Science - USA (2022-01-21)

318 21 JANUARY 2022¥VOL 375 ISSUE 6578 science.orgSCIENCE

image-enabled cell sorting

density

correlation RelA-mNG/DRAQ5

C

E

D

gene rank

statistical significance Z−score

(MAUDE)

gene symbol

IKBKA

R = -0.76

p = 0.017

IKBKG

MAP3K7

log2 FC upper bin

vs. before sort

log2 FC lower bin

vs. before sort

R = 0.91

p = 0.00079

−4

−3

−2

−1

0

0

1

2

3

0.0 0.1 0.2 0.3

coverage (cells per gRNA)

number of

1 gRNAs per gene

2

3

4

5

6

12 2448 6071108155

AUPRC

gene essentiality:

gene essentiality:

0.2

0.4

0.6

0.8

F

AT I C

IKBKA

FBXW11

HDAC3

IKBKB

IKBKG

MAP3K7

NFKBIA

PREP

RBCK1

RIPK1

SEPHS1

TNFAIP3

TNFRSF1A TRADD

TRAF2 VCPIP1

−8

−4

0

−8 −4 0

upper bin compared to plasmid library

(log2 fold change)

lower bin compared to plasmid library

(log2 fold change)

NFKBIA

(+TNFα−Dox) − (+TNFα+Dox)

G

I

TNFRFS1A

cytoplasm

nucleus

IKK complex

NFKB1RelA

IKBKBIKBKG

IKBKA

MAP3K7

TRADDTRAF2

TAB1

RIP1TRAF5

TRRAP

SAGAcomplex

canonical core NF-κB pathway

INO80complex

0

6

-3 -2 -1 0

sgRNA Z−score (MAUDE)

density

TNFRSF1A

TRADD

TRAF2

RIPK1

TRAF5

TAB1

MAP3K7

IKBKB

IKBKG

IKBKA

NFKBIA

FBXW11

RELA

significant (FDR < 1%) hit (panel G, I)not significant (panel G, I) NFKB1

012

012

-log2 fold change

0

8

−0.50 −0.25 0.00 0.25

sgRNA Z−score (MAUDE)

density

TADA1

SUPT7L

SUPT3H

TAF6L

SGF29

TAF5L

INO80C

ACTR5

INO80E

INO80B

-log2 fold change

readout (RelA-mNeonGreen)

J

ICS (pooled)

upper lower enriched in bin

ICS (individual)

microscopy

VCPIP1−3PREP−2AMBRA1−3CSDE1−1

ATIC−3IKBKG−1KAT2A−3STAG2−4

INO80E−3IKBKG−3INO80C−3DUSP1−3CRTC3−2ACTR5−3SUPT3H−2ACTR8−4TAF6L−3TAF5L−1INO80B−2INO80−3TADA1−2SGF29−1IKBKA−2IKBKA−1IKBKA−3SUPT7L−3SEPHS1−4MAP3K7−1MAP3K7−2MAP3K7−3IKBKG−2

phenotype

−4

−2

0

2

immune signaling chromatin modification others

gene silencing

H

GCN5

TADA3SGF29

TADA2b

TADA1TAF9TAF6LTAF10SUPT7L

TAF12TAF5L

SUPT3H

TRRAP SUPT20H

ACTL6A

INO80

TCF3 ACTR8

MCRS1

INO80E

NFRKB

INO80D

RUVBL1RUVBL2

INO80C

ACTR5INO80B

0

1

2

−0.5 0.0 0.5

density

−0.5 0.0 0.5 −0.5 0.0 0.5

0.40.8−0.5 0.0 0.5 −0.5 0.0 0.5

−0.5 0.0 0.5 −0.5 0.0 0.5 - 0. 4 0.0 4.0 0. 8

−Dox−TNFα −Dox+TNFα +Dox−TNFα +Dox+TNFα

0

1

2

−0.4 0.4 0.8

density

0.0 −0.50.0 0.5 −0.50.0 0.5

nt-1 nt-2 nt-3

IKBKA- 1 IKBKA- 2 IKBKA- 3

IKBKG-1 IKBKG-2 IKBKG-3

MAP3K7-1 MAP3K7-2 MAP3K7-3

correlation RelA-mNG/DRAQ5

treatment

−0.4 0.0 gRNA library synthesis lentiviral library

gRNA selection

Cas9 induction

recovery

input sample

before sort

TNFα stimulation

lower

5% bin

upper

5% bin

HeLa RelA-mNeonGreen

Tet::Cas9

A B

TRADD

TRAF2

RIPK1

FBXW11NFKBIA

TNFRSF1A

MAP3K7IKBKA

IKBKB

IKBKG

RBCK1

PDPK1

NFKBIB

TNFAIP3

−60

−40

−20

0

0 300 600 900

snoRNA processing

Fig. 3. ICS detects the effects of CRISPR perturbations and enables
pooled genetic screens of protein localization.(A) Effects of individual
CRISPR perturbations on RelA nuclear translocation. HeLa cells with Tet-
inducible Cas9 and stably expressing RelA-mNG were transduced with gRNA-1,
gRNA-2, and gRNA-3 targeting the core NF-kB pathway proteins IKBKG, IKBKA,
and MAP3K6, respectively, or with nontargeting (nt) control gRNAs. gRNA
expression was induced with doxycycline (Dox) or left uninduced. Correlation
between RelA-mNG and DRAQ5 was quantified using ICS as a measurement for
RelA nuclear translocation in the presence or absence of TNFa.(B) Overview
of the pooled CRISPR screening setup and readout using ICS. Positive regulators
of RelA nuclear translocation are enriched in the lower bin and depleted from
the upper bin. Tet::Cas9, tetracycline/doxycycline–inducible Cas9. (CtoE) Results

of the ICS-based CRISPR screen using an NF-kB pathway–focused library (n=

1068 genes). (C) The screen was performed at different library coverages,

and reads from collected samples were combined in silico to a high-coverage

(359 cells per gRNA per sorted bin) dataset. Hits were called using the software

MAUDE ( 26 ). Genes are ranked by their statistical significance and selected

positive/negative regulators are highlighted. The horizontal dashed lines indicate

an FDR of 1%, whereas genes with FDR <1% are marked in cyan and orange,

respectively. (D) Comparison of phenotypes measured in individual perturbation

experiments from (A) (xaxis) or the pooled screen (yaxis) using the same

gRNAs. For the pooled screen, differences in gRNA abundance in the upper (top

panel) and lower (bottom panel) sorted bins compared with the input sample

were determined from the high-coverage dataset in (C).Rvalues represent

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