plasmid px551 cmv cj cas9  (Addgene inc)

Journal: Nature Communications

Article Title: The therapeutic implications of all-in-one AAV-delivered epigenome-editing platform in neurodegenerative disorders

doi: 10.1038/s41467-024-50515-6

Figure Lengend Snippet: The all-in-one AAV/ d Sa Cas9- KRAB-MeCP2(TRD) repressor platform and the control vector AAV/d Sa Cas9 with no repressor were administered by stereotaxic injection into the mouse dorsal hippocampus (DH) and validated using a GFP reporter gene ( a – c ). a , b LV-GFP reporter vector was co-injected with the AAV/gRNA1-d Sa Cas9- KRAB-MeCP2(TRD) into the left DH and with the control AAV/d Sa Cas9 into the right DH. The AAV/gRNA1-d Sa Cas9- KRAB-MeCP2(TRD) vector repressed the expression of the GFP reporter gene. a Representative images of brain coronal slices at 2x magnification 14 days and 42 days post-injection, showing GFP expression and DAPI staining in the DH. b Signals were quantified using ImageJ. Box plot displays the ratios of the left DH relative to the right DH in both age groups of 16 weeks (14d post-injection n = 9, p = 0.003; 42d post-injection n = 11, p = 0.028) and 32 weeks ( n = 10, p = 0.026) mice. Each open circle represents the quantified signal left/right for a mouse. c The AAV/gRNA1-d Sa Cas9- KRAB-MeCP2(TRD) vector repressed the expression of the GFP mRNA, box plot displays mean relative expression of GFP mRNA at 14 days ( n = 5, p = 0.027) or 42 days ( n = 6, p = 0.00046) post-injection. Each open circle represents the relative expression (log2) for a mouse. Values represent mean ± SEM. * p < 0.05 ** p < 0.01 *** p < 0.001; Two-tailed paired t -test. Source data are provided as a Source Data file.

Article Snippet: The Cj Cas9 was derived from the plasmid pX551-CMV- Cj Cas9 (addgene, #107035; gift from Alex Hewitt’s lab).

Techniques: Control, Plasmid Preparation, Injection, Expressing, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: The therapeutic implications of all-in-one AAV-delivered epigenome-editing platform in neurodegenerative disorders

doi: 10.1038/s41467-024-50515-6

Figure Lengend Snippet: The all-in-one AAV/ d Sa Cas9- KRAB-MeCP2(TRD) repressor platform and the control vector AAV/d Sa Cas9 with no repressor were administered by stereotaxic injection into the mouse dorsal hippocampus (DH) and validated using the mouse endogenous Apoe gene ( a , b ) AAV/gRNA( Apoe )p-d Sa Cas9-KRAB-MeCP2(TRD) vectors with gRNA1 or gRNA2 were injected into the right DH and the control AAV/d Sa Cas9 into the left DH. Both AAV/gRNA ( Apoe )p-d Sa Cas9-KRAB-MeCP2(TRD) vectors reduced the mouse endogenous ApoE expression. a Representative images of brain coronal slices at 2× magnification 42 days post-injection, showing ApoE expression and DAPI staining in the DH; 20× magnification of DH region showing ApoE expression. b Signals were quantified using ImageJ. Box plot displays the ratios of the right DH relative to the left DH for mice injected with the repressor vector harboring gRNA1 ( n = 8, p = 0.0002) and gRNA2 ( n = 8, p = 0.0011). Each open circle represents the quantified signal right/left for a mouse. Values represent mean ± SEM. * p < 0.05 ** p < 0.01 *** p < 0.001; Two -tailed Mann–Whitney U -Test. Source data are provided as a Source Data file.

Article Snippet: The Cj Cas9 was derived from the plasmid pX551-CMV- Cj Cas9 (addgene, #107035; gift from Alex Hewitt’s lab).

