Nature Methods: doi: /nmeth Supplementary Figure 1. Schematics of SAM, dcas9-suntag and dcas9-vpr.
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- Solomon O’Connor’
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1 Supplementary Figure 1 Schematics of SAM, dcas9-suntag and dcas9-vpr. (a) Schematics of SAM. SAM consists of two chimeric proteins, dcas9 fused with VP64 and MS2-coat protein fused with p65 and HSF1 activator domains (MS2-NLS-p65-HSF1), and an sgrna having MS2 aptamers in stem loops, known as sgrna 2.0. MS2 aptamers in sgrna recruits MS2-NLS-p65-HSF1, so this system can simultaneously recruit VP64, p65 and HSF1. (b) Schematics of dcas9- Suntag. dcas9-suntag consists of two chimeric proteins, dcas9 fused with 10 copies of a GCN4 peptide and single-chain variable fragment nanobody (scfv) for GCN4 fused with VP64 activator domains (scfv-vp64), and an sgrna. Because scfv-vp64 strongly binds to GCN4 peptides, this system can recruit multiple VP64 into Cas9-targeted locus. (c) Schematics of dcas9-vpr. VPR is a potent tripartite activator consisting of VP64, p65ad and the Epstein-Barr virus R transactivator Rta.
2 Supplementary Figure 2 Design and characterization of padcas9-sam, padcas9-suntag and padcas9-vpr. (a) Schematics of padcas9-sam. padcas9-sam consists of three chimeric proteins, N-terminal fragment of dcas9 (residues 2 713, named dcas9 N ) fused with pmag (NLS-dCas9 N -pmag), C-terminal fragment of dcas9 (residues , named dcas9 c ) fused with VP64 and nmag (nmag-dcas9 c -NLS-VP64) and MS2-coat protein fused with p65 and HSF1 activator domains (MS2-NLS-p65-HSF1), and an sgrna having MS2 aptamers in stem loops, known as sgrna 2.0. MS2 aptamers in sgrna recruits MS2-NLS-p65-HSF1, so this system can simultaneously recruit VP64, p65 and HSF1. (b) Schematics of padcas9-suntag. padcas9-suntag consists of three chimeric proteins, NLS-dCas9 N -pmag, C-terminal fragment of dcas9 fused with nmag and 10 copies of a GCN4 peptide (nmag-dcas9 c -NLS-10xGCN4), and single-chain variable fragment nanobody (scfv) for GCN4 fused with VP64 activator domains (scfv-vp64), and an sgrna. Because scfv-vp64 strongly binds to GCN4 peptides, this system can recruit multiple VP64 into targeted locus by light. (c) Schematics of padcas9-vpr. VPR is a potent tripartite activator consisting of VP64, p65ad and the Epstein-Barr virus R transactivator Rta. padcas9-vpr consists of two chimeric proteins, NLS-dCas9 N -pmag and C-terminal fragment of dcas9 fused with nmag and VPR (nmag-dcas9 c -NLS-VPR), and an sgrna. (d) Light-induced endogenous ASCL1 activation in HEK293T cells with padcas9-suntag, padcas9-vpr and padcas9-sam with a single sgrna targeting the promoter region of ASCL1. Data are expressed as relative mrna amount to the negative control transfected with empty vector in the dark and represented as mean ± s.e.m. (n=3 from three individual experiments). D, Dark; L, Light.
3 Supplementary Figure 3 Optimization of padcas9-sam. (a) Optimizing subcellular localization of padcas9 fragments and replacing nmag photoactivatable dimerization domain. The nuclear localization signal (NLS) of NLS-dCas9N-pMag was removed and two copies of nuclear export signal (NES) were fused with dcas9npmag (NES-dCas9 N -pmag-nes). We also replaced nmag of nmag-dcas9 C -NLS-VP64 with nmaghigh1 for enhancing dimerization efficiency (nmaghigh1-dcas9 C -NLS-VP64). (b) Screening of MS2 effectors that have different designs. About MS2-coat protein, we tested MS2(N55K) mutant, which is originally used in SAM, and non-aggregating MS2 delta FG mutant. We also tested whether the number and position of NLS effects induction potency. (c) Gene activation efficiencies of several MS2 effectors with NES-dCas9 N - pmag-nes and nmaghigh1-dcas9 C -NLS-VP64. sgrna 2.0 targeting human ASCL1 is used. Data are expressed as relative ASCL1 mrna amount to the negative control transfected with empty vector in the dark and represented as mean ± s.e.m. (n=4 from two individual experiments with two cell culture replicates). D, Dark; L, Light.
