Schmidtet al.,Science 375 , eabj4008 (2022) 4 February 2022 5 of 12
APOBEC3C
APOBEC3D
FOXQ1OO
IFNG
IL2
IL2RB
LAT2AA
MAP4K1
OTUD7B
PIK3AP1
TNFRSF1B
TRIM21
VVAVAV1
IL−2 IFN- TNF-
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IL-2
C D
E
CD4
IL-2 IFN- TNF-
Control
VAV1
CRISPRa
68.9 9.66
17.0 4.41
63.9 14.3
20.7 1.07
50.6 25.2
20.8 3.32
30.7 45.5
18.1 5.72
55.2 21.4
13.1 10.4
22.9 51.7
14.3 11.1
sgRNA
−2 0 2 −2 0 2 −2 0 2
−2
0
2
CD4+
CD8
+
Category Control Negative both cytokines Positive both cytokines Positive IFN- only Positive IL-2 only
log 2 FoldChange(CRISPRa sgRNA / Control sgRNAs)
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
VAV1_1VAV1_2
TRIM21_1TRIM21_2
PIK3AP1_1PIK3AP1_2
OTUD7B_1OTUD7B_2
FOXQ1_1FOXQ1_2
APOBEC3C_1APOBEC3C_2
TNFRSF1A_1TNFRSF1A_2
IL1R1_1IL1R1_2
IFNG_1IFNG_2
LHX6_1LHX6_2
IL2RB_1IL2RB_2
IL2_1IL2_2
NO−TARGET_1NO−TARGET_2
MAP4K1_1MAP4K1_2
LAT2_1LAT2_2
VAV1_1VAV1_2
TRIM21_1TRIM21_2
PIK3AP1_1PIK3AP1_2
OTUD7B_1OTUD7B_2
FOXQ1_1FOXQ1_2
APOBEC3C_1APOBEC3C_2
TNFRSF1A_1TNFRSF1A_2
IL1R1_1IL1R1_2
IFNG_1IFNG_2
LHX6_1LHX6_2
IL2RB_1IL2RB_2
IL2_1IL2_2
NO−TARGET_1NO−TARGET_2
MAP4K1_1MAP4K1_2
LAT2_1LAT2_2
% Gated
Category
Positive both cytokines
Positive, IFN- specific
Positive, IL-2 specific
Negative both cytokines
Control
Stimulation
+ Stimulation
- Stimulation
IFN- TNF-
CD4
CD8
G
APOBEC3C_2
FOXQ1_1 IFNG_1
IL1R1_1
IL2_1
IL2RB_1LAT2_1
LHX6_1
MAP4K1_1
NO−TARGET_1
NO−TARGET_2
OTUD7B_1
PIK3AP1_1
TNFRSF1A_1
−5.0 VAV1_1
−2.5
0.0
2.5
5.0
−4 0 4
PC1 (40.9% explained variance)
PC2 (16.0% e
xplained va
riance)
Type 1 Type 2 IL-17 IL-10IL-9Chemokines IL3−CSF2 locus
IFN-IL−2TNF-TNF-IL−4IL−5IL−13IL−17AIL−17FIL−10IL−9MIP−1MIP−1
RANTES
IL−8
MIG/CXCL9
IP−10GM−CSFIL−3
NO−TARGET_1
NO−TARGET_2
LAT2_1
MAP4K1_1
APOBEC3C_2
PIK3AP1_1
FOXQ1_1
VAV1_1
OTUD7B_1
TNFRSF1A_1
IFNG_1
IL1R1_1
IL2_1
IL2RB_1
LHX6_1
Cytokine Category
Gene Category
−2
−1
0
1
2
ColumnZ-score
F H
**
***
**
**
*
***
**
***
IL-2 IFN- TNF-
Contr
ol
Negati
ve both cytokinesPositive, IL-2 spec
ific
Positiv
e, IFN
- spe
cific
Positiv
e both cyto
kines Control
Negative both cytoki
nes
Positi
ve both cytoki
nes Control
Negati
ve both cytoki
nes
Positiv
e both cytok
ines
0
20000
40000
60000
0
20000
40000
60000
0e+00
1e+05
2e+05
Cytokine Concentration (pg/ml)
Positiv
e, IL-
2 specific
Positi
ve, IFN
- specific
Positiv
e, IL-
2 specific
Positi
ve, IFN-
specific
Positive both cytokines
Positive, IFN- specific
Positive, IL-2 specific
Negative both cytokines
Paralog of gene in array
Screen hit category
APrimary Human
Bulk T cells
Arrayed CRISPRa T cells
Arrayed sgRNA
plasmid cloning
sgRNA Lentiviruses
dCas9-VP64
Transduction
sgRNA
Transduction
dCas9-VP64
Lentivirus
Screen validation and
further characterization
RT-qPCR overexpression
validation
Intracellular cytokine
staining
Secreted cytokines in
supernatant
B
−2
0
2
−2 0 2
log 2 FoldChange(IL-2hi/IL-2lo)
log
FoldChange (IFN- 2
hi/IFN-
lo)
CRISPRa Screens
Fig. 3. Characterization of CRISPRa screen hits by arrayed profiling.
(A) Schematic of arrayed experiments. (B)ComparisonofIL-2(inCD4+Tcells)and
IFN-g(in CD8+T cells) CRISPRa screens, with genes targeted by the arrayed sgRNA
panel indicated, as well as their screen hit categorization. Paralogs of arrayed panel
genes that were also highly ranked hits are additionally indicated. (C) Representative
intracellular cytokine staining flow cytometry for indicated cytokines in control
(NO-TARGET_1 sgRNA) orVAV1(VAV1_1 sgRNA) CRISPRa T cells after 10 hours of
stimulation. (D) Intracellular cytokine staining of full arrayed sgRNA panel, showing
the percentage of cells that gated positive for the indicated cytokines in CD4+or CD8+
T cells. Points represent the mean value of four donors, with and without stimulation.
Dashed vertical lines represent the mean no-target control sgRNA control value
with stimulation. *q< 0.05, **q< 0.01, Mann–WhitneyUtest, followed byqvalue
multiple-comparisons correction. Full data are provided in fig. S11B. The medium
stimulation dose is shown for IL-2 and IFN-g, and low-dose stimulation is shown for
TNF-a.(E) Scatter plot comparison of log 2 -fold changes in the percentage of
cytokine-positive cells for arrayed panel sgRNAs versus the mean of no-target control
sgRNAs in stimulated CD4+and CD8+cellsusingthesamedatafrom(D).(F) Secreted
cytokine staining arrayed panel grouped by indicated gene categories, with sgRNAs
targeting theIL2andIFNGgenes removed. Points represent a single gene and
donor measurement. *P< 0.05, **P< 0.01, ***P< 0.001, Mann–WhitneyUtest.
(G) Principal component analysis of secreted cytokine measurements resulting
from the indicated CRISPRa sgRNAs. (H) Heatmap of selected secreted cytokine
measurements grouped by indicated biological category. Values represent the
median of four donors, followed byZ-score scaling for each cytokine.
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