Figure 11. CRISPR mutagenesis of Trpc5 channels in Kiss1^ARH neurons.

(A) Structure of AAV1-FLEX-SaCas9-U6sgTrpc5-exon2. Exon 2 of Trpc5 is denoted with guide sequence highlighted in red, the PAM is underlined. (B) Structure of AAV1-FLEX-SaCas9-U6sgTrpc5-exon7. Exon 7 of Trpc5 is denoted with guide sequence highlighted in red, the PAM is underlined. (C1) Image of coronal section through the ARH from Kiss1-Cre::Ai32 mouse with dual co-injections of AAV-DIO-mCherry and AAV1-FLEX-SaCas9-U6-sgTrpc5. Scale = 200 µm. (C2, C3) Higher-power overlays of epifluorescence (EYFP and mCherry) images with recording pipette patched onto a Kiss1^ARH-Cre:mCherry cell (C3). Scale = 40 µm. (D) Quantitative PCR measurements of Trpc5 transcripts from 10-cell neuronal pools (three pools from each animal) in double sgRNA mutagenesis of Trpc5 (second sgRNA against pore-forming region) in Kiss1^ARH neurons. Primers were targeted to first (left panel) or second (right panel) guide, respectively (unpaired t-test for first, t₍₁₀₎ = 10.67, ****p<0.0001; for second, t₍₁₀₎ = 10.79, ****p<0.0001). Data are expressed as mean ± SEM, with data points representing individual animals.