1640 3479 9911

MYH 6

MESP 1

G AT A4 PR

D M1 4

EEF 2

G APD H

EEF 1A 1 0.001

0.01 0.1 1 10 100 1000 10000 100000

T P M

1640 3479 9911

Closed chromatin loci Open chromatin loci

Figure S1. TPM values for MYH6, MESP1, GATA4, PRDM14, EEF2, GAPDH, and EEF1A1 in human iPSCs. The human iPSC RNA-seq data (n = 6 biological replicates) were analyzed using the web-based platform Galaxy. We use the Salmon program to determine transcripts per million (TPM) by aligning the data with the human transcriptome (Gencode Release 19, GRCh37.p13). Low TPM (<1) indicates nonexpression of the gene, and the chromatin area is closed, whereas high TPM is associated with open chromatin.

0.05 0.20 0.28

52.1

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

10 20 30

HDR (%)

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

10 20 30 40

NHEJ (%)

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

20 40 60

HDR/(HDR+NHEJ) (%)

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

10 20 30 40

HDR (%)

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

10 20 30 40

NHEJ (%)

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

20 40 60 80

HDR/(HDR+NHEJ) (%)

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

10 20 30 40

HDR (%)

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

10 20 30 40

NHEJ (%)

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

20 40 60 80

HDR/(HDR+NHEJ) (%)

A

C

Schematic of HDR editing at GATA4

Cas9-sgDocut Cas9-sgDocut

Cas9-sgGATA4

Exon 7

HDR pD-GATA4(HA)-sg HA HA

E7 6bp

E7

Schematic of HDR editing at MESP1

Cas9-sgDocut Cas9-sgDocut

Cas9-sgMESP1

Exon 2

HDR pD-MESP1(HA)-sg

HA HA

Exon 2 15bp

Schematic of HDR editing at MYH6

Cas9-sgDocut Cas9-sgDocut

Cas9-sgMYH6

Exon 39

HDR pD-MYH6(HA)-sg HA HA

E39 6bp

E39

E

**** ****

**

****

ns

****

****

***

****

****

ns

*

**** ****

HDR and NHEJ editing at MESP1

HDR and NHEJ editing at MYH6

HDR and NHEJ editing at GATA4 ns

***

ns

* *

**** ****

** **

****

ns ns

ns

Figure S2. Fusion proteins of Cas9 and VP64 or EP300core decrease gene editing efficiency at closed loci. (A, C, E) Schematic of HDR editing at three closed chromatin loci (MESP1, MYH6, and GATA4). (A) sgMESP1 targets the stop codon of MESP1 gene. (C) sgMYH6 targets the stop codon of MYH6 gene. (E) sgGATA4 targets the stop codon of the GATA4gene. HDR editing will lead to the insertion of a 6- to 15-bp fragment at these loci. We used sgDocut to cut the donor. Left and right homology arms (HA): light blue (600 bp); Cas9-sgRNA cleavage site: red lightning mark. HDR and NHEJ efficiencies were determined 3 days after transfection using high-throughput sequencing; n= 5; An unpaired t-test with Welch’s correction was used for statistical analysis, ns = not significant, *P < 0.05, **P< 0.01, ***P< 0.001,

****P< 0.0001.

B

D

F

Schematic of HDR editing at GAPDH

Cas9-sgGAPDH Cas9-sgGAPDH

Cas9-sgGAPDH

Exon 8

HDR

pD-E2A-mNeonGreen-sg HA mNeonGreen

HA

Exon 9 mNeonGreen E9

C

E

Schematic of HDR editing at EEF1A1

Cas9-sgDocut Cas9-sgDocut

Cas9-sgEEF1A1

Exon 7

HDR

pD-E2A-mNeonGreen-sg HA mNeonGreen

HA

Exon 7 mNeonGreen Exon8

G

Schematic of HDR editing at PRDM14

Cas9-sgDocut Cas9-sgDocut

Cas9-sgPRDM14

Exon 8

HDR

pD-E2A-mNeonGreen-sg HA mNeonGreen

HA

Exon 8 mNeonGreen

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

20 40 60

NHEJ (%)

