px551 (cas9) plasmid  (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

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: medRxiv

Article Title: Personalized allele-specific CRISPR-Cas9 strategies for myofibrillar myopathy 6

doi: 10.1101/2024.02.03.24302252

Figure Lengend Snippet: A) A PAM-altering SNP may generate the NGG PAM site selectively on the mutant allele (red) in a given individual. The same gRNA relying on this PAM site is, therefore, expected to edit the mutant allele, leaving the normal allele (green) intact. B) If a SNP flanked by a nearby PAM site, that also serves as a target of allele-specific CRISPR-Cas9. The same gRNA designed for the mutant allele (red) may interact less efficiently with the normal allele due to a mismatch (X), permitting allele-specific gene editing. Scissors represent Cas9. Each vertical line represents a match between the target and gRNA.

Article Snippet: For NMD-CRISPR, plasmids for Cas9 (PX551; http://n2t.net/addgene:60957 ) and gRNA (PX552; http://n2t.net/addgene:60958 ) were obtained from Addgene.

Techniques: Mutagenesis, CRISPR

Journal: medRxiv

Article Title: Personalized allele-specific CRISPR-Cas9 strategies for myofibrillar myopathy 6

doi: 10.1101/2024.02.03.24302252

Figure Lengend Snippet: A) Simultaneous use of two gRNAs, one and the other respectively targeting upstream and the downstream of the transcription start site (TSS), is expected to result in genomic excision. Therefore, two gRNA CRISPR-Cas9 approaches designed to excise the TSS (i.e., Transcription Prevention-CRISPR approach) are anticipated to prevent the transcription from the edited gene. B) Alternatively, a CRISPR-Cas9 strategy designed to edit an exon is likely to generate small indels and, therefore, premature stop codon (a red asterisk), leading to nonsense-mediated decay of the mRNA from the edited gene. For illustration purposes, the BAG3 gene is depicted. Scissors represent Cas9.

Article Snippet: For NMD-CRISPR, plasmids for Cas9 (PX551; http://n2t.net/addgene:60957 ) and gRNA (PX552; http://n2t.net/addgene:60958 ) were obtained from Addgene.

Techniques: CRISPR

Journal: medRxiv

Article Title: Personalized allele-specific CRISPR-Cas9 strategies for myofibrillar myopathy 6

doi: 10.1101/2024.02.03.24302252

Figure Lengend Snippet: Since a nearby NGG PAM site flanks the disease-causing mutation (rs121918312), we designed a CRISPR-Cas9 gRNA using the rs121918312 as the PPS to achieve allele-specific NMD-CRISPR. In this scenario, a gRNA designed to target the mutant allele (19 nucleotide gRNA is displayed) will interact with the mutant allele (A) without mismatches. However, the same gRNA is expected to interact with normal BAG3 with one mismatch (B), potentially leading to mutant-specific NMD.

Article Snippet: For NMD-CRISPR, plasmids for Cas9 (PX551; http://n2t.net/addgene:60957 ) and gRNA (PX552; http://n2t.net/addgene:60958 ) were obtained from Addgene.

Techniques: Mutagenesis, CRISPR

Journal: medRxiv

Article Title: Personalized allele-specific CRISPR-Cas9 strategies for myofibrillar myopathy 6

doi: 10.1101/2024.02.03.24302252

Figure Lengend Snippet:

Article Snippet: For NMD-CRISPR, plasmids for Cas9 (PX551; http://n2t.net/addgene:60957 ) and gRNA (PX552; http://n2t.net/addgene:60958 ) were obtained from Addgene.

Techniques: CRISPR, Sequencing