6TG (µM)

1 5516TG (µM)

TSSExonType

KRAB-dCas9(pHR)

KRAB-dCas9(pLX311)

Cas9(pLX311)

HP

RT1

targ

etin

g sg

RN

A

sgRNA (FC)0 8-8

3

2

1

0

1

4 3 2 1 0 1WT Cas9

dCas

9category

exonTSS

Ref seq TSS

Cage seq TSS

0.0 0.25 0.50 0.75 1.00

0.0

0.25

0.50

0.75

1.00

1-specificity

Sen

sitiv

ity

Cage-seq-TSSRef-seq-TSS

1.0

0.5

0.0

1.5

-4 -2 0 2

Before optimizationAfter optimizationNegative Controls

Pro

babi

lity

dens

ity

sgRNA aboundance (Log2[fold change])

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-3

-2

-1

0

1

sgR

NA

Log 2

[FC

]

BRAF

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-4

-3

-2

-1

0

1

sgR

NA

Log 2

[FC

]

KRAS

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-1.5

-1.0

-0.5

0.0

0.5

1.0 CTNNB1

sgR

NA

Log 2

[FC

]

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-3

-2

-1

0

1 ERBB2

sgR

NA

Log 2

[FC

]

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-3

-2

-1

0

1

2 EGFR

sgR

NA

Log 2

[FC

]A37

5_Cas

9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-4

-3

-2

-1

0

1 CDK4

sgR

NA

Log 2

[FC

]

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-4

-3

-2

-1

0

1

sgR

NA

Log 2

[FC

]

HNF1B

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-3

-2

-1

0

1

sgR

NA

Log 2

[FC

]

MAP2K1

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-3

-2

-1

0

1

sgR

NA

Log 2

[FC

]

MDM2

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-4

-3

-2

-1

0

1 MDM4

sgR

NA

Log 2

[FC

]

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-4

-3

-2

-1

0

1

sgR

NA

Log 2

[FC

]

MTOR

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-3

-2

-1

0

1

sgR

NA

Log 2

[FC

]

PIK3CA

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-4

-3

-2

-1

0

1

sgR

NA

Log 2

[FC

]

RPTOR

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-3

-2

-1

0

1

sgR

NA

Log 2

[FC

]

TCF7L2

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-3

-2

-1

0

1

2 NFE2L2

sgR

NA

Log 2

[FC

]

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-6

-5

-4

-3

-2

-1

0

1

sgR

NA

Log 2

[FC

]

MYC

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-2

-1

0

1

sgR

NA

Log 2

[FC

]

EP300

Cell specific

Dependencies

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-4

-3

-2

-1

0

1

sgR

NA

Log 2

[FC

]

PCBP1

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-5

-4

-3

-2

-1

0

1

sgR

NA

Log 2

[FC

]

POLR1C

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-4

-3

-2

-1

0

1 POLR2D

sgR

NA

Log 2

[FC

]

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-4

-3

-2

-1

0

1

sgR

NA

Log 2

[FC

]

PSMD2

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-5

-4

-3

-2

-1

0

1 RPL19

sgR

NA

Log 2

[FC

]

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-6

-4

-2

0

2 RPL4

sgR

NA

Log 2

[FC

]

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-6

-4

-2

0

2 RPL5

sgR

NA

Log 2

[FC

]

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-6

-4

-2

0

2 RPL8

sgR

NA

Log 2

[FC

]

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-4

-3

-2

-1

0

1

sgR

NA

Log 2

[FC

]

U2AF1

Pan essential D

ependenciesCas

9 an

d KR

AB-d

Cas

9 co

nsis

tent

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-5

-4

-3

-2

-1

0

1

sgR

NA

Log 2

[FC

]

DHPS

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-3

-2

-1

0

1

2

sgR

NA

Log 2

[FC

]

APC

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-2

-1

0

1 ST20

sgR

NA

Log 2

[FC

]

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-2

-1

0

1

2

sgR

NA

Log 2

[FC

]

STK11

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-4

-3

-2

-1

0

1

2

sgR

NA

Log 2

[FC

]

TP53

Tumor

suppressors

Cas

9 an

d KR

AB-d

Cas

9 in

cons

iste

nt

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-2

-1

0

1

sgR

NA

Log 2

[FC

]

SMARCA2

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-3

-2

-1

0

1

sgR

NA

Log 2

[FC

]

