Schmidtet al.,Science 375 , eabj4008 (2022) 4 February 2022 6 of 12
IFNGIL2
No Targets
Control
DEPDC7DEF6
TAGAPRAC2
VAV1
SignalingGTPase
CEACAM1ARHGDIB
FOXL2NBEOMES
GATA3GRAP
INPPL1IKZF3
JMJD1CITPKA
MAP4K1LAT2
MUC1SLA2
RegulatorsNegative
IL1R1RELA
TRAF3IP2
SignalingNF-B
APOBEC3CABCB10
APOBEC3DAPOL2
BICDL2EMP1
MGST3EMP3
PAPOLGTRIM21
UnknownOther /
IL2RBIL2RG
IL9R
Cytokine Other
Receptor
AKAP12CBY1
NLRC3CNR2
NOTCH1P2RY14
PIK3AP1PLCG2
PRKD2
Other Signal
CD2 Transduction
CD247CD28
LCP2LAT
OTUD7AOTUD7B
Stim/Co-StimTCR
FOSBALX4
FOXD2FOSL1
FOXQ1LHX4
PRDM13LHX6
TBX21WT1
Transcription Factor
CD27LTBR
TNFRSF12ATNFRSF1A
TNFRSF1BTNFRSF9
Receptor TNF
Superfamily
~150 sgRNA / ~70 Target Gene CRISPRa Perturb-seq Library
EOMES
G ATA 3
IFNG
IL2
IL2RB
MAP4K1
VAV1
−2
0
2
−2 IL−2 0 2
IFN−
CRISPRa Genome-Wide Screens
Primary Human
T cells
Perturb-seq CRISPRa
Library
CRISPRa Modified
T Cells
Restimulation
Resting
single cell RNA-seq with sgRNA direct capture
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Cluster
−2 0 2
Z-score
CCL4CCL3
IFNG
CCL4L2
MAL
GZMALT B
GIMAP7
IL13
CSF2IL5
IL4
IL22
TNFRSF4MAF
LGMN
HIST1H1B
HIST1H4CHIST1H1D
HIST1H1E
CENPFASPM
TOP2ACCNB1
NEAT1LTA
TBX21NCR3
GNLYCCL5
LGALS1AHNAK
LRRN3F5
TNFAIP3UBR5
CD8BCD8A
COL6A3
TRERF1
EMP1
TJP2
ERRFI1
TRAF3IP2
PMAIP1ELL2
RAP1B
IL2
TAGAPIL21
PPP1CC
MRPS26
CSTBCCR7
MCM4
G0S2
SP140MYC
TRAF3IP3
Top
Markers
FOXQ1VAV1
TBX21CD28
APOBEC3COTUD7B
MAP4K1MUC1
LAT2SLA2
INPPL1GRAP
G ATA 3
FOXL2NBTNFRSF9
FOXD2LHX4
ITPKAALX4
TNFRSF1BLT B R
TNFRSF1ACD27
EOMESTBX21
FOXQ1FOXD2
FOXL2NBLT B R
CD247TBX21
APOBEC3CBICDL2
PLCG2LCP2
OTUD7BFOSL1
DEF6WT1
FOXD2
PIK3AP1FOSB
DEPDC7CNR2
EMP1
IFNGIL1R1
TNFRSF1ATRAF3IP2
TNFRSF1BBICDL2
VAV1IL2
OTUD7B
IKZF3IL2RB
BICDL2ALX4
NLRC3CD27
PRDM13CD28
Overrep-
resented
sgRNAs
CCL4CCL3
IFNG
CCL3L1
LT B
IL32
CCL5
IL13
CSF2IL5
IL4
LT B
IL22IL10
CXCL10
HMGB1
HMGB1
LT B
LTA
TNF
TNFSF10FAM3C
CCL5CSF1
CCL4CCL3
NAMPTCCL5
LIF
LTA
IFNG
NAMPTLIF
LTA
LTA
IL21IL2
TNF
LIF
LTAIL2
TNF
Upregulated
Cytokine
Genes Cluster
1: IFNG High 1
2: Negative
Regulators
3: Th2
4: IL22 High
5: Proliferative (S)
6: Proliferative
(G2/M)
7: TNF Locus High
8: GNLY High
10: CD8 Common
11: EMP1 Guides
12: IFNG High 2
13: IL2 High 1
14: CD4 Common
15: IL2 High 2
9: CCL3/4 High,
IFNG Low
CD4/CD8 Score
−1
0
1
CD4
CD8
NoTarget ControlsPerturbed Cells MAP4K1
VAV1 TNFRSF1A IL1R1
FOXQ1 G ATA 3 TBX21
IFNG IL2 LTA
LT B IL13 CCL3
A
B
F
G H
I
J
Restimulated Condition:
>28K single cells
Donor: Donor1 Donor2
UMAP 1
UMAP 2
C D
E
log 2 (CD4/CD8)
−4 −2 0 24
CD8+ CD4+
Low High
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*
***
******
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***
*
******
***
***
*
***
***
***
***
***
**
***
***
***
MAP4K1MUC1
SLA2LAT2
INPPL1GRAP
JMJD1CITPKA
ARHGDIBEOMES
FOXL2NBGATA3
