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 in primary human neurons with potential for future
clinical application for sensory diseases.