Science - USA (2022-02-04)

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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******

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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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