Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeted Transgene Activation in the Brain Tissue by Systemic Delivery of Engineered AAV1 Expressing CRISPRa

doi: 10.1016/j.omtn.2019.04.015

Figure Lengend Snippet: Constructions of Minimal CRISPRa and CRISPRi Transgenes into pAAV Vector Backbone To achieve efficient packaging of dSaCas9 fusion proteins and its sgRNA in a single AAV vector, a truncated version of the mouse Mecp2 brain-specific promoter (235 bp, pMecp2); a minimal polyadenylation signal (48 bp, spA); and the smallest version of the transcriptional regulator domain, such as VP64 activator (156 bp), VP160 activator (390 bp), KRAB repressor (227 bp), and SID4X repressor (432 bp), were used.

Article Snippet: To construct the pAAV-pSyn1-iRFP720-T2A-GFP-spA vector, the 476-bp human Syn1 promoter was amplified from the plasmid pAAV-hSyn-hChR2(H134R)-mCherry (Addgene 26976) and replaced the pMecp2 promoter in the pAAV-pMecp2-iRFP720-T2A-GFP-spA vector, using XbaI and AgeI.

Techniques: Plasmid Preparation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeted Transgene Activation in the Brain Tissue by Systemic Delivery of Engineered AAV1 Expressing CRISPRa

doi: 10.1016/j.omtn.2019.04.015

Figure Lengend Snippet: Constructions of Minimal CRISPRa and CRISPRi Transgenes into pAAV Vector Backbone To achieve efficient packaging of dSaCas9 fusion proteins and its sgRNA in a single AAV vector, a truncated version of the mouse Mecp2 brain-specific promoter (235 bp, pMecp2); a minimal polyadenylation signal (48 bp, spA); and the smallest version of the transcriptional regulator domain, such as VP64 activator (156 bp), VP160 activator (390 bp), KRAB repressor (227 bp), and SID4X repressor (432 bp), were used.

Article Snippet: To construct pAAV-pMecp2-dSaCas9-spA-pU6-sgRNA (vector B), the SaCas9 from vector A was replaced with the catalytically inactive SaCas9 (dSaCas9) from plasmid PX603 (Addgene 61594), using AgeI and EcoRI.

Techniques: Plasmid Preparation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeted Transgene Activation in the Brain Tissue by Systemic Delivery of Engineered AAV1 Expressing CRISPRa

doi: 10.1016/j.omtn.2019.04.015

Figure Lengend Snippet: Activation and Repression of Luciferase Activity in Mouse N2a Cells Mediated by Various Minimal CRISPRa and CRISPRi Transgenes (A) Luciferase reporter vector construction. Luc1 is the promoterless pGL4.10[luc2] background control. Luc2 is a modified Luc1 vector that has been inserted with 1.3-kb mouse alpha CaMKII promoter amplified from the genomic DNA of N2a cells. Luc3 is a modified Luc1 vector that has been inserted with 6.8-kb mouse alpha CaMKII promoter and intergenic super-enhancer regions amplified from the genomic DNA of N2a cells. TK-Renilla was used as an internal normalization control in all luciferase reporter assay experiments. (B) Luciferase activity with dSaCas9-fused activators and repressors targeting mouse alpha CaMKII promoter. The CRISPRa or CRISPRi transgenes targeting the mouse alpha CaMKII promoter were co-transfected with Luc2 vector and TK-Renilla control vector. Three sgRNAs were designed to target three different binding sites on the promoter (Pm). Luciferase activities were measured when the CRISPRa or CRISPRi transgenes were used singly (site 1, 2, or 3) or in combination (sites 1, 2, and 3). (C) Luciferase activity with dSaCas9-fused activators and repressors targeting mouse alpha CaMKII promoter and intergenic super-enhancer. The sgRNAs targeting mouse alpha CaMKII promoter and intergenic super-enhancer were co-transfected with the Luc3 vector and TK-Renilla control vector. A total of nine sgRNAs was designed to target three different binding sites on the promoters (Pm), three on proximal (SE P ) and three on distal intergenic super-enhancer sites (SE D ). All luciferase activities were normalized to the corresponding empty sgRNA. The statistical significance levels from control (empty sgRNA) are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. All data are presented as mean ± SD (n = 4).

