1 Data Settings
Source:
Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function, GEO accession: GSE119450.
The following analysis follows a similar approach as used in this reference paper to generate Fig 4.
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 and without T cell receptor (TCR) stimulation.
All cells were pooled together into 1 analysis.
2 scRNA-seq data
2.1 Preprocessing
There are 24955 cells with a single type of gRNA readout using the quality control criteria of % mitochondria gene expression < 10 and number of unique genes > 500.
Gene expressions were normalized as log(count per \(10^4\) + 1), and then scaled.
Library size and mitochondria percentage were corrected for, while batch effect was not.
Top 1000 most variable genes were used to perform PCA, and the first 30 PCs were used to perform UMAP.
2.2 UMAP representation
Donors and stimulation status:
Clustering of cells: 
Expression patterns of marker genes:
activation state (IL7R, CCR7), cell cycle (MKI67), and effector function (GZMB) in the reference paper. 
Cluster 1, 2 and 3: resting state
Cluste 5 and 6: proliferation
3 CRISPR Perturbations
3.1 Over/Under-Representation of gRNA Targets in Clusters
Pairs of associations that passed chi-square test FDR < 0.05:
13
Perturbations with significant enrichment or depletion in clusters: BTLA, CBLB, CD3D, HAVCR2, LCP2, NonTarget, SOCS1, TCEB2, TNFRSF9
Selected clusters and gRNA targets:
4 Stimulated cells only
4.1 Clustering of cells
Expression patterns of marker genes:
activation state (IL7R, CCR7), cell cycle (MKI67), and effector function (GZMB) in the reference paper. 
4.2 Over/Under-Representation of gRNA Targets in Clusters
Pairs of associations that passed chi-square test FDR < 0.05:
6
Perturbations with significant enrichment or depletion in clusters: BTLA, CD3D, CDKN1B, HAVCR2, LCP2