Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9
Data files
Oct 14, 2025 version files 37.28 KB
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CRISPR_article_Data.xlsx
24.40 KB
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DisciplineSpecificMetadata.json
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README.md
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Abstract
CRISPR-Cas9 is now the leading method for genome editing and is advancing for the treatment of human disease. CRISPR has promise in treating neurological diseases, but traditional viral-vector-delivery approaches have neurotoxicity, limiting their use. Here, we describe a simple method for non-viral transfection of primary human DRG (hDRG) neurons for CRISPR-Cas9 editing. We edited TRPV1, NTSR2, and CACNA1E using a lipofection method with CRISPR-Cas9 plasmids containing reporter tags (GFP or mCherry). Transfection was successfully demonstrated by the expression of the reporter two days post-administration. CRISPR-Cas9 editing was confirmed at the genome level with a T7-endonuclease-I assay; protein level with immunocytochemistry and Western blot; and functional level through capsaicin-induced Ca2+ accumulation in a high-throughput compatible fluorescent imaging plate reader (FLIPR) system. This work establishes a reliable, target-specific, non-viral CRISPR-Cas9-mediated genetic editing in primary human neurons with potential for future clinical application for sensory diseases.
Dataset DOI: 10.5061/dryad.kh18932mc
Description of the data and file structure
The data collected was from multiple experiments, including:
MTS viability assays: evaluate cell death.
Live cell imaging: Confirm and evaluate the number of cells successfully transfected with plasmid (mCherry or GFP signal vs. no signal) in culture and in neurons only.
PCR/Indel assay: these assays were run in sequence to determine insertion/deletion scars in the genome of cells from dorsal root ganglion culture, primary culture post CRISPR construct application.
Immunocytochemistry: confocal images of DRG neurons to quantify target protein expression post CRISPR editing of primary culture.
Western blot analysis: to evaluate total expression levels of target proteins in culture post CRISPR edit.
Fluorescent imaging plate reader assay (FLIPR): evaluate functional protein levels, measuring intracellular calcium response using a specific agonist of TRPV1 (targeted protein for CRISPR knockout).
Acronyms:
NC= Non-coding CRISPR control
KO= knockout
Cap= capsaicin
Files and variables
File: CRISPR_article_Data.xlsx
Description: The Excel file is organized by experiments performed (see tabs).
tab 1: Donor Demographic
List containing the age/sex number of dorsal root ganglions used from the donor and neuronal yield post tissue dissociation.
tab 2: MTS Viability Assays
TRPV1 plasmid transfection impact on cell viability. Cells were treated with either vehicle (transfection reagent no plasmid) or with the TRPV1 plasmid ( transfection reagent with plasmid) and observed over the course of 2 days. The values in the grid were the raw absorption values, which were then converted to percentages to monitor the percent decrease daily.
Each column displays the percent normalized change of cell culture viability over the course of 2 days. The groups were either NC or TRPV1 plasmid-containing cells and measured with the Lipofectamine for 2 days.
Variables
- NC, 24HRS,48HRS
tab 3: Transfection Efficiency
Proportion of neurons and other cell types determined by counting the total number of cells in 3 fields from live cell imaging, and also counting the number of cells noted to have the mCherry/GFP fluorescent tag. The cells were counted using the biorad ZOE microscope. The total number of cells was counted, and the number of cells with the fluorescent tag was counted, as were the neuronal cells and glial cells. In the Excel sheet, the rows and columns are specifically labeled.
Variables
- TRPV1, Cav2.3, NTSR2 (3-4 cultures each)
tab 4: ICC quant
fluorescent intensity values generated from ImageJ Fiji analysis of images represented in the publication, including background-adjusted values for each image.
Variables
- TRPV1, Cav2.3, NTSR2, control
tab 5: WB quantification
Western blot band quantitation values generated through ImageJ Fiji and graphed in GraphPad Prism. Values were either normalized to total protein or to GAPDH loading control.
Variables
- TRPV1, Cav2.3, NTSR2, NC (3 cultures each)
tab 6: FLIPR Raw data
values generated from the fluorescent plate reader. All values here are the normalized fluorescent values taken from the Tecan plate reader. values were used to create the figure in the original manuscript (Figure 3, panel d)
values were normalized to the NC (vehicle) group baseline for comparison
Variables
- NC (cap 800nM), TRPV1 KO (cap 800nM), NC (vehicle)
tab 7: cell titer viability assay (plate reader)
Values generated from the fluorescent plate reader. All values here are absorption values normalized to the average of the control group and presented as percentages.
Variables
- TRPV1, Cav2.3, NTSR2, NC (3-4 cultures each)
File: DisciplineSpecificMetadata.json
Code/software
Microsoft Excel
ImageJ Fiji
GraphPad Prism
Access information
NA