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

5 10 15

mNeonGreen (%)

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

10 20 30 40 50

mNeonGreen (%)

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

20 40 60 80

NHEJ (%)

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

20 40 60 80

NHEJ (%)

ns

*

****

**

****

ns

**

****

***

****

ns ns

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

10 20 30 40 50

mNeonGreen (%)

*

****

ns ns

ns

****

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

10 20 30 40

HDR/(HDR+NHEJ) (%)

ns

*** ***

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

10 20 30 40 50

HDR/(HDR+NHEJ) (%)

* **

****

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

10 20 30 40 50

HDR/(HDR+NHEJ) (%)

ns ns

***

HDR and NHEJ editing at PRDM14 HDR and NHEJ editing at EEF1A1 HDR and NHEJ editing at GAPDH

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

10 20 30 40 50

NHEJ (%)

Schematic of HDR editing at EEF2

Cas9-sgDocut Cas9-sgDocut

Cas9-sgEEF2

Exon 15

HDR

pD-E2A-mNeonGreen-sg HA mNeonGreen

HA

Exon 15 mNeonGreen

A

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

10 20 30

mNeonGreen (%)

** *

**** ****

**

****

HDR and NHEJ editing at EEF2

SpCas9 SpCas9

-VP6 4

SpCas9 -EP300

EP3 00-Sp

Cas9 0

10 20 30 40 50

HDR/(HDR+NHEJ) (%)

ns

**** ****

B

D

F

H

Figure S3. Fusion proteins of Cas9 and VP64 or EP300core decrease HDR efficiencies at different open loci. (A, C, E, G) Schematic of HDR editing at EEF2,

GAPDH, EEF1A1, and PRDM14loci. (A) sgEEF2 targets the stop codon of the EEF2gene. (C) sgGAPDH targets intron 8 of the GAPDH gene. (E) sgEEF1A1 targets intron 7 of theEEF1A1 gene. (G) sgPRDM14 targets the stop codon of the PRDM14 gene. We used a promoterless double-cut HDR donor pD-E2A-mNeonGreen-sg to guide HDR insertion of the mNeonGreen reporter. E2A is a self-cleaving linker for multicistronic expression. Left and right homology arms (HA): light blue (600 bp);

E2A: blue; Cas9-sgRNA cleavage site: red lightning mark. (B, D, F, H) Cas9 fusion proteins decrease HDR efficiency at (B) EEF2, (D) GAPDH, (F) EEF1A1, and (H) PRDM14 loci. HDR efficiency was determined three days after transfection by FACS and NHEJ efficiency by high-throughput sequencing (n = 5). An unpaired t-test with Welch’s correction was used for statistical analysis, ns = not significant, *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001.

HDAC inhibitors

(HDACi) Chemical structure

Classification by chemical

structure

HDAC specificity Treated Disorders*

Concentrati on used in this study

Valproic acid (VPA) Aliphatic fatty

acids

Class I and IIa HDAC1/2/3/8/

PD, AD, ALS, SMA, epilepsy

and bipolar disorder

1 mM

Sodium Butyrate (NaB)

Aliphatic fatty acids

Class I and IIa

HDAC1/2/3/4/5/7/8/9 PD, HD, AD, SMA 1 mM

Trichostatin A (TSA) Hydroxamates Class I and II

HDAC1/2/3/4/6/7/9

PD, AD, ALS,

SMA 0.1 μM

Vorinostat

(SAHA) Hydroxamates Pan-inhibitor

HDAC1/2/3/4/6/7/9

PD, HD, AD, SMA, cutaneous T-cell

lymphoma

5 μM

Panobinostat

(LBH589) Hydroxamates Pan-inhibitor

HDAC1/2/3/4/7/9 SMA, MM 0.1 μM

Entinostat

(MS-275) Benzamides Class I

HDAC1/9

AD, Brest cancer,

Colorectal cancer 5 μM

Figure S4. Detailed information about HDAC inhibitors used in this study. Molecular structures, functions, and other significant properties of HDAC inhibitors. The concentration of each HDAC inhibitor used in this study is also indicated. PD, Parkinson’s disease; AD, Alzheimer’s disease; ALS, Amyotrophic lateral sclerosis; SMA, Spinal muscular atrophy; HD, Huntington disease; MM, multiple myeloma.