Article Snippet: To construct pAAV-pMecp2-dSaCas9-spA-pU6-sgRNA (vector B), the SaCas9 from vector A was replaced with the catalytically inactive SaCas9 (dSaCas9) from plasmid PX603 (Addgene 61594), using AgeI and EcoRI.

Techniques: Activation Assay, Luciferase, Activity Assay, Plasmid Preparation, Modification, Amplification, Reporter Assay, Transfection, Binding Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeted Transgene Activation in the Brain Tissue by Systemic Delivery of Engineered AAV1 Expressing CRISPRa

doi: 10.1016/j.omtn.2019.04.015

Figure Lengend Snippet: Modulation of Endogenous Alpha CaMKII Expressions by Using Minimal CRISPRa and CRISPRi Transgenes Targeting Super-Enhancer and Promoter in Mouse N2a Cells (A) sgRNA target sites on super-enhancer and promoter of mouse alpha CaMKII. The locations of nine different sgRNA target sites designed to target the promoter (Pm), proximal super-enhancer (SE P ), and distal super-enhancer (SE D ) are shown. Red arrows indicate the sense or antisense orientation of sgRNAs designed to recognize target DNA sequences. (B) dSaCas9-VP160-mediated activation of endogenous alpha CaMKII mRNA level in mouse N2a cells. Nine dSaCas9-VP160 activators were designed to target the Pm, SE P , and SE D . Expression levels of alpha CaMKII in N2a cells are shown. (C) dSaCas9-KRAB-mediated repression of endogenous alpha CaMKII mRNA level in mouse N2a cells. Nine dSaCas9-KRAB repressors were designed to target the Pm, SE P , and SE D . Expression levels of alpha CaMKII in N2a cells are shown. (D) dSaCas9-mediated transcriptional interference of mouse alpha CaMKII. (E) Western blot for protein lysate of alpha CaMKII in mouse N2a cells. The monoclonal mouse alpha CaMKII- (54 kDa) specific antibody was used. Beta-actin (Actb, 42 kDa) was used as an internal control. Protein expression levels of alpha CaMKII were measured for wild-type (WT), knockout (KO), and under the effects of dSaCas9-VP160 and dSaCas9-KRAB in N2a cells. Right panel shows the normalized protein expression level of alpha CaMKII in N2a cells. (B-D) The statistical significance levels from control (wild-type N2a) are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. All data are presented as mean ± SD (n = 3).

Article Snippet: To construct pAAV-pMecp2-dSaCas9-spA-pU6-sgRNA (vector B), the SaCas9 from vector A was replaced with the catalytically inactive SaCas9 (dSaCas9) from plasmid PX603 (Addgene 61594), using AgeI and EcoRI.

Techniques: Activation Assay, Expressing, Western Blot, Knock-Out

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeted Transgene Activation in the Brain Tissue by Systemic Delivery of Engineered AAV1 Expressing CRISPRa

doi: 10.1016/j.omtn.2019.04.015

Figure Lengend Snippet: Targeted Transgene Activation in the Brain Tissue by Systemic Delivery of AAV1-PHP.B Expressing CRISPRa (A) pSyn1-iRFP720-GFP construct. The red arrows (34, 35, and 36) on the hSyn1 promoter indicate the locations of three different sgRNA target sites for dSaCas9-VP64. (B) Example of in vivo imaging of the mouse brains. AAV1-WT (left) or AAV1-PHP.B (right) expressing iRFP720 and GFP fluorescent proteins was intravenously injected into the mice. In vivo imaging was carried out 5 weeks after injection. iRFP720 signal was detected only at the head of the AAV1-PHP.B-injected mouse. (C) Ex vivo imaging of the dissected ventral brain tissues. AAV1-PHP.B expressing dSaCas9-VP64 was injected together with AAV1-PHP.B expressing iRFP720 and GFP fluorescent proteins. After seven weeks of AAVs injection, the mouse brains were harvested. Ex vivo imaging was then carried out to detect and quantify the GFP (left) and iRFP720 (right) fluorescent signals on the mouse ventral brains. The statistical significance levels from control (uninjected brain) are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. All data are presented as mean ± SD.

