dcas9vph construct  (Addgene inc)

Journal: Nature Communications

Article Title: Human pluripotent reprogramming with CRISPR activators

doi: 10.1038/s41467-018-05067-x

Figure Lengend Snippet: CRISPRa-mediated reprogramming of NSCs and EEA-motif targeting. a Schematic representation of dCas9VPH structure. b Schematic representation of NSC reprogramming into iPSCs with dCas9VPH mediated OCT4 activation. c Immunocytochemical detection of pluripotency markers in NCS-derived iPSCs (top row) and tri-lineage differentiation in plated embryoid bodies (bottom row). Nuclei stained blue. Scale bar = 200 µm. d Targeting of EGA enriched Alu-motif with SpdCas9 gRNAs. e Quantification of iPSC-like alkaline phosphatase positive colonies induced from NSCs. n = 6 independent inductions ( P = 0.053, OCT4 targeting with EEA-gRNAs vs. without EEA-gRNAs). Data presented as mean ± s.e.m., two-tailed Student’s t -test

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-P300 core domain.

Techniques: Activation Assay, Derivative Assay, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Human pluripotent reprogramming with CRISPR activators

doi: 10.1038/s41467-018-05067-x

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 Fig. 2a. Best performing guides used for plasmid cloning are marked with dotted lines. Scale bar = 400 µm. c Schematic representation of concatenated reprogramming factor gRNA plasmid construction. d Reprogramming factor activation by qRT-PCR, in HEK293 cells 3 days after transfection and HFFs 4 days after electroporation, using transiently expressed dCas9VPH effector. n = 3, data are from three independently treated samples. Data presented as mean ± s.e.m., two tailed Student’s t -test. * P < 0.05, ** P < 0.01, *** P < 0.001

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-P300 core domain.

Techniques: Activation Assay, Staining, Plasmid Preparation, Cloning, Quantitative RT-PCR, Transfection, Electroporation, Two Tailed Test