Control NaB

SAH A

TSA VPA Entinostat

Panobinostat 0

2 4 6 8

Relative mRNA expression

Control NaB

SAH A

TSA VPA Entinostat

Panobinostat 0

1 2 3

Relative mRNA expression

Control NaB

SAH A

TSA VPA Entinostat

Panobinostat 0.0

0.5 1.0 1.5 2.0

Relative mRNA expression

Figure S5. Effects of HDAC inhibitors on human iPSC survival. (A) Cell survival was determined by counting cell numbers on days 1, 2, and 3 after electroporation of editing plasmids. n= 4. The dosages used in this study are NaB (1 mM), SAHA (5 μM), TSA (0.1 μM), VPA (1 mM), entinostat (5 μM), and panobinostat (100 nM). (B) After treating with HDAC inhibitors for 24 hours, we assessed cell survival by counting cell numbers on day 3. The dosages of NaB are 0, 0.5, 1, 2 mM; SAHA are 0, 2.5, 5, 10 μM; TSA are 0, 0.05, 0.1, 0.2 μM; VPA are 0, 0.5, 1, 2 mM; entinostat are 0, 2.5, 5, 10 μM; panobinostat are 0, 50, 100, 200 nM. n = 4; sodium butyrate (NaB), vorinostat/suberoylanilohydroxamic acid (SAHA), trichostatin A (TSA), valproic acid (VPA), entinostat (MS275), panobinostat (LBH589). (C) HDAC inhibition does not attenuate iPSC pluripotency. Relative expression of OCT4, NANOG, and SOX2 was determined by RT-qPCR 72 hours after treatment with HDAC inhibitors. n = 4. An unpaired t-test with Welch’s correction was used for statistical analysis, ns = not significant, *P< 0.05, **P< 0.01.

DMSO NaB

0.5m M

NaB 1m

M NaB

2m M 0

50 100 150

Cell number (x10e4)

DMSO SAH

A 2.5μM SAH

A 5μM SAH

A 10μM 0

50 100 150

Cell number (x10e4)

DMSO TSA

0.05μM TSA

0.1μM TSA

0.2μM 0

50 100 150

Cell number (x10e4)

NaB (Butyrate) SAHA TSA

VPA Entinostat Panobinostat

DMSO VPA

0.5m M

VPA 1m

M VPA

2m M 0

50 100 150

Cell number (x10e4)

DMSO Entinost

at 2.5μM Entinostat 5μM

Entinostat 10μM 0

50 100 150

Cell number (x10e4)

DMSO

Panobinostat 50nM Panobinostat 100nM

Panobinostat 200nM 0

50 100 150

Cell number (x10e4)

B

ns ns

**

*

ns

ns

*

ns

ns

*

Control NaB

1m M SAH

A 5μM TSA

0.1μM VPA

1m M

Entinostat 5μM Panobinostat 100nM 0

20 40 60 80 100

Cell number (x10e4)

A

ns

24h

Control NaB

1mM SAH

A 5μM TSA

0.1μM VPA

1m M

Entinostat 5μM Panobinostat 100nM 0

50 100 150 200

Cell number (x10e4)

* 72h

Control NaB

1mM SAH

A 5μM TSA

0.1μM VPA

1m M

Entinostat 5μM Panobinostat 100nM 0

20 40 60 80 100

Cell number (x10e4)

48h

ns

OCT4 NANOG SOX2

ns

** **

ns

** *

ns

*

C

Figure S6. Moderate improvement of HDR/NHEJ editing efficiencies by HDAC inhibitors at open loci with mNeonGreen donors. (A-D) The effects of HDAC inhibitors on HDR efficiency (KI), NHEJ efficiency, Indels (HDR + NHEJ), and HDR percentage in all edited cells at (A)GAPDH, (B)PRDM14, (C)EEF1A1,and (D) EEF2. HDR percentage is the ratio of HDR to HDR + NHEJ. We determined HDR efficiency three days after the transfection of editing plasmids by FACS and NHEJ

efficiency by high-throughput sequencing (n = 3). A paired t-test was used for statistical analysis, ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P <

0.0001.

