sv40pa  (Addgene inc)

Journal: Bio-protocol

Article Title: Single-Particle Tracking of AMPA Receptor-Containing Vesicles

doi: 10.21769/BioProtoc.5325

Figure Lengend Snippet: (A) Constructs used to insert HaloTag into the genomic loci of Gria1 using homology-independent targeted integration (HITI). The donor construct (Donor) contains the DNA sequence coding for HaloTag (HaloTag) and the single guide RNA that will target Cas9 to Gria1 (sgRNA). Importantly, the HaloTag coding sequence is flanked by Gria1 sequences that will be targeted and cut by Cas9 (red bars). Cas9 is expressed from a second plasmid (Cas9). Black bars represent promoters driving the expression of Cas9 and the sgRNA. (B) Cas9 creates double-strand breaks in the genomic loci of Gria1 and releases HaloTag from the Donor. HaloTag can be inserted into Gria1 by non-homologous end joining (NHEJ). When expressed from the edited copy of Gria1 (Gria1-HT ), GluA1 will contain HaloTag inserted into its amino-terminal domain (ATD; GluA1-HT). SP, signal peptide; LBD, ligand-binding domain; TMD, transmembrane domain; CT, C-terminal tail. (C) Diagram of AMPAR containing a GluA1 subunit (blue) with HaloTag (green) inserted into the ATD (red). AMPARs are tetramers that can contain zero to four GluA1 subunits. As HaloTag is inserted into the ATD, it will sit on the extracellular side of the receptor. HaloTag can be labeled with fluorescent dyes that are conjugated to HaloTag ligand (HTL), such as JF 549 -HTL. (D) Representative confocal image of a cultured rat hippocampal neuron expressing GluA1 tagged with HaloTag and labeled with JF 549 -HTL (GluA1-HT-JF 549 ). Scale bar, 25 μm. (E) Representative widefield images of edited neurons expressing GluA1-HT labeled with JF 549 -HTL (GluA1-HT-JF 549 ) and a fluorescent neuron marker (in this case, miRFP670 driven by a synapsin promoter). Scale bar, 100 μm.

Article Snippet: PX551 (Cas9) plasmid (Addgene, catalog number: 60957) 3. px552-sg-gria1-HT (donor) plasmid (Addgene, catalog number: 187652) 4. rh10-PX551 AAV (Janelia Research Campus Viral Services) 5. rAAV2-px552-sg-gria1-HT AAV (Janelia Research Campus Viral Services) Reagents 1.

Techniques: Construct, Sequencing, Plasmid Preparation, Expressing, Non-Homologous End Joining, Ligand Binding Assay, Labeling, Cell Culture, Marker

Journal: bioRxiv

Article Title: Gene Editing for ATXN3 Inactivation in Machado-Joseph disease: CRISPR-Cas9 as a Therapeutic Alternative to TALEN-Induced Toxicity

doi: 10.1101/2025.02.14.637261

Figure Lengend Snippet: (A) Four guide sequences (sgKO.1, sgKO.2, sgKO.3, and sgKO.4) were designed to recognize exon 2 of the human ATXN3 gene (sgRNA target sequences are displayed in green). Sp Cas9 is recruited to the locus of interest, mediating the insertion of a DSB (colored scissors in the scheme and arrowheads in the target sequences) at approximately 3 base pairs upstream of a PAM sequence (sequence displayed in magenta). Subsequently, genome editing is achieved via NHEJ repair pathway for the permanent blocking of ATXN3 gene expression. (B) sgRNA sequences were cloned into a lentiviral expression vector (lentiCRISPRv2, addgene plasmid #52961), which also codifies for a FLAG-tagged Sp Cas9 and a puromycin resistance cassette. For the validation of the guide sequences, HEK293T cells were transfected with each of the generated plasmids and maintained in culture for 72 hours (selection medium with puromycin 10 µg/mL for 48 hours). Cells transfected with a guide sequence targeting the bacterial lacZ gene (sgCTRL) were used as a negative control. (C-D) Locus modification efficiencies were analyzed using Surveyor nuclease assay. Lane 1: DNA ladder (GeneRuler 100 bp, Thermo Fisher Scientific); Lane 2: Cells transfected with the sgCTRL construct; Lanes 3-6: Cells transfected with the sgRNA knock-out guide sequences (sgKO.1, sgKO.2, sgKO.3 and sgKO.4, respectively). Arrowheads indicate the expected DNA fragments, cleaved by Surveyor nuclease. The estimated indel occurrence within human ATXN3 locus is represented as a percentage (n=4). (E-F) Western blot analysis revealed a significant decrease (approximately 0.5-fold change) in ATXN3 protein levels after Sp Cas9 targeting of ATXN3 locus in comparison with the control sequence (n=3). Results are presented as fold change relative to cells transfected with the sgCTRL expressing plasmid. Optical densitometry analysis of ATXN3 fractions were normalized with β-actin and FLAG signals. Statistical significance was evaluated with one-way ANOVA with Dunnett’s post hoc test (*p<0.05). Data are expressed as mean ± SEM. Abbreviations : LTR (long terminal repeat); U6 (Pol III promoter); sgRNA (single guide RNA); EFS (elongation factor 1α short promoter); Sp Cas9 (Cas9 nuclease from Streptococcus pyogenes) ; NLS (nuclear localization signal); FLAG (FLAG octapeptide tag); P2A (2A self-cleaving peptide); Puro (puromycin selection marker); WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