Article Snippet: To construct pAAV-pMecp2-dSaCas9-spA-pU6-sgRNA (vector B), the SaCas9 from vector A was replaced with the catalytically inactive SaCas9 (dSaCas9) from plasmid PX603 (Addgene 61594), using AgeI and EcoRI.

Techniques: Activation Assay, Expressing, Construct, In Vivo Imaging, Injection, Ex Vivo, Imaging

Journal: Nature Communications

Article Title: Editing DNA methylation in vivo

doi: 10.1038/s41467-025-67222-5

Figure Lengend Snippet: A Design of 7 sgRNAs targeting the mouse Mecp2 promoter. sg–sgRNA, TSS–transcription start site. B Scheme of reporter cell line with Mecp2-luciferase on the Xi used to identify effective sgRNAs for Mecp2 reactivation. Luc luciferase, Hygro hygromycin resistance gene. C Luciferase assay results of Mecp2-luciferase reporter cells edited with dCas9-TET1 and an individual sgRNA or the pool of all 7 sgRNAs, or treated with GSK-3484862 (GSK). ( n = 2 (Mock, sgRNA-1, sgRNA-3, sgRNA-6, Scr) and n = 3 sgRNA-2, sgRNA-4, sgRNA-5, sgRNA-7, sgRNA-Pool, GSK) biological replicates, one-way ANOVA (Dunnett’s test Pool vs Scr), P = 1.9 × 10 −9 ) Scr Scrambled sgRNA. D DNA methylation status of the Mecp2 promoter measured by pyrosequencing of mock cells, and cells edited with dCas9-Tet1 with a non-targeting Scr sgRNA or the pool of 7 sgRNAs. ( n = 2 (Mock, Scr) and n = 3 ( Mecp2 ) biological replicates, one-way ANOVA with Tukey’s test, exact P -values found in source data). E Scheme for in vivo reactivation of Mecp2 . ICV intracerebroventricular, IF immunofluorescence. Created in BioRender. Liu, S. (2025) https://BioRender.com/4dk6z6j . F Transduction efficiency of AAV9 in ( D ). G DNA methylation status of the Mecp2 promoter in the isolated nuclei (mCherry+) from mice edited by dCas9-TET1 with Scr or Mecp2 sgRNA pool. ( n = 2 mice per group, one-way ANOVA with Tukey’s test, exact P -values found in source data). H Representative immunofluorescent staining of MeCP2, mCherry, GFP, and NeuN from Scr and Mecp2 targeted cortical sections. I Quantification of MeCP2+ cells in ( H ). ( n = 3 mice per group, two-sided t test, P = 0.0309). J Flow cytometry analysis of neuronal nuclear size of mCherry- cells from dCas9-TET1 mice injected with Scr or Mecp2 sgRNA pool. ( n = 5 (Scr) and n = 6 (Mecp2) mice, two-sided t test, P = 0.49). FSC–forward scatter. K Flow cytometry analysis of neuronal nuclear size of mCherry+ cells from dCas9-TET1 mice injected with Scr or Mecp2 sgRNA pool. ( n = 5 (Scr) and n = 6 ( Mecp2 ) mice, two-sided t test, P = 0.013). L Quantification of the relative neuronal nuclear size between mCherry+ and mCherry− cells in control (Scr) and edited ( Mecp2 ) mice. ( n = 5 (scr) and n = 6 (Mecp2) mice, two-sided t test, P = 0.0017).

Article Snippet: The AAV construct expressing Pcsk9 target sgRNA was cloned by inserting an mCherry-KASH fragment into the backbone construct (Addgene plasmid: 60229), and the AAV construct expressing Mecp2 target sgRNA was cloned by replacing the EGFP with mCherry in the backbone construct (Addgene plasmid: 60231) .

