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: 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: Neuron

Article Title: Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.

doi: 10.1016/j.neuron.2019.01.035

Figure Lengend Snippet: Figure 4. Unilateral Injection of LSL-gRNA in Rats Expressing Cre in Dopaminergic Neurons Enables Region- and Cell-Type-Specific Th Editing (A and C) Representative images demonstrate unilateral loss of TH immunoreactivity in the (A) midbrain and (C) striatum of DAT-iCre rats (bottom), but not WT animals (top), 6 weeks following co-delivery of AAV Cas9 and AAV LSL-Th gRNA to the left (L) SN. GFP fluorescence represents transduction by AAV. (B) Quantification of optical density of TH immunoreactivity relative to DAPI in the ipsilateral compared to the contralateral SN of animals described in (A). Each point represents one analyzed coronal section (n = 3–4 sections/animal), and each color represents a distinct animal (n = 5–6/group, unpaired t test, t(9) = 4.166, **p = 0.0024). (D) Quantification of optical density of TH immunoreactivity relative to DAPI in the ipsilateral compared to the contralateral striatum of animals described in (C). Each point represents one analyzed coronal section (n = 3–4 sections/animal), and each color represents a distinct animal (n = 5–6/group, unpaired t test, t(11) = 2.896, *p = 0.0177). Scale bars, 500 mm.

Article Snippet: The AAV vector encoding SpCas9 (pX551) was a gift from Feng Zhang (Addgene plasmid # 60957; Swiech et al., 2015) and the corresponding AAV is referred to as ‘‘AAV Cas9’’ herein.

Techniques: Injection, Expressing, Transduction

Journal: Neuron

Article Title: Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.

doi: 10.1016/j.neuron.2019.01.035

Figure Lengend Snippet: Figure 5. Characterization and Use of an LSL-Cas9 Transgenic Rat for Cre-Dependent Knockout of TH (A) Representative confocal images of colocalization of iCre recombinase and the FLAG-tagged Cas9 transgene in LSL-Cas9 rats. FLAG immunoreactivity is not observed following delivery of Flpo, a non-Cre recombinase. iRFP fluorescence indicates comparable delivery of Flpo- and iCre-encoding viruses. (B) Representative confocal images of unilateral TH loss in the SN of LSL-Cas9 rats 4 weeks after a midbrain injection of AAV iCre and AAV control gRNAs (right side, top) or AAV Th gRNAs (left side, bottom). Comparable EGFP fluorescence in control and Th gRNAs-injected hemispheres indicates comparable viral delivery between conditions. (C) A TH-immunostained striatal section from a rat injected as in (B) (L, left side; R, right side). (D) Quantification of optical density of TH immunoreactivity in the SN and striatum of animals described in (B) and (C). Each data point represents one analyzed coronal section (n = 3–4 sections/animal), and each color represents a different animal (n = 4). Scale bars represent 50 mm (A), 100 mm (B), and 500 mm (C).

Article Snippet: The AAV vector encoding SpCas9 (pX551) was a gift from Feng Zhang (Addgene plasmid # 60957; Swiech et al., 2015) and the corresponding AAV is referred to as ‘‘AAV Cas9’’ herein.

Techniques: Transgenic Assay, Knock-Out, Injection, Control

Journal: Neuron

Article Title: Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.

doi: 10.1016/j.neuron.2019.01.035

Figure Lengend Snippet: Figure 6. Developing an Assay for Knockout of MANF In Vivo (A) A schematic of the gRNA-binding sites and the PCR assay used to amplify the 893 nt flanking the second exon of rat Manf. (B–E) Rat primary cortical neurons were transduced with AAV Cas9 and AAV Manf gRNAs or AAV control gRNAs and harvested 1 week later for determination of mutagenesis and knockout. (B) Co-transduction with AAV Cas9 and AAV Manf gRNAs resulted in resolvase-induced cleavage of the PCR product (arrows). (C) An alignment of seven independently isolated clones of the PCR fragment shows precise +A insertions among the alleles. Knockout of Manf was verified with (D) real- time qRT-PCR and (E) Wes analyses of Manf mRNA and protein levels, respectively. (D) Manf mRNA levels were normalized to the geometric mean of reference genes and presented as 2ddCq ± upper and lower limits (n = 3, unpaired t test using dCq values, t(4) = 20.27, ****p < 0.0001). (E) The MANF protein band density was normalized to actin and presented as density relative to control gRNA (mean ± SE, n = 3, unpaired t test, t(4) = 4.999, **p = 0.0075). The arrow in the cropped blot points at the 25-kDa MANF band. (F) Representative images of unilateral loss of MANF immunoreactivity in the SN of LSL-Cas9 rats four weeks after co-injection of AAV iCre and AAV control gRNAs or AAV Manf gRNAs. GFP fluorescence represents delivery of gRNA. Scale bar 100 mm. (G) Quantification of the optical density of MANF immunoreactivity in the SN of LSL-Cas9 or WT animals injected as described in (F). Each data point represents one analyzed coronal section (n = 3–4/animal), and each color represents data from a distinct animal (n = 4/group, unpaired t test, t(6) = 5.437, **p = 0.0016).

