sgrna scaffold vector  (Addgene inc)

Journal: Science Advances

Article Title: Self-inactivating AAV-CRISPR at different ages enables sustained amelioration of Huntington’s disease deficits in BAC226Q mice

doi: 10.1126/sciadv.aea8052

Figure Lengend Snippet: ( A ) Schematic representation of targeted human HTT exon 1. sgRNAs HTTg1 and HTTg2 were designed to target the N17 domain–coding DNA sequence, which is located upstream of the CAG repeats. PAM, protospacer-adjacent motif. ( B ) Schematic representation of AAV-SaCas9-HTT sgRNA vector. U6-driven sgRNA and a CMV-driven Cas9 from S. aureus tagged with HA were packaged into a single AAV vector of 4.5 kb. ITR, inverted terminal repeat; NLS, nuclear localization signal sequence; 3×HA, three tandem repeats of the human influenza hemagglutinin (HA) tag. ( C to K ) Validation of human HTT editing in human embryonic kidney (HEK) 293T cells expressing EGFP-hHTT-20Q or EGFP-hHTT-120Q reporter system. (C) SaCas9-HTTg1 and SaCas9-HTTg2 reduced EGFP-fusion protein expression and aggregation in cells, whereas the control SaCas9-sgRNA did not. Scale bar, 100 μm. [(D) and (E)] Quantitative analyses of EGFP-20Q (D) or EGFP-120Q (E) fluorescence intensity. n = 3 or 4; ** P = 0.0083 (20Q), ** P = 0.0087 (120Q), and *** P = 0.0009 (120Q). [(F) to (K)] Western blot detecting EGFP and SaCas9 (anti-HA) protein level in EGFP-hHTT-20Q [(F) to (H)] or EGFP-hHTT-120Q [(I) to (K)] reporter system. [(F) and (I)] SaCas9-HTTg1 and SaCas9-HTTg2 reduced EGFP-hHTT protein expression compared to control SaCas9-sgRNA with equivalent SaCas9 expression level. [(G), (H), (J), and (K)] Quantification of relative EGFP [(G) and (J)] and SaCas9 [(H) and (K)] protein expression. n = 3 per group; ** P = 0.0087 (20Q) and ** P = 0.0069 (120Q). Data were shown as means ± SEM. One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test.

Article Snippet: A single-vector AAV-SaCas9 system containing Cas9 from Staphylococcus aureus (SaCas9) and its sgRNA scaffold was obtained from Addgene (plasmid no. 61591). sgRNAs targeting human HTT and SaCas9 were designed on the basis of PAM sequence (5′-NNGRRT-3′) and by CRISPR RGEN Tools ( www.rgenome.net/ ). sgRNA sequences targeting human HTT are as follows: HTTg1: 5′-TGG AAA AGC TGA TGA AGG CCT-3′; and HTTg2: 5′-GAA AAG CTG ATG AAG GCC TTC-3′.

Techniques: Sequencing, Plasmid Preparation, Biomarker Discovery, Expressing, Control, Fluorescence, Western Blot

Journal: eBioMedicine

Article Title: The rs713586 risk variant dysregulates ADCY3 rather than DNAJC27, leading to obesity through ZFP42-TET1-mediated DNA methylation

doi: 10.1016/j.ebiom.2025.106112

Figure Lengend Snippet: The rs713586 region exhibits allele specific enhancer activity. a. Plot of obesity-associated noncoding SNPs in ADCY3 SNP locus revealed by GWAS database. Dotted line indicates p -value = 1 × 10 −8 . b. Epigenetic annotations of H3K27ac, H3K4me1, and H3K4me3 marks in the rs713586 region from neural-related samples in the ENCODE database. The rs713586 locus is indicated by orange vertical lines. c. Violin plots showing normalised expression levels of ADCY3 and DNAJC27 according to rs713586 alleles in brain and adipose tissues. The information was extracted from GTEx Consortium. d. Dual-luciferase reporter assays measuring signals from cells transfected with ADCY3 promoter alone or ADCY3 promoter combined with a region containing either rs713586-T ( ADCY3 pro-586-T) or -C ( ADCY3 pro-586-C). Signals were normalised to firefly luciferase activity (n = 3). e. Dual-luciferase reporter assays measuring signals from cells transfected with DNAJC27 promoter alone or DNAJC27 promoter combined with a region surrounding either rs713586-T ( DNAJC27 pro-586-T) or -C ( DNAJC27 pro-586-C). Signals were normalised to firefly luciferase activity (n = 3). p values were calculated using one-way ANOVA in d and e. f and g. qPCR analysis of ADCY3 and DNAJC27 gene expression levels in CRISPRa- or CRISPRi-treated cells. GAPDH was used as a loading control (n = 3). h and i. Western blot analysis of CRISPRa-NC and CRISPRa-treated cell lysates, probed with anti-ADCY3. α-Tubulin was used as a loading control (n = 3). j and k. Western blot analysis of CRISPRi-NC and CRISPRi-treated cell lysates, probed with anti-DNAJC27. α-Tubulin was used as a loading control (n = 3). All data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, # p < 0.0001 (“#” representing “∗∗∗∗”). ns, no significance, and p values were calculated using Student's t test in f to k.

