Stitch-seq for genome-wide pooled genomic screening with RNA-seq readout

Stitch-seq 通过 RNA-seq 读数进行全基因组汇集基因组筛选

• 批准号: 10413630
• 负责人: Paul Clark Blainey
• 金额: $ 20.78万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-10 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10413630
• 关键词: Address; Adoption; Affect; Bar Codes; Behavior; Biological Assay; Biology; CRISPR interference; CRISPR screen; CRISPR-mediated transcriptional activation; Cell Line; Cell model; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complementary DNA; Custom; Data; Disease; Drug resistance pathway; Emulsions; Engineering; Ensure; Epithelial; Equilibrium; Expression Profiling; Gene Expression; Gene Expression Profiling; Gene Pool; Generations; Genes; Genetic Screening; Genomic approach; Genomics; Goals; Guide RNA; Health; Knock-out; Libraries; Link; Liquid substance; MCF10A cells; Malignant Neoplasms; Mammary Gland Parenchyma; Mesenchymal; Messenger RNA; Methods; Modality; Monitor; Nature; Pathway interactions; Performance; Pharmacotherapy; Problem Solving; Publishing; RNA Sequences; Reaction; Reagent; Regulator Genes; Reproducibility; Resources; Reverse Transcriptase Polymerase Chain Reaction; Running; Screening Result; Signal Transduction; Technology; Testing; Time; Transcript; Work; anticancer research; base; base editing; cost; experimental study; fitness; functional genomics; gene panel; genetic association; genetic element; genome wide screen; genome-wide; indexing; interest; novel; prime editing; screening; solution hybridization; transcriptome sequencing; wasting

PROJECT SUMMARY Large-scale pooled CRISPR screens have proven to be a powerful approach to identify genes that affect cell state and behavior. Notwithstanding the countless genetic associations discovered with proliferation and drug resistance pathways using viability or marker-based enrichment screens, there is tremendous potential for pooled screens to go further by incorporating high complexity readouts like gene expression profiles. The cancer community is already acting on this opportunity to access new pathways and immediately gain mechanistic information about the nature of screening hits by interpreting the impact of perturbations on expression profiles. However, pooled gene expression screens are underutilized and not routinely applied for large (eg. genome- wide) screens for a simple reason - the cost of processing so many cells for single-cell gene expression readout and carrying out the massive amount of sequence data generation required is too high. Here, we propose to solve this problem with a method we call Stitch-seq, an ultra-high-throughput droplet-based overlap PCR method that enables readout of the expression levels of a target gene panel in the pooled screening context. Stitch-seq is low-cost because it does not require specialized barcoded reagents like beads and operates as a massively parallel single-cell emulsion Rt-PCR that physically links CRISPR guide RNA sequences to the targeted mRNA sequences by overlap extension. In the proposed work, we will characterize the quantitative performance of the Stitch-seq reaction in detail, and demonstrate the technical and cost performance of Stitch-seq in targeted and genome-wide screens. Stitch-seq will address the significant throughput limitations associated with existing pooled CRISPR expression screening modalities by providing a facile, low-cost, and ultra-high-throughput method to physically link perturbations to gene sets of interest across a range of perturbation modalities including all manner of CRISPR perturbations (knock-out, CRISPRi, CRISPRoff, CRISPRa, base editing, prime editing, etc.) and beyond.

项目摘要 事实证明，大型合并CRISPR屏幕是识别影响细胞的基因的强大方法 状态和行为。尽管有无数的遗传关联是通过增殖和药物发现的 使用生存能力或基于标记的富集屏幕的电阻途径，具有巨大的潜力 汇总屏幕可以通过结合诸如基因表达曲线之类的高复杂性读数来进一步发展。癌症 社区已经在这一机会访问新途径并立即获得机理 通过解释扰动对表达曲线的影响，有关筛选命中的性质的信息。 然而，汇集基因表达筛选未被充分利用，并且不常规应用于大（例如，基因组 - 宽）屏幕是出于简单的原因 - 处理如此多单元的单细胞基因表达读数的成本 而且，进行大量序列数据生成的数量太高。 在这里，我们建议通过一种我们称为“超高通知液滴” stitch-seq的方法来解决此问题 重叠PCR方法，可以读取合并筛选中目标基因面板的表达水平 语境。针迹seq是低成本的，因为它不需要专门的条形码试剂，例如珠子和 作为一个大规模平行的单细胞乳液RT-PCR，可将CRISPR指南RNA链接起来 通过重叠延伸到靶向mRNA序列的序列。在拟议的工作中，我们将表征 详细详细的针迹反应的定量性能，并证明了技术和成本 针迹在靶标和全基因组筛选中的性能。针迹塞克将解决重要的问题 与现有的合并CRISPR表达筛选方式相关的吞吐量限制通过提供 轻松，低成本和超高通量方法将扰动物理上的扰动与各个兴趣的基因集联系起来 各种扰动方式包括各种CRISPR扰动（淘汰赛，Crispri， CRISPROFF，CRISPRA，基础编辑，主要编辑等）及以后。