Science - USA (2022-02-04)

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

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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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