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

Palomino, S. M.; Gabriel, K.; Mwirigi, J.; Cervantes, A.; Horton, P.; Funk, G.; Moutal, A.; Martin, L.; Khanna, R.; Price, T. J.; Patwardhan, A.

doi:10.1101/2024.04.02.587857

bioRxiv · 10.1101/2024.04.02.587857

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

Palomino, S. M.·Gabriel, K.·Mwirigi, J.·Cervantes, A.·Horton, P.·Funk, G.·Moutal, A.·Martin, L.·Khanna, R.·Price, T. J.·Patwardhan, A.

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Abstract

CRISPR-Cas9 editing is now the leading method for genome editing and is being advanced for the treatment of human disease. CRIPSR editing could have many applications for treatment of neurological diseases, including pain but traditional viral vector delivery approaches have neurotoxicity limiting their use. Overcoming these issues could open the door for genome editing treatments for diseases like intractable pain where the dorsal root ganglia (DRG) would be the desired target. To this end, we describe a simple method for viral-vector-independent transfection of primary human DRG (hDRG) neurons for CRISPR-Cas9 editing. As proof of principle, we edited TRPV1, NTSR2, and CACNA1E using a lipofection method with CRISPR-Cas9 plasmids containing reporter tags (GFP or mCherry). Transfection was successful as demonstrated by the expression of the reporters as early as two days in vitro. CRISPR-Cas9 editing was confirmed at the genome level with insertion and deletion detection system 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 intractable pain. TeaserWe describe a non-viral transfection method for CRISPR-Cas9 gene editing in human dorsal root ganglion neurons.

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Palomino, S. M., Gabriel, K., Mwirigi, J., Cervantes, A., Horton, P., Funk, G., Moutal, A., Martin, L., Khanna, R., Price, T. J., Patwardhan, A.. 2024-04-03. Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9 with functional confirmation. https://doi.org/10.1101/2024.04.02.587857

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