Engineering Novel Precision Genome Editing Tools

项目总结/摘要 已知的人类遗传变异中有一半是由于单核苷酸 多态性（SNP）。因此，迫切需要开发精确的基因组编辑工具， 以高效率和准确性校正这些SNP。当前基于CRISPR-Cas的精确基因组编辑 设计了一些工具，如DNA碱基编辑器和引物编辑器，用于进行靶向的单核苷酸改变 而不引入双链断裂并依赖于同源性定向修复途径。但是，在这方面， 在细胞中观察到这些工具有几个缺点，例如脱靶DNA和RNA编辑，效率低， 以及靶核苷酸附近的核苷酸的非预期编辑（旁观者编辑）， 不希望的基因组变化。此外，DNA碱基编辑器能够仅执行转换（互换 嘌呤（A G）或嘧啶（C T）），但不能颠换（嘌呤和嘧啶互换）。 反之亦然）。这些缺点降低了当前精确基因组编辑工具的靶向能力， 将它们用作治疗剂的关键限制。基于我们最近的工作， 针对DNA碱基编辑器的缺陷，提出了四种设计精确基因组的创新策略 编辑方法，解决当前基因组编辑工具的局限性，并扩大其靶向范围。 四种策略中的三种将产生具有双重可编程性的基本编辑器。除了可编程核酸酶 （Cas9），其将碱基编辑器引导至感兴趣的序列，这些新颖的碱基编辑器将另外容易地具有 可编程催化模块将允许仅选择一个核苷酸进行编辑。这种双重 可编程性将消除旁观者编辑，并使这些DNA碱基编辑器异常准确。 四分之二的设计将产生能够执行颠换的DNA碱基编辑器（将嘧啶互换为嘧啶）。 嘌呤（C-嘌呤G和T-嘌呤G）），并纠正额外的~25%的目前碱基无法获得的致病性SNP 编辑。此外，两个转换基编辑器中的一个将具有双重可编程性，因此将是 异常准确总的来说，这里提出的四种策略将产生下一代精确基因组 编辑工具，除了在体内直接治疗SNP的校正外，还将允许询问 多个SNP、基因表达和疾病（神经退行性疾病或各种 癌症类型）。因此，这些DNA编辑工具将为研究分子机制铺平道路 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.