Control NaB SAHA TSA VPA 0

10 20 30 40

mNeonGreen (%)

HDR and NHEJ editing at GAPDH

ns

Control NaB SAHA TSA VPA 0

50 100 150

HDR+NHEJ (%) ns

* *

Control NaB SAHA TSA VPA 0

50 100 150

NHEJ (%) ns

ns

*

Ctrl NaB SAHA TSA VPA 0

10 20 30 40

HDR/(HDR+NHEJ) (%)

ns

Control NaB SAHA TSA VPA 0

10 20 30 40

mNeonGreen (%)

HDR and NHEJ editing at PRDM14 ns

*

_{ns ns}

Control NaB SAHA TSA VPA 0

50 100 150

HDR+NHEJ (%)

ns

**

ns ns

Control NaB SAHA TSA VPA 0

50 100 150

NHEJ (%) ns

Ctrl NaB SAHA TSA VPA 0

10 20 30 40 50

HDR/(HDR+NHEJ) (%) ns

Control NaB SAHA TSA VPA 0

10 20 30 40

mNeonGreen (%)

HDR and NHEJ editing at EEF1A1

*

^{ns ns}

*

Control NaB SAHA TSA VPA

0 50 100 150

HDR+NHEJ (%) ns

*

^{ns ns}

Control NaB SAHA TSA VPA 0

20 40 60 80 100

NHEJ (%) ns

**

ns ns

Ctrl NaB SAHA TSA VPA 0

10 20 30 40 50

HDR/(HDR+NHEJ) (%) ns

Control NaB SAHA TSA VPA 0

10 20 30 40

mNeonGreen (%)

HDR and NHEJ editing at EEF2 ns

Control NaB SAHA TSA VPA 0

20 40 60 80

HDR+NHEJ (%) ns

Control NaB SAHA TSA VPA 20

30 40 50 60

NHEJ (%)

**

ns

Ctrl NaB SAHA TSA VPA 0

10 20 30 40 50

HDR/(HDR+NHEJ) (%)

ns Schematic of HDR editing at GAPDH

Cas9-sgGAPDH Cas9-sgGAPDH

Cas9-sgGAPDH

Exon 8

HDR

pD-E2A-mNeonGreen-sg HA mNeonGreen

HA

Exon 9 mNeonGreen E9

A

Schematic of HDR editing at EEF1A1

Cas9-sgDocut Cas9-sgDocut

Cas9-sgEEF1A1

Exon 7

HDR

pD-E2A-mNeonGreen-sg HA mNeonGreen

HA

Exon 7 mNeonGreen Exon8

Schematic of HDR editing at PRDM14

Cas9-sgDocut Cas9-sgDocut

Cas9-sgPRDM14

Exon 8

HDR

pD-E2A-mNeonGreen-sgmNeonGreen HA HA

Exon 8 mNeonGreen

Schematic of HDR editing at EEF2

Cas9-sgDocut Cas9-sgDocut

Cas9-sgEEF2

Exon 15

HDR

pD-E2A-mNeonGreen-sg HA mNeonGreen

HA

Exon 15 mNeonGreen

B

C

D

Control VP

A NaB

TSA SAH A Entinostat

Panobinostat 0

5 10 15 20

HDR (%)

Figure S7. HDAC inhibitors improve HDR and NHEJ efficiency at open loci with the small fragment knockin. (A-D) The effects of HDAC inhibitors on relative induction fold of HDR efficiency (KI), NHEJ efficiency (KO), Indels (HDR + NHEJ), and HDR percentage in all edited cells at four loci (A) GAPDH, (B) PRDM14, (C) EEF1A1, and (D)EEF2. HDR percentage is the ratio of HDR to HDR + NHEJ. HDR and NHEJ efficiency was determined three days after transfection by high-throughput sequencing (n= 3). A t-test was used for statistical analysis, ns = not significant, *P< 0.05, **P< 0.01, ***P< 0.001.