4 Supplementary Figure 4 Optogenetic activation of various genes with Split-CPTS2.0. Light-induced upregulation of ASCL1 (a), IL1R2 (b), NEUROD1 (c), IL1RN (d) and MYOD1 (e) in HEK293T cells using Split-CPTS2.0, CPTS, LACE, dcas9-vp64 and SAM with a single sgrna targeting each gene. Data are expressed as relative mrna amount to the negative control transfected with empty vector in the dark and represented as mean ± s.e.m. ((a d) n=4 from two individual experiments with two cell culture replicates. (e) n=6 from three individual experiments with two cell culture replicates.). D, Dark; L, Light; ND, Not determined. Welch s two-tailed t test was performed. *p < 0.05, ** < 0.01, ***p < versus the sample in the dark.
5 Supplementary Figure 5 Split-CPTS2.0 can induce potent gene activation in various human cells. Light-induced ASCL1 upregulation in HeLa cells (a) and human fetal fibroblasts (b) using Split-CPTS2.0, CPTS, LACE, dcas9-vp64 and SAM with a single sgrna. Data are expressed as relative ASCL1 mrna amount to the negative control transfected with empty vector or EGFP in the dark and represented as mean ± s.e.m. ((a) n=4 from two individual experiments with two cell culture replicates. (b) n=2 from two individual experiments). D, Dark; L, Light.
6 Supplementary Figure 6 Split-CPTS2.0-mediated optogenetic gene activation with multiple sgrnas. Light-induced upregulation of MYOD1 (a) and IL1RN (b) in HEK293T cells using Split-CPTS2.0 and CPTS with a single sgrna and quadruple sgrnas targeting MYOD1 and IL1RN, respectively. Data are expressed as relative mrna amount to the negative control transfected with empty vector in the dark and represented as mean ± s.e.m. (n=6 from three individual experiments with two cell culture replicates). D, Dark; L, Light. Welch s two-tailed t test was performed. *p < 0.05, ** < 0.01, ***p < versus the sample in the dark.
7 Supplementary Figure 7 Spatial surrogate reporter activation by Split-CPTS2.0. (a) Schematic of GAL4-UAS mcherry reporter. (b) Light-induced mcherry reporter activation of Split-CPTS2.0. HEK293T cells were transfected with Split-CPTS2.0, GAL4-UAS mcherry reporter and sgrna targeting the reporter. Twenty hours post transfection, samples were incubated under dark state, global blue light illumination for 24 h. Scale bar = 2 mm. (c) Spatial gene activation by Split- CPTS2.0. Slit-patterned mcherry expression in HEK293T cells illuminated by blue light with a spatial pattern using a photomask. EGFP is used as transfection marker. The width of slit is 2.6 mm. Close-up view within white square region is also shown. Scale bar, 2 mm (zoom-out view) and 0.5 mm (close-up view), respectively. (d) Line scan intensity profile of EGFP (turquoise) and mcherry (magenta) on dashed line in c.
8 Supplementary Figure 8 CPTS2.0-mediated optogenetic endogenous gene activation. (a,b) Light-induced upregulation of ASCL1 (a) and MYOD1 (b) in HEK293T cells. Used dcas9-activators and the number of sgrnas are indicated. (c,d) Light-induced IL1RN upregulation in HeLa cells (c) and human induced pluripotent stem cells (d) using CPTS, CPTS2.0 and Split-CPTS2.0 with a single sgrna targeting IL1RN gene. (e) Reversible and repeatable activation of endogenous ASCL1 with CPTS2.0. Data are expressed as relative mrna amount to the negative control transfected with empty vector in the dark and represented as mean ± s.e.m. (n=4 from two individual experiments with two cell culture replicates). D, Dark; L, Light. Welch s twotailed t test was performed. *p < 0.05, ** < 0.01, ***p < versus the sample in the dark.
