小柯机器人

科学家开发出新型组合式单细胞CRISPR筛选技术
2020-04-01 17:28
来源：小柯机器人

美国普林斯顿大学Britt Adamson、加州大学旧金山分校Jonathan S. Weissman等研究人员合作取得一项新成果。他们开发出直接向导RNA（sgRNA）捕获和靶向测序的组合式单细胞CRISPR筛选技术。2020年3月30日，《自然—生物技术》在线发表了这一成果。

研究人员报道了直接捕获Perturb-seq，这是一种通用的筛选方法，其中表达的sgRNA与单细胞转录组一起测序。直接捕获Perturb-seq可检测单个细胞中多个不同的sgRNA序列，因此可将合并的单细胞CRISPR筛选轻松与包含双向导表达载体的组合扰动文库配对。
 
研究人员证明了这种方法对遗传相互作用高通量研究的实用性，并利用这种能力，剖析了胆固醇生物发生与DNA修复之间的上位相互作用。使用直接捕获Perturb-seq，研究人员还发现，针对每个细胞具有多个sgRNA的单个基因，这一技术可以提高CRISPR干扰和激活的效率，从而有助于将紧凑、高效的CRISPR库用于单细胞筛选。
 
最后，研究人员证明了基于杂交的靶标富集能够对来自单细胞RNA-seq实验的有用转录本进行灵敏、特异的测序。
 
据了解，单细胞CRISPR筛选可以探索哺乳动物的基因功能和遗传调控网络。但是，由于依赖于单个sgRNA的间接索引，该技术的使用受到了限制。
 
附：英文原文

Title: Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing

Author: Joseph M. Replogle, Thomas M. Norman, Albert Xu, Jeffrey A. Hussmann, Jin Chen, J. Zachery Cogan, Elliott J. Meer, Jessica M. Terry, Daniel P. Riordan, Niranjan Srinivas, Ian T. Fiddes, Joseph G. Arthur, Luigi J. Alvarado, Katherine A. Pfeiffer, Tarjei S. Mikkelsen, Jonathan S. Weissman, Britt Adamson

Issue&Volume: 2020-03-30

Abstract: 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.

DOI: 10.1038/s41587-020-0470-y

Source: https://www.nature.com/articles/s41587-020-0470-y

Nature Biotechnology：《自然—生物技术》，创刊于1996年。隶属于施普林格·自然出版集团，最新IF：68.164
官方网址：https://www.nature.com/nbt/
投稿链接：https://mts-nbt.nature.com/cgi-bin/main.plex


本期文章：《自然—生物技术》：Online/在线发表

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