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DNA methylation averages per CpG-site and sample in the TFRC promoter region of transfected. HEK293 cells compared to untransfected HEK293 cells.
Supplementary information Supplementary figure S1. Enrichment of transfected cells with MACS. A) Representative results of a typical co-transfection experiment before sorting in which 5% pVenusNLS fluorescent protein construct was used to assay transfection efficiency. B) FACS results of transiently transfected SKOV-3 cells before and after magnetic-activated cell sorting (MACS) shows an enrichment of the proportion of fluorescent cells from 51 % to 88 % (considering only events in the top two quadrants designated as living cells).

A

B 48.7%

Before MACS

51.3%

12%

After MACS

88%

Supplementary figure S2. Expression of dCas9-Dnmt3a3L fusion proteins in HEK293 cells after transient transfection of the expression plasmids. A) Western blot with anti-Cas9 (ActiveMotif #61578) as primary antibody and anti-mouse (GE Healthcare Life Sciences NA931V) as secondary antibody shows at least equal expression of the dCas9Dnmt3a3L mutant fusion proteins compared to the wt-variants. Expected protein masses are 224 kDa for dCas9-Dnmt3a3L and 197 kDa for dCas9-Dnmt3a.

C706A R832E

R832E

C706A

WT

HEK293

dCas9-Dnmt3a3L

WT

A

dCas9-Dnmt3a

B) Maps of the dCas9-Dnmt3a3L SC and dCas9-Dnmt3a CD plasmids used for transient transfection. The sequences of the dCas9 fusion genes are provided in supplementary text.

250 kDa

dCas9-Dnmt3a3L dCas9-Dnmt3a

AB: α-Cas9 (ActiveMotif)

β-actin

AB: α-ACTNB (ActiveMotif) dCas9-Dnmt3a3L - 1950 aa, MW 224487.2 Da dCas9-Dnmt3a - 1709 aa, MW 197044.0 Da

B

dCas9-Dnmt3a3L

dCas9-Dnmt3a CD

Supplementary figure S3. Analysis of targeted methylation at the TFRC promoter. DNA methylation averages per CpG-site and sample in the TFRC promoter region of transfected HEK293 cells compared to untransfected HEK293 cells. The same data are shown in the figure 1 in the main text in a heatmap representation. The average CpG methylation is plotted (Y-axis) relative to the genomic location within the human Chr. 3 (X-axis). In the charts, the line represents the average DNA methylation of 2-4 biological samples and the shaded region denotes the SEM per site. gRNA binding sites are indicated as red arrows.

HEK293 Untransfected gRNAs 1-10 only None

gRNA 6

4

196081561

5

196081761

6

196081961

7

196082161

89

196082361

196082561

10

40 0 196081361 80

196081561

196081761

196081961

196082161

196082361

196082561

196081561

196081761

196081961

196082161

196082361

196082561

196081561

196081761

196081961

196082161

196082361

196082561

196081561

196081761

196081961

196082161

196082361

196082561

196081561

196081761

196081961

196082161

196082361

196082561

196081561

196081761

196081961

196082161

196082361

196082561

196081561

196081761

196081961

196082161

196082361

196082561

196081561

196081761

196081961

196082161

196082361

196082561

196082161

196082361

196082561

196082361

196082561

196082361

196082561

196082361

196082561

196082361

196082561

196082361

196082561

40

0 196081361 80 40 0 196081361 80

CpG methylation [%]

dCas9-Dnmt3a3L

gRNA 5

0 196081361 80

40

gRNA 2

gRNA 4

40

0 196081361 80

gRNA 1

gRNA 3

80

40 0 196081361 80

4

40 0 196081361 80 40 0 196081361 80

gRNA 10 gRNAs 1-10 dCas9-Dnmt3a3L C706A, gRNAs 1-10

196081561

196081761

196081961

8

40 0 196081361 80

gRNA 9

7

40 0 196081361 80

gRNA 8

6

40 0 196081361 80

gRNA 7

5

196081561

196081761

196081961

196082161

9

40 0 196081361 80

196081561

196081761

196081961

196082161

10

40 0 196081361 80 4

196081561

5

196081761

6

196081961

7

196082161

89

10

40 0 196081361 80

4

196081561

5

196081761

6

196081961

7

196082161

89

10

40 0 196081361

196081561

196081761

196081961

196082161

Supplementary figure S4. Differences in DNA methylation between dCas9-Dnmt3a3L wt and mutants at the TFRC promoter. A) CpG methylation averages per CpG-site and sample in the TFRC promoter region of transfected HEK293 cells compared to untransfected HEK293 cells. In the charts, the line represents the average DNA methylation of 2-4 biological samples and the shaded region denotes the SEM per site. gRNA binding sites are indicated as red arrows. B) Heatmap representation of the results shown in A.

A

80

HEK293 Untransfected

0 80 196081361 3

196081461 4

196081561 5

196081661

196081761

196081861

40

Wild type

0 80 196081361 3

196081461 4

196081561

5

196081661

196081761

196081861

196081461 4

196081561

5

196081661

196081761

196081861

196081461 4

196081561

5

196081661

196081761

196081861

196081461

196081561

196081661

196081761

196081861

40

C706A

0 80 196081361 3 40

R832E

0 80 196081361 3 40

C706A R832E

B

40

0 196081361

3

4

5

HEK293 Wild type

C706A R832E C706A R832E

0%

40%

80%

Supplementary figure S5. Analysis of targeted methylation at the CXCR4 promoter. DNA methylation averages per CpG-site and sample in the CXCR4 promoter region of transfected HEK293 cells compared to untransfected HEK293 cells. The same data are shown in the figure 1 in the main text in a heatmap representation. The average CpG methylation is plotted (Y-axis) relative to the genomic location within the human Chr. 2 (X-axis). In the charts, the line represents the average DNA methylation of 2-4 biological samples and the shaded region denotes the SEM per site. gRNA binding sites and their orientation are indicated with red arrow heads. 80

HEK293 untransfected

gRNA 2 gRNA 3 gRNA 4 gRNA 1-4

0 136116640 80

136116840

136117040

136117240

136117440

136117640

136116840

136117040

136117240

136117440

136117640

136116840

136117040

136117240

136117440

136117640

136116840

136117040

136117240

136117440

136117640

136116840

136117040

136117240

136117440

136116840

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136117240

136117440

136116840

136117040

136117240

136117440

136116840

136117040

136117240

136117440

40

CpG methylation [%]

dCas9-Dnmt3a3L

gRNA 1

0 136116640 80 40

gRNA only None

40

0 136116640 80 1 40 0 136116640 80

2

40 0 136116640 80

3

136117640

40 0 136116640 80

136117640

40

0 136116640 80 1

2

3

136117640

40 0 136116640

136117640

Supplementary figure S6. Differences in DNA methylation between dCas9-Dnmt3a3L wt and mutants at the CXCR4 promoter. A) CpG methylation averages per CpG-site and sample in the CXCR4 promoter region of transfected HEK293 cells compared to untransfected HEK293 cells. The same data are shown in the figure 1 in the main text in a heatmap representation. The average CpG methylation is plotted (Y-axis) relative to the genomic location within the human Chr. 3 (X-axis). In the charts, the line represents the average DNA methylation of 2-4 biological samples and the shaded region denotes the SEM per site. gRNA binding sites are indicated as red arrows. B) Heatmap representation of the results shown in A.

