一种DHFR介导的可调控的腺嘌呤碱基编辑器的构建  

A Tunable Adenine Base Editor Mediated by DHFR

作　　者：刘舒 彭凤 段晓悦 万晓玲 罗学廷 李潇飒 LIU Shu;PENG Feng;DUANXiao-yue;WAN Xiao-ling;LUO Xue-ting;LI Xiao-sa(Department of Ophthalmology,First People's Hospital Affiliated to Shanghai Jiao Tong University,Shanghai,201620,China)

机构地区：[1]上海交通大学附属第一人民医院眼科,上海201620

出　　处：《现代生物医学进展》2021年第23期4401-4406,共6页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(82000906)。

摘　　要：目的:单碱基编辑器作为一种高效、精确、低脱靶的编辑系统,目前被广泛应用于生物体中。然而,Cas核酸酶本身的免疫原性具有引发机体免疫反应的潜在风险,可能阻碍其在基因治疗中的有效和安全使用。因而本研究旨在构建一种由DHFR介导的可调控腺嘌呤碱基编辑器(adenine base editor,ABE)。方法:将大肠杆菌来源的不稳定结构域-二氢叶酸还原酶(dihydrofolate reductase,DHFR)的一种突变形式,融合于ABE的不同相对位置,构建数种DD-ABE载体。从已发表的内源基因靶点中挑选三个高效位点检测DD-ABE编辑水平。转染四种DD-ABE表达载体,用不同浓度TMP处理,通过Western Blot检测DD-ABE融合蛋白的表达水平。共转染DD-ABE与sg RNA质粒,通过基因组PCR扩增以及Sanger测序检测基因的编辑水平。结果:在构建的四种DD-ABE编辑器中,缺乏TMP的诱导,各组仅有非常少量的DD-ABE融合蛋白表达。TMP最佳工作浓度为10μmol/L。DD-ABE载体加药组的蛋白表达水平以及基因编辑水平均高于未加药组。ABECD未加药组的碱基编辑效率最低,加药组的蛋白表达水平和编辑效率与ABEmax无明显差异。结论:本研究成功构建ABECD作为可调控的ABE,为其体内应用提供了新的技术依据。Objective:The base editors have been widely used in many organisms,processing the advantages of accuracy,efficiency,and low off-target.However,when applying to gene therapy,the immunogenicity of Cas nuclease may trigger immune response that prevents its efficiency and safety.Therefore,the study aims to construct a tunable adenine base editor(ABE)mediated by DHFR.Methods:By fusing a destabilizing domain-a mutant form of Escherichia coli dihydrofolate reductase(DHFR)to different positions of adenine base eitor(ABE),we constructed several DD-ABE vectors.Three of the published endogenous gene targets were selected to test the editing efficiency.We transfected HEK293 T cells with the DD-ABE expression vectors in the presence of TMP at different concentrations,and detected the expression of fusing proteins utilizing Western Blot.Then we co-transfected DD-ABE vectors with sg RNAs,and detected the base editing efficiency by PCR amplification and Sanger sequencing.Results:In all the ABE-DD editing systems that we constructed,there were tiny amounts of stabilized DD-ABE protein in the absence of TMP.The optimal working concentration of TMP was 10μmol/L.The protein expression and base editing efficiency of DD-ABE treated with TMP were higher than those of untreated group.ABECD possessed the lowest gene editing efficiency in the absence of TMP,and,the protein level of ABECD stabilized by 10μmol/L TMP could be equal to wild type ABE,leading to comparable genomic editing efficiency.Conclusions:The study constructed ABECD as an inducible ABE and provided new theoretical basis for its application in vivo.

关 键 词：ABE DHFR 基因编辑 

分 类 号：R-33[医药卫生] Q78[生物学—分子生物学]