Genome engineering tools for functional screening of non-coding elements

用于非编码元件功能筛选的基因组工程工具

• 批准号: 8804084
• 负责人: Neville Sanjana
• 金额: $ 9.99万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8804084
• 关键词: Advisory Committees; Antineoplastic Agents; Architecture; Area; Beryllium; Biological; Biological Assay; Biological Models; Biological Process; Biology; Cells; Chromosomes; Chromosomes, Human, Pair 21; Clustered Regularly Interspaced Short Palindromic Repeats; Code; DNA; Data; Data Set; Development Plans; Diagnostic; Disease; Drug resistance; Elements; Environment; Gene Expression; Gene Targeting; Genes; Genetic Variation; Genome; Genome engineering; Genomics; Genotype; Goals; Growth and Development function; Guide RNA; Human; Human Genetics; Human Genome; Human Genome Project; Indium; Institutes; Intercistronic Region; Introns; Knock-out; Knowledge; Laboratories; Libraries; Life; Machine Learning; Mentors; Modeling; Modification; Mutagenesis; Mutation; National Human Genome Research Institute; Nature; Occupations; Paper; Pathway interactions; Pharmaceutical Preparations; Phase; Phenotype; Plant Roots; Play; Positioning Attribute; Postdoctoral Fellow; Proteins; RNA Interference; Reagent; Relative (related person); Repetitive Sequence; Research; Resolution; Role; Science; Seeds; Stem cells; Subfamily lentivirinae; Techniques; Technology; Testing; Training; Untranslated RNA; Untranslated Regions; Variant; Work; Writing; cancer cell; career; career development; clinically relevant; deletion library; design; experience; functional genomics; genetic element; genome editing; genome-wide; improved; innovative technologies; insertion/deletion mutation; knock-down; loss of function; loss of function mutation; new technology; novel; nuclease; overexpression; programs; promoter; public health relevance; repaired; research study; scaffold; screening; small hairpin RNA; technology development; tool

DESCRIPTION (provided by applicant): A major goal since the completion of the Human Genome Project has been to understand all functional elements in the human genome and the role they play in normal biological processes and disease. To that end, large pooled libraries of RNA interference (RNAi) reagents have been developed for genome-wide loss-of-function screens but have been hindered by 3 problems: 1) the incompleteness of protein depletion inherent in partial knock-down; 2) off-target effects from the seed sequence; and 3) genetic elements that are not transcribed are inaccessible to manipulation. Genome engineering using precisely targeted nucleases has emerged as an innovative technology to modify the genome and causally interrogate the role of different functional elements. Recently, I developed a new technology for functional genomic screening using the RNA- guided CRISPR/Cas9 nuclease (Shalem*, Sanjana*, et al., Science, 2014). Since CRISPR works on the DNA level, it is possible to manipulate non-coding elements that are inaccessible to RNAi. The research goal of this proposal is to develop new biological tools and analysis techniques for functional annotation of non-coding elements using pooled CRISPR screens. Mentored phase: First, I plan to develop and optimize high-throughput CRISPR non-coding mutagenesis libraries targeting introns, UTRs, promoters, non-coding RNAs, and intergenic regions to enable screening at high-resolution with megabase-scale coverage. Next, I will validate functional non-coding elements and use this large dataset to find unifying principles of how non-coding elements regulate gene expression. Independent phase: I plan to develop a novel CRISPR architecture for tiled deletion screens capable of deleting many segments over entire chromosomes or even entire genomes. With this technology and the increased screening throughput it enables, I will be able to develop a long-term independent research program in several possible directions, including further genome biology, personalized functional genomics, and predictive diagnostics for drug-genome interactions. The two primary areas of training needed to help me succeed in my research goals are 1) CRISPR technology development (mentor: Dr. Feng Zhang) and 2) knowledge of human genetics and non-coding variation (mentor: Dr. David Altshuler). Each mentor is an established expert in these fields. My career development plan integrates additional laboratory training, specialized tutorials in human genetics from world experts, local and national presentations of my research, and courses in scientific writing, grantsmanship and job search strategies. To assist with science- and career-related decisions, I have assembled an Advisory Committee with a team of established, senior genomics experts: Drs. Eric Lander, Steven Hyman, and David Root. The Broad Institute is an ideal environment: All Mentors and Advisors are located in one building and there are facilities for high-throughput functional screening in th RNAi Platform (Director: Dr. Root).

描述（由申请人提供）：人类基因组计划完成以来的一个主要目标是了解人类基因组中的所有功能元件及其在正常生物过程和疾病中的作用。为此，已经开发了用于全基因组功能丧失筛选的RNA干扰（RNAi）试剂的大型汇集文库，但受到3个问题的阻碍：1）部分敲低中固有的蛋白质消耗的不完全性; 2）来自种子序列的脱靶效应;以及3）未转录的遗传元件无法进行操作。使用精确靶向核酸酶的基因组工程已经成为一种创新技术，用于修饰基因组并因果地询问不同功能元件的作用。最近，我开发了一种使用RNA引导的CRISPR/Cas9核酸酶进行功能性基因组筛选的新技术（Shalem*，Sanjana*，et al. Science，2014）。由于CRISPR在DNA水平上起作用，因此可以操纵RNAi无法访问的非编码元件。该提案的研究目标是开发新的生物学工具和分析技术，用于使用池化CRISPR筛选对非编码元件进行功能注释。 指导阶段：首先，我计划开发和优化针对内含子、UTR、启动子、非编码RNA和基因间区域的高通量CRISPR非编码诱变文库，以实现高分辨率筛选和兆碱基规模的覆盖。接下来，我将验证功能性非编码元件，并使用这个大型数据集来找到非编码元件如何调节基因表达的统一原则。独立阶段：我计划开发一种新的CRISPR架构，用于平铺删除屏幕，能够删除整个染色体甚至整个基因组上的许多片段。有了这项技术和它所能实现的更高的筛选通量，我将能够在几个可能的方向上开发一个长期的独立研究计划，包括进一步的基因组生物学，个性化的功能基因组学和药物-基因组相互作用的预测诊断。 帮助我成功实现研究目标所需的两个主要培训领域是：1）CRISPR技术开发（导师：张锋博士）和2）人类遗传学和非编码变异的知识（导师：大卫·阿尔特什基博士）。每个导师都是这些领域的专家。我的职业发展计划整合了额外的实验室培训，来自世界专家的人类遗传学专业教程，我的研究的本地和国家演示，以及科学写作，granitary和求职策略课程。为了帮助做出与科学和职业相关的决定，我组建了一个咨询委员会，成员包括一个由资深基因组学专家组成的团队：埃里克·兰德博士、史蒂文·海曼博士和大卫·鲁特博士。布罗德研究所是一个理想的环境：所有的导师和顾问都位于一个建筑物，并有设施的高通量功能筛选的RNAi平台（主任：博士根）。