Study On Editing Of Seventh Coagulation Factor Gene In Mice By CRISPR / Cas9 Mediated By Tru-RGNs

CRISPR/Cas9 as a new and highly efficient gene editing technology, which is widely used in the gene transformation of microorganisms, plants and animals. In order to improve the editing efficiency of CRISPR/Cas9, there are many ways to improve it. Truncated guided RNA (tru-RGNs,17/18 nt) was reported exhibiting substantially reduce nucleases induced off-target mutations without sacrificing on-target genome editing efficiencies in human cell. But there is no study to show that the method can be used to construct gene knockout(KO) animals. This article determined activity and specificity of tru-RNGs induced genome editing in mouse cells, and its effects in generating FVⅡ KO mouse.The study chose the second exon of mice FVⅡ gene as target, and designed three loci of tru-RGNs to identify FVⅡ gene, tru-RGNs (tru-gRNA+Cas9) expression vectors were transfected into mouse NIH/3T3 cells, std-RGNs (20 nt) served as controls. Results showed tru-RGNs of ctF7-2 presented a increased editing frequencies than std-RGNs of cF7-2 (49.5% vs 30.1%), ctF7-1 and ctF7-3 exhibited a reduced editing activities comparing to their corresponding std-RGNs (ctF7-1,12.1% vs cF7-1,23.6%; ctF7-3,7.7% vs cF7-310.9%) (P<0.05). The off target detection showed that in predicted off target sites, tru-RGNs vs std-RGNs off target mutation had no significant difference. In mice, tru-gRNA, std-gRNA and Cas9 expression vector with T3 promoter by transcribing mRNA in vitro, which injected into mouse zygotes. Tru-gRNA and Cas9 mRNA were injected into zygotes, total of 98,75 and 120 embryos were injected,8,20 and 20 newborns were born in tru-RGNs groups (tF7-1, tF7-2 and tF7-3) respectively; in std-RGNs groups (F7-1, F7-2 and F7-3),78, 110 and 80 embryos were injected,18,24 and 6 newborns were obtained respectively, newborns genome were amplified by PCR F7-667-f1/F7-667-r1, then were detected by T7EI assays, results showed 19 (55.0%),15 (80.1%)and 8 (39.4%) mouse containing indels mutations at FVⅡ locus using tru-RGNs of tF7-1, tF7-2 and tF7-3, in std-RGNs groups of F7-1, F7-2 and F7-3,1 (3.7%),8 (35.8%) and 2 (27.8%) mouse containing indels mutations. The off target detection also showed that excepting one tru-RGNs (tF7-3) induced a high off-target mutagenesis at one of predicted sites (OT3-2,29.3%), none off-target mutations were induced by tru-RGNs in the rest of mouse. FO FVⅡKO resulted in the extension of prothrombin time (PT) and there was a significant difference in PT(P<0.05) compared with wild-type mice, and FVII expression to an extent lower than its 1/2WT level by Western blot. The positive mice in FO generated F1 FVII KO in mice for T7EI with mutant mice, indicating that FVII KO mice can be stable inheritance.In conclusion, truncated gRNA together with Cas9 mRNAs (tru-RGNs) can be effectively used to generate FVⅡ KO mice with a much higher efficiency than using standard RGNs, although in a site-dependent manner. RGNs editing activity can be altered to a certain extent, depending upon where truncated gRNA to 17/18 complementary nucleotides are located at different target sites. When the gene-edited KO mice are generated by RGNs microinjection into zygotes, the frequency of animals carrying off-target mutation is much lower than cells mediated by expressed RGNs and their cellular genome editing.FVII KO mice model can be studied in the treatment of disease and drug for the hereditary Coagulation factorVII deficiency by CRISPR/Cas9 technology.