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

Single-cell CRISPR screens allow for the exploration of mammalian gene function and genetic regulatory networks, but their utility has been limited in part by their reliance on indirect sgRNA indexing. 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 the detection of multiple distinct sgRNAs expressed from a single vector within individual cells and thus allows pooled single-cell CRISPR screens to be easily paired with combinatorial perturbation libraries. We demonstrate that this approach allows high-throughput investigations of genetic interactions, and we leverage this ability to dissect epistatic interactions between cholesterol biogenesis and DNA repair. We also show that targeting individual genes with multiple sgRNAs per cell improves the efficacy of CRISPR interference and activation, facilitating the use of compact, highly active CRISPR libraries for single-cell screens. Lastly, we show that hybridization-based target enrichment permits sensitive, specific sequencing of informative transcripts from single-cell RNA-seq experiments. Single-cell, pooled CRISPR screening experiments comparing the transcriptional effects of knockdown or overexpression of genes. This repository includes five discrete experiments. (1) CRISPRi knockdown of genes known to induce the UPR across 5 different Perturb-seq platforms to compare data quality. (2) Paired CRISPRi knockdown of genes involved in cholesterol biosynthesis and DNA repair, to assess the utility of direct capture Perturb-seq for epistasis analysis. (3) CRISPRi knockdown of genes with either a single sgRNA or two sgRNAs, to assess compact library design. (4) CRISPRa overexpression of genes with either a single sgRNA or two sgRNAs, to assess compact library design. (5) Hybrid-capture based target enrichment applied to the above scRNA-seq libraries. experiments.