9 Supplementary Figure 9 Comparison of light-induced MYOD1 and IL1RN activation efficiency among various sgrnas targeting different sites in the promoter regions. Light-induced upregulation of MYOD1 (a) and IL1R2 (b) in HEK293T cells using Split-CPTS2.0, CPTS2.0, CPTS and SAM with an indicated single sgrna. Maps show the sgrna targeting sites in each human locus. Data are expressed as relative mrna amount to the negative control transfected with empty vector in the dark and represented as mean ± s.e.m. (n=4 from two individual experiments with two cell culture replicates).
10 Supplementary Figure 10 DNA-targeting specificity analysis of Split-CPTS2.0 and CPTS2.0. (a) Luciferase reporter is constructed of Cas9-binding sites, minimal CMV promoter and luciferase gene. (b) DNA targeting specificity analysis of Split-CPTS2.0, CPTS2.0, dcas9-vp64 and SAM using luciferase reporter plasmid activation assay. The positions of mutations in each sgrna are indicated in red. Values are normalized to positive control with perfectly matched sgrnas. Data are shown as the mean ± s.e.m. (n=6 from two individual experiments with three cell culture replicates).
11 Supplementary Figure 11 NEUROD1 activation kinetics in human ipscs with dcas9 activators. (a) Light-induced NEUROD1 upregulation in human ipscs using indicated dcas9 activators with indicated sgrnas at day 4. The negative sgrna targets unrelated DNA sequence. (b) Time-course of NEUROD1 activation in human ipscs using indicated dcas9 activators. Data are expressed as relative mrna amount to the negative control transfected with EGFP vector in the dark and are shown as the mean ± s.e.m. (n=3 three cell culture replicates). D, Dark; L, Light; ND, Not determined. Welch s two-tailed t test was performed. ***p < versus the sample in the dark.
12 Supplementary Figure 12 Representative images of Split-CPTS2.0-induced neurons. Immunofluorescence images of Split-CPTS2.0-induced neurons from human ipscs (454E2) for beta III tubulin (magenta) and DAPI (blue). Immunofluorescence images were taken 4 days after blue light stimulation or dark incubation. Scale bar = 50 μm.
13 Supplementary Figure 13 CPTS could not induce neuronal differentiation of human ipscs. Immunofluorescence images of human ipscs for beta III tubulin (magenta), DAPI (blue) and EGFP (Green) after 96 h blue light illumination or incubation in the dark. Transfected plasmids are indicated. Images are representative from three individual experiments. Scale Bar = 50 μm.
14 Supplementary Figure 14 Split-CPTS2.0-mediated neuronal differentiation of human ipscs (201B7). (a) Immunofluorescence images of 201B7-derived neurons induced by split-cpts2.0 targeting NEUROD1. Cells were immunostained after 4 days blue light stimulation or dark incubation. (b) Immunofluorescence images of 201B7 cells expressing indicated dcas9-based photoactivatable transcription systems and sgrnas. Cells were immunostained after 4 days blue light stimulation or dark incubation. Magenta, beta III tubulin; Blue, DAPI; Green, EGFP. Scale bar = 50 μm.
15 Supplementary Figure 15 CPTS2.0 induced higher NEUROD1 expression in human ipscs than CPTS but was still insufficient to induce neuronal differentiation. (a) Light-induced NEUROD1 upregulation in human ipscs using indicated dcas9-activators with four sgrnas targeting NEUROD1 at day 1. Data are expressed as relative mrna amount to the negative control transfected with EGFP vector in the dark and represented as mean ± s.e.m. (n=3 cell culture replicates from one of two representative experiments). (b) Immunofluorescence images of human ipscs expressing CPTS2.0 targeting NEUROD1 with four sgrnas. Magenta, beta III tubulin; Blue, DAPI. In this experiment, no positive beta III tubulin signal was observed. Immunostaining were performed after 96 h blue light illumination or incubation in the dark. Scale Bar = 50 μm.