A

80

Untransfected HEK293 Wild type C706A R832E C706A R832E

B

1

40 0 80 136116640 1

136116840

136117040

0 80 136116640 1 40

136116840

136117040

0 80 136116640 1

136116840

136117040

136116840

136117040

136116840

136117040

2

136117240

136117440

136117240

136117440

136117240

136117440

136117240

136117440

136117240

136117440

3

136117640

40

2

2

3

3

136117640

136117640

40 0 80 136116640 1

2

3

136117640

40 0 136116640

2

136117640

3

HEK293 Wild type C706A R832E C706A R832E

0%

40%

80%

Supplementary figure S7. Analysis of DNA methylation within the human EpCAM promoter in SKOV3 cells. Bisulfite sequencing results of separate clones (rows) covering the human EpCAM promoter in four amplicons. The Sanger sequencing results were analyzed using BISMA (http://services.ibc.unistuttgart.de/BDPC/BISMA/). Columns represents separate CpG sites in the amplicons, rows denote separate sequences. Red squares denote methylated sites, blue unmethylated CpGs and white inconclusive methylation status. No significant change in DNA methylation is observed for cells transfected with either gRNAs or dCas9-Dnmt3a3L or dCas9-Dnmt3a alone compared to untransfected SKOV-3 cells. Deposition of DNA methylation using the targeted dCas9-Dnmt3a3L construct is presented in figure S8. Amplicon 1

Untransfected SKOV-3

Venus only

gRNAs 1-12 only

Cas9-Dnmt3a3L only Cas9-Dnmt3a only

Amplicon 2

Amplicon 3

Amplicon 4

Supplementary figure S8. Analysis of targeted DNA methylation within the human EpCAM promoter in SKOV-3 cells with single gRNAs. Bisulfite sequencing results of separate clones (rows) of the second amplicon covering the human EpCAM promoter in SKOV-3 cells. The Sanger sequencing results were analyzed using BISMA (http://services.ibc.uni-stuttgart.de/BDPC/BISMA/). Each column represents one CpG site and red squares denote methylated sites, blue means not methylated and white is inconclusive. Little or no change in DNA methylation is observed for cells transfected with either gRNAs or dCas9-Dnmt3a3L or dCas9-Dnmt3a alone compared to untransfected SKOV-3 cells whereas substantial CpG methylation can be observed for most samples transfected with both dCas9-Dnmt3a3L and a guide RNA. Human EpCAM amplicon 2 Untreated SKOV-3

mVenus

EpCAM mix

dCas9-Dnmt3a

None

6

7

dCas9-Dnmt3a3L

9

10

11

12

Supplementary figure S9. Analysis of targeted methylation at the EpCAM promoter. DNA methylation averages per CpG-site and sample in the EpCAM promoter region of transfected SKOV-3 cells compared to untransfected SKOV-3 cells. The same data are shown in the figure 2 in the main text in a heatmap representation. The average CpG methylation is plotted (Y-axis) relative to the genomic location within the human Chr. 2 (X-axis). In the charts, the line represents the average DNA methylation of 2 biological repeats (4 biological repeats for co-targeting by gRNA 6+7+9+10) and the shaded region denotes the SEM per site. The gRNA binding sites are indicated as red arrows with gRNA number present next to arrow.

80

Untransfected SKOV-3

40 0 80 47368934

All gRNAs only

0 47368934 80

0 80 47368934 1

0 80 47368934 2

0 80 47368934

0 80 47368934

gRNA 10 gRNA 11 gRNA 12 gRNAs 6+7+9+10

CpG methylation [%]

dCas9-Dnmt3a3L

47369134

47369334

47369534

47369734

47369934

47369134

47369334

47369534

47369734

47369934

47369134

47369334

47369534

47369734

47369934

3 47369134

47369334

47369534

47369734

47369934

47369134 4

47369334

47369534

47369734

47369934

47369134 5

47369334

47369534

47369734

47369934

47369134

473693346

47369534

47369734

47369934

47369134

47369334

47369534 7

47369734

47369934

47369134

47369334

473695348

47369734

47369934

47369134

47369334

47369534

9 47369734

47369934

47369134

47369334

47369534

47369734 10

47369934

47369134

47369334

47369534

47369734

47369934 11

47369134

47369334

47369534

47369734

47369134

473693346

747369534

9 47369734 10

47369934

47369134

47369334

47369534

47369734

47369934

40

gRNA 4

gRNA 9

47369934

40

gRNA 3

gRNA 8

47369734

40

gRNA 2

gRNA 7

47369534

40

gRNA 1

gRNA 6

47369334

40

None

gRNA 5

47369134

40

0 80 47368934 40 0 80 47368934 40 0 80 47368934 40 0 80 47368934 40 0 80 47368934 40 0 80 47368934 40 0 80 47368934 40 0 80 47368934

4736993412

40 0 80 47368934 40 0 47368934

Supplementary figure S10. Distribution of DNA methylation introduced by dCas9-Dnmt3a3L wt and mutants at the EpCAM promoter co-targeted with gRNA 6, 7, 9 and 10. A) XY-plot representation of the DNA methylation averages per CpG-site and sample in the EpCAM promoter region of transfected SKOV-3 cells compared to untransfected cells. In the charts, the line represents the average DNA methylation of 2-4 biological samples and the shaded region denotes the SEM per site. gRNA binding sites are indicated as red arrows. B) Heatmap representation of the results in A). This panel is the same as figure 5A in the main text.

A Untransfected SKOV-3 Wild type

80 40 0 80 47368934

0 80 47368934

R832E

C706A R832E

B

473693346

747369534

9 47369734 10

47369934

47369134

473693346

747369534

9 47369734 10

47369934

47369134

47369334 6

47369534 7

9

47369734 10

47369934

47369134

473693346

747369534

9 47369734 10

47369934

47369134

47369334

47369534

47369734

47369934

40 0 80 47368934

C706A

47369134

40

40 0 80 47368934 40

0 47368934

6

7

9

10

SKOV-3 Wild type C706A R832E C706A R832E

0%

40%

80%

Supplementary figure S11. Limited off-target activity of the dCas9-Dnmt3a3L targeted DNA methylation A) Heatmap representation of CpG methylation averages per CpG-site and sample at predicted off-target sites in untreated HEK293 cells or in cells co-transfected with gRNA (as indicated) and dCas9-Dnmt3a3L. B) Comparison of observed DNA methylation increase (ΔCpG methylation) at on-target (blue bars) and off-target (orange bars) regions show a weaker deposition of methylation at the offtarget sites. C) Heatmap representation of DNA methylation profiles at unrelated CGIs in SKOV-3 cells.

A

Chr.1: 218284797-218285104

40

gRNATFRC 2

35

HEK293 gRNATFRC 3 Chr.12: 30170448-30170694

ΔCpG methylation [%]

HEK293

Chr.11: 68016642-68016942

Off-targets

B

HEK293

30 25 20

15 10 5

gRNACXCR4 6 Chr.8: 144244724-144244962

0

HEK293 gRNACXCR4 20

C

hDSCR3 - Chr.21: 37267819-37268209 SKOV-3 dCas9-Dnmt3a3L

Unrelated CGIs

hKIAA0179 - Chr.21: 43659372-43659781 SKOV-3 dCas9-Dnmt3a3L hSUMO3 - Chr.21: 44818316-44818674 SKOV-3

dCas9-Dnmt3a3L hWRB - Chr.21: 39380511-39380746 SKOV-3 dCas9-Dnmt3a3L

gRNA TFRC2

gRNA TFRC3

Oligonucleotides used in this study Oligo name

Sequence (5’ to 3’)

Q_EpCAM_f Q_EpCAM_r Q_hTFRC_f Q_hTFRC_r Q_CXCR4_f Q_CXCR4_r Q-bactin_f Q-bactin_r

CTCCATGTGCTGGTGTGT TGTTTTAGTTCAATGATGATCCAGTA ACCGGCACCATCAAGCT TGATCACGCCAGACTTTGC AGCATGACGGACAAGTACAGG GATGAAGTCGGGAATAGTCAGC TCACCAACTGGGACGACATG ACCGGAGTCCATCACGATG

