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

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1 Supplementary Information RNA-Guided Gene Activation by CRISPR-Cas9-Based Transcription Factors Pablo Perez-Pinera 1, Daniel D. Kocak 1, Christopher M. Vockley 2,3, Andrew F. Adler 1, Ami M. Kabadi 1, Lauren R. Polstein 1, Pratiksha I. Thakore 1, Katherine A. Glass 1,4, David G. Ousterout 1, Kam W. Leong 1,5, Farshid Guilak 1,4, Gregory E. Crawford 2,6, Timothy E. Reddy 2,7, and Charles A. Gersbach 1,2,4 1 Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of America, Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, United States of America, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, United States of America, Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina, United States of America, King Abdulaziz University, Jeddah, Saudi Arabia 6 Department of Pediatrics, Division of Medical Genetics, Duke University Medical Center, Durham, North Carolina, United States of America, Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, North Carolina, United States of America, * Address for correspondence: Charles A. Gersbach, Ph.D. Department of Biomedical Engineering Room 136 Hudson Hall, Box Duke University Durham, NC Phone: Fax: charles.gersbach@duke.edu 1

2 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. 2

3 Supplementary Figure 2. Positions of grna target sites and DNAse hypersensitivity of human target genes. IL1RN: ASCL1: NANOG: HBG1: MYOD1: 3

4 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 described 1, 2. The results show that open chromatin was not a requirement for gene activation by combinations of grnas with dcas9-vp64. 4

5 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. 5

6 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 cotransfected 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 = ) and TNFRS9 (false discovery rate = 0.002). 6

7 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. 7

8 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. 8

9 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 < vs. Empty vector, Tukey s test). 9

10 Supplementary Figure 8. dcas9-vp64 protein sequence. MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGRGMDKKYSIGLAIGTNSVGWAVIT DEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKK LVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA KLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIK RYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQ TVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVL TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYK VREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKAT AKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVN IVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRM LASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQ ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK RYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDPIAGSKASPKKKRKVGRADALDDFD LDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINYPYDVPDYAS 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 10

11 Supplementary Figure 9. Sequence of grna expression cassette with U6 promoter. GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAG AGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACG TGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAA AATGGACTATCATATGCTTATCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATA TATCTTGTGGAAAGGACGAAACACCGGGTCTTCGAGAAGACCTGTTTTAGAGCTA GAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGA GTCGGTGCTTTTTTT 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 11

12 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 %. 12

13 Supplementary Table 1: Target sequences and positions of grnas. Target Name Sequence Position Relative to Transcriptional Start Site CR1 GCTGGGTGTCCCATTGAAA -43 ASCL1 CR2 CAGCCGCTCGCTGCAGCAG -103 CR3 TGGAGAGTTTGCAAGGAGC -220 CR4 GTTTATTCAGCCGGGAGTC -284 CR1 CGCCAGGAGGGGTGGGTCTA -36 NANOG CR2 CCTTGGTGAGACTGGTAGA -103 CR3 GTCTTCAGGTTCTGTTGCT -182 CR4 ATATTCCTGATTTAAAAGT -241 CR1 TTAAAAGTCGGCTGGTAGC -28 VEGFA CR2 CGGGCCGGGGGCGGGGTCC -83 CR3 GCCCGAGCCGCGTGTGGAA -135 CR4 CCTTCATTGCGGCGGGCTG -189 CR1 CCGACCCCTCCCGGGTCCC -79 TERT CR2 CAGGACCGCGCTTCCCACG -181 CR3 TGCACCCTGGGAGCGCGAG -305 CR4 CCGCACGCACCTGTTCCCA -412 CR1 AAAACAGCGAGGGAGAAAC -9 IL1B CR2 TTAACTTGATTGTGAAATC -82 CR3 AAAACAATGCATATTTGCA -227 CR4 AAAATCCAGTATTTTAATG -275 CR1 ACCCAGCACTGCAGCCTGG -28 IL1R2 CR2 AACTTATGCGGCGTTTCCT -82 CR3 TCACTTTAAAACCACCTCT -146 CR4 GCATCTTTTTCTCTTTAAT -191 CR1 TGTACTCTCTGAGGTGCTC -29 IL1RN CR2 ACGCAGATAAGAACCAGTT -180 CR3 CATCAAGTCAGCCATCAGC -113 CR4 GAGTCACCCTCCTGGAAAC -145 CR1 GCTAGGGATGAAGAATAAA -26 HBG1 CR2 TTGACCAATAGCCTTGACA -101 CR3 TGCAAATATCTGTCTGAAA -163 CR4 AAATTAGCAGTATCCTCTT -209 CR1 CCTGGGCTCCGGGGCGTTT -55 MYOD1 CR2 GGCCCCTGCGGCCACCCCG -142 CR3 CTCCCTCCCTGCCCGGTAG -214 CR4 AGGTTTGGAAAGGGCGTGC

14 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 14

15 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, (2011). 3. Guschin, D.Y. et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol 649, (2010). 15