16 Supplementary Table 1 Statistical tests for figures. See figure legends and Methods for descriptions of statistical tests used. Statistically significant differences (P < 0.05) are bolded. Figure Data set 1 Data set 2 P value 1c Split-CPTS2.0 (ASCL1, Dark) Split-CPTS2.0 (ASCL1, Light) Split-CPTS2.0 (MYOD1, Dark) Split-CPTS2.0 (MYOD1, Light) Split-CPTS2.0 (IL1R2, Dark) Split-CPTS2.0 (IL1R2, Light) Split-CPTS2.0 (HBG1, Dark) Split-CPTS2.0 (HBG1, Light) CPTS (ASCL1, Dark) CPTS (ASCL1, Light) 0.23 CPTS (MYOD1, Dark) CPTS (MYOD1, Light) CPTS (IL1R2, Dark) CPTS (IL1R2, Light) 0.13 CPTS (HBG1, Dark) CPTS (HBG1, Light) a Split-CPTS2.0 (x1 sgrna, Dark) Split-CPTS2.0 (x1 sgrna, Light) Split-CPTS2.0 (Neg. sgrna, Split-CPTS2.0 (Neg. sgrna, 0.87 Dark) Light) CPTS (x1 sgrna, Dark) CPTS (x1 sgrna, Light) CPTS (x4 sgrna, Dark) CPTS (x4 sgrna, Light) EGFP control (Dark) EGFP control (Light) 0.84 S1
17 Supplementary Table 2 Target sequences of sgrnas and oligonucleotide sequences for constructing sgrnas. See attached excel file. S2
18 Supplementary Note 1 Amino acid sequences of photoactivatable dcas9 fused with VP64 activator domain. NES-N713d-pMag-NES (Split-CPTS2.0) MGLPPLERLTLGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLAL AHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSAR LSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTY DDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHH QDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKI LTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNF DKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEE NEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVGTGGSGSSGG SGHTLYAPGGYDIMGYLRQIRNRPNPQVELGPVDTSCALILCDLKQKDTPIVYASE AFLYMTGYSNAEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTINTMRKAIDRNAE VQVEVVNFKKNGQRFVNFLTMIPVRDETGEYRYSMGFQCETEGGSGGSGGGSG SGSGGEFLPPLERLTL S3
19 nmaghigh1-c714d-nls-vp64 (Split-CPTS2.0) MGGSGSSGGSGGSGGSGHTLYAPGGYDIMGYLDQIGNRPNPQVELGPVDTSCA LILCDLKQKDTPIVYASEAFLYMTGYSNAEVLGRNCRFLQSPDGMVKPKSTRKYV DSNTINTIRKAIDRNAEVQVEVVNFKKNGQRFVNFLTIIPVRDETGEYRYSMGFQC ETEGGSGGSGGGSGSGSGGGTSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDE LVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLT RSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKS EQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSP TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAEN IIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGG DEFGGGGSGGGGSGGGGSGPKKKRKVAAAGSGRADALDDFDLDMLGSDALDD FDLDMLGSDALDDFDLDMLGSDALDDFDLDMLIN S4
20 NLS-dCas9-NLS (CPTS2.0) MYPYDVPDYASPKKKRKVEASDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVL GNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAK VDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKA DLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAK LQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIK PILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLK DNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS FIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKK AIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQ GDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQK GQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE LDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR MNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVG TALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKEL S5
21 LGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEF SKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK RYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSPKKKRKVEAS S6
22 Supplementary Note 2 Amino acid sequences of activation effectors. MS2(N55K)-NLS-p65-HSF1 MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSA QKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQG LLKDGNPIPSAIAANSGIYGGSGGSGGGSGSGSGSGPKKKRKVAAAGSPSGQIS NQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLSAPVPKSTQA GEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSEFQQLLNQGVSM SHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFSSIADM DFSALLSQISSSGQGGGGSGFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQEL LSPQEPPRPPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEG SYFSEGDGFAEDPTISLLTGSEPPKAKDPTVS S7
