Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9

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 ..., , # 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...,