CEACAM1CD247
ABCB10IL2
AKAP12ALX4
P2RY14LHX6
APOBEC3CMGST3
NO−TARGETIFNG
PLCG2CBY1
TRIM21TBX21
PAPOLGIKZF3
PRKD2CNR2
APOL2IL2RG
BICDL2IL2RB
NOTCH1TAGAP
APOBEC3DPIK3AP1
RAC2IL9R
OTUD7APRDM13
TNFRSF9LTBR
FOSL1CD2
TRAF3IP2NLRC3
EMP3CD27
TNFRSF12AFOXQ1
EMP1FOSB
LHX4WT1
FOXD2RELA
DEPDC7DEF6
IL1R1L AT
OTUD7BLCP2
TNFRSF1ACD28
TNFRSF1BVAV1
−200 0 200 400 600
Activation Score
CRISPRa Target Gene
ControlNegative RegulatorPositive Regulator
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Cluster
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Activation Score
−100^0100200300400
Fig. 4. CRISPRa Perturb-seq captures diverse T cell states driven by
genome-wide cytokine screen hits.(A) Schematic of CRISPRa Perturb-seq
experiment. (B) Categorical breakdown of genes targeted by the sgRNA library
comprising hits from our primary genome-wide CRISPRa cytokine screens as
indicated. Genes with a summed log 2 -fold change less than zero across both
screens (diagonal line) are categorized as negative regulators. (C) UMAP
projection of post–quality control filtered restimulated T cells, colored by blood
donor. (D) Distribution of CD4+and CD8+T cells across restimulated T cell
UMAP projection. Each bin is colored by the average log 2 (CD4/CD8) transcript
levels of cells in that bin. (E) Restimulated T cell UMAP colored by average cell
activation score in each bin. (F) Boxplots of restimulated T cells’activation
scores grouped by sgRNA target genes. Dashed line represents the median
activation score of no-target control cells. P< 0.05, P< 0.01, P< 0.001,
Mann–WhitneyUtest with Bonferroni correction. (G) Restimulated T cell
UMAP with cells colored by cluster. (H) Heatmap of differentially expressed
marker genes in each cluster. The top 50 statistically significant (FDR < 0.05)
differentially up-regulated genes for each cluster are shown, with genes that are
up-regulated in multiple clusters being given priority to the cluster with the
higher log 2 -fold change for the given gene. To the right of the heatmap are
(left to right), the top marker genes by log 2 -fold change in each clusters’section,
the top overrepresented sgRNAs in each cluster by odds ratio (full data are
provided in fig. S20G), and the top differentially up-regulated cytokine genes in
each cluster. Mean cell log 2 (CD4/CD8) cell transcript values in each cluster are
shown on the far right. (I) Restimulated T cell UMAP with the expression of
indicated genes shown. (J) Contour density plots of restimulated cells assigned
to indicated sgRNA targets in UMAP space. The no-target control contour is
shown in grayscale underneath.“Perturbed cells”represents all cells assigned a
single sgRNA other than no-target control sgRNAs.
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