Hide-and-Seek genome editing reveals that Gephyrin is required for axo-axonic synapse assembly.

The visualization and manipulation of proteins in neurons is widely used to deduce their functions. While every experimental approach has limitations, the concurrent knock-in and knockout of two different proteins can be especially challenging. To this end, we developed Hide-and-Seek genome editing, which allows the simultaneous visualization and knockout of proteins in neurons using Adeno-associated viral vectors and the CRISPR/Cas9 system. We demonstrate the efficacy and flexibility of this method for rapid, efficient, and simultaneous knock-in and knockout of proteins in vitro and in vivo, at the synapse, axon initial segment (AIS), nucleus, and mitochondria. Using Hide-and-Seek, we show that the scaffolding protein Gephyrin is required for the proper assembly of axo-axonic synapses at the AIS.