23 NLS-MS2(N55K)-p65-HSF1 MGPKKKRKVGGSGMASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRS QAYKVTCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIF ATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYGTPSGQISNQALALAPSSAPVLA QTMVPSSAMVPLAQPPAPAPVLTPGPPQSLSAPVPKSTQAGEGTLSEALLHLQFD ADEDLGALLGNSTDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLMEYPEA ITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFSSIADMDFSALLSQISSSGQG GGGSGFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPRPPEAEN SSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAEDPT ISLLTGSEPPKAKDPTVS NLS-MS2(deltaFG)-p65-HSF1 MGPKKKRKVGGSGMASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQ AYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQ GLLKDGNPIPSAIAANSGIYAGTPSGQISNQALALAPSSAPVLAQTMVPSSAMVPL AQPPAPAPVLTPGPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNS TDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPP DPAPTPLGTSGLPNGLSGDEDFSSIADMDFSALLSQISSSGQGGGGSGFSVDTSA LLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPRPPEAENSSPDSGKQLVHYT AQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAEDPTISLLTGSEPPKAK DPTVS S8
24 MS2(deltaFG)-NLS-p65-HSF1 MASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSA QNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIA ANSGIYAGGSGGSGGGSGSGSGSGPKKKRKVAAAGSPSGQISNQALALAPSSAP VLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLSAPVPKSTQAGEGTLSEALLHL QFDADEDLGALLGNSTDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLMEY PEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFSSIADMDFSALLSQISSSG QGGGGSGFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPRPPEA ENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAE DPTISLLTGSEPPKAKDPTVS NLS-MS2(deltaFG)-NLS-p65-HSF1 (Split-CPTS2.0) MGPKKKRKVGGSGMASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQ AYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQ GLLKDGNPIPSAIAANSGIYAGGSGGSGGGSGSGSGSGPKKKRKVAAAGSPSGQ ISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLSAPVPKST QAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSEFQQLLNQGV SMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFSSIA DMDFSALLSQISSSGQGGGGSGFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASI QELLSPQEPPRPPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFEL GEGSYFSEGDGFAEDPTISLLTGSEPPKAKDPTVS S9
25 NLSx3-MS2(deltaFG)-NLS-CIB1 (CPTS2.0) MASPKKKRKVEASASPKKKRKVEASASPKKKSKVEASGSGGGMASNFTQFVLVD NGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPK GAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYAGTGGSG SSGGSGGSGGSGMNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFA DSSMITGGEMDSYLSTAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKKRKFD TETKDCNEKKKKMTMNRDDLVEEGEEEKSKITEQNNGSTKSIKKMKHKAKKEEN NFSNDSSKVTKELEKTDYIHVRARRGQATDSHSIAERVRREKISERMKFLQDLVP GCDKITGKAGMLDEIINYVQSLQRQIEFLSMKLAIVNPRPDFDMDDIFAKEVASTPM TVVPSPEMVLSGYSHEMVHSGYSSEMVNSGYLHVNPMQQVNTSSGGSGGSGG GSGSGSGGLESRGPFEGKPIPNPLLGLDSTRTGHHHHHH S10
26 NLSx3-CRY2PHR-NLS-p65-HSF1 (CPTS2.0) MASPKKKRKVEASASPKKKRKVEASASPKKKSKVEASGSGTGGSGSSGGSGGS GGSGMKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQFYPGR ASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLYDPV SLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMS IESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNA DKLLNEFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWAR DKNSEGEESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFK AWRQGRTGYPLVDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFW DTLLDADLECDILGWQYISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELA RLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMI GAAGGSGGSGGGSGSGSGGLEPSGQISNQALALAPSSAPVLAQTMVPSSAMVP LAQPPAPAPVLTPGPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGN STDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRP PDPAPTPLGTSGLPNGLSGDEDFSSIADMDFSALLSQISSSGQGGGGSGFSVDTS ALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPRPPEAENSSPDSGKQLVH YTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAEDPTISLLTGSEPPKA KDPTVS S11
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doi: 10.1038/nature06721 SUPPLEMENTARY INFORMATION. Supplemental Figure Legends Supplemental Figure 1 The distribution of hatx-1[82q] in Cos7 cells. Cos7 cells are co-transfected with hatx-1[82q]-gfp (green)
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Supplementary Figures a HindIII XbaI pcdna3.1/v5-his A_Nfluc-VVD 1-398 37-186 Nfluc Linker 1 VVD HindIII XhoI pcdna3.1/v5-his A_VVD-Nfluc 37-186 1-398 VVD Linker 2 Nfluc HindIII XbaI pcdna3.1/v5-his A_Cfluc-VVD
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Supplemental Information Itemized List Materials and Methods, Related to Supplemental Figures S5A-C and S6. Supplemental Figure S1, Related to Figures 1 and 2. Supplemental Figure S2, Related to Figure