Supplementary table S1. Quantitative RT-PCR primers

Target

Human EpCAM

Human CXCR4

Human TFRC

Off-targets

Oligo name

Sequence (5’ -> 3’)

Bis_hEpCAM_1f

GAAGGTTTTTTGTTTGTGTTTGTATT

Bis_hEpCAM_1r

AAAAAATAAATAAACTCCCCTCCC

Bis_hEpCAM_2f

GAGGGGAGTTTATTTATTTTTTTAATTTT

Bis_hEpCAM_2r

ACCTCTTAATCCCCTCCCTATTATAC

Bis_hEpCAM_3f

TATAATAGGGAGGGGATTAAGAGGT

Bis_hEpCAM_3r

ACTAAAACTATTTTAAAACCCCAAA

Bis_hEpCAM_4f

TTTGGGGTTTTAAAATAGTTTTAGT

Bis_hEpCAM_4r

CATTACTCATATAATCCTTAAAAAAAA

Bis_hCXCR4_0f

GTGTGTGGGTTTTTTGTTATTTT

Bis_hCXCR4_0r

CTCCCCAACACCCTATAAAAC

Bis_hCXCR4_1f

GTTTTGTTTTATAGGGTGTTGGG

Bis_hCXCR4_1r

ATAATTTAACCTCCCCTTTAACACC

Bis_hCXCR4_2f

GGTGTTAAAGGGGAGGTTAAATTAT

Bis_hCXCR4_2r

ACCTAAACTAAATTCCCAAAAAAAA

Bis_hCXCR4_3f

TTTTTTTGGGAATTTAGTTTAGGTA

Bis_hCXCR4_3r

CTACATATATCTCCCCCTTAAATCC

Bis_hTFRC_1f

TGGTAAGATTAGGTTGTATTAGAGT

Bis_hTFRC_1r

AAACCAAATATACCCCCACTATC

Bis_hTFRC_2f

GATAGTGGGGGTATATTTGGTTT

Bis_hTFRC_2r

ATACTATCCAACAACCATAAAAAAC

Bis_hTFRC_3f

GTTTTTTATGGTTGTTGGATAGTAT

Bis_hTFRC_3r

AAAACTACCAAACTACCAAAATAAA

Bis_hTFRC_4f

TTTATTTTGGTAGTTTGGTAGTTTT

Bis_hTFRC_4r

CAATAACAAAAATTAAATTTTATTTATTTA

Bis_CXCR4-4-off-f

TTTGATTTAGGTGGGATTTTTGGAGTTTG

Bis_CXCR4-4-off-r

TCTAATCACAAACACAACCCAACCC

Bis_CXCR4-2-off-f

TTTATTTTTTAGGGAGAAAAGTGTTTTTG

Bis_CXCR4-2-off-r

ATCCAAAAAAACCTCTTCTCCTAAC

Bis_TFRC-2-off-f

TATTYGTTTTAGGGAAAGAAGGTGAG

Bis_TFRC-2-off-r

AATCAACRACTAAATAACCTCAACC

Bis_TFRC-3-off-f

GTTTTTGTTTTAGTATATTTGTTTTAGGGGTTG

Bis_TFRC-3-off-r

TATTCTAAACTACTACTACCTTTATAAAACCCC

Unrelated CGI

Bis_KIA0179_f

TTYGTTTTTTTTTGGTTAAGTTT

Bis_KIA0179_r

RCCCACCTATCTCCCTCTA

Bis_DSCR3_f

TTTTTAAGTTTTAGAGTTTGGGGGT

Bis_DSCR3_r

ATACCTACCCTCCTCAAATATCCTTAA

Bis_SUMO3_f

GGGTTTTGGGGATTTTTGTT

Bis_SUMO3_r

AACCTCCCAATACCTCAATTTCT

Bis_WRB_f

GTAAGTGGTTGTTTGGTTTTTTAA

Bis_WRB_r

AAACACTAAAAACTCCCAATATAAAAT

Supplementary table S2. Bisulfite amplification primers

Target

Human EpCAM

Human CXCR4

Human TFRC

gRNA name

Target sequence in the genome

hEpCAM_1

GTTCCCGCACCCTTCCCCCCAGG

hEpCAM_2

GCAGCGCTCCTCCGGTTAAAAGG

hEpCAM_3

GGAGACGAAGCACCTGGGGCGGG

hEpCAM_4

GCGCCCCAACTGCAGCGCCGGGG

hEpCAM_5

GGCCAGAGGTGAGCAGTCCCGGG

hEpCAM_6

GCCCCAGGCCTCGCGCTGCCCGG

hEpCAM_7

GCGCAGCATGGCGCCCCCGCAGG

hEpCAM_8

GTTGTGGAGCTGGGCTGGGCTGG

hEpCAM_9

GCGCTTTCCAGCGTGGAGACCGG

hEpCAM_10

GCAGCTTTGCTCGCCTTGGTAGG

hEpCAM_11

GCGCACCCACGTCCTCGGTTCGG

hEpCAM_12

GGGCCCCGGATGTGGGCCTCAGG

hCXCR4_1

TGTTTATCACTTGGCGCGTTTGG

hCXCR4_2

CCACCGACGGTTAAACATCACAA

hCXCR4_3

CCGTCGGCCGCACAGAGTTTAAC

hCXCR4_4

ATAAAAACACGCTCCGAGCGCGG

hTFRC1_1

CTTTAATCCTCTTATCAACGGGG

hTFRC1_2

GAACACCTGCGGCGTTCAGTCGG

hTFRC1_3

TCAGGTTGTACCAGAGCCGAAGG

hTFRC1_4

CCCGGAGCCCGGGGAACCGAAGG

hTFRC1_5

CAGCCCGGAGCCGGCGCACTAGG

hTFRC1_6

GGGAAGGGACGAGAGGCGAAGGG

hTFRC1_7

CCCACAGGCTCGCGGCCCCGCTC

hTFRC1_8

CCACCCGATATCCCGACGCTCTG

hTFRC1_9

TGCTCTGACAGATCGCCCGGGGG

hTFRC1_10

GAGCCGCGCGATGGGCGCACTGG

Supplementary table S3. gRNA target sequences

Oligo name

Sequence (5’ to 3’)

dCas9_vector_f dCas9_vector_r D3a3L_Cas9_f

TGAAAGGGTTCGATCCCTACCGG AGCCCGTCCGGAACCGCT AGCGGTTCCGGACGGGCTTCCCCAGGAATTCCCGGGTCGACTCGAAACCATGACCAGGAATTTGACCC C

D3a3L_Cas9_r D3a_Cas9_f D3a_Cas9_r

CCGGTAGGGATCGAACCCTTTCAGACTTTCCTTTTCTTCTTCGGATCTGCCCTGCTAAGAGGAAGTGAG TTTTGAGAAAAATACTTGAAG AGCGGTTCCGGACGGGCTTCCCCAGGAATTCCCGGGTCGACTCGAATGAACCATGACCAGGAATTTGA CCC CCGGTAGGGATCGAACCCTTTCAGACTTTCCTTTTCTTCTTCGGATCTGCCCTGCTCACACAAGCAAAAT ATTCCTTCAGCGG

Supplementary table S4. Cloning oligonucleotides

Oligo name D3a_C706A_f D3a_Cunch_f D3a_Runch_r D3a_R832E_r

Sequence (5’ to 3’) GAGGCAGTCCCGCCAATGACCTCTC GAGGCAGTCCCTGCAATGACCTCTC CTTTATAGAGTTTGACCTGGTGGTAATGGTC CTTTATAGAGTTTGACTCCGTGGTAATGGTC

Supplementary table S5. Mutagenesis oligonucleotides

Sample name

Effector

gRNA used

Average methylation [%]

SEM [%] n=2-4

None None dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L C706A dCas9-Dnmt3a

