Efficient genetic perturbation of murine sensory neurons in vivo using CRISPR/Cas9

Gene editing using CRISPR/Cas9 in vivo offers a powerful tool to investigate pain mechanisms. We generated a conditional knock-in mouse model where Streptococcus pyogenes CRISPR-associated protein 9 (Cas9) expression is restricted to cells that express SCN9A. Transgenic markers were detected in key tissues including the dorsal root ganglia (DRG) and sciatic nerve. To assess in vivo editing efficacy, RNA guides targeting TRPV1 were intrathecally administered. Two injections of guide RNAs resulted in a significant reduction of TRPV1 in both the DRG and sciatic nerve without triggering caspase-3-mediated apoptosis or motor deficits. Edited animals exhibited increased withdrawal latencies to heat and reduced nocifensive behaviors following capsaicin injection. Capsaicin-evoked thermal hyperalgesia and mechanical allodynia were diminished. This approach enables rapid and efficient sensory neuron-specific CRISPR/Cas9 gene perturbations for pain research in mice. We envisage that this method can be employed both for the exploration of molecular mechanisms underlying nociception and for the validation of therapeutic targets associated with pain. PERSPECTIVE: There are tremendous opportunities afforded by facile multi-locus genome perturbation of sensory neurons in vivo. This model and approach enables rapid and low-cost genetic depletion experiments in mice.