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

Abstract

Gene editing using CRISPR/Cas9 in vivo offers a powerful tool to investigate pain mechanisms. We validated the use of conditional knock-in mouse model expressing Streptococcus pyogenes CRISPR associated protein 9 selectively in sensory neurons by crossing with Scn10a-Cre driver. Transgene expression was confirmed in key tissues including the dorsal root ganglia (DRG) and sciatic nerve. To assess in vivo editing efficacy, RNA guides targeting GFP or TRPV1 were intrathecally administered. A dose of 3 {micro}g RNA guides significantly reduced GFP expression, and two rounds of nanoparticle delivery targeting TRPV1 resulted in [~]65% reduction in DRG and [~]55% in 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. Their responses to 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.