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

Liyou An,Fuliang Du,Jiao Liu,Lan Yang,Yanhong Liu,Xiumei Zhu,Giorgio Antonio Presicce,Fenli Zhang,Yexiang Chen,Shiwei Chang,Pingping Ling,Yeshu Hu

doi:10.1038/srep25199

Liyou An, Fuliang Du + Show 10 more

Open Access

https://doi.org/10.1038/srep25199

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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.

Highlights

Mutation frequency (%) Mean ± SEM 23.6 ± 0.5a 12.1 ± 0.2b 30.1 ± 0.9a 49.5 ± 1.0b 10.9 ± 1.3a 7.7 ± 0.9b result in a high efficiency in generating gene knockout (KO) in animal models
To determine efficiency and specificity of RNA-guided nucleases (RGNs)-mediated genome mutations, tru-RGN and std-RGN plasmids were transfected into murine NIH/3T3 cells, and on-target mutations in the factor VII (FVII) gene were determined by the T7EI assay (Fig. 1A) and confirmed by sequencing
It was reported that Std-RGNs can mediate genome modification in one-celled embryos and generate KO mice[14,15,16,17]

Summary

Results

Tru-RGNs induce genome editing in murine cells. Tru-RGNs (Site One, tF7–1, 18 nt; Site Two, tF7–2, 18 nt; and Site Three, tF7–3, 17 nt) target three different sites of at the exon 2 of FVII gene (Supplementary Fig. S1A,B), and corresponding std-RGNs (F7–1, 43–63; F7–2, 46–66; and F7–3,67–87, all 20 nt, in the exon 2) (Table 1) expression vectors were constructed as controls. In NIH/3T3 cells, off-target mutation frequencies induced by tru-RGNs at most predicated sites had been decreased in a different degree compared to std-RGNs (Table 3). Only one off-target mutation at OT3–2 site mediated by tru-RGNs of tF7–3 had a significantly increased frequency when compared to std-RGNs control (22.0 vs 3.7%, P < 0.05) (Table 3). Six mice generated with tru-RGNs of tF7–3 carried off-target mutations at one predicated site (OT3–2, Fig. 2B; Supplementary Table S2), with its off-target ratio of 29.3% in mutant mice versus newborns (n = 20). In other sites, both tru-RGNs and std-RGNs did not generate off-target mutations in FVII KO mice (Table 3). We found that the ratio of heterozygous FVII+/− offspring mice was among 49.7–55.5% which followed Mendelian hereditary pattern (Fig. 2E)

Discussion

Methods