Genetic Correction and Hepatic Differentiation of Hemophilia B-specific Human Induced Pluripotent Stem Cells

遗传上在人的导致的 pluripotent 干细胞(iPSCs ) 改正一个引起疾病的点变化的目的首先源于出血不止 B patient.Methods，引起疾病的变化被定序人的凝结因素 IX (F IX ) 的编码区域检测基因。Genomic DNA 从 iPSCs 被提取，并且教材被设计放大 F IX 的八 exons。下次，在那些 iPSCs 的点变化遗传上面对 129 核苷酸用 CRISPR/Cas9 技术被改正包含了二个同义的变化的相应修理模板。然后，顶 8 潜在的离开目标地点随后用定序的 Sanger 被分析。最后，改正的克隆被区分进象 hepatocyte 一样房间，并且 F IX 的分泌物被房间行厌烦的 immunocytochemistry 和 ELISA assay.Results 验证在 6 th 编码 exon (c.676 C > T ) F IX 基因。点变化的修正与在大约 22% 点的功效(10/45 ) 和没有离开目标效果在改正的 iPSC 克隆检测了的高度在 situ 经由 CRISPR/Cas9 技术被完成。F IX 分泌物，被 immunocytochemistry 进一步设想并且在 vitro 由 ELISA 确定了，在人的疾病特定的 iPSCs 在白天 21 区别 procedure.Conclusions 变化上到达了大约 6 ng/ml 能被 CRISPR/Cas9 技术精确改正，并且改正了仍然维持的房间肝的区别能力。我们的调查结果可能在临床的应用程序在基于 iPSC 的个性化的治疗上扔一盏灯，特别为出血不止 B。

Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.