DOI: 10.5061/dryad.kh18932mc
title: Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9

description: 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.
description: # Genetic editing of primary human dorsal root ganglion neurons using
 CRISPR-Cas9 Dataset DOI:
 [10.5061/dryad.kh18932mc](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