Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing.,Nature Biotechnology

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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

Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA.
Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.
Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, USA.
California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, CA, USA.
Program for Computational and Systems Biology, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
10x Genomics Inc., Pleasanton, CA, USA.
Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA.
Department of Molecular Biology, Princeton University, Princeton, NJ, USA.

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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