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

• Published in: Nature biotechnology Vol. 38; no. 8; pp. 954 - 961
• Main Authors: Replogle, Joseph M., Norman, Thomas M., Xu, Albert, Hussmann, Jeffrey A., Chen, Jin, Cogan, J. Zachery, Meer, Elliott J., Terry, Jessica M., Riordan, Daniel P., Srinivas, Niranjan, Fiddes, Ian T., Arthur, Joseph G., Alvarado, Luigi J., Pfeiffer, Katherine A., Mikkelsen, Tarjei S., Weissman, Jonathan S., Adamson, Britt
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.08.2020; Nature Publishing Group
• Subjects: 631/208/191/1472; 631/208/191/2018; 631/337/2019; 631/553/2490/1472; Agriculture; Bioinformatics; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Cholesterol; Combinatorial analysis; CRISPR; CRISPR-Cas Systems; Deoxyribonucleic acid; DNA; DNA repair; Epistasis; Expression vectors; Gene Expression Regulation; Gene sequencing; Gene Targeting; HEK293 Cells; High-Throughput Nucleotide Sequencing; Humans; Hybridization; Letter; Libraries; Life Sciences; Methods; Nucleic Acid Amplification Techniques - methods; Perturbation; Ribonucleic acid; RNA; RNA sequencing; RNA, Guide, CRISPR-Cas Systems - genetics; Single-Cell Analysis; Transcriptome; United States
• ISSN: 1087-0156; 1546-1696; 1546-1696
• DOI: 10.1038/s41587-020-0470-y

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.