CRISPR/Cas9-based knockout screening revealed GSK3β as a regulator of axon initial segment structural plasticity

Abstract

The axon initial segment (AIS) undergoes structural plasticity to tune neuronal excitability, yet the underlying molecular mechanisms remain unclear. Here we developed an in vivo CRISPR/Cas9 knockout platform using an all-in-one triple-guide RNA vector introduced via electroporation and employed this approach to identify molecules that regulate developmental AIS shortening in the chicken nucleus magnocellularis. We targeted fourteen molecules associated with microtubules and found that knockout of either glycogen synthase kinase 3{beta} (GSK3{beta}) or Tau impaired the AIS shortening. Conversely, overexpression of a constitutively active form of GSK3{beta} facilitated the AIS shortening in vivo. This GSK3{beta}-induced shortening was reproduced in slice cultures and suppressed by microtubule stabilization. Together, these findings identify GSK3{beta}-dependent microtubule remodeling as a mechanism underlying developmental AIS shortening and establish an in vivo genetic approach for molecular screening in chick embryos.