Development and Application of New Genome Editing Tools for the Functional Investigation of Genetic Variants of Uncertain Significance

Komor-项目摘要/摘要-“开发和应用新的基因组编辑工具 不确定意义的遗传变异的功能研究“ 我的研究计划旨在解决笼罩在精确学领域的变体解释问题 医学：在基因组聚合数据库中发现的460万个错义变体中，超过一半是 被承认为不确定意义的变体(VU)。支持解释和功能的新方法 这些VU的特征不仅可以通过更好地向患者提供信息来提高当前疗法的疗效 选择策略，也加速开发新的抗击疾病的方法与基因 组件。有针对性的基因组编辑，在基因组DNA中引入特定的修饰，具有 有可能让研究人员研究和更好地了解人类遗传病的机制，但 传统的基因组编辑方法(包括CRISPR-Cas9)的基因组编辑效率不高，因为 以及不想要的基因改变，特别是在试图引入由于依赖它们而导致的点突变时 双链DNA断裂(DSB)。最近，我开发了一类名为BASE的基因组编辑代理 不涉及DSB的编辑，而是使用催化不活跃的Cas9拴在单链 直接化学修饰基因组DNA中的目标碱基的DNA修饰酶。两类编辑 目前存在的方法是利用胞嘧啶和腺嘌呤脱氨基的化学作用来催化C·G碱的转化 碱基对分别为T·A(CBE)和A·T碱基对G·C(ABES)。我的研究项目涉及到 开发新的基本编辑方法以及利用现有的基本编辑工具 从功能上审问VU。方向1研究旨在培养新的基础编辑人员，能够促进 使用计算辅助定向进化的新的点突变。由此产生的工具将引起广泛的兴趣 给科学界，因为它们将使研究人员能够干净有效地安装其他类型的 活细胞基因组中的点突变，使研究和潜在的人类基因治疗成为可能 疾病。方向2研究致力于启动对共生病毒致病性的第一次调查 VU(即，当给定的个体在其基因组中有两个或更多VU时) 基本编辑。我们的生物信息学分析表明，错义变体的临床解释正在被 由于它们经常与其他未解释的变体同时出现而令人费解，以及正交化的发展 基础编辑将允许我们从功能上审问这些共同出现的变体，并评估它们对 人类遗传病。最后，方向3研究提出了高通量基地的发展方向 编辑，这将允许一次对数万台SNV进行功能调查。虽然我们目前 拥有在所有三个领域开始工作的工具，我们的研究方向被设计为在任何方面取得进展 一个方向可以集成到其他方向，以指数级推进研究。成功者 拟议工作的完成将证明对破译疾病机制具有变革性，并导致 开发更有效的疾病治疗方法。

Komor – Project Summary/Abstract – “Development and Application of New Genome Editing Tools for the Functional Investigation of Genetic Variants of Uncertain Significance” My research program aims to combat the variant interpretation problem that looms over the field of precision medicine: out of 4.6 million missense variants identified in the Genome Aggregation Database, over half are admitted to be variants of uncertain significance (VUS). New methods to enable the interpretation and functional characterization of these VUS would not only enhance the efficacy of current therapies by better informing patient selection strategies, but also accelerate the development of new approaches to combat diseases with a genetic component. Targeted genome editing, the introduction of a specific modification in genomic DNA, has the potential to allow researchers to study and better understand mechanisms of human genetic diseases, but traditional genome editing methods (including CRISPR-Cas9) suffer from modest genome editing efficiencies as well as unwanted gene alterations, particularly when attempting to introduce point mutations due to their reliance on double-stranded DNA breaks (DSBs). Recently, I developed a class of genome editing agents called base editors that does not involve DSBs, but rather uses a catalytically inactive Cas9 tethered to a single-stranded DNA modifying enzyme to directly chemically modify target nucleobases in genomic DNA. Two classes of editors currently exist, which use cytosine and adenine deamination chemistries to catalyze the conversion of C•G base pairs to T•A (CBEs), and A•T base pairs to G•C (ABEs), respectively. My research program involves both the development of new base editor methodologies, as well as the utilization of currently available base editor tools to functionally interrogate VUS. Direction 1 research aims to develop new base editors capable of facilitating new point mutations using computationally-aided directed evolution. The resulting tools will be of broad interest to the scientific community as they will enable researchers to cleanly and efficiently install additional types of point mutations into the genome of living cells, enabling the study and potential treatment of human genetic diseases. Direction 2 research endeavors to initiate the first investigation into the pathogenicity of co-occurring VUS (i.e. when a given individual has two or more VUS in their genome) through the development of orthogonal base editing. Bioinformatic analyses of ours suggest that the clinical interpretation of missense variants is being convoluted by their frequent co-occurrence with other uninterpreted variants, and the development of orthogonal base editing will allow us to functionally interrogate these co-occurring variants and assess their contribution to human genetic diseases. Finally, Direction 3 research proposes the development of high-throughput base editing, which will allow for the functional investigation of tens of thousands of SNVs at a time. While we currently have the tools to begin work in all three areas, our research Directions are designed such that progress in any one Direction can be integrated into the other Directions to exponentially advance the research. The successful completion of the proposed work will prove transformative for deciphering disease mechanisms and result in the development of more effective disease treatments.