Enzymatic modification of nucleobases for single base pair editing in genomic DNA

项目概要/摘要 靶向重写发育细胞的遗传密码以获得治疗益处是本领域的长期目标。 生物医学科学碱基编辑是一种新的基因组工程方法，它利用序列- 催化失活或受损的Cas9核酸酶对靶向核碱基剪裁的特异性靶向能力 酶在基因组中的特定位置，最终导致有针对性的单碱基突变。三元 在Cas9、sgRNA和靶DNA之间的复合物中，DNA的非模板化链的一部分形成一个非模板化的DNA链。 暴露的无序的单链环在碱基编辑中，与Cas9融合的酶利用了这一点。 机会并催化该环的一小段内的核碱基上的化学修饰。这些 设计修饰以改变修饰的核苷酸的碱基配对性质，并在修饰期间错误编码。 DNA复制或修复影响最终的点突变。这个项目的长期目标是发现 修饰的核碱基的生产性碱基配对相互作用，并利用这些修饰来开发 碱基编辑器能够选择性地影响点突变的每个排列。蛋白质工程 将采取优化新的核碱基氧化酶的方法，并结合化学 合成和酶学将允许评估修饰的核碱基的错误编码可能性。 预计碱基编辑器对人类健康有直接影响：大多数致病人类 基因突变是点突变，碱基编辑有可能在体细胞中逆转这些突变。 细胞

PROJECT SUMMARY/ABSTRACT Targeted rewriting of the genetic code of developed cells for therapeutic benefit is a longstanding goal in the biomedical sciences. Base editing is a new genome engineering approach that harnesses the sequence- specific targeting ability of a catalytically inactive or impaired Cas9 nuclease to target nucleobase-tailoring enzymes to specific locations in the genome, eventually causing targeted single base mutations. In the ternary complex between Cas9, sgRNA, and the target DNA, a portion of the non-templated strand of DNA forms an exposed, disordered, single-stranded loop. In base editing, an enzyme fused to Cas9 takes advantage of this opportunity and catalyzes chemical modifications on nucleobases within a small segment of this loop. These modifications are designed to alter the base pairing properties of the modified nucleotide, and miscoding during DNA replication or repair effects the eventual point mutation. The long term goal of this project is to discover productive base pairing interactions of modified nucleobases and to harness these modifications in developing base editors capable of selectively effecting every permutation of a point mutation. A protein engineering approach to optimize new nucleobase-oxidizing enzymes will be undertaken, and a combination of chemical synthesis and enzymology will allow for the evaluation of the miscoding potential of modified nucleobases. Base editors are anticipated to have a direct impact on human health: the majority of pathogenic human genetic mutations are point mutations, and base editing has the potential to reverse these mutations in somatic cells.