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Supplementary Information

RNA-Guided Gene Activation by CRISPR-Cas9-Based Transcription Factors

Pablo Perez-Pinera1, Daniel D. Kocak

1, Christopher M. Vockley

2,3, Andrew F. Adler

1, Ami M. Kabadi

1,

Lauren R. Polstein1, Pratiksha I. Thakore

1, Katherine A. Glass

1,4, David G. Ousterout

1, Kam W. Leong

1,5,

Farshid Guilak1,4

, Gregory E. Crawford2,6

, Timothy E. Reddy2,7

, and Charles A. Gersbach1,2,4

1 Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of

America, 27708

2Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, United States of

America, 27708

3Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, United States of

America, 27710

4Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina, United

States of America, 27710

5King Abdulaziz University, Jeddah, Saudi Arabia

6Department of Pediatrics, Division of Medical Genetics, Duke University Medical Center, Durham,

North Carolina, United States of America, 27710

7Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, North

Carolina, United States of America, 27710

*Address for correspondence:

Charles A. Gersbach, Ph.D.

Department of Biomedical Engineering

Room 136 Hudson Hall, Box 90281

Duke University

Durham, NC 27708-0281

Phone: 919-613-2147

Fax: 919-668-0795

Email: charles.gersbach@duke.edu

Nature Methods: doi:10.1038/nmeth.2600

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Supplementary Figure 1. Expression of dCas9-VP64

Expression of dCas9-VP64 in transfected HEK293T cells was confirmed by western blot for the

N-terminal Flag epitope tag. The wt Cas9 expression plasmid does not contain the epitope tag.

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Supplementary Figure 2. Positions of gRNA target sites and DNAse hypersensitivity of human

target genes.

IL1RN:

ASCL1:

NANOG:

HBG1:

MYOD1:

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VEGFA:

TERT:

IL1B:

IL1R2:

The four gRNA target sites (CR1-4) for each locus are designated as custom tracks above

each gene and DNase-seq data indicating DNAse-hypersensitive open chromatin regions

is shown below each gene. DNase-seq was performed in HEK293T cells to identify

DNase hypersensitive regions as previously described1, 2

. The results show that open

chromatin was not a requirement for gene activation by combinations of gRNAs with

dCas9-VP64.

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Supplementary Figure 3. Absence of detectable nuclease activity by dCas9-VP64.

Wild-type Cas9 or dCas9-VP64 (D10A, H840A) expression plasmids were co-transfected with

expression plasmids for four different guide RNAs targeting the IL1RN promoter. Nuclease

activity was determined by the Surveyor assay, which has a detection limit of approximately 1-

2% modified alleles.3 The lower molecular weight bands indicative of nuclease activity and

DNA repair by non-homologous end joining are only present following treatment with wild-type

Cas9, supporting abrogation of nuclease activity by dCas9-VP64.

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Supplementary Figure 4. RNA-seq for samples treated with gRNAs targeting HBG1 and HBG2.

RNA-seq was performed on samples treated with a control empty expression vector (n = 3) or co-

transfected with the expression plasmids for dCas9-VP64 and the four gRNAs targeting HBG1 (n = 2).

Three of these gRNAs also perfectly target HBG2. Increases in both HBG1 and HBG2 relative to control

were observed but were not statistically significant due to low expression levels. The only statistically

significant changes in gene expression between these treatments were decreases in IL32 (false discovery

rate = 0.0007) and TNFRS9 (false discovery rate = 0.002).

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Supplementary Figure 5. Upregulation of Ascl1 and γ-globin by dCas9-VP64.

HEK293T cells were transfected with dCas9-VP64 and four gRNAs targeting the ASCL1 or

HBG1 promoter. Levels of corresponding Ascl1 and γ-globin protein production were assessed

by western blot. Low levels of these proteins were detectable in HEK293T cells and increases in

expression were detectable following dCas9-VP64 treatment in two independent experiments.

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Supplementary Figure 6. Activation of Ascl1 in dCas9-VP64-treated murine embryonic

fibroblasts.

Mouse embryonic fibroblasts (MEFs) were transfected with a control GFP expression plasmid or

the dCas9-VP64 expression plasmid and a combination of four gRNA expression plasmids

targeting ASCL1 at a ratio of 50:50 or 75:25. (a) The gRNA target sites in the human ASCL1

promoter are conserved in the mouse ASCL1 promoter. Target sites are shown in yellow and the

transcribed region is shown in red. (b) ASCL1 expression in MEFs increased at four days after

dCas9-VP64/gRNA treatment as determined by qRT-PCR. n = 2 independent experiments and

data are represented as mean ± standard error of the mean.

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Supplementary Figure 7. IL32 expression in response to dCas9-VP64.

The expression of IL32 was assessed by qRT-PCR in HEK293T cells in response to transfection

with an empty vector control expression plasmid (pCMV), the dCas9-VP64 expression plasmid,

the combination of four IL1RN gRNA expression plasmids, or the dCas9-VP64 and IL1RN

expression plasmids. The downregulation of IL32 observed by RNA-seq (Fig. 1f,

Supplementary Fig. 4) is a general response to dCas9-VP64. n = 2 independent transfections

and data are represented as mean ± standard error of the mean (* P < 0.0001 vs. Empty vector,

Tukey’s test).

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Supplementary Figure 8. dCas9-VP64 protein sequence .

MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGRGMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDPIAGSKASPKKKRKVGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINYPYDVPDYAS

FLAG epitope tag = italicized Nuclear localization sequence = bold Streptococcus pyogenes Cas9 (D10A, H840A) = underlined VP64 (4x minimal VP16 domain) = italicized and bold HA epitope tag = italicized and underlined

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Supplementary Figure 9. Sequence of gRNA expression cassette with U6 promoter.

GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTATCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGTCTTCGAGAAGACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

U6 promoter = bold +1 transcription start site = underlined BbsI restriction sites to clone in guide RNA = italicized and underlined Chimeric guide RNA sequence = italicized Poly-T terminator sequence = bold and underlined

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Supplementary Figure 10. Standard curves for qRT-PCR.

For each gene, the experimental sample with the highest expression level was diluted to create a

standard curve that was assayed by qRT-PCR to ensure efficient amplification over an

appropriate dynamic range. The efficiencies of all amplification reactions were within 90-115%.

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Supplementary Table 1: Target sequences and positions of gRNAs.

Target Name Sequence Position Relative to

Transcriptional Start Site

ASCL1

CR1 GCTGGGTGTCCCATTGAAA -43

CR2 CAGCCGCTCGCTGCAGCAG -103

CR3 TGGAGAGTTTGCAAGGAGC -220

CR4 GTTTATTCAGCCGGGAGTC -284

NANOG

CR1 CGCCAGGAGGGGTGGGTCTA -36

CR2 CCTTGGTGAGACTGGTAGA -103

CR3 GTCTTCAGGTTCTGTTGCT -182

CR4 ATATTCCTGATTTAAAAGT -241

VEGFA

CR1 TTAAAAGTCGGCTGGTAGC -28

CR2 CGGGCCGGGGGCGGGGTCC -83

CR3 GCCCGAGCCGCGTGTGGAA -135

CR4 CCTTCATTGCGGCGGGCTG -189

TERT

CR1 CCGACCCCTCCCGGGTCCC -79

CR2 CAGGACCGCGCTTCCCACG -181

CR3 TGCACCCTGGGAGCGCGAG -305

CR4 CCGCACGCACCTGTTCCCA -412

IL1B

CR1 AAAACAGCGAGGGAGAAAC -9

CR2 TTAACTTGATTGTGAAATC -82

CR3 AAAACAATGCATATTTGCA -227

CR4 AAAATCCAGTATTTTAATG -275

IL1R2

CR1 ACCCAGCACTGCAGCCTGG -28

CR2 AACTTATGCGGCGTTTCCT -82

CR3 TCACTTTAAAACCACCTCT -146

CR4 GCATCTTTTTCTCTTTAAT -191

IL1RN

CR1 TGTACTCTCTGAGGTGCTC -29

CR2 ACGCAGATAAGAACCAGTT -180

CR3 CATCAAGTCAGCCATCAGC -113

CR4 GAGTCACCCTCCTGGAAAC -145

HBG1

CR1 GCTAGGGATGAAGAATAAA -26

CR2 TTGACCAATAGCCTTGACA -101

CR3 TGCAAATATCTGTCTGAAA -163

CR4 AAATTAGCAGTATCCTCTT -209

MYOD1

CR1 CCTGGGCTCCGGGGCGTTT -55

CR2 GGCCCCTGCGGCCACCCCG -142

CR3 CTCCCTCCCTGCCCGGTAG -214

CR4 AGGTTTGGAAAGGGCGTGC -274

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Supplementary Table 2: Sequences of primers used for qRT-PCR.

Target Forward primer Reverse Primer

hASCL1 GGAGCTTCTCGACTTCACCA AACGCCACTGACAAGAAAGC

NANOG GATTTGTGGGCCTGAAGAAA CAGATCCATGGAGGAAGGAA

VEGFA AAGGAGGAGGGCAGAATCAT GGGTACTCCTGGAAGATGTCC

TERT AAACCTTCCTCAGCTATGCCC GTTTGCGACGCATGTTCCTC

IL1B AGCTGATGGCCCTAAACAGA AAGCCCTTGCTGTAGTGGTG

IL1R2 CAGGAGGACTCTGGCACCTA CGGCAGGAAAGCATCTGTAT

IL1RN GGAATCCATGGAGGGAAGAT TGTTCTCGCTCAGGTCAGTG

HBG1/2 GCTGAGTGAACTGCACTGTGA GAATTCTTTGCCGAAATGGA

MYOD1 CTCTCTGCTCCTTTGCCACA GTGCTCTTCGGGTTTCAGGA

GAPDH CAATGACCCCTTCATTGACC TTGATTTTGGAGGGATCTCG

mASCL1 GGAACAAGAGCTGCTGGACT GTTTTTCTGCCTCCCCATTT

mGAPDH AACTTTGGCATTGTGGAAGG GGATGCAGGGATGATGTTCT

IL32 GCTACCTGGAGACAGTGG ATCTGTTGCCTCGGCACC

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Supplementary References

1. Song, L. & Crawford, G.E. DNase-seq: a high-resolution technique for mapping active gene regulatory

elements across the genome from mammalian cells. Cold Spring Harbor protocols 2010, pdb

prot5384 (2010).

2. Song, L. et al. Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that

shape cell-type identity. Genome Res 21, 1757-1767 (2011).

3. Guschin, D.Y. et al. A rapid and general assay for monitoring endogenous gene modification. Methods

Mol Biol 649, 247-256 (2010).

Nature Methods: doi:10.1038/nmeth.2600