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Editor's Notes

• #2: Based on the previous figures, ultrasound excites neurons via mechanical force, hypothesize…. As seen in the diagram, channels with mechanosensitivity include: TRPV1,2,4, Piezo1, TRPC1, TRPM7, TRPP1/2 complex, and indirectly through GPCR Used gadolinium(III) to change deformability of the lipid bilayer, acts as a global mechanosensitive channel inhibitor Amplitude of response decreased by 60%, validating…
• #3: Used selective blockers to inhibit specific mechanosensitive channels Ruthenium red – TRPV1/2/4, no significant difference from the control (channels not involved) Suramin – GPCR signaling, inhibits release of GDP from G alpha subunit, no significant difference Peptide inhibitor GsMTx4 to change membrane tension near the Piezo1 and TRPC1 channels, statistically significant reduction in magnitude, suggesting partial involvement
• #4: Designed guide RNAs for each channel and inserted it into a vector. All-in-one, containing single guide RNA cassette and Cas9 nuclease expression cassette, which was delivered to neurons with lentivirus Editing efficiency of 20-40%
• #5: Plotted average calcium response and the change in magnitude of the response First, used a non-targeting gRNA control, did not change response TRPM7 also did not have any significant changes Next, knockdown TRPP1/2, changed magnitude of the response for both targets Knocking down Piezo1 did not significantly change the magnitude, however knocking down TRPC1 did significantly change the response Highlights advantage of CRISPR-Cas9 system to decouple these two channels, which the peptide inhibitor GsMTx4 couldn’t Lastly, calculated relative contributions of the channels based on reduction of response and knockdown efficiency…