Journal: bioRxiv

Article Title: A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

doi: 10.1101/2023.12.12.571328

Figure Lengend Snippet: (a) Schematic of (top) the C9ORF72 gene and (bottom) the three main mRNA transcript variants expressed from it. V1 produces the short protein isoform (C9-S), while V2 and V3 produce the long protein isoform (C9-L). (b, c) Schematic of the dual-reporter system used to evaluate crRNAs. The platform consists of a (b) Renilla luciferase-encoding plasmid, pSV40-RLuc, whose 3’ untranslated region (UTR) carries a fragment of the C9ORF72 gene with 20 copies of the hexanucleotide repeat and 250-and 98-base pairs (bps) of the flanking upstream and downstream gene sequences, respectively, and (c) a firefly luciferase-encoding plasmid, pHSV-TK-FLuc, which was used as a proxy for collateral cleavage. (d) Normalized Renilla and firefly luciferase expression in HEK293T cells transfected with pSV40-RLuc, pHSV-TK-FLuc, and an expression vector encoding RfxCas13d and one of the 15 candidate crRNAs. All values were normalized to cells transfected with pSV40-RLuc, pHSV-TK-FLuc, and an expression vector encoding RfxCas13d with a non-targeted (NTG) crRNA ( n = 3). (e, f) Relative all-V and V3 mRNA in (e) HEK293T and (f) SH-SY5Y cells transfected with RfxCas13d and crRNAs 13, 7, and 1 or a NTG crRNA or one of two ASOs ( n = 3). All values from HEK293T and SH-SY5Y cells were normalized to untreated cells. Values indicate means and error bars indicate SD. **P < 0.01, ***P < 0.001, ****P < 0.0001; one-tailed unpaired t-test comparing each crRNA to the NTG crRNA. All data points are biologically independent samples.

Article Snippet: Neural progenitor cells (NPCs) derived from a 74-year-old female ALS patient with >145 copies of the hexanucleotide repeat expansion in the C9ORF72 gene were obtained from AXOL Bioscience (ax0073).

Techniques: Luciferase, Plasmid Preparation, Expressing, Transfection, One-tailed Test

Journal: bioRxiv

Article Title: A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

doi: 10.1101/2023.12.12.571328

Figure Lengend Snippet: (a) Cartoon illustrating the injection scheme. (b) Overview of the experimental plan to analyze C9ORF72 mRNA in EGFP-KASH + nuclei isolated by fluorescence-activated cell sorting (FACS). (c ) Representative immunofluorescent staining of the hippocampus (HPC) and motor cortex (MC) in C9-BACexp mice two-months after injection with 2 x 10 10 GCs each of AAV-PHP.eB-RfxCas13d-crRNA and AAV-PHP.eB-EGFP-KASH. Scale bar, 20 µm. (d, e) Relative all-V and V3 mRNA in (d) the HPC and (e) MC of EGFP-KASH + nuclei from C9-BACexp injected with AAV-PHP.eB-RfxCas13d-crRNA-13, -7, -1, or -NTG with AAV-PHP.eB-EGFP-KASH ( n ≥ 7). (f) Representative RNA FISH to detect the G 4 C 2 repeat RNA (purple) from the HPC of C9-BACexp mice injected with AAV-PHP.eB-RfxCas13d-crRNA-13 or -NTG and AAV-PHP.eB-EGFP-KASH. Arrowheads indicate representative cells. Scale bar, 15 µm. (g, h) Quantification of (g) the number of RNA foci per EGFP-KASH + cell in the HPC and (h) the percentage of EGFP-KASH + cells with 0, 1-2, 3-4, or >5 foci ( n ≥ 4). (g, h) 58-455 cells were counted per animal. 398, 845, and 467 cells total were counted for AAV-PHP.eB-RfxCas13d-crRNA-7, crRNA-13, and -NTG respectively ( n ≥ 4). RNA foci measurements were conducted by a blinded investigator. Values indicate means and error bars indicate SD. *P < 0.05, **P < 0.01, ***P < 0.001; one-tailed unpaired t-test comparing each crRNA to the NTG crRNA. All data points are biologically independent samples.