Techniques: Control, Plasmid Preparation, Injection, Expressing, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: The therapeutic implications of all-in-one AAV-delivered epigenome-editing platform in neurodegenerative disorders

doi: 10.1038/s41467-024-50515-6

Figure Lengend Snippet: The all-in-one AAV/ d Sa Cas9- KRAB-MeCP2(TRD) repressor platform and the control vector AAV/d Sa Cas9 with no repressor were administered by stereotaxic injection into the mouse dorsal hippocampus (DH) and validated using a GFP reporter gene ( a – c ). a , b LV-GFP reporter vector was co-injected with the AAV/gRNA1-d Sa Cas9- KRAB-MeCP2(TRD) into the left DH and with the control AAV/d Sa Cas9 into the right DH. The AAV/gRNA1-d Sa Cas9- KRAB-MeCP2(TRD) vector repressed the expression of the GFP reporter gene. a Representative images of brain coronal slices at 2x magnification 14 days and 42 days post-injection, showing GFP expression and DAPI staining in the DH. b Signals were quantified using ImageJ. Box plot displays the ratios of the left DH relative to the right DH in both age groups of 16 weeks (14d post-injection n = 9, p = 0.003; 42d post-injection n = 11, p = 0.028) and 32 weeks ( n = 10, p = 0.026) mice. Each open circle represents the quantified signal left/right for a mouse. c The AAV/gRNA1-d Sa Cas9- KRAB-MeCP2(TRD) vector repressed the expression of the GFP mRNA, box plot displays mean relative expression of GFP mRNA at 14 days ( n = 5, p = 0.027) or 42 days ( n = 6, p = 0.00046) post-injection. Each open circle represents the relative expression (log2) for a mouse. Values represent mean ± SEM. * p < 0.05 ** p < 0.01 *** p < 0.001; Two-tailed paired t -test. Source data are provided as a Source Data file.

Article Snippet: The Cj Cas9 was derived from the plasmid pX551-CMV- Cj Cas9 (addgene, #107035; gift from Alex Hewitt’s lab).

Techniques: Control, Plasmid Preparation, Injection, Expressing, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: The therapeutic implications of all-in-one AAV-delivered epigenome-editing platform in neurodegenerative disorders

doi: 10.1038/s41467-024-50515-6

Figure Lengend Snippet: The all-in-one AAV/ d Sa Cas9- KRAB-MeCP2(TRD) repressor platform and the control vector AAV/d Sa Cas9 with no repressor were administered by stereotaxic injection into the mouse dorsal hippocampus (DH) and validated using the mouse endogenous Apoe gene ( a , b ) AAV/gRNA( Apoe )p-d Sa Cas9-KRAB-MeCP2(TRD) vectors with gRNA1 or gRNA2 were injected into the right DH and the control AAV/d Sa Cas9 into the left DH. Both AAV/gRNA ( Apoe )p-d Sa Cas9-KRAB-MeCP2(TRD) vectors reduced the mouse endogenous ApoE expression. a Representative images of brain coronal slices at 2× magnification 42 days post-injection, showing ApoE expression and DAPI staining in the DH; 20× magnification of DH region showing ApoE expression. b Signals were quantified using ImageJ. Box plot displays the ratios of the right DH relative to the left DH for mice injected with the repressor vector harboring gRNA1 ( n = 8, p = 0.0002) and gRNA2 ( n = 8, p = 0.0011). Each open circle represents the quantified signal right/left for a mouse. Values represent mean ± SEM. * p < 0.05 ** p < 0.01 *** p < 0.001; Two -tailed Mann–Whitney U -Test. Source data are provided as a Source Data file.

Article Snippet: The Cj Cas9 was derived from the plasmid pX551-CMV- Cj Cas9 (addgene, #107035; gift from Alex Hewitt’s lab).