Article Snippet: The AAV vector encoding SpCas9 (pX551) was a gift from Feng Zhang (Addgene plasmid # 60957; Swiech et al., 2015) and the corresponding AAV is referred to as ‘‘AAV Cas9’’ herein.

Techniques: Knock-Out, In Vivo, Binding Assay, Transduction, Control, Mutagenesis, Isolation, Clone Assay, Quantitative RT-PCR, Injection

Journal: Neuron

Article Title: Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.

doi: 10.1016/j.neuron.2019.01.035

Figure Lengend Snippet: Figure 8. Characterization and Use of the LSL-Cas9 3 DAT-iCre Double-Transgenic Rat for Knockout of MANF in the Midbrain Dopaminergic Neurons (A) Representative confocal images of TH and FLAG-tagged Cas9 colocalization in the SN of LSL-Cas9 3 DAT-iCre double-transgenic rats. (B) Representative midbrain images depicting unilateral loss of MANF immunoreactivity (white arrowheads) in TH+ dopaminergic cells in LSL-Cas9 3 DAT-iCre rats 4 weeks following injection of AAV control or AAV Manf gRNAs into the SN. (C) High-magnification confocal images acquired 4 weeks after AAV injection demonstrating selective loss of MANF immunoreactivity in TH+ cells that received Manf gRNAs (right), but not in those that received control gRNAs (left). GFP fluorescence indicates viral transduction. Open arrowheads signal to GFP+TH cells in which MANF immunoreactivity is maintained in both control and experimental conditions. Closed arrowheads signal to GFP+TH+ cells in which MANF immunoreactivity is lost in the hemisphere receiving Manf gRNAs, but not control.

Article Snippet: The AAV vector encoding SpCas9 (pX551) was a gift from Feng Zhang (Addgene plasmid # 60957; Swiech et al., 2015) and the corresponding AAV is referred to as ‘‘AAV Cas9’’ herein.

Techniques: Transgenic Assay, Knock-Out, Injection, Control, Transduction

Journal: The Journal of Neuroscience

Article Title: Mouse γ-Synuclein Promoter-Mediated Gene Expression and Editing in Mammalian Retinal Ganglion Cells

doi: 10.1523/JNEUROSCI.0102-20.2020

Figure Lengend Snippet: In vitro assays to select effective Ddit3 and Sarm1 gRNAs. A, Schematic illustration of AAV vectors for U6 promoter driven gRNA expression. The C/S gRNAs include four U6 promoters that drive 2 Ddit3 and Sarm1 gRNAs individually. B, Sequences of 4 Ddit3 gRNAs and Sarm1 gRNAs that we tested in vitro. The red coded sequences are selected to make C/S gRNAs. C, Schematic illustration of mCHOP-mCherry and Sarm1-mCherry constructs and corresponding targeting regions of gRNAs. Note, 49 bp in between of C2 and C3 sgRNAs and 147 bp in between of S3 and S4 sgRNAs are potential truncations by paired sgRNAs. D, Co-transfection of SpCas9, gRNAs and reporters (Ddit3-mCherry or Sarm1-mCherry) in HEK293T cells to determine knock-down effects. Scale bar, 50 µm.

Article Snippet: Constructs for Cas9 and sgRNAs The plasmid containing SpCas9 with N-terminal HA tag was purchased from Addgene (PX551, plasmid #60957).

Techniques: In Vitro, Expressing, Construct, Cotransfection, Knockdown

Journal: The Journal of Neuroscience

Article Title: Mouse γ-Synuclein Promoter-Mediated Gene Expression and Editing in Mammalian Retinal Ganglion Cells

doi: 10.1523/JNEUROSCI.0102-20.2020

Figure Lengend Snippet: Ddit3 and Sarm1 knock-down by AAV-mSncg-CRISPR/Cas9 in mouse RGCs. A, Confocal images of wholemount retinas showing AAV-mSncg-HA-SpCas9 expression in retina (upper panel) and the expression of C/S gRNAs-GFP in RGCs (GFP-positive cells) and Ddit3-mCherry inhibition. B, Confocal images of wholemount retinas showing AAV-mSncg-Cre-mediated Flag-SpCas9 expression in retinas of LSL-Flag-SpCas9 mice, and Sarm1-mCherry inhibition by C/S gRNAs-GFP. Scale bar, 20 µm. C, Confocal images of cross sections of retinas showing Ddit3 and Sarm1 mRNA levels in GCL by ISH. Scale bar, 20 µm. D, Confocal images of wholemount retinas showing Ddit3 expression in RGCs. For Ddit3-positive RGCs counting, four random fields from each retina were imaged using a 40× oil lens, and the RGCs doubly positive for RBPMS and Ddit3 were counted by masked observer. The percentage of Ddit3+ RGCs was calculated as the ratio of Ddit3+ RGCs compared with total RGCs in the field. Data are presented as mean ± SEM, n = 5; ***p < 0.001, Student's t test.

Article Snippet: Constructs for Cas9 and sgRNAs The plasmid containing SpCas9 with N-terminal HA tag was purchased from Addgene (PX551, plasmid #60957).

Techniques: Knockdown, CRISPR, Expressing, Inhibition