None 1-10 None 1 2 3 4 5 6 7 8 9 10 1-10 1-10 3

3.8 3.8 3.6 15.3 29.4 38.5 27.1 30.3 35.1 34.0 23.6 27.1 14.2 28.3 4.2 11.4

0.3 0.2 0.1 1.1 1.7 2.2 3.5 0.5 6.5 2.6 1.9 0.7 1.4 1.5 0.1 2.1

None None dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L C706A dCas9-Dnmt3a

None 1-4 None 1 2 3 4 1-4 1-4 2

0.8 0.7 0.6 28.3 35.3 31.2 13.3 31.8 1.9 7.9

0.02 0.04 0.1 1.0 1.8 0.3 1.5 0.4 0.4 0.9

None None dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L

None 1-12 None 1 2

6.4 6.0 11.2 28.0 33.7

1.8 0.4 0.1 1.2 1.9

TFRC HEK293 All None 1 2 3 4 5 6 7 8 9 10 All WT All C 3 Dnmt3a CD

CXCR4 HEK293 All None 1 2 3 4 All WT All C 2 Dnmt3a CD

EpCAM SKOV-3 All None 1 2

3 4 5 6 7 8 9 10 11 12 6+7+9+10 WT 6+7+9+10 C 5 Dnmt3a CD

dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L dCas9-Dnmt3a3L C706A dCas9-Dnmt3a

3 4 5 6 7 8 9 10 11 12 6+7+9+10 6+7+9+10 5

27.8 19.4 33.9 26.2 31.3 28.0 25.0 32.1 13.2 9.5 35.8 5.9 13.7

4.9 1.7 0.5 0.7 9.2 1.3 4.1 8.5 0.3 0.9 2.3 0.5 0.9

None dCas9-Dnmt3a3L None dCas9-Dnmt3a3L None dCas9-Dnmt3a3L None dCas9-Dnmt3a3L

None TFRC 2 None TFRC 3 None CXCR4 None CXCR4

16.3 25.5 34.8 45.9 89.4 88.7 92.1 91.5

0.8 1.7 0.7 1.4 0.1 0.2 0.6 0.3

None dCas9-Dnmt3a3L None dCas9-Dnmt3a3L None dCas9-Dnmt3a3L None dCas9-Dnmt3a3L

None None None None None None None None

20.4 21.0 2.7 2.7 0.5 0.5 1.6 2.7

0.7 1.0 0.1 0.1 0.1 0.1 0.1 0.2

Off-targets TFRC2 off-target TFRC2 off-target TFRC 3 off-target TFRC 3 off-target CXCR4 2 off-target CXCR4 2 off-target CXCR4 4 off-target CXCR4 4 off-target

Unrelated CGIs SUMO3 SUMO3 KIAA0179 KIAA0179 DSCR3 DSCR3 WRB WRB

Supplementary table S6. Summary of the targeted DNA methylation results. Reported methylation averages are calculated as mean methylation measured over all the CpG sites in the analyzed regions. SEM is calculated from the average methylation of the regions analyzed in 2-4 biological repeats.

Bisulfite sequencing amplicons Human EpCAM promoter (gRNAs target sites are underlined) >hEpCAM_1st [298 bps; 18 CGs] GAAGGTTCTCTGCCTGTGTTTGTATTTCCTTTAGCCTCCACGTTCCTCTATCCAGTTCCCGCACCCTTCCCCCCAG G(1)CCCCATTCTTCAAGGCTTCAGAGCAGCGCTCCTCCGGTTAAAAGG(2)AAGTCTCAGCACAGAATCTTCAA ACCTCCTCGGAGGCCACCAAAGATCCCTAACGCCGCCATGGAGACGAAGCACCTGGGGCGGG(3)GCGGAGC GGGGCGCGCGGGCCCACACCTGTGGAGAGGGCCGCGCCCCAACTGCAGCGCCGGGG(4)CTGGGGGAGGGG AGCCTACTCACTCCC >hEpCAM_2nd [519 bps; 66 CGs] GAGGGGAGCCTACTCACTCCCCCAACTCCCGGGCGGTGACTCATCAACGAGCACCAGCGGCCAGAGGTGAGC AGTCCCGGG(5)AAGGGGCCGAGAGGCGGGGCCGCCAGGTCGGGCAGGTGTGCGCTCCGCCCCGCCGCGCG CACAGAGCGCTAGTCCTTCGGCGAGCGAGCACCTTCGACGCGGTCCGGGGACCCCCTCGTCGCTGTCCTCCCG ACGCGGACCCGCGTGCCCCAGGCCTCGCGCTGCCCGG(6)CCGGCTCCTCGTGTCCCACTCCCGGCGCACGCCC TCCCGCGAGTCCCGGGCCCCTCCCGCGCCCCTCTTCTCGGCGCGCGCGCAGCATGGCGCCCCCGCAGG(7)TCC TCGCGTTCGGGCTTCTGCTTGCCGCGGCGACGGCGACTTTTGCCGCAGCTCAGGAAGGTGAGGCGCGGATTG GAGCAGAGTTGTGGAGCTGGGCTGGGCTGG(8)GGGGCAGCGGCCCCCGGCCCTCGGCCCCCGAAACGGGCA TAATAGGGAGGGGACCAAGAGGC >hEpCAM_3rd [298 bps; 29 CGs] CATAATAGGGAGGGGACCAAGAGGCCGCGCTTTCCAGCGTGGAGACCGG(9)ACGGTGCGGCCGTGCTCCGG CTCAGGCCCTCCGCGCGGTAGGAAACGGCGAGGGCCGTCCCGGGGAGCAGCCTCACTTCGCAGCTTTGCTCG CCTTGGTAGG(10)GAAATGGCCTTGGGCGGAGGCGGGGGACAGGCAGGGAACGGAGTGGCCACGTCCAGG TTTCCTGCGGCCACCGAACCGGTGCCTCGCGCCCTGGCGCACCCACGTCCTCGGTTCGG(11)GGTGGACTTGG GGTTCCAAAACAGCCCCAGC >hEpCAM_4th [221 bps; 17 CGs] CTTGGGGTTCCAAAACAGCCCCAGCCGGTGGCGGAGTCTTTACGACAGGGACCAGCGGGCTCGCCCTTGTCC TTGCAGCGGGCCCCGGATGTGGGCCTCAGG(12)CGGGGACAGGCGCCCGCAGGGAGGCCTCCAGGGCCGCT ATGCACCTGCGCGCGGCAGGCGGCCCGGACCACACAGGGCGTGTGGGTGTTTTCCCTTTTCTAAGGATCATAT GAGTAATG

Human CXCR4 promoter >hCXCR4_1st [351 bps, 28 CGs] TGTGTGGGTCTCTTGCCATCCTCGTGTTTATCACTTGGCGCGTTTGG(1)GACGTTAGGGAGCGGGGCATTTTCC TGGGTGGAGAAGGTAACGGGGTCTGCACCCGTGGTCCTCGCCCCAAGTTTCATTTCCTCACTCTCCCGGGTGG CTTCCCATTACCCCGCCACTGATCCAGTTAACCCGGCCGGAGGTGGGCAGCTGGAAGCCTCCAGGCGGTGGG CACGCGGGGGGCCGGGTCGTCCAGCCCCGGGCCGCCGCGGCTGCCCACTACACCCACGCCAACCGCCCGCAA GCAGCGCTGCAGGGGCTCCGCTGGGCGACACGCCAGGCTCTGTCCCACAGGGTGCTGGGGAG >hCXCR4_2nd [325 bps, 28 CGs] GCTCTGTCCCACAGGGTGCTGGGGAGCGACTGGGCGGCTCCGCCGCGAGCGTCTTTGAATTGCGCGCCGCTG CAGGAAACCAAAAACTCCCTAGCAAGAGGGTTTCAAAAGGTTTCTGGAAA(2)CCACCGACGGTTAAACATCAC AACTGGACTCGGAGAGAGCCAAACGGTTTCCCCACTTGCACCTGCCAGTCTTCGCGGCGGCGACCTGGCAGC