Techniques: Luciferase, DNA Methylation Assay, In Vivo, Immunofluorescence, Transduction, Isolation, Staining, Flow Cytometry, Injection, Control

Journal: Nature Communications

Article Title: Editing DNA methylation in vivo

doi: 10.1038/s41467-025-67222-5

Figure Lengend Snippet: A IGV browser track of dCas9 ChIP-seq data demonstrating binding of dCas9-TET1 to the Mecp2 promoter. ChIP – chromatin immunoprecipitation. B Manhattan plot of genome-wide binding sites of dCas9-TET1 with the pool of 7 sgRNAs. C Representative IGV track of WGBS data at the Mecp2 promoter of nuclei isolated from Scr sgRNA and Mecp2 pool sgRNA injected mice. The differentially methylated region (DMR) at the Mecp2 promoter is highlighted in red. WGBS whole genome bisulfite sequencing. D DNA methylation levels of dCas9-TET1 binding sites in mCherry+ nuclei isolated from Scr sgRNA or Mecp2 pool sgRNAs. Average methylation was calculated by averaging methylation of differentially methylated cytosines (DMCs) within binding sites if DMCs were identified within, or an average of all CpGs within the binding site if no DMCs were identified. Mecp2 is labeled by a red dot and other sites are labeled by black dots. The dashed lines represent a 15% change of DNA methylation level. E RNA-seq of Mecp2 -luciferase reporter cells edited with dCas9-TET1 + a Scr sgRNA or the pool of Mecp2 sgRNAs. Genes associated with dCas9-TET1 binding sites are labeled in black. Mecp2 reporter gene expression is labeled in red. The dashed lines represent a fourfold change in expression between the conditions. CPM counts per million.

Article Snippet: The AAV construct expressing Pcsk9 target sgRNA was cloned by inserting an mCherry-KASH fragment into the backbone construct (Addgene plasmid: 60229), and the AAV construct expressing Mecp2 target sgRNA was cloned by replacing the EGFP with mCherry in the backbone construct (Addgene plasmid: 60231) .

Techniques: ChIP-sequencing, Binding Assay, Chromatin Immunoprecipitation, Genome Wide, Isolation, Injection, Methylation, Methylation Sequencing, DNA Methylation Assay, Labeling, RNA Sequencing, Luciferase, Gene Expression, Expressing

Journal: Cancer cell

Article Title: The osteogenic niche is a calcium reservoir of bone micrometastases and confers unexpected therapeutic vulnerability

doi: 10.1016/j.ccell.2018.10.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Western blots were performed using antibodies against NFAT1(Cell Signalling, 5861S), pS6K(T389) (Cell Signaling, 9234S), pCamKII(T286) (Cell Signalling, 12716S), MeCP2 (Cell Signalling, 3456S), pMeCP2(S421)(Rockland, 600-401-X14), Firefly luciferase (Thermo, PA5-32209).

Techniques: Luciferase, Recombinant, Expressing, In Vivo, Software

Journal: Nature microbiology

Article Title: Engineering Toxoplasma gondii secretion systems for intracellular delivery of multiple large therapeutic proteins to neurons.

doi: 10.1038/s41564-024-01750-6

Figure Lengend Snippet: Fig. 4 | Probing the functionality of T. gondii-delivered MeCP2 via heterochromatin binding, pull-down assay and single-cell sequencing in human cortical organoids. a, Mouse primary neurons inoculated with GRA16-MeCP2. Top images show a close-up view of the soma and T. gondii (2 independent repeats). Blue arrowheads mark co-localization of GRA16-MeCP2 with foci of heterochromatic DNA. Scale bars, 10 μm. b, Mouse neuroblastoma cells inoculated with GRA16-MeCP2 and GRA16-HA T. gondii (2 independent repeats). Scale bars, 10 μm. c, Pull-down assay of protein lysates from HFF cells infected with GRA16-MeCP2 or WT ME49 T. gondii. L, protein ladder; In, Input lysate; Me(+), protein pull down with methylated DNA probes; Me(−), protein pull down with non-methylated DNA probes; primary antibody, anti-MeCP2. Representative blot from 3 independent repeats. d, UMAP of single cells based on human+T. gondii gene quantification, coloured by percentage of Toxoplasma and human transcript counts. e, Same UMAP as in d but coloured by exogenous constructs transcript counts. f, UMAP of single cells based on human gene expression. Main clusters were identified and coloured by cell subtypes.

Article Snippet: Ten male C57BL/6J mice (Envigo RMS Israel) aged 4 weeks were injected through the lateral tail vein with 100 μl of UPS-grade PBS (N = 5) or 50,000 freshly syringe-lysed tachyzoites of ME49 GRA16-MeCP2 T. gondii diluted in PBS (N = 5).

Techniques: Binding Assay, Pull Down Assay, Sequencing, Infection, Methylation, Construct, Gene Expression