Techniques: Control, Plasmid Preparation, Injection, Expressing, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Plasmid-mediated gene transfer of Cas9 induces vector-related but not Sp Cas9-related immune responses in human retinal pigment epithelial cells

doi: 10.1038/s41598-022-17269-x

Figure Lengend Snippet: Sp Cas9 plasmid transfection of IMhu cells results in Cas9 expression but does not induce cytokine responses. ( A ) Experimental Streptococcus pyogenes ( Sp ) Cas9 expression plasmid ( Sp Cas9 plasmid) and non-coding control plasmid (NC plasmid) used to assess the effect of Cas9 on IMhu cells. In the NC plasmid the Cas9 encoding sequence is removed. ( B ) Immunostaining of Cas9 in transfected IMhu cells. Cells were separately transfected with either the Sp Cas9 plasmid or the NC plasmid. At 24 h post-transfection, cells were stained with Cas9 antibodies (red) and DAPI (nuclei, blue). Sp Cas9 plasmid-transfected IMhu cells expressed Cas9 after Sp Cas9 plasmid transfection, whereas NC plasmid-transfected cells did not express Cas9. Data are representative of three independent experiments. Scale bar 100 µm. NLS nuclear localization sequence, ori origin of replication, AmpR ampicillin resistance gene, FLAG protein tag. ( C ) Heatmap of cytokine levels in IMhu supernatant 24 h after transfection with the Sp Cas9 plasmid. Cytokine levels were determined using Proteome Profiler Antibody Arrays. Colors represent cytokine ratios of Sp Cas9 plasmid-transfected versus L3000 treated cells (left column) or NC plasmid-transfected versus L3000 treated cells (right column) respectively.

Article Snippet: The PCR product was digested with restriction enzymes MluI-HF (New England Biolabs, Ipswich, MA, USA) and Nsil-HF (New England Biolabs, Ipswich, MA, USA) and inserted into the backbone of the cj Cas9 plasmid (#89752, Addgene, Watertown, MA, USA).

Techniques: Plasmid Preparation, Transfection, Expressing, Control, Sequencing, Immunostaining, Staining

Journal: Scientific Reports

Article Title: Plasmid-mediated gene transfer of Cas9 induces vector-related but not Sp Cas9-related immune responses in human retinal pigment epithelial cells

doi: 10.1038/s41598-022-17269-x

Figure Lengend Snippet: Sp Cas9-, EGFP- Sp Cas9-, and EGFP-stuffer-plasmid transfection results in Cas9 and/or EGFP expression in ARPE-19 cells. ( A ) Sp Cas9 plasmid, stuffer plasmid, EGFP- Sp Cas9 plasmid, and EGFP-stuffer plasmid used to evaluate the effect of Cas9 on ARPE-19 cells. In the stuffer plasmid and the EGFP-stuffer plasmid the Cas9 encoding sequence is replaced by an equally sized non-coding stuffer sequence. NLS nuclear localization sequence, ori origin of replication, AmpR ampicillin resistance gene, FLAG protein tag. ( B ) Microscopic evaluation of Cas9 and EGFP expression in transfected ARPE-19 cells. Cells were separately transfected with either the Sp Cas9 plasmid, stuffer plasmid, EGFP- Sp Cas9 plasmid, or the EGFP-stuffer plasmid. After 24 h cells were stained with Cas9 antibodies (red) and DAPI (blue). Data are representative of three independent experiments. Scale bar 100 µm. ( C – E ) Flow cytometric evaluation of the transfection rates of EGFP- Sp Cas9 plasmid or the EGFP-stuffer plasmid-transfected cells. ( C , D ) ARPE-19 cells were gated as single, 7-AAD negative living cells ( C ) and analyzed for the expression of EGFP ( D ). ( E ) Percentages of EGFP positive ARPE-19 cells. Bars represent means + SD of mean values pooled from three independent experiments measured in duplicates.

Article Snippet: The PCR product was digested with restriction enzymes MluI-HF (New England Biolabs, Ipswich, MA, USA) and Nsil-HF (New England Biolabs, Ipswich, MA, USA) and inserted into the backbone of the cj Cas9 plasmid (#89752, Addgene, Watertown, MA, USA).