CCAGGTGCGGTCTTAACCGCCCCCGCCCCTCACCCCGTACCCGCTCCTATCCCCGGAGCGCAAATCTCAGGGCT GGCAGCTGCGCGGTGTCAAAGGGGAGGTCAAACCAC >hCXCR4_3rd [365 bps, 30 CGs] GTGTCAAAGGGGAGGTCAAACCACTCCGCTGACCTCTGCACGACCCCAAACTCTCGAACTGCAGGACCCACTC GCGGCCGTGGGGAAGAGGCGCGCTTCGGACGGCGGGAAGGTTTTCCCCCTCAAACCCAAAGCGCGCGGGCG GATCAACTCCTAGCTGCTGCCACCACTCGATCCCCTCAGAGGATCGGCGCGGTGGGTCCACCCGCCTCTCCCG CCCTCTGCCTACTGTGCTGGGAGACTGGCACAGCT(3)CCGTCGGCCGCACAGAGTTTAACAAACACGCACCCA GTGTCAAGAACAGTCACCAGGCGCTTAACCCCGAAGTTAAAGCGGGCGCAATCTCCTCCTGGGAACTCAGCCC AGGC >hCXCR4_4th [221 bps, 18 CGs] TCCTCCTGGGAACTCAGCCCAGGCACGCCGCCCTCCGCCTCTAAATTCAGACAATGTAACTCGCTCCAAGACAT CCCCGCTTCCCCAAGGAAGAGACCGGTGGTCTGAGTCCCGAGGCAGCGCGCACGCCTTCTCTGCACTTGTGCA CAGAATGTTCTTACGTTTGCAAACAGCGTGCAAGCCGCCGCGCGCGGCGGGACTCAAGGGGGAGACACATGC AG

Human TFRC promoter >hTFRC1_1st [400 bps, 35 CGs] CACACACAAGGCGAACAGGCAGAGCATGGCAAGATCAGGTTGTACCAGAGCCGAAGG(1)GTAAGTTTACAG AAACCCGTATCTAATTAATGCCTGTAACACGCTAGTAACCGAATCGGGGACAGCTTTGTCCCTTTCCGCAGTGA GAACGGGGCCCGCCCGGAGCCCGGGGAACCGAAGG(2)TTTAGGAGCCCAAGGTCACCCACGAATCCCAGCC GTTCCCTGGGGCGACTTCCTGGAGCAGCCACGTGTTCCCCCGGCGCCGCACGCCGCGCAGCTGCACCTTGGCT GCGCCCGCGCTCCCGGACCCGCAGCCCGGAGCCGGCGCACTAGGGCCTGCGGCCTTCAAGGGCGAGGACCC GAAGTCCCCGGGGCTGAGAGCGACAGTGGGGGTACACCTGGTCT >hTFRC1_2nd [276 bps, 30 CGs] ACAGTGGGGGTACACCTGGTCTCGTCATTCGTCGCTCTGTGACCGAGGAGGCGGGCAAGAAGGCAGAGAGA AGGGAAGGGACGAGAGGCGAAGGGACGAGAGGCGCAGGGACGCCTCTGCGCACAGCCGCCGACCCCGTGT ACCTGCAGCCGCTCGAGGCGCCGCCACAGCCCCTTCCCCACAGGCTCGCGGCCCCGCTCCATCCCGGCCGCCC CGCCTGGGGCCGCGACCGCTTTCCCGCTCCCCGTGCGGCTCCCTATGGCTGCTGGACAGCAC >hTFRC1_3rd [440 bp, 46 CGs] GCTCCCTATGGCTGCTGGACAGCACCGGAGCGGCCGCTCCAGCCAGCCCCGCAGGATGAAGGGAGGACACG AGGGTCGGTGTAGTTCTAGAAGCCCGCACTCACACTAGCGCGTCCTCCGTCCCGAGCCGCCACCCGATATCCC GACGCTCTGAGGGGATGGCGGCCCCTAACCGGCGGTTTATAGCCTGGCCCCGCCCCCGGCCCCCAGGGGGGC TGTGCGTCACTTCCTGAGGCACGTACGCTCGCGAGGTGCTCTGACAGATCGCCCGGGGGGCGGGGATGCGC GCGCAGCGGGCGGGGCCCTATGCGGAGGGCGCTGCGGGCGAGGCGAAGGCCAAGGGAAAAGGCGCCAAG GCAGCAGGTGCCCCGAGGAGCCGCGCGATGGGCGCACTGGCCTCTGCCTCGGCGCCTTCCTCCACCCTGGTA GCCTGGCAGCCT >hTFRC1_4th [440 bp, 9 CGs] TCCACCCTGGTAGCCTGGCAGCCTCCGCCTGACCTTGACCAACCTCCAGTCCCTGCAGTGCTAGGTCCAATCTG TTGGCGTTGACGCTTAAATGTTGGATATTGCACTGCGGCAGTGCCTTGAAATGTACGTGCAGGATGGAATGTT AGGTAACTATTAAAGCCACTCTTGTGAAGATGTATTTTGCAGCCTCAGAAATACAAAAAGGAAAAGAAAAGCA AAATGCAAATTTTTATTCAGAATGTATACAAGCTGCACTTTAACCAAGACTAGCGGAGTGCTTTGCGTAATGTT

TAATGCATGAGGGAATAAAAGGGAAAATAGAAACAAGTGACTTAATAGGGTGATAGGATTGTAGATTAACTT TCGTTTTCATTGCCATCGCTGTTATTGGATTGTGCAATGAATAACAAATAAATAAAATTCAATTCTTGTCACTG *CG sites are highlighted in yellow

Off-targets >hCXCR4 4 off-target [239 bps; 7 CGs] CCTGACCCAGGTGGGACCTCTGGAGCCTGTCCTCTCCCGCCACTGCATGGCGCGGGGATGGAGGGGCCCACG GGTACCCATGGCTCCTGACTGGGGGCCGCTACCAGGGTGGTGCTAGGGTGGGGCCGACCC(CXCR4-4offsite)CCATGCTCGGAGTGTGTTCTTCTAGGACCAAGGTGGCCTCCACCCTGTCCCTCCACAGTCCTGGCTGG CCCTGGATCCACCTGGGCTGGGTTGTGTCTGTGACCAGA >hCXCR4 2 off-target [247 bps; 15 CGs] CCCACCTTCCAGGGAGAAAAGTGCCTCTGCTTCTCGGGAAGTTCCCGGCTTTTCAGCACCCGGGACAACAGCA GCGGCACCTGGGGAGAGGCTCTCCACCCCGCCCCGCGCTCGCTTCCAGTCGCCAGCTTCGTGATCTTCTGCCC GGACTTTCTCTCACCCGCCGGGCGCTGCGTCCTGCAGTCCCAGCCTCCCTTGGGGACAGGGAGCACCTTCCCC AAAGCCAGGAGAAGAGGTCTCCCTGGAC >hTFRC 2 off-target [335 bps; 24 CGs] CACCCGCCCCAGGGAAAGAAGGTGAGTCTATTCGGTTTGCAAGAAAGGTCTCGCGTGTTATCTTGGGCGGGG GGATGATACGATCGTATTCTAATTATCGGCCCCCAGTGGATCTGTCCCGCGTCCTAGCTGCTGCGGCGTTCAGT GGG(2)CACGTAAGAGACACCTGAGGAGGACACTAAACCAACATTAAGGCAGCAGACAAGGGAAGCCGCCGA GGCTAGGAAATCAAAGTGGACCGCGCTGGGCTGGGGACGGGCTGGGGACGCACTGGGGCGGAGGACCAGG GCACAGGCGAGCTAGCGGAGGAGTCGGCTGAGGCCATTCAGCCGCTGATT >hTFRC 3 off-target [301 bps; 8 CGs] GCCCCTGCCTCAGCACATCTGCCCCAGGGGCTGCCCCCGGCTCCTATTCTCTGGACAAAATATCTCCTCCCTTT GCCTTGAAGGCTGTAGTGTTCAGACCTTCCCCCAGCCCATTGGGCTCCCTGGGGACTGACTCCCCCGCCACCAC TGAGCTTCTGCCACCGGCCGCCTGCCGGCCTTCATGAGACTTGACCTGACGTCTGTCTGGTCCCTTTTCT(3)CC CTTCGGCTCTGATTCCACCTCCTGCCTTCCCATCCCAGCTGCAGGAGCGGGGCCCTACAAAGGCAGCAGTAGC CCAGAACA

