Targeted Perturb-seq enables genome-scale genetic screens in single cells
| Autor: | Petra Jakob, Daniel R. Leonce, Lukas Mathur, Jan O. Korbel, Lars Velten, Jennifer Milbank, Christoph A. Merten, Andreas R Gschwind, Daniel Schraivogel, Lars M. Steinmetz |
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| Předmět: |
genetic processes
Computational biology Biology Biochemistry Genome Article Transcriptome 03 medical and health sciences Humans Clustered Regularly Interspaced Short Palindromic Repeats natural sciences Epigenetics RNA-Seq Enhancer Molecular Biology Gene 030304 developmental biology 0303 health sciences Gene map Genome Human Cell Biology Human genome Single-Cell Analysis Biotechnology Genetic screen |
| Zdroj: | Nature methods Nature Methods |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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