Techniques: Plasmid Preparation, Transfection, Expressing, Sequencing, Staining

Journal: Scientific Reports

Article Title: Plasmid-mediated gene transfer of Cas9 induces vector-related but not Sp Cas9-related immune responses in human retinal pigment epithelial cells

doi: 10.1038/s41598-022-17269-x

Figure Lengend Snippet: Plasmid-mediated gene transfer of Sp Cas9 induces vector-related IL-6 and IL-8 release in ARPE-19 cells. ( A ) Heatmap of cytokine levels in the supernatant of ARPE-19 cells 24 h after transfection with the Sp Cas9 plasmid as measured by Proteome Profiler Antibody Arrays. Colors show cytokine ratios of Sp Cas9 plasmid-transfected versus LTX treated cells. ( B , C ) ELISA analysis of IL-8 and IL-6 concentrations in the supernatant of ARPE-19 cells transfected with the Sp Cas9 plasmid, the stuffer plasmid, the EGFP- Sp Cas9 plasmid, and the EGFP-stuffer plasmid, or treated with the SpCas9 plasmid or LTX alone or left unstimulated. Poly(I:C) (10 µg/ml) served as a positive stimulation control. ( B ) Transfection with both the Sp Cas9-encoding plasmids and the stuffer control plasmids induced a significant IL-8 release. ( C ) Transfection with both the Sp Cas9-encoding plasmids and the stuffer control plasmids also triggered a significant production of IL-6. Data was analyzed via one-way ANOVA followed by Bonferroni’s comparison tests for selected pairs of columns. Asterisks indicate significant differences in comparison to unstimulated ARPE-19 cells. Bars represent means + SD of pooled mean values from three independent experiments measured in duplicates. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The PCR product was digested with restriction enzymes MluI-HF (New England Biolabs, Ipswich, MA, USA) and Nsil-HF (New England Biolabs, Ipswich, MA, USA) and inserted into the backbone of the cj Cas9 plasmid (#89752, Addgene, Watertown, MA, USA).

Techniques: Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Control, Comparison

Journal: Scientific Reports

Article Title: Plasmid-mediated gene transfer of Cas9 induces vector-related but not Sp Cas9-related immune responses in human retinal pigment epithelial cells

doi: 10.1038/s41598-022-17269-x

Figure Lengend Snippet: Plasmid-mediated gene transfer of Cas9 induces HLA-ABC and CD54 upregulation in ARPE-19 cells. ARPE 19 cells were transfected with the Sp Cas9-encoding plasmids or the corresponding stuffer plasmids, or treated with the SpCas9 plasmid or LTX alone or left unstimulated. IFN-γ (1000 IU/ml) served as a positive stimulation control. At 24 h after transfection cells were analyzed by flow cytometry. Living ARPE-19 cells were gated as shown in Fig. C and analyzed for the expression of the surface markers HLA-ABC, HLA-DR and CD54. Surface marker expression following transfection with the Sp Cas9 plasmid or the stuffer plasmid ( A , B ) and the EGFP- Sp Cas9 plasmid or the EGFP-stuffer plasmid ( C , D ). ( A , C ) Surface marker expression presented as overlays of single-color histograms of log10 mean fluorescence intensity (MFI) obtained with fluorescence minus one (FMO) control (filled histograms) and specific antibodies against respective surface marker (open histograms). ( B , D ) Graphical analysis of the surface marker expression showing increases of HLA-ABC and CD54 after transfection with the Sp Cas9-encoding plasmids or the corresponding stuffer plasmids. Bars represent means + SD of pooled mean values from three independent experiments measured in duplicates. Data was analyzed by one-way ANOVA followed by Bonferroni’s comparison tests. Asterisks indicate differences in comparison to unstimulated ARPE-19 cells. Kruskal–Wallis-test followed by pairwise comparisons was used to analyze non-normally distributed data. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The PCR product was digested with restriction enzymes MluI-HF (New England Biolabs, Ipswich, MA, USA) and Nsil-HF (New England Biolabs, Ipswich, MA, USA) and inserted into the backbone of the cj Cas9 plasmid (#89752, Addgene, Watertown, MA, USA).