Article Snippet: The U6 promoter and rs713586 sgRNA scaffold of the cloned pSpCas9(BB)-2A-Puro (PX459, RRID: Addgene_48139, Addgene, 48139) plasmids (pre-linked with rs713586 sgRNA) were subcloned into the pEASY -Blunt Zero Cloning vector (TRAN, CB501-01).

Techniques: Activity Assay, Expressing, Luciferase, Transfection, Gene Expression, Control, Western Blot

Journal: eBioMedicine

Article Title: The rs713586 risk variant dysregulates ADCY3 rather than DNAJC27, leading to obesity through ZFP42-TET1-mediated DNA methylation

doi: 10.1016/j.ebiom.2025.106112

Figure Lengend Snippet: The rs713586 variant impairs ADCY3 and DNAJC27 expression and leads to shorter primary cilia. a. Schematic illustrating the ABE mediated targeting of the rs713586 region. The rs713586 locus is marked in red, and the PAM sequences are highlighted in purple. b. Sanger sequencing of the rs713586 locus in ARPE-19 cells following ABE editing. c and d. Western blot probing with anti-ADCY3 and anti-DNAJC27 antibodies in cells carrying different rs713586 alleles. α-Tubulin was used as a loading control (n = 3). p values were calculated using one-way ANOVA. e–g. Monotonic regression analysis of 4C-seq peak calling for the rs713586 region in rs713586-T and rs713586-C cells (e). The blue line indicates the ADCY3 promoter region, and the orange line indicates the DNAJC27 promoter. The significance of the peaks in the promoter of ADCY3 (f) and DNAJC27 (g) was carried out using ChIPseeker in rs713586-T and rs713586-C cells (n = 2). p values were calculated using Student's t -test in f and g. h–j. rs713586-T and rs713586-C cells were starved for 48 h and stained with anti-ARL13B (red) and anti-α-AC-Tubulin (green) antibodies. DAPI (blue) was used to stain nuclei. Scale bar, 5 μm (h). Percentage of ciliated cells (i) and quantitative analysis of cilium length (j) in rs713586-T and rs713586-C cells (n = 3). p values were calculated using Student's t -test in i and j. All data are presented as mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, # p < 0.0001. ns, no significance.

Article Snippet: The U6 promoter and rs713586 sgRNA scaffold of the cloned pSpCas9(BB)-2A-Puro (PX459, RRID: Addgene_48139, Addgene, 48139) plasmids (pre-linked with rs713586 sgRNA) were subcloned into the pEASY -Blunt Zero Cloning vector (TRAN, CB501-01).

Techniques: Variant Assay, Expressing, Sequencing, Western Blot, Control, Staining

Journal: eBioMedicine

Article Title: The rs713586 risk variant dysregulates ADCY3 rather than DNAJC27, leading to obesity through ZFP42-TET1-mediated DNA methylation

doi: 10.1016/j.ebiom.2025.106112

Figure Lengend Snippet: The Enh region mediates rs713586 regulation of ADCY3. a. Epigenetic annotations of H3K27ac, H3K4me1, and H3K4me3 marks in the Enh region from neural-related samples in the ENCODE database. The Enh region is indicated by the orange shaded area. b. Dual-luciferase reporter assays measuring signals from cells transfected with pGL4.7, ADCY3 promoter alone, ADCY3 promoter containing the region containing either rs713586-T ( ADCY3 pro-586-T) or -C ( ADCY3 pro-586-C), the ADCY3 promoter containing the Enh region, or ADCY3 promoter containing both the Enh and the region surrounding either rs713586-T ( ADCY3 pro-586-T) or -C ( ADCY3 pro-586-C). Signals were normalised to firefly luciferase activity (n = 6). p values were calculated using one-way ANOVA. c. The significance of the peaks in Enh region was carried out using ChIPseeker in rs713586-T and rs713586-C cells. d. qPCR analysis of ADCY3 transcript levels in CRISPRi-NC and CRISPRi-Enh cells following Enh knockout (n = 3). p values were calculated using Student's t -test. e. Sanger sequencing of the Enh region CRISPR/Cas9 in ARPE-19 cells following CRISPR/Cas9-mediated editing. f. Agarose gel electrophoresis analysis of the amplified target band after knockout the Enh region. g and h. Western blot probing with anti-ADCY3 in cells that were treated with 586-T-KO-NC or 586-T-KO-Enh. 586-T-KO-NC: control group, cells with rs713586-T allele transfected with a negative vector; 586-T-KO-Enh: cells with rs713586-T allele transfected with an Enh knockout vector. α-Tubulin was used as a loading control (n = 3). p values were calculated using Student's t -test. i and j. Western blot probing with anti-ADCY3 in cells that were treated with 586-C-KO-NC or 586-C-KO-Enh. 586-C-KO-NC: control group, cells with rs713586-C allele transfected with a negative vector; 586-C-KO-Enh: cells with rs713586-C allele transfected with an Enh knockout vector. α-Tubulin was used as a loading control (n = 3). p values were calculated using Student's t -test. All data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, # p < 0.0001. ns, no significance.

