Georg Kosche

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.

In vivo single-cell gene editing using RNA electroporation reveals sequential adaptation of cortical neurons to excitatory-inhibitory imbalance The balance between excitatory and inhibitory neurotransmission is fundamental for normal brain function, yet the adaptation of individual neurons to disrupted excitatory-inhibitory balance is not wel...

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...