Techniques: Plasmid Preparation, Transfection, Control, Flow Cytometry, Expressing, Marker, Fluorescence, Comparison

Journal: Scientific Reports

Article Title: Plasmid-mediated gene transfer of Cas9 induces vector-related but not Sp Cas9-related immune responses in human retinal pigment epithelial cells

doi: 10.1038/s41598-022-17269-x

Figure Lengend Snippet: Viability rates of ARPE-19 cells after plasmid transfection. Viability rates were analyzed via flow cytometry 24 h after plasmid transfection. Percentages of viable ARPE-19 cells following transfection with the Sp Cas9 plasmid or the stuffer plasmid ( A ) and the EGFP- Sp Cas9 plasmid or the EGFP-stuffer plasmid ( B ). ARPE-19 cells receiving LTX or Sp Cas9 plasmid treatment only and unstimulated ARPE-19 cells served as control groups. Bars represent means + SD of pooled mean values from three independent experiments measured in duplicates. Normally distributed data was analyzed by one-way ANOVA followed by Bonferroni’s comparison tests for selected pairs of columns. Kruskal–Wallis-test followed by pairwise comparisons was used to analyze non-normally distributed data. Asterisks indicate differences in comparison to unstimulated ARPE-19 cells. *p < 0.05 **p < 0.01.

Article Snippet: The PCR product was digested with restriction enzymes MluI-HF (New England Biolabs, Ipswich, MA, USA) and Nsil-HF (New England Biolabs, Ipswich, MA, USA) and inserted into the backbone of the cj Cas9 plasmid (#89752, Addgene, Watertown, MA, USA).

Techniques: Plasmid Preparation, Transfection, Flow Cytometry, Control, Comparison

Journal: Nucleic Acids Research

Article Title: MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in vivo

doi: 10.1093/nar/gkab174

Figure Lengend Snippet: Activation of endogenous genes with a d Cj Cas9-SunTag-VPR system. ( A ) Schematic of the d Cj Cas9-SunTag-VPR system. 10× SunTag was fused to the N- or C-terminus of a DNase-dead d Cj Cas9 (termed S-D and D-S, respectively). By binding to the SunTag, scFv-GCN4-sfGFP-VPR could be recruited to the sgRNA target site and activate transcription. VPR, the truncated tripartite activation domains of VP64, p65 and RTA. ( B ) The subcellular localization of the d Cj Cas9-SunTag fusion proteins in HEK293T cells revealed by immunofluorescence staining. An HA tag was fused to the C-terminus of d Cj Cas9. ( C ) Targeted gene activation guided by pooled sgRNAs (four sgRNAs for each gene) with the d Cj Cas9-SunTag-VPR system. ( D ) Targeted gene activation guided by a single sgRNA with the S–D system. ( E ) Multiplexed gene activation with the d Cj Cas9-SunTag-VPR system. ( F ) The gene activation specificity of the S-D system. Gene expression plot generated from RNA-seq data from HEK293T cells co-transfected with S-D and the sgRNA2 targeting MYOD compared to that transfected with the corresponding S–D plasmid only. R indicates Pearson's correlation coefficient. Average of two biological replicates was shown. For C–E, quantitative RT-PCR revealed relative mRNA expression of IL1RN , HBG , and MYOD in HEK293T cells co-transfected with either S-D or D-S plasmids and four sgRNAs targeting the promoter region of each gene ( C ) or with S-D plasmid and four single sgRNAs targeting the promoter region of each gene ( D ) or with either S–D or D–S plasmids and the pooled three sgRNAs (sgRNA1, sgRNA2 and sgRNA2 for IL1RN , HBG , and MYOD , respectively) ( E ). Mean values are presented with S.D., n = 3 independent experiments. For each experiment, fold changes of mRNA expression in tested samples (transfected with the plasmids encoding S–D/D–S and sgRNA) versus that in control samples (transfected with plasmids encoding S–D/D–S and the backbone plasmid for sgRNA, herein termed ‘without sgRNA’) were shown. * P <0.05, ** P <0.01, *** P <0.001 (Student's t -test for C and E, one-way ANOVA test for D, tested sample versus control sample).