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Supplementary Figures and Figure legends 3 Supplementary Figure 1. Conditional targeting construct for the murine Satb1 locus with a modified FLEX switch. Schematic of the wild type Satb1 locus; the conditional
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Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing
Published online 9 December 2017 Nucleic Acids Research, 2018, Vol. 46, No. 5 e25 doi: 10.1093/nar/gkx1222 Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing
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Supplementary Information Sam68 modulates the promoter specificity of NF-κB and mediates expression of CD25 in activated T cells Kai Fu 1, 6, Xin Sun 1, 6, Wenxin Zheng 1, 6, Eric M. Wier 1, Andrea Hodgson
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Supplementary
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SUPPLEMENTARY INFORMATION Supplementary figure 1: List of primers/oligonucleotides used in this study. 1 Supplementary figure 2: Sequences and mirna-targets of i) mcherry expresses in transgenic fish used
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DOI: 10.1038/ncb3240 Supplementary Figure 1 GBM cell lines display similar levels of p100 to p52 processing but respond differentially to TWEAK-induced TERT expression according to TERT promoter mutation
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doi:.38/nature899 Supplementary Figure Suzuki et al. a c p7 -/- / WT ratio (+)/(-) p7 -/- / WT ratio Log X 3. Fold change by treatment ( (+)/(-)) Log X.5 3-3. -. b Fold change by treatment ( (+)/(-)) 8
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Supplementary Figure 1 Supplementary Figure 1. Activation capacity of tettale-ad compared to tet trans-activator (ttas) using different teto variants. (A) HeLa cells were co-transfected with activation
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embryos. Asterisk represents loss of or reduced expression. Brackets represent
Supplemental Figures Supplemental Figure 1. tfec expression is highly enriched in tail endothelial cells (A- B) ISH of tfec at 15 and 16hpf in WT embryos. (C- D) ISH of tfec at 36 and 38hpf in WT embryos.
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GFP CCD2 GFP IP:GFP
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Mannen et al., http :// /cgi /content /full /jcb /DC1
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Supplemental Table 1 Primers used in study. Human. Mouse
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Supplementary Figure 1 Generation of the AARE-Gene system construct. (a) Position and sequence alignment of AAREs extracted from human Trb3, Chop or Atf3 promoters. AARE core sequences are boxed in grey.
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Supplementary Fig. 1. Schematic structure of TRAIP and RAP80. The prey line below TRAIP indicates bait and the two lines above RAP80 highlight the
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Nanog-Luc. R-Luc. 40 HA-Klf Relative protein level of Klf USP21. Vector USP2 USP21 *** *** *** ***
:Flag Relative protein level of Relative protein level of : Flag Vec 21LV 21SV 2 : HA HA- HA- HA- HA-Klf4 Relative mrna expression Relative protein level of Relative protein level of Relative protein level
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File name: Supplementary Information Description: Supplementary figures and supplementary tables. File name: Peer review file Description:
File name: Supplementary Information Description: Supplementary figures and supplementary tables. File name: Peer review file Description: Supplementary Figure 1. dcas9-mq1 fusion protein induces de novo
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SUPPORTING INFORMATION: Targeted m 6 A reader proteins to study epitranscriptomic regulation of single RNAs Simone Rauch, Chuan He,,, and Bryan C. Dickinson *, Department of Biochemistry and Molecular
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/04/$15.00/0 Molecular Endocrinology 18(3): Copyright 2004 by The Endocrine Society doi: /me
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doi:10.1038/nature12119 SUPPLEMENTARY FIGURES AND LEGENDS pre-let-7a- 1 +14U pre-let-7a- 1 Ddx3x Dhx30 Dis3l2 Elavl1 Ggt5 Hnrnph 2 Osbpl5 Puf60 Rnpc3 Rpl7 Sf3b3 Sf3b4 Tia1 Triobp U2af1 U2af2 1 6 2 4 3