Journal: bioRxiv

Article Title: Human pluripotent reprogramming with CRISPR activators

doi: 10.1101/206144

Figure Lengend Snippet: CRISPRa mediated reprogramming of NSCs and EEA-motif targeting. (a) Different dCas9 activator constructs tested. (b) Staining for OCT4 activation with DDdCas9 activators in HEK293 after 5 days of TMP treatment. Nuclei stained blue. (c) Schematic representation of NSC reprogramming into iPSC with dCas9VPH mediated OCT4 activation. (d) Immunocytochemical detection of pluripotency markers in NCS-derived iPSCs (top row) and tri-lineage differentiation in plated embryoid bodies (bottom row). Nuclei stained blue. (e) Targeting of EGA enriched Alu-motif with SpdCas9 gRNAs. (f) Alkaline phosphatase positive cells induced from NSC by dCas9VPH mediated OCT4 activation with and without EEA-motif targeting gRNAs. (g) Quantification of iPSC-like alkaline phosphatase positive colonies induced from NSC. n = 6 independent inductions (P=0.053). Data points connected with lines are from the same batch of NSCs. (h) Quantification of iPSC-like alkaline phosphatase positive colonies induced from skin fibroblasts by transgenic reprogramming with GFP control plasmid or EEA-motif targeting gRNA plasmid. n = 5 independent inductions (P=0.034). Data presented as mean ± SEM, two-tailed Student’s t-test. * P<0.05

Article Snippet: dCas9VPH construct was cloned by adding a P65-HSF1 containing fragment from lenti-MS2-P65-HSF1_Hygro (gift from Feng Zhang, Addgene Plasmid #61426) after the VP192 domain by PCR. dCas9VPP300 was cloned by PCR amplifying the P300 core domain from human cDNA and cloning it after the VP192 domain, as described by Hilton et al. . dCas9VPPH was cloned by adding the P65-HSF1 domain in fusion after the VP192-P300core domain.

Techniques: Construct, Staining, Activation Assay, Derivative Assay, Transgenic Assay, Control, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: Human pluripotent reprogramming with CRISPR activators

doi: 10.1101/206144

Figure Lengend Snippet: Optimization of dCas9 activator and gRNA targeting in HEK293 for reprogramming factor activation. (a) Locations of promoter targeting gRNAs for reprogramming factors ( OCT4 , SOX2 , KLF4 , C-MYC , LIN28A and NANOG ) in relation to transcription start site. (b) Immunocytochemical staining of reprogramming factors after single gRNA activation and pooled mixture of five guides in HEK293 with dCas9VPH. Pictures are in similar order to guides in . Best performing guides used for plasmid cloning are marked with dotted lines. (c) Schematic representation of concatenated reprogramming factor gRNA plasmid construction. (d) Reprogramming factor activation, using constitutively expressed DDdCas9 effectors with different activation domains, in HEK293 by qPCR after TMP addition. n = 3, data are from 3 independent experiments. Error bars represent standard deviation. One way Anova with Tukey HSD test used for statistical comparisons. * P<0.05

Article Snippet: dCas9VPH construct was cloned by adding a P65-HSF1 containing fragment from lenti-MS2-P65-HSF1_Hygro (gift from Feng Zhang, Addgene Plasmid #61426) after the VP192 domain by PCR. dCas9VPP300 was cloned by PCR amplifying the P300 core domain from human cDNA and cloning it after the VP192 domain, as described by Hilton et al. . dCas9VPPH was cloned by adding the P65-HSF1 domain in fusion after the VP192-P300core domain.

Techniques: Activation Assay, Staining, Plasmid Preparation, Cloning, Standard Deviation

Journal: bioRxiv

Article Title: Human pluripotent reprogramming with CRISPR activators

doi: 10.1101/206144

Figure Lengend Snippet: Pluripotency characterization of iPCS lines, (a) Episomal plasmid derived CRISPR iPSCs from HFFs (HEL136, 140, 141) and F72 fibroblasts (HEL139) are positive for pluripotency associated factors. Scale bar 400μm (b) Embryoid body differentiation of the cell lines into three embryonic germ layers: ectoderm (TUBB3), mesoderm (VIMENTIN) and endoderm (SOX17) (green). Scale bar 200μm (c) Karyotypes of HEL139.5 (46, XX), HEL139.8 (46, XX), HEL140.1 (46, XY, 75% of analysed cells), HEL141 (46, XY, abn(3q)) and SeV control line HEL46.11 (46, XX). (d) Episome detection by PCR with EBNA primers of CRISPR dCas9VPH reprogrammed iPSCs at passage 3. (e) Pluripotency, differentiation and absence of viral episome characterization of control SeV iPSC line HEL46.11. Scale bar 200μm. Nuclei stained blue.

Article Snippet: dCas9VPH construct was cloned by adding a P65-HSF1 containing fragment from lenti-MS2-P65-HSF1_Hygro (gift from Feng Zhang, Addgene Plasmid #61426) after the VP192 domain by PCR. dCas9VPP300 was cloned by PCR amplifying the P300 core domain from human cDNA and cloning it after the VP192 domain, as described by Hilton et al. . dCas9VPPH was cloned by adding the P65-HSF1 domain in fusion after the VP192-P300core domain.

Techniques: Plasmid Preparation, Derivative Assay, CRISPR, Control, Staining