Article Snippet: The following antibodies were used in this study: anti-HA-tag antibody (MBL, M180–3) for Cj Cas9, β-tubulin mouse antibody (Proteintech, 66240-1-Ig), β-actin antibody (Sigma-Aldrich, A3854), UCP1 antibody (Abcam, ab10983), FGF21 (Abcam, ab171941).

Techniques: Activation Assay, Binding Assay, Immunofluorescence, Staining, Gene Expression, Generated, RNA Sequencing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing, Control

Journal: Nucleic Acids Research

Article Title: MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in vivo

doi: 10.1093/nar/gkab174

Figure Lengend Snippet: Activation of endogenous genes with a VPR-d Cj Cas9 system. ( A ) Schematic of the VPR-d Cj Cas9 system. VPR was fused to the N- or C-terminus of d Cj Cas9 (termed V–D and D–V, respectively). ( B ) The subcellular localization of the VPR-d Cj Cas9 fusion proteins in HEK293T cells revealed by immunofluorescence staining. ( C ) Targeted gene activation guided by pooled sgRNAs with the VPR-d Cj Cas9 system. ( D ) Targeted gene activation guided by a single sgRNA with the V-D activator. ( E ) Multiplexed gene activation with the VPR-d Cj Cas9 system. ( F ) The gene activation specificity of the V-D activator. Average of two biological replicates was shown. For C–E, mean values are presented with S.D., n = 3 independent experiments. The experiments in Figure were similar to that in Figure except using the VPR-d Cj Cas9 system instead of the d Cj Cas9-SunTag-VPR system. * P <0.05, ** P <0.01, *** P <0.001 (Student's t -test for C and E, one-way ANOVA test for D, tested sample versus control sample).

Article Snippet: The following antibodies were used in this study: anti-HA-tag antibody (MBL, M180–3) for Cj Cas9, β-tubulin mouse antibody (Proteintech, 66240-1-Ig), β-actin antibody (Sigma-Aldrich, A3854), UCP1 antibody (Abcam, ab10983), FGF21 (Abcam, ab171941).

Techniques: Activation Assay, Immunofluorescence, Staining, Control

Journal: Nucleic Acids Research

Article Title: MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in vivo

doi: 10.1093/nar/gkab174

Figure Lengend Snippet: Multiplexed orthogonal genome editing and transcriptional activation with a VPR- Cj Cas9 fusion nuclease. ( A ) Genome editing efficiency of the VPR- Cj Cas9 fusion nuclease at the IL1RN site revealed by Deep-seq. ( B ) Relative mRNA expression of IL1RN revealed by qRT-PCR. V-D, VPR-d Cj Cas9 fusion protein. V-WT, VPR- Cj Cas9 fusion protein. For a & b, human HEK293T cells were co-transfected with either V-D or V-WT plasmids and ILRN targeting sgRNAs with indicated length. Half of the cells was used to extract RNA for RT-PCR and half was used to extract DNA for Deep-seq. Ctrl, V-WT transfection only (without sgRNA). ( C , D ) Genome editing and gene activation mediated by long and short sgRNAs at the Fgf21 site in mouse cells. The experiments in C & D were similar to that in A & B except using B16 cells. (E, F) Multiplexed orthogonal genome editing at the IL1RN and HBG sites and gene activation of MYOD in HEK293T cells. Indel formation was revealed by Deep-seq ( E ), and relative mRNA expression was revealed by qRT-PCR ( F ). gI22H22M15, a combination of three sgRNAs including a 22-nt g IL1RN , a 22-nt g HBG , and a 15-nt g MYOD . P.C., positive control, HEK293T cells transfected with WT Cj Cas9 and corresponding 22-nt sgRNA plasmids. ( G ) The gene activation specificity of the V-WT activator. Average of two biological replicates was shown. ( H ) Schematic of the VPR- Cj Cas9 based orthogonal system. 15-nt sgRNAs activate gene expression, while 22-nt sgRNAs induce gene editing. For B, D and F, mean values are presented with S.D., n = three independent experiments. ** P <0.01, *** P <0.001 (Student's t -test, V–D sample versus corresponding V-WT sample).

