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CRISPR Plus Single-Cell Sequencing Combines Benefits of Pooled, Arrayed Screens

NEW YORK (GenomeWeb) – Researchers have published a new study on BiorXiv in which they combined CRISPR/Cas9 screening with single-cell transcriptome sequencing to combine the benefits of both pooled and arrayed screens.

Led by first author Paul Datlinger and senior author Christoph Bock, researchers at the CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences described how they combined targeted genome editing and droplet-based RNA-seq to dissect T cell signaling pathways.

Pooled genetic screens can edit cells in bulk but are limited to a simple readout like cell survival.  Arrayed screens can give much more specific data, but are tedious and expensive because each sample has to be processed separately, the researchers said in a statement. So they came up with a method to get the benefits of high-throughput and rich data, called CRISPR Drop-seq (CROP-seq), which uses the guide RNA (gRNA) expression as a barcode to identify which gene was edited.

"This approach allowed us to link guide-RNA expression to the associated transcriptome responses in thousands of single cells using a straightforward and broadly applicable screening workflow," the authors wrote.

The method involves a special gRNA vector that enables researchers to detect the individual gRNA through RNA-seq. The researchers also had to develop a bioinformatics pipeline to assign single-cell transcriptomes to unique gRNAs.

They generated three HEK293T cell lines by knocking out one of three genes with a single gRNA. "By performing the infections independently, we ensured that each cell received only one gRNA — even in the rare case of multiple infection events."

The researchers then pooled the cell lines in an equal ratio mix and performed Drop-seq. "We observed high sequencing coverage at the gRNA, which allowed us to assign between 30 percent and 60 percent of high-quality transcriptomes to one gRNA," they said.

After establishing that the method could work, they applied it to a screen for T cell receptor activation in Jurkat cells, targeting six genes regulating signaling and 23 transcription factors.

"We observed that gRNAs for target genes immediately downstream of the TCR (such as the kinases LCK and ZAP70 and the adapter protein LAT) had a strong negative effect on the TCR activation signature, consistent with their crucial role in signal propagation and amplification," they wrote. "Targeting of RUNX2, NR4A1, and EGR2 strongly decreased the TCR activation signature in our model, indicating a potential role of these proteins in TCR signaling. This is in line with recent reports identifying RUNX2 as a potential activator of an early CD8 cytokine and effector signature and with the surprising finding that EGR2 can have activating functions during the differentiation of naïve peripheral T cells and influenza infection."

"With falling single-cell sequencing costs, this technology could give rise to the first comprehensive maps of gene-by-gene and combinatorial knockout maps of complex vertebrate genomes," they said.