Unrelated CGIs >hKIAA0179 [410 bp; 55 CGs] TAACCCTGGCTAACCTCGCCGTCTACAGCCTGAATTTTGGCAACCGAAAGGCAGCGCCGGCGCCACGTGCACA CGGGCTGGGCCGCTCCGCCAGCTGCCAGGGCCACTGCCGCGCTCACTCCCAGAGCGCGCTGCGAGCCGCGGC GCCTTTGTGACGCCATCAGCCCGCGCGCCGCCGCCGCCGCCTTCTGTGCAGTCGCGGCCCGGGCGGACGGTG GCTGGCTGCTCCGCAGCGCTCGGCTGGCTGCAGCGGCACCGCGGGTTGCGCGGCCGGGGATGCTCCAGCGG GCGCGATGGCCCCCGCCATGCAGCCGGCCGAGATCCAATTTGCCCAGCGGCTGGCGTCCAGCGAGAAGGGC ATCCGGGACCGAGCGGTGAAGAAGCTGCGCCAGTACATCAGCGTGAAGACG >hDSCR3 [391 bp; 41 CGs] CGTGCCTTTCCACCCTTCACGCGGGGCGCACGGATCTGAATATTTAACACCCGGTTCCAGGACACGCGCCTGG GGAACACCCACAATCCCTCGCGGCCCGCGGAGCCCGGCTGGCGGGTGACCCAGCGCCCGCGGCTCCGAGAG GAGCGAGGTCACCCGGCGCTTCCCGAGACCTTGGCCCTAGGCCCAGCTGGCGCTGGGACGCGAGGCAACCG AGCCGCCGGCCAATCAGCGCTTTGCAGGACTAGCACGCAGGGGCCGCACCCCTGAGGGTGACCGGAACCGCT

CCCGCGGGAAGGGCAGGGTGCGGCGCCTGGCCGCAGACAGACAAGGGGGCGCCGGCTCTTCAGAACCCCA GGGAGAAAGCGGTGGAGGGCTAGAGTTGAGGAGA >hSUMO3 [359 bp; 42 CGs] CGCCTCCCCGCGTCGCCCGGGCCCCGACTCCTTTCCTGGGGGCCGCGTTCCTGGGGCGCCGGGAAGTGAGGC CGGGGCCGCCGCCCATGGCCGCAGGGACCTGGGGCGGAGGAGGGAGGACGCTGGGGGTGACGTTCCCTCT CAGGAGCCGCGGACAGAGCGGAGAAGGCGGCCCGCGGGGTCCCGTCCGCGGGGGCCGCGCGAATCGGGCC AGCACCCTAGACGATTGGGGGCGCCACCCGCCTCCCTGCCGCCGCGGTTTCCTGTCAGCCCCAGGCCGGGCTC CTTTGGCCAACGCTCCTACGGGGCTGCCCCGGGCCCTCCACACCCGTCCTCAAGAAGACCCGTTTTACCTGTGA >hWRB [236 bp; 16 CGs] GGGCCGGTGGGGATGCCGCCCCAGTCCCCCGGCGGGTCTGGCGTAGGTACAGGGGTCTCAACTGGGCGACT GAAGGCCGTAGTAGCGTCTTGGTTGGTCCGTAAGCTTTTTTGAGATAGTTGTTAGCGTCTAAAAGGTACGACG CTTTACCCCAAAATCAGACTTTCTGGGTTCTAGGGGGTTGGGAGAAACACCGGGCAACCCTGGACTCTTCTGG CTGTGGGTGGCGGCGGCGA

Sequence of the Cas9-Dnmt3a and Cas9-Dnmt3a3L single-chain protein and gene dCas9-NLS-linker-Dnmt3a-NLS MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNR ICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAP LSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLN REDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERM KRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTR SDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQIL DSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKG SPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSRADPKKKRKVEASGSGRASPGIPGSTRMNHDQEFDP PKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQE WGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFL ESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFP VFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSRADPKKKRKV atggacaagaagtactccattgggctcgctatcggcacaaacagcgtcggctgggccgtcattacggacgagtacaaggtgccgagcaaaaaa ttcaaagttctgggcaataccgatcgccacagcataaagaagaacctcattggcgccctcctgttcgactccggggagacggccgaagccacgc ggctcaaaagaacagcacggcgcagatatacccgcagaaagaatcggatctgctacctgcaggagatctttagtaatgagatggctaaggtgg atgactctttcttccataggctggaggagtcctttttggtggaggaggataaaaagcacgagcgccacccaatctttggcaatatcgtggacgag gtggcgtaccatgaaaagtacccaaccatatatcatctgaggaagaagcttgtagacagtactgataaggctgacttgcggttgatctatctcgc