Article Snippet: Neural progenitor cells (NPCs) derived from a 74-year-old female ALS patient with >145 copies of the hexanucleotide repeat expansion in the C9ORF72 gene were obtained from AXOL Bioscience (ax0073).

Techniques: Injection, Isolation, Fluorescence, FACS, Staining, One-tailed Test

Journal: bioRxiv

Article Title: Altered metabolic function induced by amyloid-beta oligomers and PSEN1-mutations in iPSC-derived astrocytes

doi: 10.1101/2023.08.23.554346

Figure Lengend Snippet: A) Heatmap displaying differential compounds identified using metabolomic analysis. Summary of Pathway analysis for the comparison of control and B) A246E, C) L286V and D) R278 fAD patient derived astrocytes. The pathway analysis results of PSEN1 astrocytes compared with control: the colour graduated from white to and red indicates the degree of significance, the size of bubble represents the number of metabolites hit in the pathway. E) Bar charts indicating intensity changes in key metabolites represented within metabolic pathway that were detected in astrocytes carrying PSEN1 mutations. Data shown is expressed as ± SD, n=6.

Article Snippet: Healthy control (ax0018) and PSEN1 human neural precursor cells (hNPCs) carrying L286V (ax0112) and A246E (ax0114) mutations were purchased from Axol Bioscience (Cambridge, UK) and plated as previously described in parallel to the R278I generated hNPCs (see above).

Techniques: Comparison, Control, Derivative Assay

Journal: Lab on a Chip

Article Title: Development of two-photon polymerised scaffolds for optical interrogation and neurite guidance of human iPSC-derived cortical neuronal networks

doi: 10.1039/C9LC01209E

Figure Lengend Snippet: Figure 3. Candidate material biocompatibility for long-term adherent cultures. (A) i) Representative images of Phalloidin (green) and DAPI (blue) staining 24 hours post-plating on Polystyrene and DClear with human neural progenitor cells. Scale bars = 500 µm. ii) F-actin expression with thresholded image below. iii) Nuclei localisation with thesholded image below. (B) 24 hours post-plating adherent nuclei were quantified by number of thresholded DAPI+ regions of interest. (C) Mean surface area per cell (µm2) quantified by

Article Snippet: Human iPSC-derived NPC plating and differentiation Human iPSC-derived neural progenitor cells (ax0016, Axol Bioscience Ltd., Cambridge, UK) were plated from frozen at 1 x 105 cells cm-2 onto SureBond (Axol Bioscience) coated 6-well plates using Neural Maintenance Medium (Axol Bioscience) and Y-27632 2HCl (10 μM, Selleck Chemicals, Texas, USA) and cultured for 24 hours at 37 °C in a 5% CO2 environment 54.

Techniques: Staining, Expressing

Journal: Lab on a Chip

Article Title: Development of two-photon polymerised scaffolds for optical interrogation and neurite guidance of human iPSC-derived cortical neuronal networks

doi: 10.1039/C9LC01209E

Figure Lengend Snippet: Figure 4. Assessment of neural progenitor efficiency in differentiation into functionally active post-mitotic neurons. (A) Representative images of fluorescence immunocytochemistry for protein markers. Mitotically active neural progenitors which are (i) SOX2+ (magenta) and PAX6+ (green), (ii) Nestin+ (magenta) and

Article Snippet: Human iPSC-derived NPC plating and differentiation Human iPSC-derived neural progenitor cells (ax0016, Axol Bioscience Ltd., Cambridge, UK) were plated from frozen at 1 x 105 cells cm-2 onto SureBond (Axol Bioscience) coated 6-well plates using Neural Maintenance Medium (Axol Bioscience) and Y-27632 2HCl (10 μM, Selleck Chemicals, Texas, USA) and cultured for 24 hours at 37 °C in a 5% CO2 environment 54.

Techniques: Fluorescence, Immunocytochemistry