Targeted Perturb-seq enables genome-scale genetic screens in single cells

• Published in: Nature methods Vol. 17; no. 6; pp. 629 - 635
• Main Authors: Schraivogel, Daniel, Gschwind, Andreas R., Milbank, Jennifer H., Leonce, Daniel R., Jakob, Petra, Mathur, Lukas, Korbel, Jan O., Merten, Christoph A., Velten, Lars, Steinmetz, Lars M.
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01-06-2020; Nature Publishing Group
• Subjects: 631/208/177; 631/208/191; 631/208/200; 631/61/191; 631/61/212/2019; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CRISPR; Enhancers; Epigenetic inheritance; Epigenetics; Gene expression; Gene mapping; Gene sequencing; Genes; Genetic screening; Genome, Human; Genomes; Genomics; Humans; Life Sciences; Perturbation; Phenotypes; Proteomics; Ribonucleic acid; RNA; RNA-Seq - methods; Sensitivity; Single-Cell Analysis - methods; Transcriptome - genetics; Transcriptomics
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/s41592-020-0837-5

The transcriptome contains rich information on molecular, cellular and organismal phenotypes. However, experimental and statistical limitations constrain sensitivity and throughput of genetic screening with single-cell transcriptomics readout. To overcome these limitations, we introduce targeted Perturb-seq (TAP-seq), a sensitive, inexpensive and platform-independent method focusing single-cell RNA-seq coverage on genes of interest, thereby increasing the sensitivity and scale of genetic screens by orders of magnitude. TAP-seq permits routine analysis of thousands of CRISPR-mediated perturbations within a single experiment, detects weak effects and lowly expressed genes, and decreases sequencing requirements by up to 50-fold. We apply TAP-seq to generate perturbation-based enhancer–target gene maps for 1,778 enhancers within 2.5% of the human genome. We thereby show that enhancer–target association is jointly determined by three-dimensional contact frequency and epigenetic states, allowing accurate prediction of enhancer targets throughout the genome. In addition, we demonstrate that TAP-seq can identify cell subtypes with only 100 sequencing reads per cell.
Targeted sequencing of perturbation effects offers a sensitive approach to capture genes of interest in CRISPR-mediated screens, enabling genome-scale screens at higher scale and lower cost than whole-transcriptome Perturb-seq.