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

• 成果类型: Article
• 署名作者: Ogawa, Yuki; Nguyen, Duc V. M.; Ogawa, Ayano; Rasband, Matthew N.
• 署名单位: Baylor College of Medicine
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10534
• DOI: 10.1073/pnas.2500726122
• 发表日期: 2025-08-12
• 关键词: receptor; mechanisms; neurons; cortex

摘要

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.