【作者】 张梅；

【导师】 李大力；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2021， 硕士

【摘要】 碱基编辑技术是基于CRISPR/Cas系统和碱基脱氨酶衍生出的一种新的基因组修饰方法,该技术主要包括:胞嘧啶碱基编辑器(Cytosine Base Editor,CBE)和腺嘌呤碱基编辑器(Adenine Base Editor,ABE)。其中,CBE是通过核酸酶活性缺失的Cas蛋白携带胞嘧啶脱氨酶将靶位点的胞嘧啶(C)脱氨成尿嘧啶(U),然后在尿嘧啶糖苷酶抑制剂(UGI)的保护下,实现C到胸腺嘧啶(T)的转换。CBE既可以用于模拟和修复致病突变位点,又可以通过产生终止密码子实现靶基因的敲除,已广泛用于动物模型构建和疾病治疗的研究。然而,研究人员发现由于真核细胞基因组结构复杂,CBE在基因组不同位置的碱基编辑效率差异较大。另外,CBE作为疾病治疗的有效工具在造血干细胞中的应用意义重大,而高质量CBE蛋白的表达纯化和在造血干细胞中的高效碱基编辑是制约该应用的关键因素。因此,本研究希望通过增强Cas9的结合能力构建出高效率的CBE工具(efficiency enhanced Cytosine Base Editor,ee CBE),建立CBE蛋白表达和纯化体系,并在造血干细胞的治疗性靶点上实现高效的碱基编辑。首先,本研究将Cas9单切口酶(Cas9n)与DNA双链结合蛋白融合来增强CBE在靶位点处的结合能力,从而提高C到T的编辑效率。据此,本研究构建出效率平均提高1.3倍,且高活性窗口、产物纯度和indels比率都没有明显变化的高效胞嘧啶碱基编辑器(ee BE4max)。为了拓展ee BE4max的靶向范围,本研究将Sp Cas9n核酸酶更换为PAM更简单的Sp Cas9n变体,构建了一系列靶向“NGN”PAM和几乎无PAM限制的ee BE4max变体。另外,为了进一步提高CBE碱基编辑器的效率,本研究将ee BE4max的APOBEC1脱氨酶换成活性更高的APOBEC3A,构建了本研究中效率最高的ee A3A-BE4max,在真核细胞中C到T的转换效率高达80%。接下来,为了将ee CBE工具应用到造血干细胞的碱基编辑中,本研究建立了CBE蛋白的原核表达和纯化体系,通过镍柱纯化和分子筛柱纯化,获得了高质量的ee CBE蛋白。最后,本研究通过电转将ee CBE工具的核糖核蛋白(RNP)复合物递送至造血干细胞中,显著提高BCL11A增强子区和HBG1/2调控区C突变为T的频率,其中在BCL11A上C到T的编辑效率高达70%,γ-珠蛋白的相对表达量达到了α-珠蛋白的50%以上,且ee CBE与CBE的脱靶效率没有显著差异。总的来说,本研究通过增强Cas9n的DNA结合能力开发了多种高效率的ee CBE,同时也建立了CBE蛋白的原核表达与纯化体系,获得了高质量的ee CBE蛋白,并在造血干细胞中实现了C到T的高效转换和γ-珠蛋白的重新激活,为β-地中海贫血疾病的治疗提供了技术支持。更多还原

【Abstract】 Base editing technology is a new genome modification method based on CRISPR/Cas system and base deaminase,which mainly includes: cytosine base editor(CBE)and adenine base editor(ABE).Cytosine base editor(CBE)can catalyze the deamination of cytosine(C)and result in uracil(U)through catalytically disabled Cas nuclease carrying deaminase,thereby generating a C to thymine(T)base conversion.CBE not only could introduce or repair the pathogenic mutation,but also achieve targeted gene knockout by generating a stop codon,which has been widely used in animal model construction and disease treatment.However,the base editing efficiency of CBE varies considerably with different genomic sites due to the complexity of chromatin structures of eukaryotic genomic DNA.In addition,as an effective tool for disease treatment,CBE shows encouraging application potential in hematopoietic stem cells,while the expression and purification of high-quality CBE protein and efficient base editing in hematopoietic stem cells are the key factors to restrict its application.Therefore,we hope to build an efficiency enhanced CBE(ee CBE)tools by enhancing the binding ability of Cas9 nickase(Cas9n)nuclease,and express and purify the CBE protein to achieve efficient base editing in the therapeutic target of hematopoietic stem cells.Firstly,we fused the DNA double stranded domain into Cas9 n nuclease to enhance the DNA binding ability,and lead to improvement of C-to-T editing efficiency.Accordingly,we developed an ee BE4 max tool to enhance base editing efficiency up to1.3-fold,while the editing window,product purity and indels rates have no obvious change.In order to expand the targeting scope of ee BE4 max,we replaced Sp Cas9 n nuclease with other Sp Cas9 n variants,and constructed a series of ee BE4 max variants targeting "NGN" PAM and near-PAMless.We also replaced the deaminase of ee BE4 max with more active APOBEC3 A to generate the most efficient ee A3ABE4 max in our study,and ee A3A-BE4 max can achieve up to 80% C to T conversion rates.Then,we established a prokaryotic expression system for CBE protein,and purified high quality A3A-BE3 and ee A3A-BE3 proteins by nickel column and molecular sieve column.Finally,we electroporated ee CBE and sg RNA as RNP complex into hematopoietic stem cells to edit the C base of BCL11 A enhancer region and HBG1/2 promoter region,results show that ee A3A-BE3 improve base editing efficiency significantly,and C to T conversion rates up to 70% at BCL11 A site,which reactivated γ-globin expression levels up to 50% relative to α-globin levels.We also detected the off-target effect caused by ee A3A-BE3 was no significant difference when compared with A3A-BE3.Overall,we developed a variety of efficiency enhanced CBE tools by improving the binding ability of Cas9 n nuclease.We also purified the high quality ee A3A-BE3 protein to generate C to T conversion and reactivate γ-globin expression in hematopoietic stem cells,which laid a theoretical and technical foundation for treatment of β-thalassemia disease.更多还原