Engineering Novel Precision Genome Editing Tools

项目摘要/摘要 已知的导致疾病的人类基因变异中有一半是由单核苷酸引起的 多态(SNPs)。因此，迫切需要开发能够 以高效、准确地纠正这些SNP。当前基于CRISPR-CAS的精确基因组编辑 诸如DNA碱基编辑和主编辑之类的工具被设计用于执行有针对性的单核苷酸改变 而不引入双链断裂和依赖同源导向的修复途径。然而， 这些工具在细胞中观察到了几个缺点，例如脱靶DNA和RNA编辑，效率低， 以及目标核苷酸附近的非故意编辑(旁观者编辑) 到不受欢迎的基因组变化。此外，DNA碱基编辑只能执行转换(互换 嘌呤(AG)或嘧啶(CT))，但不是转位(将嘧啶互换为嘌呤和黄嘌呤 反之亦然。这些缺点降低了当前精确基因组编辑工具的定位能力，并 将它们用作治疗剂的关键限制。在我们最近解释分子的工作的基础上 根据DNA碱基编辑的不足，我们提出了四种设计精确基因组的创新策略 解决当前基因组编辑工具的局限性并扩大其目标范围的编辑方法。 四种战略中的三种将产生具有双重可编程性的基础编辑。除了可编程核酸酶 (Cas9)将基础编辑引导到感兴趣的序列，这些新颖的基础编辑将另外轻松地 可编程的催化模块，只允许选择一个核苷酸进行编辑。这种双重奏 可编程性将消除旁观者的编辑，并使这些DNA碱基编辑异常准确。 四分之二的设计将产生能够执行颠换的DNA碱基编辑程序(互换嘧啶用于 嘌呤(CG和TG))，并纠正当前碱基无法获得的额外~25%的致病SNP 编辑。此外，两个基于转换的编辑器中的一个将具有双重编程能力，因此将 非常准确。总体而言，这里提出的四种策略将产生下一代精密基因组 编辑工具，除了体内SNPs的直接治疗纠正外，还将允许审问 多个SNPs、基因表达与疾病(神经退行性疾病或各种疾病)的相关性 癌症的类型)。因此，这些DNA编辑工具将为研究分子机制铺平道路 研究多种遗传性疾病，并使我们能够开发新的治疗策略。

PROJECT SUMMARY/ABSTRACT Half of the known human genetic variations that contribute to disease are due to single nucleotide polymorphisms (SNPs). Thus, there is a pressing need to develop precision genome editing tools that are able to correct these SNPs with high efficiency and accuracy. Current CRISPR-Cas based precision genome editing tools, such as DNA base editors and prime editors, were designed to perform targeted single nucleotide changes without introducing double stranded breaks and relying on the homology-directed repair pathway. However, these tools have several drawbacks observed in cells, such as off-target DNA and RNA editing, low efficiency, and unintended editing of nucleotides within the neighborhood of the target nucleotide (bystander editing) leading to undesired genomic changes. Moreover, DNA base editors are able to perform only transitions (interchanging purines (AG) or pyrimidines (CT)) but not transversions (interchanging pyrimidines for purines and vice versa). These shortcomings reduce the targeting capabilities of current precision genome editing tools and are the key limitations of using them as therapeutic agents. Building on our recent work that explains the molecular basis of the DNA base editors’ drawbacks, we propose four innovative strategies to design precision genome editing approaches that address the limitations of current genome editing tools and expand their targeting scope. Three of the four strategies will yield base editors with dual programmability. Besides the programable nuclease (Cas9) that guides base editors to the sequence of interest, these novel base editors will additionally have easily programmable catalytic modules that will allow selecting only one nucleotide for editing. This dual programmability will eliminate the bystander editing and make these DNA base editors exceptionally accurate. Two out of four designs will yield DNA base editors able to perform transversions (interchanging pyrimidines for purines (CG and TG)) and to correct additional ~25% of pathogenic SNPs inaccessible by current base editors. Moreover, one of the two transversion base editors will possess dual programmability, hence will be exceptionally accurate. Overall, the four strategies proposed here will yield the next generation precision genome editing tools that, besides the direct therapeutic corrections of SNPs’ in vivo, will also allow interrogating the association between multiple SNPs, gene expression and diseases (neurodegenerative diseases or various types of cancers). Thus, these DNA editing tools will pave the way for investigating the molecular mechanisms of multiple genetic disorders and enable us to develop new therapeutic strategies.