Intersectional CRISPR-Cas9 genetic targeting reveals acute role of Nav1.1 in proprioceptive behavior and function

Abstract

Proprioceptors, a specialized subset of mechanosensory neurons that relay sensory feedback from muscles and tendons, are required for precise, goal-directed movement. Like all neurons, proprioceptors rely on voltage-gated ion channels to generate and transmit electrical signals. Investigating ion channel function in proprioceptors in vivo is technically challenging because current approaches to selectively target proprioceptors require the generation of triple transgenic models and creation of both loxP- and Frt-flanked alleles. To facilitate selective targeting of genes within proprioceptors, we employed an intersectional cell-specific gene editing approach that leverages CRISPR/Cas9 and sensory-neuron selective viral capsids. This approach combines single-guide RNA (sgRNA) delivery in sensory neuron-selective adeno-associated viral (AAV) capsids in mice with parvalbumin-driven Cas9 expression. We tested this approach by targeting the voltage-gated sodium (NaV) channel NaV1.1. Targeting NaV1.1 using this approach led to significant motor coordination deficits as early as 3 weeks following sgRNA delivery. Furthermore, whole-cell current clamp recordings from transduced proprioceptors revealed NaV1.1 is required for maintaining short-duration action potentials, which would support high-frequency firing typically observed in proprioceptors. Collectively, this study establishes a versatile platform for precise spatiotemporal gene manipulation in otherwise hard-to-access sensory neuron populations, while also providing evidence that NaV1.1 is essential for proprioceptor function in adulthood. Significance StatementProprioceptors are sensory neurons that relay information about muscle length and force to enable coordinated movement and motor reflexes. Investigating how ion channels contribute to proprioceptor function has been limited by the lack of straightforward and selective genetic tools, which can also confound interpretation of behavioral phenotypes. Here, we developed an intersectional CRISPR/Cas9 strategy that combines sensory-neuron specific viral delivery of sgRNAs with spatially restricted Cas9 expression in mice. Using this method we targeted the voltage-gated sodium channel, NaV1.1, which led to persistent motor coordination deficits and impaired proprioceptor action potential waveform, establishing a direct, cell-autonomous role for NaV1.1 in proprioceptor function. Thus, our approach provides a flexible platform for spatially and temporally precise gene manipulation in proprioceptors.