Article Snippet: Due to constraints related with AAV packaging capacity, a two-vector system was adopted for in vivo applications : i) AAV- Sp Cas9 (vector pX551, plasmid #60957, Addgene) and ii) AAV- Sp Guide (vector pX552, plasmid #60958, Addgene).

Techniques: Sequencing, Blocking Assay, Expressing, Clone Assay, Plasmid Preparation, Transfection, Generated, Selection, Negative Control, Modification, Nuclease Assay, Construct, Knock-Out, Western Blot, Comparison, Control, FLAG-tag, Marker, Virus

Journal: bioRxiv

Article Title: Gene Editing for ATXN3 Inactivation in Machado-Joseph disease: CRISPR-Cas9 as a Therapeutic Alternative to TALEN-Induced Toxicity

doi: 10.1101/2025.02.14.637261

Figure Lengend Snippet: (A-B) Schematic representation of the stereotaxic co-injection of viral vectors in the striatum of C57BL/6 mice. Lentivirus encoding for the human mutant ATXN3 protein with 72Q (Myc-tagged), rAAV1/2 encoding for Sp Cas9 (HA-tagged) and rAAV1/2 encoding for the CTRL guide sequence (EGFP-KASH co-expression) were injected in the left hemisphere, serving as experimental control. In the contralateral hemisphere rAAV1/2 encoding for the sgKO.2 sequence were injected, along with LV-PGK- ATXN3 72Q and the rAAV1/2- Sp Cas9. Four weeks after surgery animals were sacrificed. (C) Western blot analysis of striatal homogenates demonstrates that CRISPR- ATXN3 KO system promotes a reduction of mutant ATXN3 species in treated hemispheres, when compared with the contralateral control hemispheres (data not quantified). (D-E) Immunohistochemical peroxidase staining upon labelling of striatal sections with anti-ubiquitin antibody, 4 weeks after stereotaxic surgery. Scale bar, 50 µm. (F) CRISPR- ATXN3 KO injected hemispheres display a drastic reduction in the number of ubiquitin-positive inclusions in comparison with the contralateral control hemisphere, injected with CRISPR-CTRL. (G-H) Immunohistochemical analysis using anti-DARPP-32 antibody for expanded ATXN3-derived lesion identification. Treated hemispheres, injected with CRISPR- ATXN3 KO showed a statistically significant reduction of DARPP-32 depleted volume, as quantified in (I) . Scale bar, 200 µm. (J-K) Iba-1 immunoreactivity in mouse striata. No statistically significant differences are observed between control (J) and CRISPR-edited hemispheres (K), as quantified in (L) . Scale bar, 200 µm in general view images and 50 µm in detail magnifications. (M-N) Gfap immunoreactivity in mouse striata. No statistically significant differences in the Gfap immunoreactivity are observed between non-edited (M) and CRISPR-edited striata (N), as quantified in (O) . Scale bar, 200 µm in general view images and 50 µm in detail magnifications. Statistical significance was evaluated with paired Student’s t-test (**p<0.01, n=5). Data are expressed as mean ± SEM. Abbreviations : ITR (invert terminal repeat); pMecp2 (mouse methyl CpG binding protein 2 promoter); HA (hemagglutinin tag); NLS (nuclear localization signal); Sp Cas9 (Cas9 nuclease from Streptococcus pyogenes) ; U6 (Pol III promoter); sgRNA (single guide RNA); hSyn1 (human synapsin 1 promoter); EGFP (enhanced green fluorescent protein); KASH (Klarsicht, ANC1, Syne Homology nuclear transmembrane domain); hGHpA (human growth hormone gene polyadenylation signal).

Article Snippet: Due to constraints related with AAV packaging capacity, a two-vector system was adopted for in vivo applications : i) AAV- Sp Cas9 (vector pX551, plasmid #60957, Addgene) and ii) AAV- Sp Guide (vector pX552, plasmid #60958, Addgene).

Techniques: Injection, Mutagenesis, Sequencing, Expressing, Control, Western Blot, CRISPR, Immunohistochemical staining, Staining, Comparison, Derivative Assay, Binding Assay