Source:
Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function, GEO accession: GSE119450.
Perturbations:
CRISPR knock-out of 20 genes (2 gRNAs per gene) + 8 non-targeting gRNAs. Guide conditions were defined on the target gene level; target genes were either found to regulate T cell responses in the genome-wide screens, or known checkpoint genes.
Guide RNAs were introduced into T cells through a novel procedure called sgRNA lentiviral infection with Cas9 protein electroporation (SLICE).
Cells:
Primary human CD8+ T cells from two healthy donors, with T cell receptor (TCR) stimulation.
Cells from 2 donors were pooled together into 1 analysis.
There are 13983 cells with a single type of gRNA readout using the quality control criteria of % mitochondria gene expression < 10 and total UMI count < 2e+4.
Only genes detected in > 10% of cells were kept, resulting in 6062 genes.
Seurat “LogNormalize”: log(count per \(10^4\) + 1).
Batch effect, unique UMI count, library size, and mitochondria percentage were all corrected for.
Cells are colored by the mean expression of signature genes at the corresponding cell cycle stage:
From the reference paper: activation state (IL7R, CCR7), cell cycle (MKI67), and effector function (GZMB).
Cells are colored by the mean expression of signature genes at the corresponding cell cycle stage:
From the reference paper: activation state (IL7R, CCR7), cell cycle (MKI67), and effector function (GZMB).
The expression of MKI67 coincides with cells at the mitotic stage.
KO | ARID1A | BTLA | C10orf54 | CBLB | CD3D | CD5 | CDKN1B |
DE_genes | 14 | 0 | 0 | 15 | 5 | 6 | 1 |
KO | DGKA | DGKZ | HAVCR2 | LAG3 | LCP2 | MEF2D | NonTarget |
DE_genes | 0 | 0 | 0 | 1 | 39 | 0 | 0 |
KO | PDCD1 | RASA2 | SOCS1 | STAT6 | TCEB2 | TMEM222 | TNFRSF9 |
DE_genes | 0 | 9 | 0 | 5 | 73 | 0 | 1 |