Pooled CRISPR screening with single-cell transcriptome read-out

By Paul Datlinger, André F. Rendeiro, Christian Schmidl, Thomas Krausgruber, Peter Traxler, Johanna Klughammer, L. Schuster, Amelie Kuchler, D. Alpár, Christoph Bock. Nature Methods, 2017

998 citations recorded by the source corpus.

CRISPR-based genetic screens are accelerating biological discovery, but current methods have inherent limitations. Widely used pooled screens are restricted to simple readouts including cell proliferation and sortable marker proteins. Arrayed screens allow for comprehensive molecular readouts such as transcriptome profiling, but at much lower throughput. Here we combine pooled CRISPR screening with single-cell RNA sequencing into a broadly applicable workflow, directly linking guide RNA expression to transcriptome responses in thousands of individual cells. Our method for CRISPR droplet sequencing (CROP-seq) enables pooled CRISPR screens with single-cell transcriptome resolution, which will facilitate high-throughput functional dissection of complex regulatory mechanisms and heterogeneous cell populations.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/17885ef18992e52763ed7692da1e50e12603cc0a

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Molecular Biology

Citations in the corpus

Predicting cellular responses to complex perturbations in high‐throughput screens (2023, background): “To study such genetic interactions (GIs), recent perturbation scRNA-seq assays allow us to measure the gene expression response of a cell to the perturbation of genes alone or in combination (Dixit et al , 2016; Datlinger et al , 2017).”
Multiplexed droplet single-cell RNA-sequencing using natural genetic variation (2017, background): “…natural genetic variation reduces the per-sample and per-cell library preparation cost of single-cell RNA-sequencing, does not require synthetic barcodes or split-pool strategies 36–40 , and captures biological variability among individual samples while limiting unwanted technical variability.”
Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq (2021, background): “, Perturbseq, CROP-seq) (Adamson et al., 2016; Datlinger et al., 2017; Dixit et al., 2016; Jaitin et al., 2016; Replogle et al., 2020), technical advances have enabled their application to epigenetic (Rubin et al.”
Computational principles and challenges in single-cell data integration (2021, methodology): “Similar methods can be applied to test for regulatory effects of genetic perturbations (rather than natural variation) on different molecular readouts, through the use of assays such as Perturb-seq 58,59 ,CROP-seq 60 and TAP-seq 61 .”
Spatial components of molecular tissue biology (2022, positive): “Finally, recent advances in pooled perturbation screens with high content readouts, either optically 42,43 or by single-cell profiling 16,44 , open the way to inform and test such causal models, by observing both cell biology and molecular features under a large number of perturbations in cell…”
CRISPR technology: A decade of genome editing is only the beginning (2023, background): “We are only beginning to see the impact of combining CRISPR screens and single-cell multiomics modalities with the rapidly advancing infrastructure for big data collection and analysis (64, 66, 67).”
Falco: high-speed FastQC emulation for quality control of sequencing data (2019, neutral): “New sequencing protocols are constantly being introduced 7,8 , and as the cost of sequencing per base decreases, sequencing data is growing in abundance, dataset size and read length 9 .”