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在精确度领域中出现的变体解释问题 医学：在基因组聚合数据库中确定的460万个错义变体中，超过一半是 被认为是不确定意义的变体（VUS）。新的方法，使解释和功能 这些VUS的表征不仅可以通过更好地告知患者， 选择策略，而且还加速开发新的方法来对抗遗传性疾病， 成分靶向基因组编辑，即在基因组DNA中引入特定修饰，具有 有可能让研究人员研究和更好地了解人类遗传疾病的机制，但 传统的基因组编辑方法（包括CRISPR-Cas9）遭受适度的基因组编辑效率， 以及不需要的基因改变，特别是当试图引入点突变时，由于它们依赖于 双链DNA断裂（DSB）。最近，我开发了一类基因组编辑代理， 不涉及DSB的编辑器，而是使用与单链DNA连接的无催化活性的Cas9， DNA修饰酶直接化学修饰基因组DNA中的靶核碱基。两类编辑 目前存在的，其使用胞嘧啶和腺嘌呤脱氨基化学来催化C·G碱基的转化 A·T碱基对与G·C碱基对（ABEs）的对应关系。我的研究项目包括 开发新的基础编辑方法，以及利用现有的基础编辑工具 来审问VUS方向1研究旨在开发新的基础编辑器，能够促进 使用计算辅助定向进化的新点突变。由此产生的工具将引起广泛的兴趣 因为它们将使研究人员能够清洁有效地安装其他类型的 点突变到活细胞的基因组中，使人类遗传病的研究和潜在治疗成为可能。 疾病方向2研究致力于启动对共同发生的致病性的首次调查 VUS（即当给定个体在其基因组中具有两个或更多个VUS时）通过开发正交 基地编辑我们的生物信息学分析表明，错义变异的临床解释正在进行中。 由于它们与其他未解释的变体频繁共存，以及正交的发展， 碱基编辑将使我们能够在功能上询问这些共同出现的变体，并评估它们对 人类遗传疾病。最后，方向3研究提出了高通量基地的发展 编辑，这将允许一次对数万个SNV进行功能调查。虽然我们目前 有工具开始在所有三个领域的工作，我们的研究方向的设计，使任何进展 一个方向可以集成到其他方向中，以指数地推进研究。成功 完成拟议的工作将证明对破译疾病机制具有变革性意义， 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.