Article Snippet: The U6 promoter and rs713586 sgRNA scaffold of the cloned pSpCas9(BB)-2A-Puro (PX459, RRID: Addgene_48139, Addgene, 48139) plasmids (pre-linked with rs713586 sgRNA) were subcloned into the pEASY -Blunt Zero Cloning vector (TRAN, CB501-01).

Techniques: Luciferase, Transfection, Activity Assay, Knock-Out, Sequencing, CRISPR, Agarose Gel Electrophoresis, Amplification, Western Blot, Control, Plasmid Preparation

Journal: eBioMedicine

Article Title: The rs713586 risk variant dysregulates ADCY3 rather than DNAJC27, leading to obesity through ZFP42-TET1-mediated DNA methylation

doi: 10.1016/j.ebiom.2025.106112

Figure Lengend Snippet: The rs713586-C risk allele exhibits reduced ZFP42 binding affinity, leading to decreased ADCY3 expression. a. Schematics of ZFP42 binding to the rs713586-T non-risk allele. b. Dual-luciferase reporter assay detecting ZFP42 binding affinity to the rs713586-T and -C alleles. The horizontal axis represents the grouping of alleles and TFs (n = 5). p values were calculated using one-way ANOVA. c. qPCR analysis of ZFP42 and ADCY3 expression levels in ARPE-19 cells with ZFP42 overexpression and Enh region knockout. GAPDH was used as a loading control (n = 3). p values were calculated using one-way ANOVA. d. ChIP-qPCR analysis of ZFP42 binding to DNA fragments containing either the rs713586-T non-risk or -C risk alleles (n = 3). p values were calculated using Student's t -test. e and f. Western blot determining ZFP42 and ADCY3 expression levels in ARPE-19 cells overexpressing ZFP42. oeNC: control group, oeZFP42: ZFP42 overexpression group. α-Tubulin was used as a loading control (n = 3). p values were calculated using Student's t -test. g. qPCR detecting ZFP42 and ADCY3 gene expression levels in ZFP42 knockdown cells (n = 3). p values were calculated using Student's t -test. h and i. Western blot probing with anti-ZFP42 in control and ZFP42 knockdown cells. Control group: shRNA-NC, ZFP42 knockdown group: shRNA-ZFP42. α-Tubulin was used as a loading control (n = 3). p values were calculated using Student's t -test. All data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01 ∗∗∗ p < 0.001, # p < 0.0001 ns, no significance.

Article Snippet: The U6 promoter and rs713586 sgRNA scaffold of the cloned pSpCas9(BB)-2A-Puro (PX459, RRID: Addgene_48139, Addgene, 48139) plasmids (pre-linked with rs713586 sgRNA) were subcloned into the pEASY -Blunt Zero Cloning vector (TRAN, CB501-01).

Techniques: Binding Assay, Expressing, Luciferase, Reporter Assay, Over Expression, Knock-Out, Control, ChIP-qPCR, Western Blot, Gene Expression, Knockdown, shRNA

Journal: eBioMedicine

Article Title: The rs713586 risk variant dysregulates ADCY3 rather than DNAJC27, leading to obesity through ZFP42-TET1-mediated DNA methylation

doi: 10.1016/j.ebiom.2025.106112

Figure Lengend Snippet: TET1 mediates the effect of the rs713586 locus on ADCY3 expression. a. Proteins from rs713586-T and rs713586-C ARPE-19 cells were immunoprecipitated with anti-ZFP42. Input protein (100 μg) and immunoprecipitates were analysed by Western blot with the indicated antibodies. IgG antibodies were used as the negative control. b and c. qPCR analysis of the ZFP42 and TET1 expression levels in ZFP42 knockdown cells (b) or ZFP42 overexpressing cells (c) (n = 3) groups from three independent experiments. p values were calculated using Student's t -test. d. qPCR determining TET1 and ADCY3 expression levels in ARPE-19 cells overexpressing TET1. oeNC: control group, oeTET1: TET1 overexpression group (n = 3) groups from three independent experiments. p values were calculated using Student's t -test. e and f. Methylation levels of the ADCY3 promoter region (e) and Enh region (f) in rs713586-T and rs713586-C cells (n = 3). CpG island was marked in red. p values were calculated using Student's t -test. The data are presented as mean ± SD. ∗ p < 0.05. ns, no significance.

Article Snippet: The U6 promoter and rs713586 sgRNA scaffold of the cloned pSpCas9(BB)-2A-Puro (PX459, RRID: Addgene_48139, Addgene, 48139) plasmids (pre-linked with rs713586 sgRNA) were subcloned into the pEASY -Blunt Zero Cloning vector (TRAN, CB501-01).

Techniques: Expressing, Immunoprecipitation, Western Blot, Negative Control, Knockdown, Control, Over Expression, Methylation