27.8%) (P &gt; 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.</jats:p

Liyou An

Yeshu Hu

Shiwei Chang

Xiumei Zhu

Pingping Ling

Fenli Zhang

Jiao Liu

Yanhong Liu

Yexiang Chen

Lan Yang

Giorgio Antonio Presicce

Fuliang Du

Crossref

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

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

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