Journal: Nature Communications

Article Title: RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors

doi: 10.1038/s41467-018-04048-4

Figure Lengend Snippet: Targeted gene silencing in adult wild-type mice with S. aureus dCas9 KRAB . a A dual AAV vector system was designed to deliver dSaCas9 KRAB and Pcsk9-targeting gRNA to adult-wild type mice via tail-vein injection. qRT-PCR for b dSaCas9 KRAB and c Pcsk9 expression was performed on mRNA from livers harvested from treated mice at 6 weeks post-injection (mean ± s.e.m, n = 4 mice). P < 0.05 is indicated by *, determined by Student’s t- test compared to controls. Serial serum collections were assayed for secreted d Pcsk9 protein levels and e low-density lipoprotein cholesterol (mean ± s.e.m., n = 4 mice, * indicates P < 0.05 by mixed design ANOVA with Tukey’s post-hoc analysis)

Article Snippet: The AAV and lentivirus plasmids expressing dSaCas9 KRAB are available on Addgene (Addgene #106249 and #106219).

Techniques: Plasmid Preparation, Injection, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors

doi: 10.1038/s41467-018-04048-4

Figure Lengend Snippet: Genome-wide analysis of gene expression in dSaCas9 KRAB -treated mice. a – d RNA-sequencing and differential expression analysis was performed comparing liver tissue from mice treated with a , b AAVs expressing dSaCas9 KRAB and Pcsk9 -targeting gRNA vs. dSaCas9 KRAB alone, c AAVs expressing dSaCas9 KRAB with Pcsk9 -targeting gRNA to PBS, or d AAV expressing dSaCas9 KRAB vs. PBS. Red data points indicate FDR < 0.05 by differential-expression analysis with a negative binomial generalized linear model and Wald test for significance ( n = 4, mice 6–8 weeks old at time of treatment). The data point representing the Pcsk9 transcript is highlighted in blue (FDR > 0.05). Extended gene lists found in Supplementary Data , ,

Article Snippet: The AAV and lentivirus plasmids expressing dSaCas9 KRAB are available on Addgene (Addgene #106249 and #106219).

Techniques: Genome Wide, Gene Expression, RNA Sequencing, Quantitative Proteomics, Expressing

Journal: Nature Communications

Article Title: RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors

doi: 10.1038/s41467-018-04048-4

Figure Lengend Snippet: Transient effects of AAV-mediated dSaCas9 KRAB gene silencing on host liver. a Secreted alanine transaminase as a measure of liver toxicity was assayed in the serum of treated mice over 6 weeks post-treatment (mean ± s.e.m., n = 4 mice at 6–8 weeks old when treated). P < 0.05 is indicated by * and calculated by mixed design ANOVA with Tukey’s post-hoc analysis compared to day 0 controls. Dotted line marks the upper limit of physiological levels of ALT in adult C57Bl/6 mice. b H&E staining was performed on sections from livers of mice harvested 6 weeks post-treatment (representative images shown, scale bars = 100 μm)

Article Snippet: The AAV and lentivirus plasmids expressing dSaCas9 KRAB are available on Addgene (Addgene #106249 and #106219).

Techniques: Staining

Journal: Nature Communications

Article Title: RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors

doi: 10.1038/s41467-018-04048-4

Figure Lengend Snippet: Long-term efficacy of targeted transcriptional silencing with dSaCas9 KRAB . In mice treated with AAVs expressing dSaCas9 KRAB and Pcsk9 -targeting gRNA at 2×10 11 and 4×10 11 viral genomes/vector/mouse, serum was collected over 168 days post-treatment and analyzed for a alanine transanimase secretion, b Pcsk9 protein concentration, and c low-density lipoprotein-cholesterol concentration (mean ± s.e.m., n = 4 mice at 6–8 weeks old when treated). * and # mark P < 0.05 in 2e11 and 4e11 doses respectively, calculated by mixed design ANOVA with Tukey’s post-hoc analysis compared to PBS controls

Article Snippet: The AAV and lentivirus plasmids expressing dSaCas9 KRAB are available on Addgene (Addgene #106249 and #106219).

Techniques: Expressing, Plasmid Preparation, Protein Concentration, Concentration 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: 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