Article Snippet: The following antibodies were used in this study: anti-HA-tag antibody (MBL, M180–3) for Cj Cas9, β-tubulin mouse antibody (Proteintech, 66240-1-Ig), β-actin antibody (Sigma-Aldrich, A3854), UCP1 antibody (Abcam, ab10983), FGF21 (Abcam, ab171941).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Transfection, Reverse Transcription Polymerase Chain Reaction, Positive Control, Gene Expression

Journal: Nucleic Acids Research

Article Title: MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in vivo

doi: 10.1093/nar/gkab174

Figure Lengend Snippet: Minimization and optimization of the VPR-d Cj Cas9 system. ( A ) Schematic of VPR-d Cj Cas9 fusion protein. ( B ) Screening the transcription factors that fused to d Cj Cas9. PR, p65-RTA; VR, VP64-RTA; VP, VP64-p65; VNR, VP64-NANOG-RTA; VRN, VP64-RTA-NANOG; PH, p65-HSF1; VPR-S, VPR with a more shortened p65 and shortened linkers between the three transcription factors. ( C ) Shortening the linker between VPR and d Cj Cas9. Linker-d1, deleting 33 aa including 2xSV40 NLS; Linker-d2, deleting 11 aa. ( D ) Minimization of d Cj Cas9. The HNH domain (495–609 aa) was deleted and the remained N- and C-terminal domains were ligated with no linker, GGGSGG linker, or GSK linker. ( E ) Codon optimization of VPR-d Cj Cas9 fusion protein. Upper panel, Western blotting of d Cj Cas9 fusion proteins in transfected HEK293T cells. Bottom panel, gene activation with the indicate activators. The fusion proteins with codon-optimized VP64 were indicated with asterisks. VPS-S-HNH-d1* was termed as miniCAFE. ( F ) Optimization of sgRNA scaffold. The structures of these sgRNAs were illuminated in . ( G ) Optimization of the molar ratio of the transfected gene activator plasmid to sgRNA plasmid. ( H ) The gene activation specificity of miniCAFE. ( I ) The comparison of gene expression profile between miniCAFE and V-D. For B–G, qRT-PCR revealed relative mRNA expression of IL1RN in HEK293T cells and of Fgf21 in B16 cells. Mean values are presented with S.D., n = 2–3 independent experiments. * P <0.05, ** P <0.01, *** P <0.001 (One-way ANOVA test for B–D, tested sample VS V-D sample; Student's t -test for E, V–D* versus V–D, and miniCAFE VS VPR-S-HNH-d1; one-way ANOVA test for F, tested sample versus WT sgRNA sample; Student's t -test for G, miniCAFE/F gRNA VS corresponding V-D/WT gRNA). For H and I, gene expression plots generated from RNA-seq data from HEK293T cells co-transfected with miniCAFE and 22-nt g IL1RN 1 with F scaffold. Ctrl, miniCAFE transfected only. Average of two biological replicates was shown.

Article Snippet: The following antibodies were used in this study: anti-HA-tag antibody (MBL, M180–3) for Cj Cas9, β-tubulin mouse antibody (Proteintech, 66240-1-Ig), β-actin antibody (Sigma-Aldrich, A3854), UCP1 antibody (Abcam, ab10983), FGF21 (Abcam, ab171941).

Techniques: Western Blot, Transfection, Activation Assay, Plasmid Preparation, Comparison, Gene Expression, Quantitative RT-PCR, Expressing, Generated, RNA Sequencing