CRISPR-Cas9基因编辑报道系统于dbDNA来源的iPSC AAVS1安全港的定点整合研究  

The Targeted Integration of a Reporter System for CRISPR-Cas9 Gene Editing into dbDNA-derived AAVS1 Safe Harbor

作　　者：何佳佳 陈耀 曹淳 鞠小丽 周小明 He Jiajia;Chen Yao;Cao Chun(Medical School,Jiangsu University,Zhenjiang 212013,China;InnoZyme Biotech Co.Ltd.,Nanjing 211800,China)

机构地区：[1]江苏大学医学院基础医学系,镇江212013 [2]依诺赞(江苏)生物科技有限公司,南京211800

出　　处：《华中科技大学学报(医学版)》2025年第2期151-158,共8页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.81571546)。

摘　　要：目的建立简便的doggybone DNA(dbDNA)制备技术路线,探讨dbDNA与传统质粒DNA基因表达与诱导免疫反应的差异;利用dbDNA诱导人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)重编程为诱导性多能干细胞(induced pluripotent stem cell,iPSC);探索在iPSC AAVS1位点插入超长DNA片段的方案,并同时将Cas9表达框定点插入dbDNA来源的iPSC腺相关病毒位点1(adeno-associated virus site 1,AAVS1)安全港内,构建仅需输入目的基因靶向单向导RNA(single guide RNA,sgRNA)即可实现iPSC基因编辑的稳定细胞株。方法利用Phi29 DNA聚合酶多重置换扩增、原核端粒酶TelN酶切及共价连接、限制性内切酶和外切酶去除骨架环,从而建立简便的dbDNA制备技术路线,将dbDNA-GFP载体与pMax-dbDNA-GFP传统质粒载体分别电转至PBMC,检测绿色荧光蛋白(green fluorescent protein,GFP)表达效率,利用qPCR检测两组细胞干扰素-γ(interferon-γ,IFN-γ)的mRNA表达水平;制备dbDNA重编程载体dbDNA-MOS、dbDNA-KLF4、dbDNA-MYC及dbDNA-BCL并核转PBMC诱导重编程,通过形态学、碱性磷酸酶染色、RT-PCR以及干细胞多能性标志物检测来鉴定dbDNA-iPSC细胞系;在iPSC AAVS1位点利用ZFN与TALEN基因编辑技术插入11.5 kb的含有Cas9表达框、序列中引入终止密码子的EGFP读码框、真核细胞筛选标记等元件的超长DNA片段,通过红色荧光检测、基因组PCR反应以及Western blot等方法鉴定Cas9整合入AAVS1位点的iPSC细胞株,将EGFP单链同源修复模板与EGFP sgRNA共转染阳性iPSC细胞株,靶向修复EGFP基因中错误终止密码子,正确表达绿色荧光蛋白,从而验证Cas9蛋白的基因编辑能力。结果成功制备dbDNA载体并利用其实现基因表达,dbDNA表达GFP基因效率与传统质粒DNA表达效率相近,IFN-γ的mRNA表达水平dbDNA组小于传统质粒DNA组;成功获取形态似人胚胎干细胞,碱性磷酸酶染色、多能性相关基因表达及干细胞多能性标志物阳性的iPSC细胞Objective To establish a simple technical procedure for preparing doggybone DNA(dbDNA),and to discuss the distinctions in gene expression and immune response between dbDNA and conventional plasmid DNA.Induced pluripotent stem cell(iPSC)was generated from human peripheral blood mononuclear cells(PBMCs)by using dbDNA.To investigate a protocol for inserting ultra-long DNA fragments into the iPSC adeno-associated virus site 1(AAVS1),concurrently with the integration of the Cas9 expression frame point into the dbDNA-derived iPSC AAVS1 safe harbor,so as to establish a stable cell line that can achieve iPSC gene editing exclusively through the introduction of the single guide RNA(sgRNA)for the targeted gene.Methods A simple dbDNA preparation technique was established through utilization of Phi29 DNA polymerase multiple displacement amplification,protelomerase TelN digestion and covalent ligation,as well as the subsequent removal of the backbone loop through the action of a restriction enzyme and exonuclease.The efficiency of green fluorescent protein(GFP)was evaluated following electroporation of the dbDNA-GFP vector and the pMax-dbDNA-GFP traditional plasmid vector into PBMCs.The mRNA expression of interferon-γ(IFN-γ)in the two groups was quantified by qPCR.The dbDNA reprogramming vectors dbDNA-MOS,dbDNA-KLF4,dbDNA-MYC and dbDNA-BCL were prepared and subsequently transferred into PBMCs to induce reprogramming.The dbDNA-iPSC cell line was identified through morphological analysis,alkaline phosphatase staining,RT-PCR,and detection of pluripotent stem cell markers.The ZFN and TALEN gene editing techniques were employed to insert an 11.5 kb ultra-long DNA fragment containing the Cas9 expression cassette,EGFP reading frame with stop codons in the sequence,a eukaryotic cell screening marker and other elements at the iPSC AAVS1.The identification of the iPSC lines with Cas9 integrated into AAVS1 was conducted through the utilization of red fluorescence detection,genomic PCR reaction and Western blot.The EGFP single-strand h

关 键 词：诱导性多能干细胞 dbDNA CRISPR-Cas9 腺相关病毒位点1 

分 类 号：Q78[生物学—分子生物学]