title: [A quick and efficient method to generate hemophilia B mouse models by the CRISPR/Cas system].
abstract: Hemophilia B, or the Christmas disease, is a common human disease caused by coagulation factor Ⅸ (FⅨ) deficiency. It is an X-linked recessive hereditary disease. Here we obtained FⅨ-knockout mouse strains with phenotype of hemophilia B with the CRISPR/Cas system efficiently. We chose the 8th exon as the target locus, and co-injected codon-optimized Cas9 mRNA with sgRNA of FⅨ into C57BL/6 mice zygotes. We obtained 60 mice in total and genotyped them by high resolution melting (HRM) and sequencing. The results showed the mutation rate was 85.0% in total, and 79.5% and 95.2% in males and females, respectively. No off-targets were detected in the similar locus by HRM. We future measured the FⅨ activity of each mice. The FⅨ: C of mutant mice were significantly below the normal level and reduced to 6.82% of wild-type mice. The activity assay demonstrated that all the mutant mice were lack of FⅨ. In summary, we have generated hemophilia B model mice with extreme efficiency, using the RNA-guided Cas9 nuclease gene editing system.
authors.affiliation: State Key Laboratory of Genetic Engineering, School of Life Science, Fudan University, Shanghai 200433, China.
authors.forename: Qi-han
authors.initials: QH
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Genetic Engineering, School of Life Science, Fudan University, Shanghai 200433, China.
authors.forename: Cong
authors.initials: C
authors.lastname: Huai
authors.affiliation: Shanghai Research Center for Model Organisms, Shanghai 201203, China.
authors.forename: Rui-lin
authors.initials: R
authors.lastname: Sun
authors.affiliation: Shanghai Research Center for Model Organisms, Shanghai 201203, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Zhuang
authors.affiliation: State Key Laboratory of Genetic Engineering, School of Life Science, Fudan University, Shanghai 200433, China.
authors.forename: Hong-yan
authors.initials: HY
authors.lastname: Chen
authors.affiliation: Shanghai Research Center for Model Organisms, Shanghai 201203, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Fei
authors.affiliation: State Key Laboratory of Genetic Engineering, School of Life Science, Fudan University, Shanghai 200433, China.
authors.forename: Da-ru
authors.initials: DR
authors.lastname: Lu
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004195:Disease Models, Animal; D005260:Female; D005838:Genotype; D002836:Hemophilia B / Q000235:genetics*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems