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Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing.
Nature Biotechnology ( IF 33.1 ) Pub Date : 2020-03-30 , DOI: 10.1038/s41587-020-0470-y
Joseph M Replogle 1, 2, 3, 4, 5 , Thomas M Norman 3, 4, 5, 6 , Albert Xu 1, 3, 4, 5 , Jeffrey A Hussmann 3, 4, 5, 7 , Jin Chen 3, 4, 5 , J Zachery Cogan 3, 4, 5 , Elliott J Meer 8 , Jessica M Terry 8 , Daniel P Riordan 8 , Niranjan Srinivas 8 , Ian T Fiddes 8 , Joseph G Arthur 8 , Luigi J Alvarado 8 , Katherine A Pfeiffer 8 , Tarjei S Mikkelsen 8 , Jonathan S Weissman 3, 4, 5 , Britt Adamson 9, 10
Affiliation  

Single-cell CRISPR screens enable the exploration of mammalian gene function and genetic regulatory networks. However, use of this technology has been limited by reliance on indirect indexing of single-guide RNAs (sgRNAs). Here we present direct-capture Perturb-seq, a versatile screening approach in which expressed sgRNAs are sequenced alongside single-cell transcriptomes. Direct-capture Perturb-seq enables detection of multiple distinct sgRNA sequences from individual cells and thus allows pooled single-cell CRISPR screens to be easily paired with combinatorial perturbation libraries that contain dual-guide expression vectors. We demonstrate the utility of this approach for high-throughput investigations of genetic interactions and, leveraging this ability, dissect epistatic interactions between cholesterol biogenesis and DNA repair. Using direct capture Perturb-seq, we also show that targeting individual genes with multiple sgRNAs per cell improves efficacy of CRISPR interference and activation, facilitating the use of compact, highly active CRISPR libraries for single-cell screens. Last, we show that hybridization-based target enrichment permits sensitive, specific sequencing of informative transcripts from single-cell RNA-seq experiments.

中文翻译:


通过直接引导 RNA 捕获和靶向测序进行组合单细胞 CRISPR 筛选。



单细胞 CRISPR 筛选能够探索哺乳动物基因功能和遗传调控网络。然而,该技术的使用因依赖单引导 RNA (sgRNA) 的间接索引而受到限制。在这里,我们提出了直接捕获 Perturb-seq,这是一种多功能筛选方法,其中表达的 sgRNA 与单细胞转录组一起进行测序。直接捕获 Perturb-seq 能够检测来自单个细胞的多个不同 sgRNA 序列,从而允许合并的单细胞 CRISPR 筛选轻松与包含双引导表达载体的组合扰动文库配对。我们展示了这种方法在遗传相互作用的高通量研究中的实用性,并利用这种能力,剖析胆固醇生物发生和 DNA 修复之间的上位相互作用。使用直接捕获 Perturb-seq,我们还表明,每个细胞使用多个 sgRNA 靶向单个基因可提高 CRISPR 干扰和激活的功效,从而促进使用紧凑、高活性的 CRISPR 文库进行单细胞筛选。最后,我们证明基于杂交的靶标富集可以对单细胞 RNA-seq 实验中的信息转录本进行灵敏、特异的测序。
更新日期:2020-04-24
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