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.


CROP-seq overview

News and updates

Date Description
2017/01/18

CROP-seq paper published in Nature Methods

We are excited to share our full article on CROP-seq, which was published on the Nature Methods website today (DOI: 10.1038/nmeth.4177) and will be part of the March print issue. It is accompanied by a complete step-by-step protocol and software to help interested laboratories get started using CROP-seq. If you are interested in our technology, we would like to hear from you: crop-seq@medical-epigenomics.org

No journal subscription? Read it online for free or ask us to send you the PDF.

Protocols and reagents

Updated protocols for CROP-seq and links to wet lab reagents.

Date Description
2017/01/18

CROP-seq step-by-step protocol (Version: 2017/01/18)

Detailed description of the CROP-seq method, separated into three chapters:

  1. Pooled, amplification-free cloning of gRNA libraries
  2. Lentivirus production and cell culture for CROP-seq screens
  3. Single-cell RNA-seq based on Drop-seq

The protocol contains a detailed listing of all required reagents and oligonucleotide sequences.
For each of the major steps, we present and discuss the expected results.

Download CROP-seq protocol

2017/01/18

CROPseq-Guide-Puro vector

Our CROPseq-Guide-Puro vector has been designed to make CRISPR gRNAs detectable by single-cell RNA-seq. We placed the gRNA expression cassette directly into the 3'LTR, so that the antibiotic resistance mRNA acts as a vehicle to detect gRNAs in single-cell transcriptome data. Derived from one of the most popular constructs for pooled CRISPR screening (LentiGuide-Puro), it retains compatibilty with established, one-step library cloning methods.


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CROPseq-Guide-Puro is now available from Addgene (86708. While the Addgene team is still expanding our plasmid feel free to contact us directly)
Please visit our lab's Addgene page for future updates, such as CROP-seq plasmids with fluorescent markers for in vivo and ex vivo use.

Data and software

Links to raw data archives and related resources.

Name Description and link
Raw and processed data GEO accession: GSE92872
Reference genome spiked with gRNA sequences GRCh38 with TCR library: download here.
Software Git repository at Github: https://github.com/epigen/crop-seq/

Citation

If you use this method in your research, please cite:

Paul Datlinger, André F Rendeiro*, Christian Schmidl*, Thomas Krausgruber, Peter Traxler, Johanna Klughammer, Linda C Schuster, Amelie Kuchler, Donat Alpar, Christoph Bock (2016). Pooled CRISPR screening with single-cell transcriptome readout. Nature Methods. DOI: 10.1038/nmeth.4177. No journal subscription? Read it online for free or ask us to send you the PDF.