NAMPT

A375_

Cas9

A375_

KRAB-dCas

9

HT29_C

as9

HT29_K

RAB-dCas

9

A549_

Cas9

A549_

KRAB-dCas

9

MIAPACA2_

Cas9

MIAPACA2_

KRAB-dCas

9-2

-1

0

1

2

sgR

NA

Log 2

[FC

]

HPRT1 Non Essential

Cell specific

Dependencies

Pan essential D

ependencies

Beforeoptimization

Afteroptimization

0

20

40

60

80

Effe

ctiv

e sg

RN

As ta

rget

ing

core

ess

entia

l gen

es (%

)A B

C

D

G

F

E

H

Supplementary Figure 1: Loss of function screens with Cas9 and KRAB-dCas9 using a

tiling sgRNA library. (A) Heat-map showing unsupervised hierarchal clustering of

proliferation changes induced by suppression of HPRT1 using different concentrations of 6TG

and Cas9 or KRAB-dCas9 (in pLX311 or pHR). (B) Comparison of proliferation changes

induced by Cas9 and KRAB-dCas9 following introduction of HPRT1 targeting sgRNAs in A375

cells treated with 15 µM of 6TG. TSS targeting sgRNAs are colored in blue and exon targeting

sgRNAs are colored in red. (C) Proliferation changes induced by sgRNAs targeting the

indicated genes, in cells expressing Cas9 (red) or KRAB-dCas9 (blue). Only efficient sgRNAs

(as determined by the sgRNA predictive algorithm) are shown for KRAB-dCas9 and only exon

targeting sgRNAs are shown for Cas9. (D) Performance of sgRNA predictive model on core

cell essential genes using NCBI ref seq TSS annotations or (E) FAMTOM CAGE-Seq TSS

annotations. (F) ROC curve of sgRNA predictive model using TSS annotations from RefSeq

(blue) or CAGE-Seq (red). (G) Distribution of sgRNAs targeting core cell essential genes

before and after using the sgRNA optimization algorithm. (H) Percent of effective sgRNAs

targeting core cell essential genes before and after sgRNA optimization algorithm.

Cas9

p < 0.0010.001 < p < 0.010.01 < p < 0.05p > 0.05

KRAB-dCas9

p < 0.0010.001 < p < 0.010.01 < p < 0.05p > 0.05

Distance to nearest essential gene (kbp)

esse

ntia

l gen

es in

C

RIS

PRi s

cree

n (%

)

<0.2 0.2-0.5 >10050-10020-5010-205-100.5-1 1-2 2-50

25

50

100

75

*** ******

High confidence Low confidence Not significant

C

0 200 400 600 800 1000 12000

3

6

9

12

15

Distance threshold for defining bidirectional promoters

Ref

seq

gene

s (%

)

D

E

sgR

NA

Log 2

[FC

]

Number of perfect alignments

−2

−1

0

1

1 2−4 5−9 >9

18 bp alignment

Cas9KRAB-dCas9

sgR

NA

Log 2

[FC

]

Number of perfect alignments

−2

−1

0

1

1 2−4 5−9 >9

17 bp alignment

Cas9KRAB-dCas9

A B

F

G

1 2−4 5−9 >9

−2

−1

0

1

Number of perfect alignments

sgR

NA

Log 2

[FC

]

1 missmatch

Cas9KRAB-dCas9

Supplementary Figure 2: Comparison of Cas9 and KRAB-dCas9 experiments in

identifying core essential genes. P-values were computed based on z-test, where the null

distribution were computed based on sgRNAs targeting AAVS locus. (A) sgRNAs targeting

exons were used for Cas9 analysis. (B) sgRNAs targeting primary CAGE-Seq TSSs were

used in KRAB-dCas9 analysis. (C-E) Violin plot showing distribution of proliferation changes

induced by intron-targeting sgRNAs that are aligned to multiple genomic loci, in Cas9 (red) or

KRAB-dCAs9 (blue) expressing cells. (C) 1 mismatch was allowed when aligning these

sgRNAs to the human genome. (D) Only 17 bp in 3’ of the spacer are used for alignment. (E)

Only 18 bp in 3’ of the spacer are used for alignment. (F) Percent of genes that are scored to

be cell essential in CRISPRi. Genes were categorized based on their distance to nearest cell

essential gene scored in CRISPRc. (G) Percent of RefSeq genes that are associated with

bidirectional promoters, which were defined based on the threshold of distance between the

TSSs of two proximal genes transcribed towards opposite directions.