We are excited by the potential benefits of single-cell RNA electroporation in elucidating the functions of hard-to-study ion channels or cell-type specific proteins in vivo, with the ability to precisely pre-select the neurons to be edited or modified.
Using whole-cell recordings in awake animals, we discovered a sequential program of adaptation to the disruption of E/I balance: After an initial increase in spiking and reduction in excitatory input (2-3d), neurons ultimately silenced themselves (7d+) through changes in membrane excitability.
We used this method to investigate how single L2/3 neurons respond to a loss of fast synaptic inhibition in vivo, when the rest of the network is unaffected. We delivered CRISPR/Cas9 against an essential subunit of the GABA-A receptor, which led to a reliable loss of inhibition in targeted cells.
We found RNA based single-cell electroporation to allow for high success rates (>85%) in protein expression as early as day 1 after electroporation. Success rate does not decrease when using many RNAs for CRISPR/Cas9 editing, resulting in very reliable in vivo gene editing.
Single-cell electroporation is performed via patch pipette and under 2-photon visual guidance in vivo. A short voltage pulse train is applied through the pipette to deliver RNA molecules into a targeted cell. The video below shows this process in cortical neurons expressing GCaMP6s.
Gene editing of single, targeted neurons in vivo is now feasible. We are proud to present our preprint for highly efficient single-cell electroporation using RNA. With @alex-fratzl.bsky.social, @munzlab.bsky.social, Botond Roska @iobswiss.bsky.social
#neuroskyence
www.biorxiv.org/content/10.1...
