Stitch-seq for genome-wide pooled genomic screening with RNA-seq readout
Stitch-seq for genome-wide pooled genomic screening with RNA-seq readout
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
关键词:
AddressAdoptionAffectBar CodesBehaviorBiological AssayBiologyCRISPR interferenceCRISPR screenCRISPR-mediated transcriptional activationCell LineCell modelCellsClustered Regularly Interspaced Short Palindromic RepeatsCommunitiesComplementary DNACustomDataDiseaseDrug resistance pathwayEmulsionsEngineeringEnsureEpithelialEquilibriumExpression ProfilingGene ExpressionGene Expression ProfilingGene PoolGenerationsGenesGenetic ScreeningGenomic approachGenomicsGoalsGuide RNAHealthKnock-outLibrariesLinkLiquid substanceMCF10A cellsMalignant NeoplasmsMammary Gland ParenchymaMesenchymalMessenger RNAMethodsModalityMonitorNaturePathway interactionsPerformancePharmacotherapyProblem SolvingPublishingRNA SequencesReactionReagentRegulator GenesReproducibilityResourcesReverse Transcriptase Polymerase Chain ReactionRunningScreening ResultSignal TransductionTechnologyTestingTimeTranscriptWorkanticancer researchbasebase editingcostexperimental studyfitnessfunctional genomicsgene panelgenetic associationgenetic elementgenome wide screengenome-wideindexinginterestnovelprime editingscreeningsolution hybridizationtranscriptome sequencingwasting
中文摘要
项目总结
大规模集合CRISPR筛查已被证明是识别影响细胞的基因的一种有效方法
状态和行为。尽管发现了无数与增殖和毒品有关的基因
使用生存能力或基于标记的增菌筛选的抗性途径,有巨大的潜力
通过整合像基因表达谱这样的高复杂性读数,集合屏幕可以走得更远。癌症
社区已经利用这个机会采取行动,进入新的途径,并立即获得机械化
关于通过解释扰动对表达谱的影响来筛选命中的性质的信息。
然而,汇集的基因表达筛选没有得到充分的利用,并且不是常规地应用于大的(例如,基因组-
Wide)筛查的原因很简单-处理如此多的细胞以进行单细胞基因表达读出的成本
并且执行所需的海量序列数据生成太高。
在这里,我们提出了一种称为Stitch-seq的方法来解决这个问题,这是一种基于超高吞吐量液滴的方法
重叠聚合酶链式反应方法,能够在集合筛选中读出目标基因面板的表达水平
背景。Stich-seq成本低,因为它不需要特殊的条码试剂,如珠子和
作为一种大规模平行的单细胞乳剂RT-PCR,物理连接CRISPR引导RNA
通过重叠延伸将序列连接到靶基因序列上。在提议的工作中,我们将描述
详细介绍了Sstich-Seq反应的定量性能,并对其技术和成本进行了论证
Stitch-seq在靶向和全基因组筛查中的表现。Stich-seq将解决重要的
与现有集合CRISPR表达筛选模式相关的吞吐量限制
简便、低成本和超高通量的方法,将扰动与感兴趣的基因集物理联系起来
一系列扰动模式,包括所有形式的CRISPR扰动(敲除、CRISPRi、
CRISPRoff、CRISPRa、基本编辑、素数编辑等)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.
期刊论文(0)
专著(0)
科研奖励(0)
会议论文
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海外基金