Next-generation Functional Genetic Screening of Un-screenable Traits

不可筛选性状的下一代功能遗传筛选

• 批准号: 9379760
• 负责人: Prashant Mali
• 金额: $ 105.77万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-25 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9379760
• 关键词: Affinity; Alleles; Biological; Biological Assay; Biological Models; CRISPR screen; CRISPR/Cas technology; Calcium; Catalogs; Cell Differentiation process; Cell Line; Cell Separation; Cell Survival; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Computing Methodologies; Coupled; DNA sequencing; Development; Disease; Elements; Engineering; Enhancers; Future; Gene Expression Regulation; Gene Targeting; Generations; Genetic; Genetic Annotation; Genetic Screening; Genetic Transcription; Genetic Variation; Genetic screening method; Genome; Genome engineering; Genomic DNA; Genomics; Germ Layers; Human Genome; In Vitro; Individual; Link; Mali; Methodology; Methods; Modeling; Modification; Mus; Mutation; Phenotype; Physiological; Pluripotent Stem Cells; Proxy; RNA; System; Technology; Teratoma; Tissues; Untranslated RNA; Ursidae Family; Variant; base; cell type; cellular imaging; design; experimental study; functional genomics; functional outcomes; genetic variant; genome editing; genome wide association study; high throughput screening; human pluripotent stem cell; in vivo; induced pluripotent stem cell; insight; knockout gene; next generation; novel; programs; screening; trait; transcription factor; transcriptome; transcriptome sequencing; transcriptomics; variant of unknown significance

Project Summary/Abstract With the rapid advances in DNA sequencing, we now have a near-complete human genome, a fairly comprehensive catalog of germline and somatic variants, as well as rich annotations of functional genomic elements. The next challenge in the field is to obtain a complete functional annotation of genetic variants and genomic elements at the cellular and organismal levels. Genome editing technology, in particularly the CRISPR/Cas9 system, has allowed rapid and precise modifications of the genome and connecting of these to functional outcomes. However, all current high-throughput screening approaches rely on phenotypes that can be coupled to cell survival, cell imaging, fluorescent cell sorting, or affinity enrichments. Genetic variants that have more subtle phenotypic consequences, which might represent the majority, are not amenable to such screens. Furthermore, screening of natural genetic variation via assaying of individual cell lines under in vitro culture conditions also has limited throughput and might miss functional differences that depend on specific physiological contexts. In this project we seek to develop a next-generation functional genetic screening method that can overcome these current limitations. Utilizing induced pluripotent stem cell differentiation as an exemplar model system, we will develop and demonstrate our integrated experimental and computational methodology to enable comprehensive and en masse functional screening of coding and non-coding genomic elements. We expect our platform will greatly accelerate the functional annotation of genetic variants, including many variants of unknown significance, across various normal and diseased cell types and tissues.

项目总结/摘要 随着DNA测序技术的快速发展，我们现在有了一个接近完整的人类基因组，一个相当完整的人类基因组。 生殖系和体细胞变异的全面目录，以及功能基因组的丰富注释 元素该领域的下一个挑战是获得遗传变异的完整功能注释， 在细胞和生物体水平上的基因组元件。基因组编辑技术，特别是 CRISPR/Cas9系统允许对基因组进行快速和精确的修饰，并将其连接到 功能成果。然而，目前所有的高通量筛选方法都依赖于表型， 与细胞存活、细胞成像、荧光细胞分选或亲和富集相结合。的基因变异 有更微妙的表型后果，这可能代表大多数，不服从这种 卡位此外，通过在体外条件下测定单个细胞系来筛选天然遗传变异， 培养条件也具有有限的通量，并且可能错过依赖于特定细胞的功能差异。 生理背景在这个项目中，我们寻求开发下一代功能性遗传筛选 这种方法可以克服目前的局限性。利用诱导的多能干细胞分化作为一种 范例模型系统，我们将开发和展示我们的综合实验和计算 能够对编码和非编码基因组进行全面和功能性筛选的方法 元素我们希望我们的平台将大大加快遗传变异的功能注释，包括 许多未知意义的变体，跨越各种正常和患病的细胞类型和组织。