Schematic of HDR editing at EEF2

Cas9-sgDocut Cas9-sgDocut

Cas9-sgEEF2

Exon 15

HDR pD-EEF2(HA)-sg

HA HA

Exon15

+6bp

D

HDR and NHEJ editing at EEF2 ns

* **

Control VPA

NaB TSA SAH A Entinostat

Panobinostat 0

20 40 60

HDR+NHEJ (%)

*

^ns

**

ns

** ***

Control VPA

NaB TSA SAH A Entinostat

Panobinostat 0

10 20 30 40 50

NHEJ (%)

** **

^nsns

** ***

Control VPA

NaB TSA SAH

A Entinostat

Panobinostat 0

10 20 30

HDR/(HDR+NHEJ) (%)

Control VP

A

NaB TSA SAH

A Entinostat

Panobinostat 0

5 10 15 20

HDR (%)

Schematic of HDR editing at GAPDH

Cas9-sgDocut Cas9-sgDocut

Cas9-sgGAPDH

Exon 8

HDR pD-GAPDH(HA)-sg

HA HA

Exon8 E9

E9

A

-6bp, +12bp

HDR and NHEJ editing at GAPDH ns _{ns ns}

* * *

Control VP

A NaB

TSA SAH A Entinost

at

Panobinostat 0

20 40 60 80

HDR+NHEJ (%) ns

*

nsns

* **

Control VPA

NaB TSA SAH

A Entinostat

Panobinostat 0

10 20 30 40 50

NHEJ (%)

* * * **

^nsns

Control VPA

NaB TSA SAH

A Entinostat

Panobinostat 0

10 20 30

HDR/(HDR+NHEJ) (%) ns

*

Control VP

A

NaB TSA SAH

A Entinostat

Panobinostat 0

20 40 60 80

HDR (%)

Schematic of HDR editing at EEF1A1

Cas9-sgDocut Cas9-sgDocut

Cas9-sgEEF1A1

Exon 7

HDR pD-EEF1A1(HA)-sg HA HA

-6bp, +12bp

Exon7 E8

E8

C

HDR and NHEJ editing at EEF1A1

* * ** * * **

Control VP

A

NaB TSA SAH

A Entinostat

Panobinostat 0

20 40 60 80 100

HDR+NHEJ (%)

**

* ** * * **

Control VPA

NaB TSA SAH

A Entinostat

Panobinostat 0

10 20 30

NHEJ (%)

**

^ns

*

ns

Control VP

A

NaB TSA SAH

A Entinostat

Panobinostat 0

20 40 60 80 100

HDR/(HDR+NHEJ) (%)

ns

ns

Control VPA

NaB

TSA SAH A Entinost

at

Panobinostat 0

5 10 15 20 25

HDR (%)

Schematic of HDR editing at PRDM14

Cas9-sgDocut Cas9-sgDocut

Cas9-sgPRDM14

Exon 8

HDR pD-PRDM14(HA)-sg

HA HA

Exon8

B

-6bp, +12bp

HDR and NHEJ editing at PRDM14

ns

*

Control VPA

NaB TSA SAH

A Entinostat

Panobinostat 0

20 40 60 80 100

HDR+NHEJ (%)

ns

**

^ns

*

Control VPA

NaB TSA SAH A Entinostat

Panobinostat 0

20 40 60 80

NHEJ (%)

ns

Control VPA

NaB TSA SAH A Entinostat

Panobinostat 0

10 20 30

HDR/(HDR+NHEJ) (%)

ns

Schematic of HDR editing at MESP1

Cas9-sgDocut Cas9-sgDocut

Cas9-sgMESP1

Exon 2

HDR pD-MESP1(HA)-sg

HA HA

Exon 2 15bp

Schematic of HDR editing at MYH6

Cas9-sgDocut Cas9-sgDocut

Cas9-sgMYH6

Exon 39

HDR pD-MYH6(HA)-sg HA HA

E39 6bp

E39

Schematic of HDR editing at GATA4

Cas9-sgDocut Cas9-sgDocut

Cas9-sgGATA4

Exon 7

HDR pD-GATA4(HA)-sg HA HA

E7 6bp

E7

Control VPA

NaB TSA SAH

A Entinostat

Panobinostat 0

20 40 60

HDR (%)

