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

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

Research output: Contribution to journalArticlepeer-review

156 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)954-961
Number of pages8
JournalNature biotechnology
Issue number8
StatePublished - Aug 1 2020

All Science Journal Classification (ASJC) codes

  • Applied Microbiology and Biotechnology
  • Bioengineering
  • Molecular Medicine
  • Biotechnology
  • Biomedical Engineering


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