gctggcgcatatgatcaaatttcggggacacttcctcatcgagggggacctgaacccagacaacagcgatgtcgacaaactctttatccaactgg ttcagacttacaatcagcttttcgaagagaacccgatcaacgcatccggagttgacgccaaagcaatcctgagcgctaggctgtccaaatcccgg cggctcgaaaacctcatcgcacagctccctggggagaagaagaacggcctgtttggtaatcttatcgccctgtcactcgggctgacccccaactt taaatctaacttcgacctggccgaagatgccaagcttcaactgagcaaagacacctacgatgatgatctcgacaatctgctggcccagatcggc gaccagtacgcagacctttttttggcggcaaagaacctgtcagacgccattctgctgagtgatattctgcgagtgaacacggagatcaccaaagc tccgctgagcgctagtatgatcaagcgctatgatgagcaccaccaagacttgactttgctgaaggcccttgtcagacagcaactgcctgagaagt acaaggaaattttcttcgatcagtctaaaaatggctacgccggatacattgacggcggagcaagccaggaggaattttacaaatttattaagccc atcttggaaaaaatggacggcaccgaggagctgctggtaaagcttaacagagaagatctgttgcgcaaacagcgcactttcgacaatggaagc atcccccaccagattcacctgggcgaactgcacgctatcctcaggcggcaagaggatttctacccctttttgaaagataacagggaaaagattga gaaaatcctcacatttcggataccctactatgtaggccccctcgcccggggaaattccagattcgcgtggatgactcgcaaatcagaagagacca tcactccctggaacttcgaggaagtcgtggataagggggcctctgcccagtccttcatcgaaaggatgactaactttgataaaaatctgcctaac gaaaaggtgcttcctaaacactctctgctgtacgagtacttcacagtttataacgagctcaccaaggtcaaatacgtcacagaagggatgagaa agccagcattcctgtctggagagcagaagaaagctatcgtggacctcctcttcaagacgaaccggaaagttaccgtgaaacagctcaaagaag actatttcaaaaagattgaatgtttcgactctgttgaaatcagcggagtggaggatcgcttcaacgcatccctgggaacgtatcacgatctcctga aaatcattaaagacaaggacttcctggacaatgaggagaacgaggacattcttgaggacattgtcctcacccttacgttgtttgaagataggga gatgattgaagaacgcttgaaaacttacgctcatctcttcgacgacaaagtcatgaaacagctcaagaggcgccgatatacaggatgggggcg gctgtcaagaaaactgatcaatgggatccgagacaagcagagtggaaagacaatcctggattttcttaagtccgatggatttgccaaccggaac ttcatgcagttgatccatgatgactctctcacctttaaggaggacatccagaaagcacaagtttctggccagggggacagtcttcacgagcacat cgctaatcttgcaggtagcccagctatcaaaaagggaatactgcagaccgttaaggtcgtggatgaactcgtcaaagtaatgggaaggcataa gcccgagaatatcgttatcgagatggcccgagagaaccaaactacccagaagggacagaagaacagtagggaaaggatgaagaggattgaa gagggtataaaagaactggggtcccaaatccttaaggaacacccagttgaaaacacccagcttcagaatgagaagctctacctgtactacctgc agaacggcagggacatgtacgtggatcaggaactggacatcaatcggctctccgactacgacgtggctgctatcgtgccccagtcttttctcaaa gatgattctattgataataaagtgttgacaagatccgataaagctagagggaagagtgataacgtcccctcagaagaagttgtcaagaaaatga aaaattattggcggcagctgctgaacgccaaactgatcacacaacggaagttcgataatctgactaaggctgaacgaggtggcctgtctgagtt ggataaagccggcttcatcaaaaggcagcttgttgagacacgccagatcaccaagcacgtggcccaaattctcgattcacgcatgaacaccaa gtacgatgaaaatgacaaactgattcgagaggtgaaagttattactctgaagtctaagctggtctcagatttcagaaaggactttcagttttataa ggtgagagagatcaacaattaccaccatgcgcatgatgcctacctgaatgcagtggtaggcactgcacttatcaaaaaatatcccaagcttgaa tctgaatttgtttacggagactataaagtgtacgatgttaggaaaatgatcgcaaagtctgagcaggaaataggcaaggccaccgctaagtactt cttttacagcaatattatgaattttttcaagaccgagattacactggccaatggagagattcggaagcgaccacttatcgaaacaaacggagaaa caggagaaatcgtgtgggacaagggtagggatttcgcgacagtccggaaggtcctgtccatgccgcaggtgaacatcgttaaaaagaccgaag tacagaccggaggcttctccaaggaaagtatcctcccgaaaaggaacagcgacaagctgatcgcacgcaaaaaagattgggaccccaagaaa tacggcggattcgattctcctacagtcgcttacagtgtactggttgtggccaaagtggagaaagggaagtctaaaaaactcaaaagcgtcaagg aactgctgggcatcacaatcatggagcgatcaagcttcgaaaaaaaccccatcgactttctcgaggcgaaaggatataaagaggtcaaaaaag acctcatcattaagcttcccaagtactctctctttgagcttgaaaacggccggaaacgaatgctcgctagtgcgggcgagctgcagaaaggtaac gagctggcactgccctctaaatacgttaatttcttgtatctggccagccactatgaaaagctcaaagggtctcccgaagataatgagcagaagca gctgttcgtggaacaacacaaacactaccttgatgagatcatcgagcaaataagcgaattctccaaaagagtgatcctcgccgacgctaacctc gataaggtgctttctgcttacaataagcacagggataagcccatcagggagcaggcagaaaacattatccacttgtttactctgaccaacttggg cgcgcctgcagccttcaagtacttcgacaccaccatagacagaaagcggtacacctctacaaaggaggtcctggacgccacactgattcatcag tcaattacggggctctatgaaacaagaatcgacctctctcagctcggtggagacagcagggctgaccccaagaagaagaggaaggtggaggc cagcggttccggacgggcttccccaggaattcccgggtcgactcgaatgaaccatgaccaggaatttgaccccccaaaggtttacccacctgtgc cagctgagaagaggaagcccatccgcgtgctgtctctctttgatgggattgctacagggctcctggtgctgaaggacctgggcatccaagtggac cgctacattgcctccgaggtgtgtgaggactccatcacggtgggcatggtgcggcaccagggaaagatcatgtacgtcggggacgtccgcagcg tcacacagaagcatatccaggagtggggcccattcgacctggtgattggaggcagtccctgcaatgacctctccattgtcaaccctgcccgcaag ggactttatgagggtactggccgcctcttctttgagttctaccgcctcctgcatgatgcgcggcccaaggagggagatgatcgccccttcttctggc tctttgagaatgtggtggccatgggcgttagtgacaagagggacatctcgcgatttcttgagtctaaccccgtgatgattgacgccaaagaagtgt ctgctgcacacagggcccgttacttctggggtaaccttcctggcatgaacaggcctttggcatccactgtgaatgataagctggagctgcaagag tgtctggagcacggcagaatagccaagttcagcaaagtgaggaccattaccaccaggtcaaactctataaagcagggcaaagaccagcatttc cccgtcttcatgaacgagaaggaggacatcctgtggtgcactgaaatggaaagggtgtttggcttccccgtccactacacagacgtctccaacat

gagccgcttggcgaggcagagactgctgggccgatcgtggagcgtgccggtcatccgccacctcttcgctccgctgaaggaatattttgcttgtgt gagcagggcagatccgaagaagaaaaggaaagtc

dCas9-NLS-linker-Dnmt3a-Dnmt3L-NLS MGGRRVRWEVYISRARLVNRQIAWRRHPRCFDLHRRHRDRSSLRTLEDRTLATMDKKYSIGLAIGTNSVGWAVIT DEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSD VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAE DAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALV RQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLG ELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIEC FDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSP AIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQ NEKLYLYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNY WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFF YSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNS DKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLII KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY ETRIDLSQLGGDSRADPKKKRKVEASGSGRASPGIPGSTRNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLV LKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYE GTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPG MNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTD VSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAW KRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWY MFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSK HAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLSRADPKKKRKV atggacaagaagtactccattgggctcgctatcggcacaaacagcgtcggctgggccgtcattacggacgagtacaaggtgccgagcaaaaaa ttcaaagttctgggcaataccgatcgccacagcataaagaagaacctcattggcgccctcctgttcgactccggggagacggccgaagccacgc ggctcaaaagaacagcacggcgcagatatacccgcagaaagaatcggatctgctacctgcaggagatctttagtaatgagatggctaaggtgg atgactctttcttccataggctggaggagtcctttttggtggaggaggataaaaagcacgagcgccacccaatctttggcaatatcgtggacgag gtggcgtaccatgaaaagtacccaaccatatatcatctgaggaagaagcttgtagacagtactgataaggctgacttgcggttgatctatctcgc gctggcgcatatgatcaaatttcggggacacttcctcatcgagggggacctgaacccagacaacagcgatgtcgacaaactctttatccaactgg ttcagacttacaatcagcttttcgaagagaacccgatcaacgcatccggagttgacgccaaagcaatcctgagcgctaggctgtccaaatcccgg cggctcgaaaacctcatcgcacagctccctggggagaagaagaacggcctgtttggtaatcttatcgccctgtcactcgggctgacccccaactt taaatctaacttcgacctggccgaagatgccaagcttcaactgagcaaagacacctacgatgatgatctcgacaatctgctggcccagatcggc gaccagtacgcagacctttttttggcggcaaagaacctgtcagacgccattctgctgagtgatattctgcgagtgaacacggagatcaccaaagc tccgctgagcgctagtatgatcaagcgctatgatgagcaccaccaagacttgactttgctgaaggcccttgtcagacagcaactgcctgagaagt acaaggaaattttcttcgatcagtctaaaaatggctacgccggatacattgacggcggagcaagccaggaggaattttacaaatttattaagccc atcttggaaaaaatggacggcaccgaggagctgctggtaaagcttaacagagaagatctgttgcgcaaacagcgcactttcgacaatggaagc atcccccaccagattcacctgggcgaactgcacgctatcctcaggcggcaagaggatttctacccctttttgaaagataacagggaaaagattga gaaaatcctcacatttcggataccctactatgtaggccccctcgcccggggaaattccagattcgcgtggatgactcgcaaatcagaagagacca tcactccctggaacttcgaggaagtcgtggataagggggcctctgcccagtccttcatcgaaaggatgactaactttgataaaaatctgcctaac

