| 刘 舒,彭 凤,段晓悦,万晓玲,罗学廷,李潇飒.一种DHFR介导的可调控的腺嘌呤碱基编辑器的构建[J].,2021,(23):4401-4406 |
| 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 |
| 投稿时间：2021-03-30 修订日期：2021-04-27 |
| DOI：10.13241/j.cnki.pmb.2021.23.001 |
| 中文关键词: ABE DHFR 基因编辑 |
| 英文关键词: ABE DHFR Gene editing |
| 基金项目:国家自然科学基金项目（82000906） |
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| 中文摘要: |
| 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sgRNA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10 μmol/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。 |
| 英文摘要: |
| ABSTRACT 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 HEK293T 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 sgRNAs, 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 10 μmol/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 10 μmol/L TMP could be equal to wild type ABE, leading to comparable genomic editing efficiency. Conclusion: The study constructed ABECD as an inducible ABE and provided new theoretical basis for its application in vivo. |
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