title: 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). 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 < 0.05), but not at Site three (39.4 vs 27.8%) (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.
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, P R China.
authors.forename: Liyou
authors.initials: L
authors.lastname: An
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, P R China.
authors.forename: Yeshu
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, P R China.
authors.forename: Shiwei
authors.initials: S
authors.lastname: Chang
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, P R China.
authors.forename: Xiumei
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, P R China.
authors.forename: Pingping
authors.initials: P
authors.lastname: Ling
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, P R China.
authors.forename: Fenli
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, P R China.
authors.forename: Jiao
authors.initials: J
authors.lastname: Liu
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, P R China.
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, P R China.
authors.forename: Yexiang
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Lannuo Biotechnologies Wuxi Inc., Wuxi 214000, P R China.
authors.forename: Lan
authors.initials: L
authors.lastname: Yang
authors.affiliation: ARSIAL, Rome, Italy.
authors.forename: Giorgio Antonio
authors.initials: GA
authors.lastname: Presicce
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, P R China.|Renova Life, Inc., College Park, Maryland 20742, USA.
authors.forename: Fuliang
authors.initials: F
authors.lastname: Du
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D004720:Endonucleases / Q000378:metabolism; D005167:Factor VII / Q000235:genetics* / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D051379:Mice; D041681:NIH 3T3 Cells; D011516:Prothrombin / Q000378:metabolism; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D005167:Factor VII; D011516:Prothrombin; D004720:Endonucleases