CAJ | 학술논문

血友病乙是由凝血因子Ⅸ(Factor Ⅸ，FⅨ)缺乏或功能缺陷导致的出血性疾病，为伴X染色体隐性遗传病。小鼠模型对于血友病乙的研究具有十分重要的意义，而基因组编辑技术又为小鼠模型的构建提供了一种快捷而且高效的途径。本文利用CRISPR/Cas系统，在小鼠FⅨ基因第8外显子上选择靶位点，将Cas9 mRNA和带有靶位点的sgRNA显微注射到C57BL/6品系小鼠的受精卵中，获得基因修饰的小鼠。利用高分辨率熔解曲线分析(High resolution melting, HRM)技术进行精确基因分型，并通过测序验证，在60只小鼠中，总共有51只小鼠的靶位点发生了突变，突变率高达85%，其中雄鼠的突变率为79.5%，雌鼠的突变率为95.2%；未检测到非目标位置的基因编辑脱靶。凝血活性实验显示，突变小鼠的FⅨ活性值(FactorⅨ coagulant activity, FⅨ： C)是非突变小鼠的6.82%，大大低于非突变小鼠，表明突变小鼠的凝血活性缺失。本研究表明，利用 CRISPR/Cas 系统成功构建了人类血友病乙遗传病小鼠模型。
혈우병을시유응혈인자Ⅸ(Factor Ⅸ，FⅨ)결핍혹공능결함도치적출혈성질병，위반X염색체은성유전병。소서모형대우혈우병을적연구구유십분중요적의의，이기인조편집기술우위소서모형적구건제공료일충쾌첩이차고효적도경。본문이용CRISPR/Cas계통，재소서FⅨ기인제8외현자상선택파위점，장Cas9 mRNA화대유파위점적sgRNA현미주사도C57BL/6품계소서적수정란중，획득기인수식적소서。이용고분변솔용해곡선분석(High resolution melting, HRM)기술진행정학기인분형，병통과측서험증，재60지소서중，총공유51지소서적파위점발생료돌변，돌변솔고체85%，기중웅서적돌변솔위79.5%，자서적돌변솔위95.2%；미검측도비목표위치적기인편집탈파。응혈활성실험현시，돌변소서적FⅨ활성치(FactorⅨ coagulant activity, FⅨ： C)시비돌변소서적6.82%，대대저우비돌변소서，표명돌변소서적응혈활성결실。본연구표명，이용 CRISPR/Cas 계통성공구건료인류혈우병을유전병소서모형。
Hemophilia B, or the Christmas disease, is a common human disease caused by coagulation factorⅨ (FⅨ) deficiency. It is an X-linked recessive hereditary disease. Here we obtainedFⅨ-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 ofFⅨ 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 sim-ilar locus by HRM. We future measured the FⅨ activity of each mice. The FⅨ: C of mutant mice were significant-ly 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.