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

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:
依托单位国家:
美国
项目类别:
财政年份:
2022
资助国家:
美国
项目状态:
未结题
起止时间:
2022-05-10 至 2025-03-31

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中文摘要
翻译
项目摘要 大规模合并CRISPR筛选已被证明是鉴定影响细胞增殖的基因的强大方法。 状态和行为。尽管发现了无数的基因与增殖和药物 使用生存力或基于标记的富集筛选的抗性途径, 通过整合高复杂性的读数,如基因表达谱,将筛选结果合并到一起,从而进一步提高效率。癌症 社区已经在这个机会采取行动,以获得新的途径,并立即获得机械 通过解释扰动对表达谱的影响来获得关于筛选命中的性质的信息。 然而,合并的基因表达筛选未得到充分利用,并且未常规应用于大型(例如,基因组- 宽)屏幕的一个简单的原因-处理这么多细胞的单细胞基因表达读出的成本 并且需要进行大量的序列数据生成。 在这里,我们建议用一种我们称之为Stitch-seq的方法来解决这个问题,这是一种超高通量的基于液滴的方法。 重叠PCR方法,能够读出合并筛选中靶基因组的表达水平 上下文Stitch-seq是低成本的,因为它不需要专门的条形码试剂,如珠, 作为大规模平行的单细胞乳液RT-PCR, 通过重叠延伸将靶向mRNA序列与靶向mRNA序列连接。在拟议的工作中,我们将描述 详细介绍了Stitch-seq反应的定量性能,并论证了其技术和成本 Stitch-seq在靶向和全基因组筛选中的性能。Stitch-seq将解决 与现有的合并CRISPR表达筛选模式相关的通量限制, 一种简便、低成本和超高通量的方法,将扰动与跨 一系列扰动模式,包括所有形式的CRISPR扰动(敲除,CRISPRi, CRISPRoff、CRISPRa、碱基编辑、引物编辑等)以及更远的地方
英文摘要
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.
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会议论文
Targeting Dysregulated RNA Splicing in Neurodegenerative Diseases
  • 批准号:
    10729566
  • 项目类别:
  • 资助金额:
    $207.29万
  • 财政年份:
    2023
  • 负责人:
    Paul Clark Blainey
  • 依托单位:
Stitch-seq for genome-wide pooled genomic screening with RNA-seq readout
  • 批准号:
    10620301
  • 项目类别:
  • 资助金额:
    $16.73万
  • 财政年份:
    2022
  • 负责人:
    Paul Clark Blainey
  • 依托单位:
Innovative technologies to transform antibiotic discovery. Project 2 Engineering antibiotic sensitization therapies
  • 批准号:
    10463689
  • 项目类别:
  • 资助金额:
    $168.76万
  • 财政年份:
    2019
  • 负责人:
    Paul Clark Blainey
  • 依托单位:
Innovative technologies to transform antibiotic discovery. Project 2 Engineering antibiotic sensitization therapies
  • 批准号:
    10242004
  • 项目类别:
  • 资助金额:
    $158.93万
  • 财政年份:
    2019
  • 负责人:
    Paul Clark Blainey
  • 依托单位:
海外基金