Figure 1.

Editing of Nf1 gene in rat dorsal root ganglia (DRG) neurons leads to an increase in calcium currents via voltage-gated N-type calcium (CaV2.2) channels. Exon/intron organization of rat Nf1 gene. Sequence of the single guide RNA (gRNA) against exon 39 is shown. Blue line, protospacer adjacent motif (PAM) sequence. The gRNA (sequence underlined in green) pairs with its DNA target followed by a 59NGG sequence. Cas9 catalyzes double stranded cleavage on the genomic DNA 3 bp before PAM sequence. Nucleotide positions indicated are based on the DNA sequence on the Nf1 gene. kbp: kilo base pair. (B) Representative micrographs of DRG sensory neurons transfected with either pSpCas9(BB)-2A-green fluorescent protein (GFP) (control sgRNA) or pSpCas9(BB)-2A-GFP-Nf1 sgRNA. Green fluorescent protein fluorescence identifies transfected neurons. In this experiment, neuron without GFP has robust expression of neurofibromin, whereas the adjacent neuron with GFP fluorescence (circled) demonstrates significantly decreased neurofibromin expression (marked by an arrow). (C) Representative family of current traces is illustrated for neurons transfected with control or Nf1 sgRNA. (D) Summary of current density (pA/pF) vs membrane potential curves from sensory neurons transfected with control or Nf1 sgRNA. (E) Peak current density, at −10 mV, for the indicated conditions (n = 15 cells for control sgRNA and n = 16 cells for Nf1 sgRNA). N-type calcium currents were pharmacologically isolated with toxins and blockers against the other channel subtypes (see Methods). Asterisks indicate significance compared with control sgRNA transfected cells (*P < 0.05, Student’s t test). (F) Boltzmann fits for activation for DRG treated as indicated. Values for V_1/2, the voltage of half-activation, and slope factors (k) were not different between the 2 conditions. Error bars represent mean ± SEM.