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

新型基因组编辑工具的开发和应用，用于意义不明的遗传变异的功能研究

• 批准号: 10224276
• 负责人: Alexis C. Komor
• 金额: $ 34.98万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10224276
• 关键词: Adenine; Area; Base Pairing; Bioinformatics; CRISPR/Cas technology; Cells; Chemicals; Chemistry; Clinical; Communities; Cytosine; DNA Double Strand Break; Databases; Deamination; Development; Directed Molecular Evolution; Disease; Enzymes; Gene Mutation; Genetic; Genetic Diseases; Genetic Variation; Genome; Genomic DNA; Human Genetics; Individual; Investigation; Methodology; Methods; Modification; Pathogenicity; Patient Selection; Point Mutation; Research; Research Personnel; Single-Stranded DNA; Time; Variant; Work; base; clinically relevant; combat; design; genetic variant; genome editing; human disease; interest; novel strategies; nucleobase; precision medicine; programs; tool; variant of unknown significance

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.

Komor -项目摘要/摘要-“新型基因组编辑工具的开发与应用”