Control VPA

NaB

TSA SAH A Entinostat

Panobinostat 0

20 40 60

HDR (%)

Control VP

A NaB

TSA SAH A Entinost

at

Panobinostat 0

20 40 60

HDR (%)

A

B

C

Figure S8. HDAC inhibitors significantly improve HDR and NHEJ efficiency at close loci. (A-C) The effects of HDAC inhibitors on relative induction fold of HDR efficiency (KI), NHEJ efficiency (KO), Indels (HDR + NHEJ), and HDR percentage in all edited cells at three loci (A) MESP1, (B) MYH6, and (C) GATA4. HDR percentage is the ratio of HDR to HDR + NHEJ. HDR and NHEJ efficiency was determined three days after transfection by high-throughput sequencing (n= 3). A paired t-test was used for statistical analysis, ns = not significant, *P< 0.05, **P< 0.01, ***P< 0.001.

*

ns

** * * *

HDR and NHEJ editing at MESP1

HDR and NHEJ editing at MYH6

HDR and NHEJ editing at GATA4

* * ** ***

^ns

****

* *

ns ns ns

*

Control VPA

NaB TSA SAH

A Entinostat

Panobinostat 0

20 40 60 80 100

HDR+NHEJ (%)

Control VPA

NaB

TSA SAH A Entinostat

Panobinostat 0

20 40 60 80 100

HDR+NHEJ (%)

Control VPA

NaB

TSA SAH A Entinost

at

Panobinostat 0

20 40 60 80 100

HDR+NHEJ (%)

* * **

ns

* **

ns ^ns

* **

^ns

**

ns ^ns

* * * **

Control VPA

NaB TSA SAH

A Entinostat

Panobinostat 0

10 20 30 40 50

NHEJ (%)

Control VPA

NaB

TSA SAH A Entinostat

Panobinostat 0

10 20 30 40

NHEJ (%)

Control VPA

NaB

TSA SAH A Entinostat

Panobinostat 0

10 20 30 40 50

NHEJ (%)

ns

*

*

ns ^ns

** *

^ns

*

ns

** *

Control VPA

NaB TSA SAH

A Entinostat

Panobinostat 0

20 40 60 80

HDR/(HDR+NHEJ) (%)

Control VPA

NaB TSA SAH

A Entinostat

Panobinostat 0

20 40 60 80

HDR/(HDR+NHEJ) (%)

Control VPA

NaB

TSA SAH A Entinost

at

Panobinostat 0

20 40 60 80

HDR/(HDR+NHEJ) (%)

ns ns

* * *

^ns

** *** **

ns

**

** **

mNeonGreen FL3(irrelevant channel)

Control DMSO NaB(Butyrate) SAHA

TSA VPA Entinostat Panobinostat A 24h

Figure S9. Treatment with HDAC inhibitors increases the expression of Cas9 and sgRNA.(A-B) The expression of mNeonGreen was assessed by flow cytometry 24 hours (A) and 48 hours (B) after electroporation.n = 5. (C) Relative expression of Cas9 and mNeonGreen by RT-qPCR; n= 4. We conducted the RT-qPCR analysis 48 hours after transfection. A t-test was used for statistical analysis, ns = not significant, **P< 0.01.

C

0 5 10 15 20 25

0 5 10 15 20

Relative Cas9 mRNA expresison Relative mNeonGreen mRNA expression

R²=0.96

48h

mNeonGreen FL3(irrelevant channel)

Control DMSO NaB(Butyrate) SAHA

TSA VPA Entinostat Panobinostat 48h

B

Control NaB

SAH

A TSA VPA MS

275 LBH589 0

20 40 60 80 100

mNeonGreen (%)

48h

** ** **

^ns

** **

Control NaB

SAH

A TSA VPA MS

275 LBH589 0

20 40 60 80 100

mNeonGreen (%)

