期刊
NATURE BIOTECHNOLOGY
卷 38, 期 8, 页码 954-+出版社
NATURE PORTFOLIO
DOI: 10.1038/s41587-020-0470-y
关键词
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资金
- National Institutes of Health [P50 GM102706, U01 CA168370, R01 DA036858, RM1HG009490]
- Defense Advanced Research Projects Agency (DARPA) [HR0011-19-2-0007]
- Chan Zuckerberg Initiative
- Princeton University
- NIH/NINDS Ruth L. Kirschstein National Research Service Award [F31 NS115380]
- Damon Runyon Cancer Research Foundation [DRG-(2211-15), DRG-2262-16]
- Jane Coffin Childs Memorial Fund for Medical Research
- NIH K99/R00 Pathway to Independence Award [GM134154]
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. Single-cell CRISPR screens are readily multiplexed and scaled with an improved version of Perturb-seq.
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