Science - USA (2022-02-04)

Schmidtet al.,Science 375 , eabj4008 (2022) 4 February 2022 3 of 12

TAF4TT

PTPRC

CD3G

VVAVAV1IL2

ZAP70

CD3D

CD3E

LCP2

CD2CD28

CD247

ITK

WASWW DEF6RAC2

GRAP2

PLCG1

L A TA

JAK3

IL2RG

NRF1

SNRPC RNF40

HGS

RPL38

CD5

CPSF4

CBLB

PDGFRA

EIF3D

CD3G

VVAVAV1

ZAP70 CD3D

LCP2 CD3E

CD28CD2

CD247 ITK

WASWW

RAC2

PLCG1

L A TA

PTPRC

SRP68

TRAF6

SPTLC2

SRP19

PI4KB

MAP3K7

ST ATTAT3

PCYT2

JAK1

IFNGLCK

NMT1 NFKB2

TSC1

RNF40

RNF20

RPL19

CD5

CBLB

A B

D E

−10

−5

0

5

10

15

−10 0 10

CRISPRi IL-2 Screen Z-score (IL-2lo/IL-2hi)

CRISPRa IL-2 Screen Z−score (IL-2

hi/IL-2

lo)

IL-2 Screens:

KEGG T CELL RECEPTOR SIGNALING PATHWAY

−5

0

5

10

−5 0 5 10

CRISPRi IFN- Screen Z−score (IFN-lo/IFN-hi)

CRISPRa IFN−

Screen Z−score (IFN-

hi/IFN-

lo)

IFN- Screens: NF-B Pathway Regulators

−2

−1

0

1

2

−4 −2 0 2

Donor 1, log 2 FoldChange

Donor 2, log

FoldChange 2

IL-2 CRISPRi Screen

−4

−2

0

2

−4 −2 0 2

Donor 1, log 2 FoldChange

Donor 2, log

FoldChange 2

IFN- CRISPRi Screen

Screen Hit

CRISPRa Only

CRISPRi and CRISPRaCRISPRi OnlyNot a Hit

Not KEGG TCR path

Screen Hit

CRISPRa Only

CRISPRi and CRISPRa

CRISPRi Only

Not a Hit

Hit Category

Positive regulator

Not a hit

Negative regulator

Hit Category

Positive regulator

Not a hit

Negative regulator

BCL10

CARD11

CD27

CD40

CHUK

IKBKB

IKBKG

IL1R1

LT B R

MALT1

MAP3K7

NFKB1

NFKBIA

RELA

TNFRSF12A

TNFRSF1A

TNFRSF1B

TNFRSF4

TNFRSF8

TNFRSF9

TRAF6

CBLB

CD247

CD28

CD3D

CD3ECD3G

CD4

GRAP2

GRB2

ICOS IL2

ITK

L AT

LCK

LCP2

NFATC2

PIK3CA

PLCG1

PPP3CC

PTPN6

PTPRC

RASGRP1

VAV1

VAV3 ZAP70

way

G H

ANXA2R

APOL2

EBF2 BRD9

EMP1

EP400

FOXD2 FOXF1

FOXL2

FOXO4

FOXQ1 HELZ2

HGS

IRX4

JMJD1C

LHX6

MUC1 MUC21

NMT1

NR4A3

PDGFRA

RNF20

RNF40

TRIM21

ZEB2

EMP3

Transcription Factors ChromatinRemodeling

E3 Ubiquitin Ligases

Receptors

GCSAM GCSAML

KIDINS220 PIK3AP1

Signal Transduction

APOBEC3A

APOBEC3D

APOBEC3C

RNA Base Editors

IL2RB

CD28

CD247

CD3E CD3G CD3D LAT

ZAP70

LCP2

VAV1

ITK

PLCG 1

PLCG2

LCK

DEF6 RAC2

WAS

CD2

SCRIB

FOSL1

JUN NFATC2

GRB2

MAP4K1

GRAP2

PIP2

IP 3

DAG

PTPRC

CD5 CBLB

PI

PI 4 KB

TNFRSF 1 A TNFRSF 1 B

TRAF6

TRAF3IP2

MAP3K7

PRKCB

MALT1

BCL10

CHUK

IKBKB

IKBKG

NFKBIA

NFKB1 RELA

OTUD7B

IL2RG

IL1R1

IL9R

JAK1

JAK3

STAT3

Ca^2 +

FOSB

Cytoskeleton

Cell polarization Reorganization

SLA2

NFKB2

IFNGR2

IL2RA

PRKD2

CD27

TNFRSF9

TNFRSF8

TNFRSF12A

LTBR

TBX21 GATA3

PTPN6

− 10

− 5

0

5

10

Z-score

regulatorPositive

Negativeregulator

CRISPRi

IFN-

CRISPRa

IL-2

MAP4K1 CD3E TNFRSF 1 A

IL-2IFN- IL-2IFN-

Core Negative Regulator Positive RegulatorCRISPRi SpecificPositive RegulatorIFN-Specific

PTPRC

IL-2IFN-

Discordant Hit

SOCS3

C

TCR Stimulation /

Co-Stimulation

0

5

10

CRISPRi CRISPRa

log

(TPM+0.1) 2

F

CARD11

TRAF6

MALT1 BCL10 MAP3K7

CHUK

TNFRSF1ATNFRSF1B TNFRSF4

TNFRSF9

TNFRSF12A

LTBR

CD27

IL1R1

IKBKB

IFNG

CRISPRi only

CRISPRa only

Both CRISPRi/a

Not a hit

IFN- Screens Hit

IKBKG

TNFRSF8

CD40

RELA NFKB1

NFKBIA

Fig. 2. Integrated CRISPRa and CRISPRi screens mapping the genetic circuits
underlying T cell cytokine response in high resolution.(AandB) Median
sgRNA log 2 -fold change (high/low sorting bins) for each gene, comparing CRISPRi
screens in two donors, for IL-2 (A) and IFN-g(B) screens. (C) Distributions of
gene mRNA expression for CRISPRa and CRISPRi cytokine screen hits in resting
CD4+T cells (this study). (D) Comparison of IL-2 CRISPRi and CRISPRa screens with
genes belonging to the TCR signaling pathway (KEGG pathways) indicated in
colors other than gray. (E) Comparison of IFN-gCRISPRi and CRISPRa screens with
manually selected NF-kB pathway regulators labeled. All other genes are shown
in gray. (F) Map of NF-kB pathway regulators labeled in (D). (G) Map of screen hits
with previous evidence of defined function in T cell stimulation and costimulation

signal transduction pathways. Genes shown are significant hits in at least one screen

and were selected based on review of the literature and pathway databases (e.g.,

KEGG and Reactome). Tiles represent proteins encoded by indicated genes with the

caveat that, because of space constraints, subcellular localization is inaccurate

because many of the components shown in the cytoplasm occur at the plasma

membrane. Tiles are colored according to log 2 -fold changeZscore, as shown

in the subpanel, with examples of different hits. Large arrows at the top represent

stimulation/costimulation sources. (H) Select screen hits with less well-described

functions in T cells in the same format as (G). For (H), only significant hits from

the top 20 positive and negative ranked genes by log 2 -fold change for each screen

were candidates for inclusion.

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