24h

ns

Table S2. Primers used for deep sequencing

Primer Sequence Length

1^stPCR

GADPH-Hout-F ATGTTCGTCATGGGTGTGAA 1589 bp GADPH-Hout-R CCAGGCTGAGCTCCACTAAC

PRDM14-Hout-F ATGCAAAGACACTGGCCGAT 1922 bp PRDM14-Hout-R TGTAACACGTGATGACCGCA

EEF1A1-Hout-F CCACCAACTCGTCCAACTGA 1609 bp EEF1A1-Hout-R CCCACGTTTCAACATGCACA

EEF2-Hout-F CATCTACGGGGTTTTGAACAGGA 1414 bp EEF2-Hout-R CACCATTCCAGAACGTTCCACCT

GATA4-Hout-F GAGGCCGTGTCTTAGTGAGC 1433 bp GATA4-Hout-R GTGTGACACGGTGAACGAAG

MYH6-Hout-F ACCTCAGGTTGAGAGGCTGA 1400 bp MYH6-Hout-R CCAGACTGTCCCCAGTAGGA

MESP1-Hout-F CCAGAGCCTGACCAAGATCGAG 1456 bp MESP1-Hout-R CCAACATGTATGGAGCTGGCAAAA

2^ndPCR

GADPH-150PE-F CTGACTTCAACAGCGACACC 277 bp GADPH-150PE-R GGTGGTCCAGGGGTCTTACT

PRDM14-150PE-F CCCATGACAGCCATGTCC 251 bp PRDM14-150PE-R TTTGGTAGAGACAGGATTTCACC

EEF1A1-150PE-F ACGAACAGCAAAGCGACCTA 235 bp EEF1A1-150PE-R GCCGTTCTGGTAAAAAGCTG

EEF2-150PE-F GTTTGACCACTGGCAGATCC 228 bp EEF2-150PE-R CAGTCTCCAGGTGTCGTCTG

MESP1-150PE-F CTCTCACCCGCAGGAATG 237 bp MESP1-150PE-R TCCGTCTCTCCCTACAGCTC

MYH6-150PE-F CTCCTCTCAGGCCCCCTACT 245 bp MYH6-150PE-R CATCTCCCTTAGGCCTCTGA

GATA4-150PE-F CAGCGTGTAAAGGCATCTGA 261 bp GATA4-150PE-R CCAAGCAGGACTCTTGGAAC

Table S1. Target sequences of sgRNAs

Target site sgRNA Sequence

sgPRDM14 GAAGACTACTAGCCCTGCC sgGAPDH GACTGGCTCTTAAAAAGTGC sgEEF2 gTTCCTGGACAAATTGTAGG sgEEF1A1 GTAGTCATCCTTACCCAA

sgGATA4 GTCCGTGCAGGAATTTGAGG

sgMYH6 GGCAAGAGTGAGGTTCCCG

sgMESP1 GTCAGTTGTCCCTTGTCACT sgDocut gGGTGCAGATGAACTTCA sgEEF1A1-S833 gATACAACTGAACAGTACTTT

Table S3. Primers used for RT-qPCR

Primer Sequence Length

Cas9-F ACGTGGATCAGGAACTGGAC 218 bp

Cas9-R CCGTTGTGTGATCAGTTTGG

sgRNA-F TGCTGGAAACAGCATAGCAA 68 bp sgRNA-R ACTCGGTGCCACTTTTTCAA

mNeonGreen-F CATCAACGGTGTGGACTTTG 211 bp mNeonGreen-R GTATCCGGAGCCATCTACCA

OCT4-F CTTGCTGCAGAAGTGGGTGGAGGAA 169 bp OCT4-R CTGCAGTGTGGGTTTCGGGCA

SOX2-F ACACCAATCCCATCCACACT 224 bp

SOX2-R GCAAACTTCCTGCAAAGCTC

NANOG-F TTCCTTCCTCCATGGATCTG 213 bp NANOG-R TCTGCTGGAGGCTGAGGTAT

ACTB-F TCGTGCGTGACATTAAGGAG 106 bp

ACTB-R GGCAGCTCGTAGCTCTTCTC