gaaaaggtgcttcctaaacactctctgctgtacgagtacttcacagtttataacgagctcaccaaggtcaaatacgtcacagaagggatgagaa agccagcattcctgtctggagagcagaagaaagctatcgtggacctcctcttcaagacgaaccggaaagttaccgtgaaacagctcaaagaag actatttcaaaaagattgaatgtttcgactctgttgaaatcagcggagtggaggatcgcttcaacgcatccctgggaacgtatcacgatctcctga aaatcattaaagacaaggacttcctggacaatgaggagaacgaggacattcttgaggacattgtcctcacccttacgttgtttgaagataggga gatgattgaagaacgcttgaaaacttacgctcatctcttcgacgacaaagtcatgaaacagctcaagaggcgccgatatacaggatgggggcg gctgtcaagaaaactgatcaatgggatccgagacaagcagagtggaaagacaatcctggattttcttaagtccgatggatttgccaaccggaac ttcatgcagttgatccatgatgactctctcacctttaaggaggacatccagaaagcacaagtttctggccagggggacagtcttcacgagcacat cgctaatcttgcaggtagcccagctatcaaaaagggaatactgcagaccgttaaggtcgtggatgaactcgtcaaagtaatgggaaggcataa gcccgagaatatcgttatcgagatggcccgagagaaccaaactacccagaagggacagaagaacagtagggaaaggatgaagaggattgaa gagggtataaaagaactggggtcccaaatccttaaggaacacccagttgaaaacacccagcttcagaatgagaagctctacctgtactacctgc agaacggcagggacatgtacgtggatcaggaactggacatcaatcggctctccgactacgacgtggctgctatcgtgccccagtcttttctcaaa gatgattctattgataataaagtgttgacaagatccgataaagctagagggaagagtgataacgtcccctcagaagaagttgtcaagaaaatga aaaattattggcggcagctgctgaacgccaaactgatcacacaacggaagttcgataatctgactaaggctgaacgaggtggcctgtctgagtt ggataaagccggcttcatcaaaaggcagcttgttgagacacgccagatcaccaagcacgtggcccaaattctcgattcacgcatgaacaccaa gtacgatgaaaatgacaaactgattcgagaggtgaaagttattactctgaagtctaagctggtctcagatttcagaaaggactttcagttttataa ggtgagagagatcaacaattaccaccatgcgcatgatgcctacctgaatgcagtggtaggcactgcacttatcaaaaaatatcccaagcttgaa tctgaatttgtttacggagactataaagtgtacgatgttaggaaaatgatcgcaaagtctgagcaggaaataggcaaggccaccgctaagtactt cttttacagcaatattatgaattttttcaagaccgagattacactggccaatggagagattcggaagcgaccacttatcgaaacaaacggagaaa caggagaaatcgtgtgggacaagggtagggatttcgcgacagtccggaaggtcctgtccatgccgcaggtgaacatcgttaaaaagaccgaag tacagaccggaggcttctccaaggaaagtatcctcccgaaaaggaacagcgacaagctgatcgcacgcaaaaaagattgggaccccaagaaa tacggcggattcgattctcctacagtcgcttacagtgtactggttgtggccaaagtggagaaagggaagtctaaaaaactcaaaagcgtcaagg aactgctgggcatcacaatcatggagcgatcaagcttcgaaaaaaaccccatcgactttctcgaggcgaaaggatataaagaggtcaaaaaag acctcatcattaagcttcccaagtactctctctttgagcttgaaaacggccggaaacgaatgctcgctagtgcgggcgagctgcagaaaggtaac gagctggcactgccctctaaatacgttaatttcttgtatctggccagccactatgaaaagctcaaagggtctcccgaagataatgagcagaagca gctgttcgtggaacaacacaaacactaccttgatgagatcatcgagcaaataagcgaattctccaaaagagtgatcctcgccgacgctaacctc gataaggtgctttctgcttacaataagcacagggataagcccatcagggagcaggcagaaaacattatccacttgtttactctgaccaacttggg cgcgcctgcagccttcaagtacttcgacaccaccatagacagaaagcggtacacctctacaaaggaggtcctggacgccacactgattcatcag tcaattacggggctctatgaaacaagaatcgacctctctcagctcggtggagacagcagggctgaccccaagaagaagaggaaggtggaggc cagcggttccggacgggcttccccaggaattcccgggtcgactcgaaaccatgaccaggaatttgaccccccaaaggtttacccacctgtgccag ctgagaagaggaagcccatccgcgtgctgtctctctttgatgggattgctacagggctcctggtgctgaaggacctgggcatccaagtggaccgc tacattgcctccgaggtgtgtgaggactccatcacggtgggcatggtgcggcaccagggaaagatcatgtacgtcggggacgtccgcagcgtca cacagaagcatatccaggagtggggcccattcgacctggtgattggaggcagtccctgcaatgacctctccattgtcaaccctgcccgcaaggg actttatgagggtactggccgcctcttctttgagttctaccgcctcctgcatgatgcgcggcccaaggagggagatgatcgccccttcttctggctct ttgagaatgtggtggccatgggcgttagtgacaagagggacatctcgcgatttcttgagtctaaccccgtgatgattgacgccaaagaagtgtct gctgcacacagggcccgttacttctggggtaaccttcctggcatgaacaggcctttggcatccactgtgaatgataagctggagctgcaagagtg tctggagcacggcagaatagccaagttcagcaaagtgaggaccattaccaccaggtcaaactctataaagcagggcaaagaccagcatttccc cgtcttcatgaacgagaaggaggacatcctgtggtgcactgaaatggaaagggtgtttggcttccccgtccactacacagacgtctccaacatga gccgcttggcgaggcagagactgctgggccgatcgtggagcgtgccggtcatccgccacctcttcgctccgctgaaggaatattttgcttgtgtgt ctagcggcaatagtaacgctaacagccgcgggccgagcttcagcagcggcctggtgccgttaagcttgcgcggcagccatatgggccctatgga gatatacaagacagtgtctgcatggaagagacagccagtgcgggtactgagcctttttagaaatattgataaagtactaaagagtttgggcttttt ggaaagcggttctggttctgggggaggaacgctgaagtacgtggaagatgtcacaaatgtcgtgaggagagacgtggagaaatggggcccctt tgacctggtgtacggctcgacgcagcccctaggcagctcttgtgatcgctgtcccggctggtacatgttccagttccaccggatcctgcagtatgc gctgcctcgccaggagagtcagcggcccttcttctggatattcatggacaatctgctgctgactgaggatgaccaagagacaactacccgcttcct tcagacagaggctgtgaccctccaggatgtccgtggcagagactaccagaatgctatgcgggtgtggagcaacattccagggctgaagagcaa gcatgcgcccctgaccccaaaggaagaagagtatctgcaagcccaagtcagaagcaggagcaagctggacgccccgaaagttgacctcctgg tgaagaactgccttctcccgctgagagagtacttcaagtatttttctcaaaactcacttcctcttagcagggcagatccgaagaagaaaaggaaa gtc

catalytically inactive SPn-Cas9 (D10A, H840A) NLS Linker (together with NLS – 28 aa linker) Dnmt3a CD Dnmt3L domain