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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